Biochemical characterisation of Pfj2, a Plasmodium falciparum heat shock protein 40 chaperone potentially involved in protein quality control in the endoplasmic reticulum
- Authors: Afolayan, Omolola Folasade
- Date: 2013
- Subjects: Plasmodium falciparum Endoplasmic reticulum Heat shock proteins Malaria , Mosquito-borne infectious disease
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3883 , http://hdl.handle.net/10962/d1001617
- Description: Plasmodium falciparum is a protozoan parasite that causes a severe form of malaria, a mosquito-borne infectious disease in humans. P. falciparum encodes a number of proteins to facilitate its life-cycle, including a type II heat shock protein 40 (Hsp40), Pfj2. Pfj2 shows a degree of homology to human ERdj5, a resident protein of the endoplasmic reticulum (ER) that promotes protein quality control by facilitating the degradation of misfolded proteins. The overall aim of this study was to further understand the function of Pfj2 in the P. falciparum cell by characterising it biochemically. A bioinformatic analysis of Pfj2 was carried out to enable the identification of a potential ER signal sequence and cleavage site. Furthermore, an analysis of Pfj2 protein sequence was performed to compare domain similarities and identities with typical type II Hsp40s namely, human ERdj5, S. cerevisiae Sis1, human Hsj1a and human DnaJB4. The method used included the insertion of the codon-optimised coding sequence for the processed ER form of Pfj2 into the prokaryotic expression vector, pQE30, to enable overproduction of a histidine-tagged protein. A 62 kDa His₆-Pfj2 was successfully expressed in Escherichia coli and purified using denaturing nickel affinity chromatography. ATPase assays were performed to determine the ability of His₆- Pfj2 to stimulate the chaperone activity of the ER Hsp70, also called immunoglobulin binding protein (BiP). Initial studies were conducted on readily available mammalian His₆-BiP as a control, which was shown to have an intrinsic activity of 12.07±3.92 nmolPi/min/mg. His₆- Pfj2 did not stimulate the ATPase activity of mammalian His₆-BiP, suggesting that it either could not act as a co-chaperone of mammalian His₆-BiP (specificity), or it required a misfolded substrate in the system. Therefore, ongoing studies are addressing the interaction of Pfj2 and misfolded substrates with P. falciparum BiP. The results of these studies will further our understanding of a poorly-studied parasite chaperone that represents a potential drug target for development of novel strategies for the control of a serious human disease
- Full Text:
- Date Issued: 2013
- Authors: Afolayan, Omolola Folasade
- Date: 2013
- Subjects: Plasmodium falciparum Endoplasmic reticulum Heat shock proteins Malaria , Mosquito-borne infectious disease
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3883 , http://hdl.handle.net/10962/d1001617
- Description: Plasmodium falciparum is a protozoan parasite that causes a severe form of malaria, a mosquito-borne infectious disease in humans. P. falciparum encodes a number of proteins to facilitate its life-cycle, including a type II heat shock protein 40 (Hsp40), Pfj2. Pfj2 shows a degree of homology to human ERdj5, a resident protein of the endoplasmic reticulum (ER) that promotes protein quality control by facilitating the degradation of misfolded proteins. The overall aim of this study was to further understand the function of Pfj2 in the P. falciparum cell by characterising it biochemically. A bioinformatic analysis of Pfj2 was carried out to enable the identification of a potential ER signal sequence and cleavage site. Furthermore, an analysis of Pfj2 protein sequence was performed to compare domain similarities and identities with typical type II Hsp40s namely, human ERdj5, S. cerevisiae Sis1, human Hsj1a and human DnaJB4. The method used included the insertion of the codon-optimised coding sequence for the processed ER form of Pfj2 into the prokaryotic expression vector, pQE30, to enable overproduction of a histidine-tagged protein. A 62 kDa His₆-Pfj2 was successfully expressed in Escherichia coli and purified using denaturing nickel affinity chromatography. ATPase assays were performed to determine the ability of His₆- Pfj2 to stimulate the chaperone activity of the ER Hsp70, also called immunoglobulin binding protein (BiP). Initial studies were conducted on readily available mammalian His₆-BiP as a control, which was shown to have an intrinsic activity of 12.07±3.92 nmolPi/min/mg. His₆- Pfj2 did not stimulate the ATPase activity of mammalian His₆-BiP, suggesting that it either could not act as a co-chaperone of mammalian His₆-BiP (specificity), or it required a misfolded substrate in the system. Therefore, ongoing studies are addressing the interaction of Pfj2 and misfolded substrates with P. falciparum BiP. The results of these studies will further our understanding of a poorly-studied parasite chaperone that represents a potential drug target for development of novel strategies for the control of a serious human disease
- Full Text:
- Date Issued: 2013
Polyunsaturated fatty acid metabolism and effects on colon cancer cell biology in vitro.
- Authors: Bulcao, Candice
- Date: 2013
- Subjects: Unsaturated fatty acids , Unsaturated fatty acids in human nutrition , Colon (Anatomy)-- Cancer , Cancer -- Nutritional aspects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4137 , http://hdl.handle.net/10962/d1016128
- Description: Colon cancer is a leading cause of cancer related deaths worldwide. Lifestyle factors such as diet and exercise have been implicated as important agents in colon cancer development and progression. Epidemiological, in vivo and in vitro studies have found that n-3 polyunsaturated fatty acids (PUFAs) reduce colon carcinoma. The role of n-6 PUFAs remains a controversial topic, with studies indicating both promoting and preventing capabilities published. In order to better understand the effects of PUFAs on colon carcinoma, it is important to have an understanding of how they will be broken down in the body. During this study, in silico metabolism of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) predicted the formation of hydroxy-, di-hydroxy- and epoxy-FAs. A gas chromatography-mass spectrometry method was developed and validated for the detection of these PUFAs and their cytochrome P450 (CYP) metabolites. A human liver microsomal system for the in vitro metabolism of EPA, DHA and AA was optimised in terms of microsomal and PUFA concentration. The system resulted in the metabolism of the positive control, lauric acid, to 12-hydroxy-lauric acid but was unable to metabolise the PUFAs of interest. EPA, DHA and AA reduced cell viability in the colon carcinoma cell lines SW480 and SW620 in the micromolar concentration range (25 – 200 μM). The CYP epoxidation metabolite of EPA, 17, 18-epoxyeicosatetraenoic acid (17, 18-EpETE) resulted in a significant reduction in SW480 cell viability relative to the parent compound at lower concentrations (25 and 50 μM). Annexin V apoptosis analysis revealed that EPA and 17, 18- EpETE did not result in apoptosis in SW480 cells at a concentration of 25 μM and over an incubation period of 24 hours. A significant reduction in reactive oxygen species production was seen in SW480 cells after incubation with 25 μM 17, 18-EpETE for 24 hours. EPA and 17, 18-EpETE were implicated in the reduction of colon cancer metastasis since they were able to reduce SW480 migration and anchorage independent cell growth. These results indicate that the dietary intake of EPA, DHA and AA may be beneficial to one’s health due to the negative effects that these PUFAs had on colon carcinoma. Future studies are needed to confirm these benefits and compare the effects of the PUFAs to their CYP-metabolites.
- Full Text:
- Date Issued: 2013
- Authors: Bulcao, Candice
- Date: 2013
- Subjects: Unsaturated fatty acids , Unsaturated fatty acids in human nutrition , Colon (Anatomy)-- Cancer , Cancer -- Nutritional aspects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4137 , http://hdl.handle.net/10962/d1016128
- Description: Colon cancer is a leading cause of cancer related deaths worldwide. Lifestyle factors such as diet and exercise have been implicated as important agents in colon cancer development and progression. Epidemiological, in vivo and in vitro studies have found that n-3 polyunsaturated fatty acids (PUFAs) reduce colon carcinoma. The role of n-6 PUFAs remains a controversial topic, with studies indicating both promoting and preventing capabilities published. In order to better understand the effects of PUFAs on colon carcinoma, it is important to have an understanding of how they will be broken down in the body. During this study, in silico metabolism of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) predicted the formation of hydroxy-, di-hydroxy- and epoxy-FAs. A gas chromatography-mass spectrometry method was developed and validated for the detection of these PUFAs and their cytochrome P450 (CYP) metabolites. A human liver microsomal system for the in vitro metabolism of EPA, DHA and AA was optimised in terms of microsomal and PUFA concentration. The system resulted in the metabolism of the positive control, lauric acid, to 12-hydroxy-lauric acid but was unable to metabolise the PUFAs of interest. EPA, DHA and AA reduced cell viability in the colon carcinoma cell lines SW480 and SW620 in the micromolar concentration range (25 – 200 μM). The CYP epoxidation metabolite of EPA, 17, 18-epoxyeicosatetraenoic acid (17, 18-EpETE) resulted in a significant reduction in SW480 cell viability relative to the parent compound at lower concentrations (25 and 50 μM). Annexin V apoptosis analysis revealed that EPA and 17, 18- EpETE did not result in apoptosis in SW480 cells at a concentration of 25 μM and over an incubation period of 24 hours. A significant reduction in reactive oxygen species production was seen in SW480 cells after incubation with 25 μM 17, 18-EpETE for 24 hours. EPA and 17, 18-EpETE were implicated in the reduction of colon cancer metastasis since they were able to reduce SW480 migration and anchorage independent cell growth. These results indicate that the dietary intake of EPA, DHA and AA may be beneficial to one’s health due to the negative effects that these PUFAs had on colon carcinoma. Future studies are needed to confirm these benefits and compare the effects of the PUFAs to their CYP-metabolites.
- Full Text:
- Date Issued: 2013
Investigation into the technical feasibility of biological treatment of precious metal refining wastewater
- Authors: Moore, Bronwyn Ann
- Date: 2013
- Subjects: Sewage -- Purification -- Biological treatment -- South Africa Sewage -- Purification -- Activated sludge process -- South Africa Water reuse -- South Africa Flotation -- South Africa Platinum mines and mining -- Waste disposal -- South Africa Platinum mines and mining -- Economic aspects -- South Africa Mine water -- Environmental aspects -- South Africa Platinum mines and mining -- Waste minimization -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3888 , http://hdl.handle.net/10962/d1002013
- Description: The hydrometallurgical refining of platinum group metals results in large volumes of liquid waste that requires suitable treatment before any disposal can be contemplated. The wastewater streams are characterized by extremes of pH, high inorganic ion content (such as chloride), significant residual metal loads and small amounts of entrained organic compounds. Historically these effluents were housed in evaporation reservoirs, however lack of space and growing water demands have led Anglo Platinum to consider treatment of these effluents. The aim of this study was to investigate whether biological wastewater treatment could produce water suitable for onsite reuse. Bench-scale activated sludge and anaerobic digestion for co-treatment of an acidic refinery waste stream with domestic wastewater were used to give preliminary data. Activated sludge showed better water treatment at lab scale in terms of removal efficiencies of ammonia (approximately 25%, cf. 20% in anaerobic digestion) and COD (70% cf. 43% in digestion) and greater robustness when biomass health was compared. Activated sludge was consequently selected for a pilot plant trial. The pilot plant was operated on-site and performed comparably with the bench-scale system, however challenges in the clarifier design led to losses of biomass and poor effluent quality (suspended solids washout). The pilot plant was unable to alter the pH of the feed, but a two week maturation period resulted in the pH increasing from 5.3 to 7.0. Tests on algal treatment as an alternative or follow-on unit operation to activated sludge showed it not to be a viable process. The activated sludge effluent was assessed for onsite reuse in flotation and it was found that there was no significant difference between its flotation performance and that of the process water currently used, indicating the effluent generated by the biological treatment system can be used successfully for flotation. Flotation is the method whereby minerals refining operations recover minerals of interest from ore through the addition of chemicals and aeration of the ore slurry. Target minerals adhere to the bubbles and can be removed from the process.
- Full Text:
- Date Issued: 2013
- Authors: Moore, Bronwyn Ann
- Date: 2013
- Subjects: Sewage -- Purification -- Biological treatment -- South Africa Sewage -- Purification -- Activated sludge process -- South Africa Water reuse -- South Africa Flotation -- South Africa Platinum mines and mining -- Waste disposal -- South Africa Platinum mines and mining -- Economic aspects -- South Africa Mine water -- Environmental aspects -- South Africa Platinum mines and mining -- Waste minimization -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3888 , http://hdl.handle.net/10962/d1002013
- Description: The hydrometallurgical refining of platinum group metals results in large volumes of liquid waste that requires suitable treatment before any disposal can be contemplated. The wastewater streams are characterized by extremes of pH, high inorganic ion content (such as chloride), significant residual metal loads and small amounts of entrained organic compounds. Historically these effluents were housed in evaporation reservoirs, however lack of space and growing water demands have led Anglo Platinum to consider treatment of these effluents. The aim of this study was to investigate whether biological wastewater treatment could produce water suitable for onsite reuse. Bench-scale activated sludge and anaerobic digestion for co-treatment of an acidic refinery waste stream with domestic wastewater were used to give preliminary data. Activated sludge showed better water treatment at lab scale in terms of removal efficiencies of ammonia (approximately 25%, cf. 20% in anaerobic digestion) and COD (70% cf. 43% in digestion) and greater robustness when biomass health was compared. Activated sludge was consequently selected for a pilot plant trial. The pilot plant was operated on-site and performed comparably with the bench-scale system, however challenges in the clarifier design led to losses of biomass and poor effluent quality (suspended solids washout). The pilot plant was unable to alter the pH of the feed, but a two week maturation period resulted in the pH increasing from 5.3 to 7.0. Tests on algal treatment as an alternative or follow-on unit operation to activated sludge showed it not to be a viable process. The activated sludge effluent was assessed for onsite reuse in flotation and it was found that there was no significant difference between its flotation performance and that of the process water currently used, indicating the effluent generated by the biological treatment system can be used successfully for flotation. Flotation is the method whereby minerals refining operations recover minerals of interest from ore through the addition of chemicals and aeration of the ore slurry. Target minerals adhere to the bubbles and can be removed from the process.
- Full Text:
- Date Issued: 2013
Nutrient supplementation and secondary metaolites in melanoma cells
- Authors: Stoll, Karin Elisabeth
- Date: 1994
- Subjects: Vitamin C -- Therapeutic use Cancer -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4049 , http://hdl.handle.net/10962/d1004110
- Description: Considerable interest exists with regard to the putative therapeutic role of ascorbic acid in various conditions. A condition which has received much attention is cancer, as it is reported that ascorbic acid may be a prophylactic against cancer development. However, the actual involvement of ascorbic acid, an oxidizing/reducing agent, in the development and progression of tumours is presently a subject of much speculation. This study initially addressed the effect of ascorbic acid supplementation over a nutritional concentration range (0 - 100 μg/ml) on the in vitro growth of non-malignant LLCMK and malignant B16 cells. Ascorbic acid supplementation of these two cell types resulted in an overall decrease in the growth of both types of cells. The actual inhibitory mechanism of ascorbic acid on cell growth was not clear. Further study attempted to define and explain a mechanism responsible for this effect. Ascorbic acid has a role in the maintenance of tissue integrity and host defences, thus providing a rational basis for examining its relationship to cancer. Ascorbic acid is lcnown to be essential for the structural integrity of the intercellular matrix of the cells, the latter being a complex aqueous gel containing, amongst other compounds, fats and prostaglandins. Fats and prostaglandins have diverse effects on. membrane stability, enzyme activity and secondary messengers within cells. Hence, this study investigated the effect of ascorbic acid supplementation on certain enzymes and secondary metabolites within the cells, which had the potential to be involved in the control of cell growth. Throughout this study, emphasis was placed on the Bl6 melanoma cells as ascorbic acid supplementation did not significantly affect levels of secondary metabolites within the non-malignant LLCMK cells. Ascorbic acid supplementation of the B16 cells resulted in significant increases in adenylate cyclase activity and cyclic adenosine monophosphate levels, witb a significant decrease in Bl6 cell growth in that particular experiment. As cyclic adenosine monophosphate has a regulatory role in the cell cycle, this study suggested that the inhibitory effect of ascorbic acid supplementation on cell growth was mediated tbrough a final effect provided by the second messenger, cyclic adenosine monophosphate. However, clarification of tbe mechanism of tbe effect of ascorbic acid on adenylate cyclase activity was required. Hence, a further study investigated prostaglandin E₂ levels, as tbese affect adenylate cyclase activity. Prostaglandin E₂ levels were also found to be inversely related to Bl6 cell growth with ascorbic acid supplementation. It thus appeared tbat adenylate cyclase activity was dependent on prostaglandin E₂ levels in the B16 cells, and further study showed that tbis was indeed the case. Here, higher levels of prostaglandin E₂ supplementation of the Bl6 cells inhibited cell growth significantly and also significantly increased adenylate cyclase activity. Arachidonic acid is the precursor of prostaglandin E₂. In the presence of ascorbic acid supplementation, the percentage arachidonic acid composition of the Bl6 cells was inversely correlated with cell growth. Hence, prostaglandin E₂ levels in ascorbic acid supplemented B16 cells appeared dependent on tbe amount of precursor present. This was confirmed when Bl6 cells were supplemented with arachidonic acid. The latter had an inhibitory effect on Bl6 cell growth and also stimulated prostaglandin E₂ production. The cause of tbe inverse relationship between B16 cell growth and arachidonic acid composition with ascorbic acid supplementation was furtber investigated and found to be dependent on tbe uptake of arachidonic acid and other essential fatty acids from tbe medium. The enzymes phospholipase A₂ delta-5 and delta-6-desaturase, and elongase which could influence arachidonic acid levels were not affected to any extent by ascorbic acid supplementation and therefore did not influence the inverse relationship between B16 cell growth and arachidonic acid. Hence, it can be concluded that the effect of ascorbic acid supplementation on the BI6 cells is mediated, in part at least, by cyclic adenosine monophosphate. However, this is not the result of a direct effect of ascorbic acid supplementation. The initial effect of ascorbic acid supplementation concerns fatty acid - in particular arachidonic acid - uptake from the medium, with subsequent cascade effects On secondary metabolites, ultimately affecting the cellular levels of cyclic adenosine monophosphate.
- Full Text:
- Date Issued: 1994
- Authors: Stoll, Karin Elisabeth
- Date: 1994
- Subjects: Vitamin C -- Therapeutic use Cancer -- Research
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4049 , http://hdl.handle.net/10962/d1004110
- Description: Considerable interest exists with regard to the putative therapeutic role of ascorbic acid in various conditions. A condition which has received much attention is cancer, as it is reported that ascorbic acid may be a prophylactic against cancer development. However, the actual involvement of ascorbic acid, an oxidizing/reducing agent, in the development and progression of tumours is presently a subject of much speculation. This study initially addressed the effect of ascorbic acid supplementation over a nutritional concentration range (0 - 100 μg/ml) on the in vitro growth of non-malignant LLCMK and malignant B16 cells. Ascorbic acid supplementation of these two cell types resulted in an overall decrease in the growth of both types of cells. The actual inhibitory mechanism of ascorbic acid on cell growth was not clear. Further study attempted to define and explain a mechanism responsible for this effect. Ascorbic acid has a role in the maintenance of tissue integrity and host defences, thus providing a rational basis for examining its relationship to cancer. Ascorbic acid is lcnown to be essential for the structural integrity of the intercellular matrix of the cells, the latter being a complex aqueous gel containing, amongst other compounds, fats and prostaglandins. Fats and prostaglandins have diverse effects on. membrane stability, enzyme activity and secondary messengers within cells. Hence, this study investigated the effect of ascorbic acid supplementation on certain enzymes and secondary metabolites within the cells, which had the potential to be involved in the control of cell growth. Throughout this study, emphasis was placed on the Bl6 melanoma cells as ascorbic acid supplementation did not significantly affect levels of secondary metabolites within the non-malignant LLCMK cells. Ascorbic acid supplementation of the B16 cells resulted in significant increases in adenylate cyclase activity and cyclic adenosine monophosphate levels, witb a significant decrease in Bl6 cell growth in that particular experiment. As cyclic adenosine monophosphate has a regulatory role in the cell cycle, this study suggested that the inhibitory effect of ascorbic acid supplementation on cell growth was mediated tbrough a final effect provided by the second messenger, cyclic adenosine monophosphate. However, clarification of tbe mechanism of tbe effect of ascorbic acid on adenylate cyclase activity was required. Hence, a further study investigated prostaglandin E₂ levels, as tbese affect adenylate cyclase activity. Prostaglandin E₂ levels were also found to be inversely related to Bl6 cell growth with ascorbic acid supplementation. It thus appeared tbat adenylate cyclase activity was dependent on prostaglandin E₂ levels in the B16 cells, and further study showed that tbis was indeed the case. Here, higher levels of prostaglandin E₂ supplementation of the Bl6 cells inhibited cell growth significantly and also significantly increased adenylate cyclase activity. Arachidonic acid is the precursor of prostaglandin E₂. In the presence of ascorbic acid supplementation, the percentage arachidonic acid composition of the Bl6 cells was inversely correlated with cell growth. Hence, prostaglandin E₂ levels in ascorbic acid supplemented B16 cells appeared dependent on tbe amount of precursor present. This was confirmed when Bl6 cells were supplemented with arachidonic acid. The latter had an inhibitory effect on Bl6 cell growth and also stimulated prostaglandin E₂ production. The cause of tbe inverse relationship between B16 cell growth and arachidonic acid composition with ascorbic acid supplementation was furtber investigated and found to be dependent on tbe uptake of arachidonic acid and other essential fatty acids from tbe medium. The enzymes phospholipase A₂ delta-5 and delta-6-desaturase, and elongase which could influence arachidonic acid levels were not affected to any extent by ascorbic acid supplementation and therefore did not influence the inverse relationship between B16 cell growth and arachidonic acid. Hence, it can be concluded that the effect of ascorbic acid supplementation on the BI6 cells is mediated, in part at least, by cyclic adenosine monophosphate. However, this is not the result of a direct effect of ascorbic acid supplementation. The initial effect of ascorbic acid supplementation concerns fatty acid - in particular arachidonic acid - uptake from the medium, with subsequent cascade effects On secondary metabolites, ultimately affecting the cellular levels of cyclic adenosine monophosphate.
- Full Text:
- Date Issued: 1994
Synthesis of silver nanoparticles and their role against a thiazolekinase enzyme from Plasmodium falciparum
- Yao, Jia
- Authors: Yao, Jia
- Date: 2014
- Subjects: Silver , Nanoparticles , Thiazoles , Plasmodium falciparum , Antimalarials , Malaria -- Chemotherapy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4168 , http://hdl.handle.net/10962/d1020894
- Description: Malaria, a mosquito-borne infectious disease, caused by the protozoan Plasmodium genus, is the greatest health challenges worldwide. The plasmodial vitamin B1 biosynthetic enzyme PfThzK diverges significantly, both structurally and functionally from its counterpart in higher eukaryotes, thereby making it particularly attractive as a biomedical target. In the present study, PfThzK was recombinantly produced as 6×His fusion protein in E. coli BL21, purified using nickel affinity chromatography and size exclusion chromatography resulting in 1.03% yield and specific activity 0.28 U/mg. The enzyme was found to be a monomer with a molecular mass of 34 kDa. Characterization of the PfThzK showed an optimum temperature and pH of 37°C and 7.5 respectively, and it is relatively stable (t₁/₂=2.66 h). Ag nanoparticles were synthesized by NaBH₄/tannic acid, and characterized by UV-vis spectroscopy and transmission electron microscopy. The morphologies of these Ag nanoparticles (in terms of size) synthesized by tannic acid appeared to be more controlled with the size of 7.06±2.41 nm, compared with those synthesized by NaBH₄, with the sized of 12.9±4.21 nm. The purified PfThzK was challenged with Ag NPs synthesized by tannic acid, and the results suggested that they competitively inhibited PfThzK (89 %) at low concentrations (5-10 μM) with a Ki = 6.45 μM.
- Full Text:
- Date Issued: 2014
- Authors: Yao, Jia
- Date: 2014
- Subjects: Silver , Nanoparticles , Thiazoles , Plasmodium falciparum , Antimalarials , Malaria -- Chemotherapy
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4168 , http://hdl.handle.net/10962/d1020894
- Description: Malaria, a mosquito-borne infectious disease, caused by the protozoan Plasmodium genus, is the greatest health challenges worldwide. The plasmodial vitamin B1 biosynthetic enzyme PfThzK diverges significantly, both structurally and functionally from its counterpart in higher eukaryotes, thereby making it particularly attractive as a biomedical target. In the present study, PfThzK was recombinantly produced as 6×His fusion protein in E. coli BL21, purified using nickel affinity chromatography and size exclusion chromatography resulting in 1.03% yield and specific activity 0.28 U/mg. The enzyme was found to be a monomer with a molecular mass of 34 kDa. Characterization of the PfThzK showed an optimum temperature and pH of 37°C and 7.5 respectively, and it is relatively stable (t₁/₂=2.66 h). Ag nanoparticles were synthesized by NaBH₄/tannic acid, and characterized by UV-vis spectroscopy and transmission electron microscopy. The morphologies of these Ag nanoparticles (in terms of size) synthesized by tannic acid appeared to be more controlled with the size of 7.06±2.41 nm, compared with those synthesized by NaBH₄, with the sized of 12.9±4.21 nm. The purified PfThzK was challenged with Ag NPs synthesized by tannic acid, and the results suggested that they competitively inhibited PfThzK (89 %) at low concentrations (5-10 μM) with a Ki = 6.45 μM.
- Full Text:
- Date Issued: 2014
Progestin receptor heterogeneity in a breast cancer cell line
- Authors: Levy, Anita Rochelle
- Date: 1995
- Subjects: Breast -- Cancer , Hormone receptors , Cancer cells -- Growth -- Regulation , Progesterone -- Receptors , Cellular control mechanisms
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4039 , http://hdl.handle.net/10962/d1004100 , Breast -- Cancer , Hormone receptors , Cancer cells -- Growth -- Regulation , Progesterone -- Receptors , Cellular control mechanisms
- Description: Anti-oestrogens act via the oestrogen receptor whether they compete with the hormone for binding to the receptor and therefore interfere with DNA binding or inhibit transcriptional activity. These receptors exist as a large 85 complex and/or a small 45 form on sucrose density gradients. High performance ion-exchange chromatography has confirmed that the oestrogen and progestin complex is present in various isoforms. Progestin receptor heterogeneity could be influenced by the presence of oestrogens and anti-oestrogens in the culture media of hormone-dependent neoplastic cells. Cell culture methods offer the opportunity to test effects of specified components in repeated experiments on a homogeneous population of cells. MCF-7 and T47-D human breast cancer cell lines were conditioned to grow in a serum-free environment. There was no difference in cell proliferation rates, nor in their oestrogen or progestin receptor levels when compared to the same cells grown in conventional media. Receptors were present mainly in the large molecular 85 form. Both the MCF-7 and T47-D breast cancer cells showed an increase in proliferation rate with the addition of oestrogen or diethylstilbestrol. There was a corresponding loss of progestin receptor levels and an alteration in the high performance ion-exchange isoforms. Flow cytometry confirmed differences in the S-phase components of the cells following exposure to oestrogens. The proliferation rates of the cell lines as well as their progestin receptor levels decreased when treated with tamoxifen or the hydroxylated tamoxifen. There were marked changes on high performance ion-exchange chromatography profiles. DNA ploidy and S-phase showed signs of toxicity and there was an increase in cellular debris. The MCF-7 and T47-D human breast cancer cell line retained response to antioestrogen saturation.
- Full Text:
- Date Issued: 1995
- Authors: Levy, Anita Rochelle
- Date: 1995
- Subjects: Breast -- Cancer , Hormone receptors , Cancer cells -- Growth -- Regulation , Progesterone -- Receptors , Cellular control mechanisms
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4039 , http://hdl.handle.net/10962/d1004100 , Breast -- Cancer , Hormone receptors , Cancer cells -- Growth -- Regulation , Progesterone -- Receptors , Cellular control mechanisms
- Description: Anti-oestrogens act via the oestrogen receptor whether they compete with the hormone for binding to the receptor and therefore interfere with DNA binding or inhibit transcriptional activity. These receptors exist as a large 85 complex and/or a small 45 form on sucrose density gradients. High performance ion-exchange chromatography has confirmed that the oestrogen and progestin complex is present in various isoforms. Progestin receptor heterogeneity could be influenced by the presence of oestrogens and anti-oestrogens in the culture media of hormone-dependent neoplastic cells. Cell culture methods offer the opportunity to test effects of specified components in repeated experiments on a homogeneous population of cells. MCF-7 and T47-D human breast cancer cell lines were conditioned to grow in a serum-free environment. There was no difference in cell proliferation rates, nor in their oestrogen or progestin receptor levels when compared to the same cells grown in conventional media. Receptors were present mainly in the large molecular 85 form. Both the MCF-7 and T47-D breast cancer cells showed an increase in proliferation rate with the addition of oestrogen or diethylstilbestrol. There was a corresponding loss of progestin receptor levels and an alteration in the high performance ion-exchange isoforms. Flow cytometry confirmed differences in the S-phase components of the cells following exposure to oestrogens. The proliferation rates of the cell lines as well as their progestin receptor levels decreased when treated with tamoxifen or the hydroxylated tamoxifen. There were marked changes on high performance ion-exchange chromatography profiles. DNA ploidy and S-phase showed signs of toxicity and there was an increase in cellular debris. The MCF-7 and T47-D human breast cancer cell line retained response to antioestrogen saturation.
- Full Text:
- Date Issued: 1995
Isolation and characterization of genes encoding heat shock protein 70s (hsp 70s) from two species of the coelacanth, Latimeria chalumnae and Latimeria menadoensis
- Modisakeng, Keoagile William
- Authors: Modisakeng, Keoagile William
- Date: 2007
- Subjects: Coelacanth Coelacanth -- Genetics Heat shock proteins Molecular chaperones Proteins -- Analysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3971 , http://hdl.handle.net/10962/d1004030
- Description: The extant coelacanths have a close resemblance to the coelacanth fossil records dating back to 230mya. Like their predecessors, the extant coelacanths inhabit rocky caves at a depth of 100-300m below sea level. In the Comoros, the water temperature at these depths is estimated to fluctuate between 14-20°C. High-level adaptation to these environment and lack of competition are thought to have led to the morphological uniformity and slow change throughout the history of the coelacanths. Under stress conditions, proteins unfold or misfold leading to the formation of aggregates. Molecular chaperones facilitate the correct folding of other proteins so that they can attain a stable tertiary structure. In addition, molecular chaperones aid the refolding of denatured proteins and the degradation of terminally misfolded protein after cellular stress. Heat shock proteins form one of the major classes of molecular chaperones. Here we show that, despite high-level adaptation to a unique habitat and slow change, the genome of the coelacanth encodes the major and highly conserved molecular chaperone, Hsp70. Latimeria menadoensis and Latimeria chalumnae contain intronless hsp70 genes encoding Hsp70 proteins archetypal of known Hsp70s. Based on the coelacanth codon usage, we have shown that bacterial protein expression systems, particularly Escherichia coli, may not be appropriate for the overproduction of coelacanth Hsp70s and coelacanth proteins in general. Also interesting, was the discovery that like the rat Hsc70, the L. menadoensis Hsp70 could not reverse thermal sensitivity in a temperate sensitive E. coli DnaK mutant strain, BB2362. We also report the successful isolation of a 1.2 kb region of L. menadoensis hsp70 upstream regulatory region. This region contain three putative heat shock elements, a TATA- box and two CAAT-boxes. This regulatory region resembled the Xenopus, mouse, and particularly tilapia hsp70 promoters, all of which have been shown to drive the expression of reporter genes in a heat dependent manner. Taken together, this data is the first to strongly suggest an inducible Hsp70-base cytoprotection mechanism in the coelacanth. It further provides basis to formulate testable predictions about the regulation, structure and function of Hsp70s in the living fossil, Latimeria.
- Full Text:
- Date Issued: 2007
- Authors: Modisakeng, Keoagile William
- Date: 2007
- Subjects: Coelacanth Coelacanth -- Genetics Heat shock proteins Molecular chaperones Proteins -- Analysis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3971 , http://hdl.handle.net/10962/d1004030
- Description: The extant coelacanths have a close resemblance to the coelacanth fossil records dating back to 230mya. Like their predecessors, the extant coelacanths inhabit rocky caves at a depth of 100-300m below sea level. In the Comoros, the water temperature at these depths is estimated to fluctuate between 14-20°C. High-level adaptation to these environment and lack of competition are thought to have led to the morphological uniformity and slow change throughout the history of the coelacanths. Under stress conditions, proteins unfold or misfold leading to the formation of aggregates. Molecular chaperones facilitate the correct folding of other proteins so that they can attain a stable tertiary structure. In addition, molecular chaperones aid the refolding of denatured proteins and the degradation of terminally misfolded protein after cellular stress. Heat shock proteins form one of the major classes of molecular chaperones. Here we show that, despite high-level adaptation to a unique habitat and slow change, the genome of the coelacanth encodes the major and highly conserved molecular chaperone, Hsp70. Latimeria menadoensis and Latimeria chalumnae contain intronless hsp70 genes encoding Hsp70 proteins archetypal of known Hsp70s. Based on the coelacanth codon usage, we have shown that bacterial protein expression systems, particularly Escherichia coli, may not be appropriate for the overproduction of coelacanth Hsp70s and coelacanth proteins in general. Also interesting, was the discovery that like the rat Hsc70, the L. menadoensis Hsp70 could not reverse thermal sensitivity in a temperate sensitive E. coli DnaK mutant strain, BB2362. We also report the successful isolation of a 1.2 kb region of L. menadoensis hsp70 upstream regulatory region. This region contain three putative heat shock elements, a TATA- box and two CAAT-boxes. This regulatory region resembled the Xenopus, mouse, and particularly tilapia hsp70 promoters, all of which have been shown to drive the expression of reporter genes in a heat dependent manner. Taken together, this data is the first to strongly suggest an inducible Hsp70-base cytoprotection mechanism in the coelacanth. It further provides basis to formulate testable predictions about the regulation, structure and function of Hsp70s in the living fossil, Latimeria.
- Full Text:
- Date Issued: 2007
Molecular and biochemical analysis of the diet of the black rhinoceros
- Authors: Kgopa, Ananias Hodi
- Date: 2009 , 2013-07-15
- Subjects: Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- South Africa -- Eastern Cape -- Analysis , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants , Plant ecology -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4064 , http://hdl.handle.net/10962/d1004721 , Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- South Africa -- Eastern Cape -- Analysis , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants , Plant ecology -- South Africa -- Eastern Cape
- Description: The black rhinoceros, Diceros bicornis, is listed as critically endangered. The black rhinoceros population in the Great Fish River Reserve (GFRR) has increased steadily to a current estimate of one hundred animals since the re-introduction of four animals in 1986. In an effort to contribute to the effective conservation and management of this species, dietary composition was studied in the medium Portulcaria thicket vegetation of the GFRR. This study used a molecular approach to determine the diet of the black rhinoceros of the GFRR by sequencing the ribulose bisphosphate carboxylase large subunit (rbcL) gene in plants and dung. Twenty-three plant species were collected from the reserve, and 802 bp of the rbcL gene were sequenced. These plant sequences were used as a reference database for the identification of plant sequences generated from black rhinoceros dung. Initial studies investigated the amplification, cloning and sequencing of DNA extracted from the dung samples which indicated the viability of the molecular approach. Thereafter, dung generated rbcL DNA was analyzed by GS FLX sequencing. Of the plant sequences identified by comparison to the GenBank database, Carissa bispinosa was the most prevalent. The study further characterized the antioxidant activities and phenolic content of plants eaten by the black rhinoceros using four different assays. Phyllanthus verrucosus, Putterlickia pyracantha, Maytenus capitata, Euclea undulata and Ozoroa mucrunata consistently had high antioxidant activities when assayed against 2,2-azinobis (3-ethyl benzothiazolium-6-sulfonic acid) (ABTSʹ⁺), 2,2-diphenyl-1-picrylhydrazyl (DPPHʹ), and ferric reducing antioxidant potentials (FRAP) and phenolic content when evaluated using the Folin-Ciocalteu assay. The majority of plants investigated showed low antioxidant potentials and low phenolic content. The extent to which antioxidants influenced the browse selection by the black rhinoceros remains inconclusive.
- Full Text:
- Date Issued: 2009
- Authors: Kgopa, Ananias Hodi
- Date: 2009 , 2013-07-15
- Subjects: Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- South Africa -- Eastern Cape -- Analysis , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants , Plant ecology -- South Africa -- Eastern Cape
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4064 , http://hdl.handle.net/10962/d1004721 , Black rhinoceros -- South Africa -- Eastern Cape , Browse (Animal food) -- South Africa -- Eastern Cape -- Analysis , Black rhinoceros -- Manure -- Analysis , Phenols , Antioxidants , Plant ecology -- South Africa -- Eastern Cape
- Description: The black rhinoceros, Diceros bicornis, is listed as critically endangered. The black rhinoceros population in the Great Fish River Reserve (GFRR) has increased steadily to a current estimate of one hundred animals since the re-introduction of four animals in 1986. In an effort to contribute to the effective conservation and management of this species, dietary composition was studied in the medium Portulcaria thicket vegetation of the GFRR. This study used a molecular approach to determine the diet of the black rhinoceros of the GFRR by sequencing the ribulose bisphosphate carboxylase large subunit (rbcL) gene in plants and dung. Twenty-three plant species were collected from the reserve, and 802 bp of the rbcL gene were sequenced. These plant sequences were used as a reference database for the identification of plant sequences generated from black rhinoceros dung. Initial studies investigated the amplification, cloning and sequencing of DNA extracted from the dung samples which indicated the viability of the molecular approach. Thereafter, dung generated rbcL DNA was analyzed by GS FLX sequencing. Of the plant sequences identified by comparison to the GenBank database, Carissa bispinosa was the most prevalent. The study further characterized the antioxidant activities and phenolic content of plants eaten by the black rhinoceros using four different assays. Phyllanthus verrucosus, Putterlickia pyracantha, Maytenus capitata, Euclea undulata and Ozoroa mucrunata consistently had high antioxidant activities when assayed against 2,2-azinobis (3-ethyl benzothiazolium-6-sulfonic acid) (ABTSʹ⁺), 2,2-diphenyl-1-picrylhydrazyl (DPPHʹ), and ferric reducing antioxidant potentials (FRAP) and phenolic content when evaluated using the Folin-Ciocalteu assay. The majority of plants investigated showed low antioxidant potentials and low phenolic content. The extent to which antioxidants influenced the browse selection by the black rhinoceros remains inconclusive.
- Full Text:
- Date Issued: 2009
Development of integrated biological processing for the biodesalination of sulphate- and metal-rich wastewaters
- Authors: Boshoff, Genevieve Ann
- Date: 1999
- Subjects: Sewage -- Purification -- Biological treatment Sulfates Mineral industries -- Environmental aspects
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3899 , http://hdl.handle.net/10962/d1003958
- Description: The substantial pollution threat to the South African environment from acid mine drainage (AMD) effluents has been well documented. Due to the juvenile nature of acidity in these flows, any remediation strategies implemented will need to function effectively and at low cost for long periods of time. The widespread use of sulphate reducing biological systems for the treatment of such effluents, and in particular large volume flows, has been limited. The supply of inexpensive electron donor and carbon sources, as well as appropriate reactor designs capable of handling large volume flows, have been identified as among the principal factors limiting development of this technology. The broad aim of the research programme reported here was to undertake an evaluation of the feasibility of an algal-bacterial integrated ponding system for the treatment of AMD, and the waste stabilisation pond (WSP) as an appropriate reactor design for this application. The study attempted to demonstrate the feasibility of individual unit operations in a proposed process train using complex organic carbon serving as the electron donor source for the sulphate reducing bacteria (SRB). Studies were undertaken as laboratory and pilot-scale investigations. Tannery effluent was shown to be a functional carbon source for biological sulphate reduction, with effective removal of sulphate and organics being recorded. In turn, the use of biological sulphate reduction for the treatment of tannery effluent was demonstrated. Algal biomass was shown in laboratory studies to function as an effective carbon source for biological sulphate reduction. It is known that micro-algae produce large quantities of photosynthate which is released to the growth medium under conditions of physiological stress. The potential for the use of photosynthate production in high rate algal ponding systems and its manipulation and use as a sustainable carbon source for sulphate reduction was investigated. Growth of a mixed culture of Dunaliella under conditions of light, temperature and salinity stress demonstrated production of large quantities of organic carbon. However, growth was inhibited at high temperatures. An elevation of salinity levels led to a decrease in growth of Dunaliella, but to increased organic carbon production. Spirulina spp., on the other hand, grew well at higher temperatures but showed the highest organic carbon production, and release to the medium, under low light conditions. These results led to a proposed process for the integration of algal ponding into an integrated system for the treatment of AMD. The algal biomass may be fed into the anaerobic digester as a carbon source, or it may be passed into a High Rate Algal Pond (HRAP) where it is stressed to enhance the organic carbon content. This can then be fed into the anaerobic digester as a carbon source. The impact of high levels of sulphide in the water feeding to the algal growth compartment was investigated. Spirulina spp. isolated from a tannery waste stabilisation pond was shown to be a sulphidophilic strain of cyanobacterium, capable of being adapted to high concentrations of sulphide. Dunaliella salina on the other hand was less tolerant. These results demonstrated the practical use of algal biomass providing an oxygen-rich cap for odour control on the surface of the facultative pond as well for the secondary treatment of sulphide-rich overflow to the High Rate Algal Pond. The ability of micro-algae to elevate the pH of their surrounding environment was evaluated as a functional precipitant and neutralisation reagent for acidic metal containing wastewater. Spirulina spp. was shown to perform effectively. D. salina was less functional in this environment. Anacystis spp. was effective in elevating the pH of a defined medium as well as a zinc-rich effluent. These results indicated the practicality of a neutralising function for algal ponds in the treatment of AMD. Metal removal in the system was found to be a combined function of sulphide precipitation, removal by binding to micro-algal biomass and extracellular polymeric substances. The feasibility of waste stabilisation ponding technology use for the treatment of large volume AMD effluents was provisionally demonstrated. It was shown that complex carbon sources would be used as efficient electron donors for sulphate reduction. The integration of algal ponding into the system provides for the generation of a sustainable carbon source, odour control with the recycling of oxygen-rich water onto the top of the facultative pond, secondary treatment of the anaerobic digester overflow, and the neutralisation of the incoming acidic effluents and removal of heavy metals. Integration of the individual unit operations, the feasibility of which has been provisionally demonstrated in this study, into a continuous process train is being investigated in follow-upstudies.
- Full Text:
- Date Issued: 1999
- Authors: Boshoff, Genevieve Ann
- Date: 1999
- Subjects: Sewage -- Purification -- Biological treatment Sulfates Mineral industries -- Environmental aspects
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3899 , http://hdl.handle.net/10962/d1003958
- Description: The substantial pollution threat to the South African environment from acid mine drainage (AMD) effluents has been well documented. Due to the juvenile nature of acidity in these flows, any remediation strategies implemented will need to function effectively and at low cost for long periods of time. The widespread use of sulphate reducing biological systems for the treatment of such effluents, and in particular large volume flows, has been limited. The supply of inexpensive electron donor and carbon sources, as well as appropriate reactor designs capable of handling large volume flows, have been identified as among the principal factors limiting development of this technology. The broad aim of the research programme reported here was to undertake an evaluation of the feasibility of an algal-bacterial integrated ponding system for the treatment of AMD, and the waste stabilisation pond (WSP) as an appropriate reactor design for this application. The study attempted to demonstrate the feasibility of individual unit operations in a proposed process train using complex organic carbon serving as the electron donor source for the sulphate reducing bacteria (SRB). Studies were undertaken as laboratory and pilot-scale investigations. Tannery effluent was shown to be a functional carbon source for biological sulphate reduction, with effective removal of sulphate and organics being recorded. In turn, the use of biological sulphate reduction for the treatment of tannery effluent was demonstrated. Algal biomass was shown in laboratory studies to function as an effective carbon source for biological sulphate reduction. It is known that micro-algae produce large quantities of photosynthate which is released to the growth medium under conditions of physiological stress. The potential for the use of photosynthate production in high rate algal ponding systems and its manipulation and use as a sustainable carbon source for sulphate reduction was investigated. Growth of a mixed culture of Dunaliella under conditions of light, temperature and salinity stress demonstrated production of large quantities of organic carbon. However, growth was inhibited at high temperatures. An elevation of salinity levels led to a decrease in growth of Dunaliella, but to increased organic carbon production. Spirulina spp., on the other hand, grew well at higher temperatures but showed the highest organic carbon production, and release to the medium, under low light conditions. These results led to a proposed process for the integration of algal ponding into an integrated system for the treatment of AMD. The algal biomass may be fed into the anaerobic digester as a carbon source, or it may be passed into a High Rate Algal Pond (HRAP) where it is stressed to enhance the organic carbon content. This can then be fed into the anaerobic digester as a carbon source. The impact of high levels of sulphide in the water feeding to the algal growth compartment was investigated. Spirulina spp. isolated from a tannery waste stabilisation pond was shown to be a sulphidophilic strain of cyanobacterium, capable of being adapted to high concentrations of sulphide. Dunaliella salina on the other hand was less tolerant. These results demonstrated the practical use of algal biomass providing an oxygen-rich cap for odour control on the surface of the facultative pond as well for the secondary treatment of sulphide-rich overflow to the High Rate Algal Pond. The ability of micro-algae to elevate the pH of their surrounding environment was evaluated as a functional precipitant and neutralisation reagent for acidic metal containing wastewater. Spirulina spp. was shown to perform effectively. D. salina was less functional in this environment. Anacystis spp. was effective in elevating the pH of a defined medium as well as a zinc-rich effluent. These results indicated the practicality of a neutralising function for algal ponds in the treatment of AMD. Metal removal in the system was found to be a combined function of sulphide precipitation, removal by binding to micro-algal biomass and extracellular polymeric substances. The feasibility of waste stabilisation ponding technology use for the treatment of large volume AMD effluents was provisionally demonstrated. It was shown that complex carbon sources would be used as efficient electron donors for sulphate reduction. The integration of algal ponding into the system provides for the generation of a sustainable carbon source, odour control with the recycling of oxygen-rich water onto the top of the facultative pond, secondary treatment of the anaerobic digester overflow, and the neutralisation of the incoming acidic effluents and removal of heavy metals. Integration of the individual unit operations, the feasibility of which has been provisionally demonstrated in this study, into a continuous process train is being investigated in follow-upstudies.
- Full Text:
- Date Issued: 1999
Genetic variation within and between some rare and common taxa of Cape Proteaceae and the implications for their conservation
- Authors: Brown, Susan Ann
- Date: 2000
- Subjects: Proteaceae -- South Africa Nature conservation -- South Africa Plant conservation -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3905 , http://hdl.handle.net/10962/d1003964
- Full Text:
- Date Issued: 2000
- Authors: Brown, Susan Ann
- Date: 2000
- Subjects: Proteaceae -- South Africa Nature conservation -- South Africa Plant conservation -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3905 , http://hdl.handle.net/10962/d1003964
- Full Text:
- Date Issued: 2000
The culture of Dunaliella salina and the production of β-carotene in tannery effluents
- Authors: Laubscher, Richard Keith
- Date: 1992
- Subjects: Dunaliella , Carotenes , Tanneries -- Waste disposal , Recycling (Waste, etc.)
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4055 , http://hdl.handle.net/10962/d1004116 , Dunaliella , Carotenes , Tanneries -- Waste disposal , Recycling (Waste, etc.)
- Description: The problems of waste disposal in the tanning industry are unique in that the effluents are highly saline, have a high organic loading and contain heavy metals. Methods are available for the safe treatment and disposal of the latter two components, but the saline component requires the expensive outlay of evaporation ponds. This study has identified a possible use for the saline effluents, turning a problematic waste product into a potentially valuable by-product. A range of tannery effluents were identified and tested for their suitability for the mass cultivation of Dunaliella salina (bardawil strain). The bardawil strain was preferred over a local isolate because of its higher production of β-carotene. Ponded tannery effluents and combined processes effluent proved unsuitable for realistic propagation of the alga. Anaerobic digestion of combined processes effluent did not improve its suitability significantly. Anaerobic digestion of hide-soak effluent may remove persistent antimicrobial agents which influence algal growth, but its contribution to enhancing algal growth is equivocal. Undigested hide-soak effluent lacking in persistent antimicrobial agents was found to be an ideal culture medium, as no additional nutrients needed to be added. Significantly higher biomass was obtained in this effluent compared to chemically defined media. Induction of β-carotene was achieved in nitrogen-deficient defined media after culture in tannery effluent. This suggests that a two-stage system using hide-soak effluent for cell propagation and nitrogen deficient media for β-carotene induction, could be possible for the mass cultivation of D. salina for β-carotene production.
- Full Text:
- Date Issued: 1992
- Authors: Laubscher, Richard Keith
- Date: 1992
- Subjects: Dunaliella , Carotenes , Tanneries -- Waste disposal , Recycling (Waste, etc.)
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4055 , http://hdl.handle.net/10962/d1004116 , Dunaliella , Carotenes , Tanneries -- Waste disposal , Recycling (Waste, etc.)
- Description: The problems of waste disposal in the tanning industry are unique in that the effluents are highly saline, have a high organic loading and contain heavy metals. Methods are available for the safe treatment and disposal of the latter two components, but the saline component requires the expensive outlay of evaporation ponds. This study has identified a possible use for the saline effluents, turning a problematic waste product into a potentially valuable by-product. A range of tannery effluents were identified and tested for their suitability for the mass cultivation of Dunaliella salina (bardawil strain). The bardawil strain was preferred over a local isolate because of its higher production of β-carotene. Ponded tannery effluents and combined processes effluent proved unsuitable for realistic propagation of the alga. Anaerobic digestion of combined processes effluent did not improve its suitability significantly. Anaerobic digestion of hide-soak effluent may remove persistent antimicrobial agents which influence algal growth, but its contribution to enhancing algal growth is equivocal. Undigested hide-soak effluent lacking in persistent antimicrobial agents was found to be an ideal culture medium, as no additional nutrients needed to be added. Significantly higher biomass was obtained in this effluent compared to chemically defined media. Induction of β-carotene was achieved in nitrogen-deficient defined media after culture in tannery effluent. This suggests that a two-stage system using hide-soak effluent for cell propagation and nitrogen deficient media for β-carotene induction, could be possible for the mass cultivation of D. salina for β-carotene production.
- Full Text:
- Date Issued: 1992
Isolation, purification and characterization of a 'factor' from Fusarium oxysporum responsible for platinum nanoparticle formation
- Authors: Govender, Yageshni
- Date: 2008
- Subjects: Nanoparticles , Platinum , Fusarium oxysporum , Fungi , Hydragenase
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3923 , http://hdl.handle.net/10962/d1003982 , Nanoparticles , Platinum , Fusarium oxysporum , Fungi , Hydragenase
- Description: Nanoparticles are microscopic particles in the nanometre range of between 1-100 nm. A wide variety of metal nanoparticles have been found to be produced by prokaryotic and eukaryotic organisms including several fungal species, when exposed to solutions containing metal salts. Previous studies have suggested that this bioreduction of metal particles may occur via an active reductase/hydrogenase enzyme process where H2 is the electron donor and positively charged platinum species act as the electron acceptors becoming reduced to a neutral metal nanoparticle. In view of this on going research, the current study investigated the “factors” in the fungus Fusarium oxysporum which were responsible for platinum nanoparticle formation. The fungus F.oxysporum was used in this study as it has been previously shown to produce a variety of nanoparticles including gold and silver. During exposure of the biomass to H2PtCl6 the initial response to the platinum salts was metal internalisation and subsequent reduction of H2PtCI6 to produce platinum nanoparticles. The observed localization and distribution of platinum precipitates provided some evidence for a hydrogenase mediated bioreduction of platinum salts to produce nanoparticles. Factors secreted by the fungus into the extracellular fluids, were shown to be responsible for platinum nanoparticle formation. From the identification, purification and characterisation studies it was concluded that a hydrogenase and other “factors” were responsible for platinum nanoparticle formation in F.oxysporum. Purification of the hydrogenase by freeze-drying and Sephacryl S200 size exclusion- ion exchange chromatography revealed the enzyme to be a dimer with a 29.4 and 44.5 kDa when analysed by a 10 % SDS-PAGE. Characterisation of the enzyme revealed optimal activity at a pH of 7.5 and temperature of 38 °C while it exhibited a poor thermal stability with a half life of 36 minutes. The kinetic parameters Vmax and Km were 3.16 U ml-1 and 3.64 mM respectively. The purified hydrogenase was used in subsequent experiments for the reduction of platinum salts, H2PtCl6 and PtCl2. the results indicated an over 90 % reduction of the platinum salts and TEM micrographs indicated the production of platinum nanoparticles under the various experimental conditions.
- Full Text:
- Date Issued: 2008
- Authors: Govender, Yageshni
- Date: 2008
- Subjects: Nanoparticles , Platinum , Fusarium oxysporum , Fungi , Hydragenase
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3923 , http://hdl.handle.net/10962/d1003982 , Nanoparticles , Platinum , Fusarium oxysporum , Fungi , Hydragenase
- Description: Nanoparticles are microscopic particles in the nanometre range of between 1-100 nm. A wide variety of metal nanoparticles have been found to be produced by prokaryotic and eukaryotic organisms including several fungal species, when exposed to solutions containing metal salts. Previous studies have suggested that this bioreduction of metal particles may occur via an active reductase/hydrogenase enzyme process where H2 is the electron donor and positively charged platinum species act as the electron acceptors becoming reduced to a neutral metal nanoparticle. In view of this on going research, the current study investigated the “factors” in the fungus Fusarium oxysporum which were responsible for platinum nanoparticle formation. The fungus F.oxysporum was used in this study as it has been previously shown to produce a variety of nanoparticles including gold and silver. During exposure of the biomass to H2PtCl6 the initial response to the platinum salts was metal internalisation and subsequent reduction of H2PtCI6 to produce platinum nanoparticles. The observed localization and distribution of platinum precipitates provided some evidence for a hydrogenase mediated bioreduction of platinum salts to produce nanoparticles. Factors secreted by the fungus into the extracellular fluids, were shown to be responsible for platinum nanoparticle formation. From the identification, purification and characterisation studies it was concluded that a hydrogenase and other “factors” were responsible for platinum nanoparticle formation in F.oxysporum. Purification of the hydrogenase by freeze-drying and Sephacryl S200 size exclusion- ion exchange chromatography revealed the enzyme to be a dimer with a 29.4 and 44.5 kDa when analysed by a 10 % SDS-PAGE. Characterisation of the enzyme revealed optimal activity at a pH of 7.5 and temperature of 38 °C while it exhibited a poor thermal stability with a half life of 36 minutes. The kinetic parameters Vmax and Km were 3.16 U ml-1 and 3.64 mM respectively. The purified hydrogenase was used in subsequent experiments for the reduction of platinum salts, H2PtCl6 and PtCl2. the results indicated an over 90 % reduction of the platinum salts and TEM micrographs indicated the production of platinum nanoparticles under the various experimental conditions.
- Full Text:
- Date Issued: 2008
Investigating the role of Hsp90 and LRP1 in FN matrix dynamics
- Authors: Boël, Natasha Marie-Eraine
- Date: 2016
- Subjects: Extracellular matrix , Molecular chaperones , Heat shock proteins , Cancer , Fibronectins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/2713 , vital:20319
- Description: Fibronectin (FN), a matrix protein responsible for regulating processes including migration and differentiation, is secreted as a soluble dimer which is assembled into an insoluble extracellular matrix. The dynamics of FN matrix assembly and degradation play a large role in cell migration and invasion contributing to the metastatic potential of cancer cells. Previous studies from our group have shown the direct binding of Hsp90 and FN in vitro and that inhibition of Hsp90 with novobiocin (NOV) caused internalisation of the FN matrix. However, the receptor mediating this internalisation is currently unknown. Low density lipoprotein 1 (LRP1) is a likely candidate as it is a ubiquitous receptor responsible for regulating internalisation of diverse ligands and is known to bind both Hsp90 and FN. We used wild type and knockout LRP1 cell lines to study the endocytosis of FN via this receptor. Here, we demonstrate that LRP1-deficient cells accumulated greatly increased levels of FN and were found to be less sensitive to pharmacological inhibition of Hsp90 by NOV. LRP1-expressing MEF-1 and Hs578T breast cancer cells experienced an increase in total FN in response to NOV, at concentrations below the EC50 value, followed by a dose-dependent loss of FN. We attributed greater FN levels to a loss of extracellular FN matrix coupled with increased internalisation of FN. Cell-surface biotinylation and DOC assays showed that loss of extracellular FN was specific to LRP1-expressing MEF-1 cells. Furthermore, we demonstrate that the loss of extracellular FN is not affected by changes in FN mRNA levels as determined by qRT-PCR, and that treatment with NOV resulted in the accelerated degradation of FN in the presence of cycloheximide. Immunoprecipitation studies reveal a putative complex exists between FN, Hsp90 and LRP1 in both cancer and non-cancer cells which is not perturbed by NOV. Western analyses revealed increased proteolytic processing of LRP1 in response to NOV which we proposed, based on literature, to modulate signalling pathways as a potential mechanism for regulating FN turnover. Moreover, using wound healing assays we identified increased migration to be one of the consequences associated with loss of extracellular FN by Hsp90 inhibition but only in cells containing LRP1. In summary, this study provides new insights into the Hsp90-LRP1 mediated loss of FN matrix and also reveals for the first time the functional consequence related to FN turnover by NOV was an increase in migration in LRP1-expressing cells.
- Full Text:
- Date Issued: 2016
- Authors: Boël, Natasha Marie-Eraine
- Date: 2016
- Subjects: Extracellular matrix , Molecular chaperones , Heat shock proteins , Cancer , Fibronectins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/2713 , vital:20319
- Description: Fibronectin (FN), a matrix protein responsible for regulating processes including migration and differentiation, is secreted as a soluble dimer which is assembled into an insoluble extracellular matrix. The dynamics of FN matrix assembly and degradation play a large role in cell migration and invasion contributing to the metastatic potential of cancer cells. Previous studies from our group have shown the direct binding of Hsp90 and FN in vitro and that inhibition of Hsp90 with novobiocin (NOV) caused internalisation of the FN matrix. However, the receptor mediating this internalisation is currently unknown. Low density lipoprotein 1 (LRP1) is a likely candidate as it is a ubiquitous receptor responsible for regulating internalisation of diverse ligands and is known to bind both Hsp90 and FN. We used wild type and knockout LRP1 cell lines to study the endocytosis of FN via this receptor. Here, we demonstrate that LRP1-deficient cells accumulated greatly increased levels of FN and were found to be less sensitive to pharmacological inhibition of Hsp90 by NOV. LRP1-expressing MEF-1 and Hs578T breast cancer cells experienced an increase in total FN in response to NOV, at concentrations below the EC50 value, followed by a dose-dependent loss of FN. We attributed greater FN levels to a loss of extracellular FN matrix coupled with increased internalisation of FN. Cell-surface biotinylation and DOC assays showed that loss of extracellular FN was specific to LRP1-expressing MEF-1 cells. Furthermore, we demonstrate that the loss of extracellular FN is not affected by changes in FN mRNA levels as determined by qRT-PCR, and that treatment with NOV resulted in the accelerated degradation of FN in the presence of cycloheximide. Immunoprecipitation studies reveal a putative complex exists between FN, Hsp90 and LRP1 in both cancer and non-cancer cells which is not perturbed by NOV. Western analyses revealed increased proteolytic processing of LRP1 in response to NOV which we proposed, based on literature, to modulate signalling pathways as a potential mechanism for regulating FN turnover. Moreover, using wound healing assays we identified increased migration to be one of the consequences associated with loss of extracellular FN by Hsp90 inhibition but only in cells containing LRP1. In summary, this study provides new insights into the Hsp90-LRP1 mediated loss of FN matrix and also reveals for the first time the functional consequence related to FN turnover by NOV was an increase in migration in LRP1-expressing cells.
- Full Text:
- Date Issued: 2016
Removal of lead from solution by the non-viable biomass of the water fern Azolla filiculoides
- Authors: Sanyahumbi, Douglas
- Date: 1999
- Subjects: Azolla , Heavy metals -- Absorption and adsorption , Lead , Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3901 , http://hdl.handle.net/10962/d1003960 , Azolla , Heavy metals -- Absorption and adsorption , Lead , Water -- Purification -- Biological treatment
- Description: The removal of lead from aqueous solution and lead-acid battery manufacturing waste-water by the non-viable biomass of the water fern Azolla filiculoides was investigated in both batch and column reactors. The maximum lead uptake by the Azolla biomass at a pH value of approximately 5, was found to be 100 mg lead/g biomass from aqueous solution. Lead removal varied from 30% of the initial lead concentration at pH 1.5 to approximately 95% at pH values of 3.5 and 5.6. Lead removal from aqueous solution decreased to 30% of the initial lead concentration if the lead concentration was initially over 400 mg/l. At initial lead concentrations of less than 400 mg/l, percentage lead removal was found to be over 90% of the initial lead concentration. Lead removal remained at approximately 90% between 10°C and 50°C. Biomass concentration (4-8 mg/l) had little effect on lead removal. The presence of iron (Fe) and lead, copper (Cu) and lead or all three metal ions in solution at varying ratios to each other did not appear to have any significant effect on lead removal. Percentage lead, copper and iron removal from aqueous solution was 80-95, 45-50 and 65-75% respectively for the different multiple-metal solutions studied. No break-through points were observed for lead removal from aqueous solutions in column reactors, with initial lead concentrations of less than 100 mg/l at varying flow rates of 2, 5 and 10 ml/min. This suggested that flow rate, and therefore retention time, had little effect on percentage lead removal from aqueous solution, which was more that 95%, at low initial lead concentrations (less than 100 mg/l). At initial lead concentrations of 200 mg/l or more, an increase in flow rate, which equates to a decrease in column retention time, resulted in break-through points occurring earlier in the column run. Percentage lead removal values, from lead-acid battery efiluent in column systems, of over 95% were achieved. Desorption of approximately 30% and 40% of bound lead was achieved, with 0.5 M HNO₃ in a volume of 50 ml, from two lead-acid battery. Repeated adsorption and desorption of lead by the Azalia biomass over 10 cycles did not result in any decrease in the percentage lead removal from effluent, which strongly suggested that the Azalla biomass could be re-used a number of times without deterioration in its physical integrity, or lead removal capacity. No evidence of deterioration in the Azolla biomass's physical integrity after 10 successive adsorption and desorption procedures was observed using scanning electron microscopy. The Azolla filiculoides biomass was, therefore, found to be able to effectively remove lead from aqueous solution and lead-acid battery effluent repeatedly, with no observed reduction in it's uptake capacity or physical integrity.
- Full Text:
- Date Issued: 1999
- Authors: Sanyahumbi, Douglas
- Date: 1999
- Subjects: Azolla , Heavy metals -- Absorption and adsorption , Lead , Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3901 , http://hdl.handle.net/10962/d1003960 , Azolla , Heavy metals -- Absorption and adsorption , Lead , Water -- Purification -- Biological treatment
- Description: The removal of lead from aqueous solution and lead-acid battery manufacturing waste-water by the non-viable biomass of the water fern Azolla filiculoides was investigated in both batch and column reactors. The maximum lead uptake by the Azolla biomass at a pH value of approximately 5, was found to be 100 mg lead/g biomass from aqueous solution. Lead removal varied from 30% of the initial lead concentration at pH 1.5 to approximately 95% at pH values of 3.5 and 5.6. Lead removal from aqueous solution decreased to 30% of the initial lead concentration if the lead concentration was initially over 400 mg/l. At initial lead concentrations of less than 400 mg/l, percentage lead removal was found to be over 90% of the initial lead concentration. Lead removal remained at approximately 90% between 10°C and 50°C. Biomass concentration (4-8 mg/l) had little effect on lead removal. The presence of iron (Fe) and lead, copper (Cu) and lead or all three metal ions in solution at varying ratios to each other did not appear to have any significant effect on lead removal. Percentage lead, copper and iron removal from aqueous solution was 80-95, 45-50 and 65-75% respectively for the different multiple-metal solutions studied. No break-through points were observed for lead removal from aqueous solutions in column reactors, with initial lead concentrations of less than 100 mg/l at varying flow rates of 2, 5 and 10 ml/min. This suggested that flow rate, and therefore retention time, had little effect on percentage lead removal from aqueous solution, which was more that 95%, at low initial lead concentrations (less than 100 mg/l). At initial lead concentrations of 200 mg/l or more, an increase in flow rate, which equates to a decrease in column retention time, resulted in break-through points occurring earlier in the column run. Percentage lead removal values, from lead-acid battery efiluent in column systems, of over 95% were achieved. Desorption of approximately 30% and 40% of bound lead was achieved, with 0.5 M HNO₃ in a volume of 50 ml, from two lead-acid battery. Repeated adsorption and desorption of lead by the Azalia biomass over 10 cycles did not result in any decrease in the percentage lead removal from effluent, which strongly suggested that the Azalla biomass could be re-used a number of times without deterioration in its physical integrity, or lead removal capacity. No evidence of deterioration in the Azolla biomass's physical integrity after 10 successive adsorption and desorption procedures was observed using scanning electron microscopy. The Azolla filiculoides biomass was, therefore, found to be able to effectively remove lead from aqueous solution and lead-acid battery effluent repeatedly, with no observed reduction in it's uptake capacity or physical integrity.
- Full Text:
- Date Issued: 1999
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
Regulation of the indoleamines by sex steroids
- Authors: Awah, Edmund Kpabi
- Date: 1992
- Subjects: Steroids -- Research , Steroid drugs -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4053 , http://hdl.handle.net/10962/d1004114 , Steroids -- Research , Steroid drugs -- Research
- Description: Alteration of serum tryptophan leads to parallel alterations in brain tryptophan levels. Such changes in brain tryptophan levels has been shown to lead to mood disturbances. The primary enzyme responsible for altering serum tryptophan levels is the liver cytosolic enzyme, tryptophan pyrrolase. Activation of this enzyme is responsible for the enhanced catabolism of circulating tryptophan. The purpose of the present study was firstly to establish whether there is a link between sex steroids and tryptophan pyrrolase activity especially since sex steroids are also known to cause mood disturbances and secondly to determine the effects of sex steroids on brain indolamine metabolism. The results show that all three sex steroids induce the activity of tryptophan pyrrolase implying that they decrease serum tryptophan levels by the activation of tryptophan pyrrolase, thus making less tryptophan available for uptake by the brain. It was also shown that the sex steroids enhance the uptake of ¹⁴C-tryptophan by brain synatopsomes. In addition, the sex steroids influenced the pattern of metabolism of serotonin by organ cultures of rat pineal glands. It is possible that the sex steroids regulate the availability and uptake of indoleamines in the brain.
- Full Text:
- Date Issued: 1992
- Authors: Awah, Edmund Kpabi
- Date: 1992
- Subjects: Steroids -- Research , Steroid drugs -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4053 , http://hdl.handle.net/10962/d1004114 , Steroids -- Research , Steroid drugs -- Research
- Description: Alteration of serum tryptophan leads to parallel alterations in brain tryptophan levels. Such changes in brain tryptophan levels has been shown to lead to mood disturbances. The primary enzyme responsible for altering serum tryptophan levels is the liver cytosolic enzyme, tryptophan pyrrolase. Activation of this enzyme is responsible for the enhanced catabolism of circulating tryptophan. The purpose of the present study was firstly to establish whether there is a link between sex steroids and tryptophan pyrrolase activity especially since sex steroids are also known to cause mood disturbances and secondly to determine the effects of sex steroids on brain indolamine metabolism. The results show that all three sex steroids induce the activity of tryptophan pyrrolase implying that they decrease serum tryptophan levels by the activation of tryptophan pyrrolase, thus making less tryptophan available for uptake by the brain. It was also shown that the sex steroids enhance the uptake of ¹⁴C-tryptophan by brain synatopsomes. In addition, the sex steroids influenced the pattern of metabolism of serotonin by organ cultures of rat pineal glands. It is possible that the sex steroids regulate the availability and uptake of indoleamines in the brain.
- Full Text:
- Date Issued: 1992
Hydrogenases from sulphate reducing bacteria and their role in the bioremediation of textile effluent
- Mutambanengwe, Cecil Clifford Zvandada
- Authors: Mutambanengwe, Cecil Clifford Zvandada
- Date: 2007
- Subjects: Bioremediation , Dyes and dyeing -- Waste disposal , Sulfur bacteria , Hydragenase , Factory and trade waste -- Purification , Textile waste
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3960 , http://hdl.handle.net/10962/d1004019 , Bioremediation , Dyes and dyeing -- Waste disposal , Sulfur bacteria , Hydragenase , Factory and trade waste -- Purification , Textile waste
- Description: The continuing industrial development has led to a corresponding increase in the amount of waste water generation leading to a consequential decline in levels and quality of the natural water in the ecosystem. Textile industries consume over 7 x 10[superscript 5] tons of dyes annually and use up to 1 litre of water per kg of dye processed and are third largest polluters in the world, the problem being aggravated by the inefficiencies of the dye houses. An abundance of physio-chemical methods are in use world wide, however, there is increasing concern as to their impact in effectively treating textile effluents as they introduce secondary pollutants during the ‘remediation’ process which are quite costly to run, maintain and clean up. Research on biological treatment has offered simple and cost effective ways of bioremediating textile effluents. While aerobic treatment of textile dyes and their effluents has been reported, its major draw back is commercial up-scaling and as such anaerobic systems have been investigated and shown to degrade azo dyes, which form the bulk of the dyes used world wide. However, the mechanisms involved in the bioremediation of these dyes are poorly understood. The aims of this study were to identify and investigate the role of enzymes produced by sulphate reducing bacteria (SRB) in bioremediating textile dye and their effluents. Sulphate reducing bacteria were used in this study because they are tolerant to harsh environmental conditions and inhibit the proliferance of pathogenic micro-organisms. The appearance of clear zones in agar plates containing azo dye concentrations ranging from 10 – 100 mgl[superscript -1] showed the ability of SRB to decolourize dyes under anaerobic conditions. Assays of enzymes previously reported to decolourise azo dyes were not successful, but led to the identification of hydrogenase enzyme being produced by SRB. The enzyme was found to be localised in the membrane and cytoplasm. A surface response method was used to optimize the extraction of the enzyme from the bacterial cells resulting in approximately 3 fold increase in hydrogenase activity. Maximum hydrogenase activity was found to occur after six days in the absence of dyes but was found to occur after one day in the presence of azo dyes. A decline in hydrogenase activity thereafter, suggested inhibition of enzymatic activity by the putative aromatic amines produced after azo cleavage. Purification of the hydrogenase by freeze drying, poly ethylene glycol, and Sephacryl – 200 size exclusion- ion exchange chromatography revealed the enzyme to have a molecular weight of 38.5 kDa when analyzed by a 12 % SDS-PAGE. Characterisation of the enzyme revealed optimal activity at a pH of 7.5 and temperature of 40 °C while it exhibited a poor thermal stability with a half-life of 32 minutes. The kinetic parameters V[subscript max] and K[subscript m] were 21.18 U ml[superscript -1} and 4.57 mM respectively. Application of the cell free extract on commercial dyes was not successful, and only whole SRB cells resulted in decolourisation of the dyes. Consequently trials on the industrial dyes and effluents were carried out with whole cells. Decolourisation rates of up to 96 % were achieved for the commercial dyes and up to 93 % for the industrial dyes over a period of 10 days.
- Full Text:
- Date Issued: 2007
- Authors: Mutambanengwe, Cecil Clifford Zvandada
- Date: 2007
- Subjects: Bioremediation , Dyes and dyeing -- Waste disposal , Sulfur bacteria , Hydragenase , Factory and trade waste -- Purification , Textile waste
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3960 , http://hdl.handle.net/10962/d1004019 , Bioremediation , Dyes and dyeing -- Waste disposal , Sulfur bacteria , Hydragenase , Factory and trade waste -- Purification , Textile waste
- Description: The continuing industrial development has led to a corresponding increase in the amount of waste water generation leading to a consequential decline in levels and quality of the natural water in the ecosystem. Textile industries consume over 7 x 10[superscript 5] tons of dyes annually and use up to 1 litre of water per kg of dye processed and are third largest polluters in the world, the problem being aggravated by the inefficiencies of the dye houses. An abundance of physio-chemical methods are in use world wide, however, there is increasing concern as to their impact in effectively treating textile effluents as they introduce secondary pollutants during the ‘remediation’ process which are quite costly to run, maintain and clean up. Research on biological treatment has offered simple and cost effective ways of bioremediating textile effluents. While aerobic treatment of textile dyes and their effluents has been reported, its major draw back is commercial up-scaling and as such anaerobic systems have been investigated and shown to degrade azo dyes, which form the bulk of the dyes used world wide. However, the mechanisms involved in the bioremediation of these dyes are poorly understood. The aims of this study were to identify and investigate the role of enzymes produced by sulphate reducing bacteria (SRB) in bioremediating textile dye and their effluents. Sulphate reducing bacteria were used in this study because they are tolerant to harsh environmental conditions and inhibit the proliferance of pathogenic micro-organisms. The appearance of clear zones in agar plates containing azo dye concentrations ranging from 10 – 100 mgl[superscript -1] showed the ability of SRB to decolourize dyes under anaerobic conditions. Assays of enzymes previously reported to decolourise azo dyes were not successful, but led to the identification of hydrogenase enzyme being produced by SRB. The enzyme was found to be localised in the membrane and cytoplasm. A surface response method was used to optimize the extraction of the enzyme from the bacterial cells resulting in approximately 3 fold increase in hydrogenase activity. Maximum hydrogenase activity was found to occur after six days in the absence of dyes but was found to occur after one day in the presence of azo dyes. A decline in hydrogenase activity thereafter, suggested inhibition of enzymatic activity by the putative aromatic amines produced after azo cleavage. Purification of the hydrogenase by freeze drying, poly ethylene glycol, and Sephacryl – 200 size exclusion- ion exchange chromatography revealed the enzyme to have a molecular weight of 38.5 kDa when analyzed by a 12 % SDS-PAGE. Characterisation of the enzyme revealed optimal activity at a pH of 7.5 and temperature of 40 °C while it exhibited a poor thermal stability with a half-life of 32 minutes. The kinetic parameters V[subscript max] and K[subscript m] were 21.18 U ml[superscript -1} and 4.57 mM respectively. Application of the cell free extract on commercial dyes was not successful, and only whole SRB cells resulted in decolourisation of the dyes. Consequently trials on the industrial dyes and effluents were carried out with whole cells. Decolourisation rates of up to 96 % were achieved for the commercial dyes and up to 93 % for the industrial dyes over a period of 10 days.
- Full Text:
- Date Issued: 2007
Purification and characterization of TbHsp70.c, a novel Hsp70 from Trypanosoma brucei
- Authors: Burger, Adélle
- Date: 2014
- Subjects: African trypanosomiasis -- Research Heat shock proteins -- Research Trypanosoma brucei -- Research Mycobacterial diseases -- Research -- Africa Parasitic diseases -- Africa -- Prevention Parasites -- Physiology Developing countries -- Economic conditions
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4105 , http://hdl.handle.net/10962/d1011618
- Description: One of Africa’s neglected tropical diseases, African Trypanosomiasis, is not only fatal but also has a crippling impact on economic development. Heat shock proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. The expression of heat shock proteins increases during these heat shock conditions, and this is considered to play a role in differentiation of these vector-borne parasites. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is involved in protein homeostasis, Hsp40 acts as a co-chaperone and stimulates its intrinsically weak ATPase activity. In silico analysis of the T. brucei genome has revealed the existence of 12 Hsp70 proteins and 65 Hsp40 proteins to date. A novel Hsp70, TbHsp70.c, was recently identified in T. brucei. Different from the prototypical Hsp70, TbHsp70.c contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. By implication the substrate range and mechanism by which the substrates are recognized may be novel. The ability of a Type I Hsp40, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective of this study was to biochemically characterize TbHsp70.c and its partnership with Tbj2 to further enhance our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed chaperone activities in their ability to suppress aggregation of thermolabile MDH. TbHsp70.c also suppressed aggregation of rhodanese. ATPase assays revealed that the ATPase activity of TbHsp70.c was stimulated by Tbj2. The targeted inhibition of the function of heat shock proteins is emerging as a tool to combat disease. The small molecule modulators quercetin and methylene blue are known to inhibit the ATPase activity of Hsp70. However, methylene blue did not significantly inhibit the ATPase activity of TbHsp70.c; while quercetin, did inhibit the ATPase activity. In vivo heat stress experiments indicated an up-regulation of the expression levels of TbHsp70.c. RNA interference studies showed partial knockdown of TbHsp70.c with no detrimental effect on the parasite. Fluorescence microscopy studies of TbHsp70.c showed a probable cytoplasmic subcellular localization. In this study both TbHsp70.c and Tbj2 demonstrated chaperone activity and Tbj2 possibly functions as a co-chaperone of TbHsp70.c.
- Full Text:
- Date Issued: 2014
- Authors: Burger, Adélle
- Date: 2014
- Subjects: African trypanosomiasis -- Research Heat shock proteins -- Research Trypanosoma brucei -- Research Mycobacterial diseases -- Research -- Africa Parasitic diseases -- Africa -- Prevention Parasites -- Physiology Developing countries -- Economic conditions
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4105 , http://hdl.handle.net/10962/d1011618
- Description: One of Africa’s neglected tropical diseases, African Trypanosomiasis, is not only fatal but also has a crippling impact on economic development. Heat shock proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. The expression of heat shock proteins increases during these heat shock conditions, and this is considered to play a role in differentiation of these vector-borne parasites. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is involved in protein homeostasis, Hsp40 acts as a co-chaperone and stimulates its intrinsically weak ATPase activity. In silico analysis of the T. brucei genome has revealed the existence of 12 Hsp70 proteins and 65 Hsp40 proteins to date. A novel Hsp70, TbHsp70.c, was recently identified in T. brucei. Different from the prototypical Hsp70, TbHsp70.c contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. By implication the substrate range and mechanism by which the substrates are recognized may be novel. The ability of a Type I Hsp40, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective of this study was to biochemically characterize TbHsp70.c and its partnership with Tbj2 to further enhance our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed chaperone activities in their ability to suppress aggregation of thermolabile MDH. TbHsp70.c also suppressed aggregation of rhodanese. ATPase assays revealed that the ATPase activity of TbHsp70.c was stimulated by Tbj2. The targeted inhibition of the function of heat shock proteins is emerging as a tool to combat disease. The small molecule modulators quercetin and methylene blue are known to inhibit the ATPase activity of Hsp70. However, methylene blue did not significantly inhibit the ATPase activity of TbHsp70.c; while quercetin, did inhibit the ATPase activity. In vivo heat stress experiments indicated an up-regulation of the expression levels of TbHsp70.c. RNA interference studies showed partial knockdown of TbHsp70.c with no detrimental effect on the parasite. Fluorescence microscopy studies of TbHsp70.c showed a probable cytoplasmic subcellular localization. In this study both TbHsp70.c and Tbj2 demonstrated chaperone activity and Tbj2 possibly functions as a co-chaperone of TbHsp70.c.
- Full Text:
- Date Issued: 2014
Analysis of the interaction of Hsp90 with the extracellular matrix protein fibronectin (FN)
- Authors: Hunter, Morgan Campbell
- Date: 2014
- Subjects: Heat shock proteins , Fibronectins , Extracellular matrix proteins , Breast -- Cancer
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4170 , http://hdl.handle.net/10962/d1020960
- Description: Mounting evidence suggests that Hsp90 is present and functionally active in the extracellular space. The biological function of extracellular Hsp90 (eHsp90) remains relatively uncharacterized compared to that of intracellular Hsp90. eHsp90 has been shown to interact with a finite number of extracellular proteins, however, despite the identification of eHsp90 interacting proteins, the function of eHsp90 in these complexes is unknown. Several reports suggest a role for eHsp90α in cell migration and invasion. Reported targets for eHsp90 stimulated cell migration include MMPs, LRP-1, tyrosine kinase receptors and possible others unidentified. Limited studies report a role for eHsp90β. Recently, Hsp90α and Hsp90β were isolated in a complex containing fibronectin (FN) on the surface of MDA-MB-231 breast cancer cells. Herein, we report direct binding of Hsp90α and Hsp90β to FN using a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. SPR spectroscopy showed that Hsp90β bound the 70 kDa amino-terminal fragment of FN (FN70), but that binding of FN to Hsp90β was not limited to FN70. Confocal microscopy showed regions of colocalization of Hsp90 with extracellular FN matrix fibrils in Hs578T breast cancer cell lines. Treatment of Hs578T breast cancer cells with novobiocin (an Hsp90 inhibitor) and an LRP-1 blocking antibody resulted in a loss of FN matrix and FN endocytosis (novobiocin treated). Addition of exogenous Hsp90β was able to recover such effect after both treatments. FN was shown to colocalize with intracellular LRP-1 in novobiocin treated Hs578T cells. Immunoprecipitation of an LRP-1 containing complex showed the presence of Hsp90 and 70 and 120+ kDa FN fragments. Treatment of Hs578T cells with novobiocin increased the level of FN120+ bound in LRP-1 immunoprecipitate. Exogenous Hsp90β decreased the level of low and high molecular weight FN fragments in a complex with LRP-1, despite the fact that higher levels of lower molecular weight FN fragments were detected in this cell lysate compared to the other treatments. We report FN as a novel interacting protein of eHsp90. Taken together, we provide evidence for a direct role of eHsp90β in FN matrix remodeling. We suggest that Hsp90 plays a direct role in FN matrix dynamics through interaction with FN and LRP-1. The identification of FN as a novel interacting protein of eHsp90 suggests a role for Hsp90 in FN matrix remodeling, which is important for a number of fundamental cellular processes including cell migration and metastasis.
- Full Text:
- Date Issued: 2014
- Authors: Hunter, Morgan Campbell
- Date: 2014
- Subjects: Heat shock proteins , Fibronectins , Extracellular matrix proteins , Breast -- Cancer
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4170 , http://hdl.handle.net/10962/d1020960
- Description: Mounting evidence suggests that Hsp90 is present and functionally active in the extracellular space. The biological function of extracellular Hsp90 (eHsp90) remains relatively uncharacterized compared to that of intracellular Hsp90. eHsp90 has been shown to interact with a finite number of extracellular proteins, however, despite the identification of eHsp90 interacting proteins, the function of eHsp90 in these complexes is unknown. Several reports suggest a role for eHsp90α in cell migration and invasion. Reported targets for eHsp90 stimulated cell migration include MMPs, LRP-1, tyrosine kinase receptors and possible others unidentified. Limited studies report a role for eHsp90β. Recently, Hsp90α and Hsp90β were isolated in a complex containing fibronectin (FN) on the surface of MDA-MB-231 breast cancer cells. Herein, we report direct binding of Hsp90α and Hsp90β to FN using a solid phase binding assay and surface plasmon resonance (SPR) spectroscopy. SPR spectroscopy showed that Hsp90β bound the 70 kDa amino-terminal fragment of FN (FN70), but that binding of FN to Hsp90β was not limited to FN70. Confocal microscopy showed regions of colocalization of Hsp90 with extracellular FN matrix fibrils in Hs578T breast cancer cell lines. Treatment of Hs578T breast cancer cells with novobiocin (an Hsp90 inhibitor) and an LRP-1 blocking antibody resulted in a loss of FN matrix and FN endocytosis (novobiocin treated). Addition of exogenous Hsp90β was able to recover such effect after both treatments. FN was shown to colocalize with intracellular LRP-1 in novobiocin treated Hs578T cells. Immunoprecipitation of an LRP-1 containing complex showed the presence of Hsp90 and 70 and 120+ kDa FN fragments. Treatment of Hs578T cells with novobiocin increased the level of FN120+ bound in LRP-1 immunoprecipitate. Exogenous Hsp90β decreased the level of low and high molecular weight FN fragments in a complex with LRP-1, despite the fact that higher levels of lower molecular weight FN fragments were detected in this cell lysate compared to the other treatments. We report FN as a novel interacting protein of eHsp90. Taken together, we provide evidence for a direct role of eHsp90β in FN matrix remodeling. We suggest that Hsp90 plays a direct role in FN matrix dynamics through interaction with FN and LRP-1. The identification of FN as a novel interacting protein of eHsp90 suggests a role for Hsp90 in FN matrix remodeling, which is important for a number of fundamental cellular processes including cell migration and metastasis.
- Full Text:
- Date Issued: 2014
Investigation of the bioconversion of constituents of olive effluents for the production of valuable chemical compounds
- Authors: Notshe, Thandiwe Loretta
- Date: 2002
- Subjects: Phenols , Sewage -- Purification , Effluent quality
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4078 , http://hdl.handle.net/10962/d1007446 , Phenols , Sewage -- Purification , Effluent quality
- Description: Olive mill wastewater is produced in large quantities during the production of olive oil and olive production effluents are produced during the processing of olives. This project was planned to find a use for constituents found in olive production wastewater. The task was carried out by first characterizing the olive effluents, then screening microorganisms for growth in the effluents and reduction of the pollutant properties of the effluents. An investigation into the biotransformation of aromatic compounds present in the effluents into useful chemicals, was carried out. The olive production effluents were collected from different stages in the process for treating olive wastewater, viz, a fermentation tank (FB), the surface of a digester (LV) and an evaporation pond (SO). The three effluents were characterized by investigating their phenolic composition. Protocatechuic acid, vanillic acid, syringic acid, hydroxyphenyl acetic acid, coumaric acid and ferulic acid were identified in an olive effluent, FB, using thin layer chromatography (TLC) and High perfomance liquid chromatography (HPLC). Hydroxyphenyl acetic acid constitutes almost 60% of the organics in olive effluent FB. Five bacteria, namely RU-LV1; RU-FBI and RU-FB2; RU-SOI and RU-S02, were isolated from the olive effluents LV, FB and SO respectively. These isolates were found to be halotolerant and were able to grow over a broad temperature and pH range, with the maximum temperature and pH for growth being 28°C and pH 7 respectively. A range of microorganisms were evaluated for their ability to grow and reduce the total phenolic content of the olive effluents. Among these Neurospora crassa showed the highest potential for the biological reduction of total phenolics in olive effluents. Approximately 70% of the total phenolic content was removed by N. crassa. Trametes verscilor, Pseudomonas putida strains, RU-KMI and RU-KM3s, and the bacteria isolated from olive effluents could also degrade the total phenolic content of olive effluents, but to a lesser extent. The ability of the five bacterial isolates to grow and degrade aromatic compounds was assessed by growing them in medium with standard aromatic compounds. RU-L V1 degraded 96%, 100%, 73% and 100% of caffeic acid, protocatechuic acid, p-coumaric acid and vanillic acid respectively. The other isolates degraded caffeic acid and protocatechuic acid, but their ability to degraded p-coumaric acid and vanillic acid was found to be lesser than the ability of RU-LV1 to degrade the same aromatic compounds. Whole cells of RU-LV1 degraded vanillic acid but no metabolic products were observed on HPLC analysis. Resting cells, French pressed extract, cell free extracts and cell debris from RU-LV1 cells induced with vanillic acid degraded vanillic acid, ferulic acid and vanillin at rates higher than those obtained from non-induced cultures. No products were observed during the degradation of vanillic acid. Ferulic acid was converted into vanillic acid by French pressed extract, cell free extract and cell debris of RU-LV1. The maximum yield of vanillic acid as a product (0 .23 mM, 50 %yield) was obtained when cell free extracts of RU-LVI, grown in glucose and induced by vanillic acid, were used for the degradation of 0.4 mM ferulic acid. Vanillin was rapidly converted into vanillic acid by resting cells, cell free extracts and French pressed extract of RU-LVI. Using molecular techniques, the similarity ranking of the RU-LVI 16S rRNA gene and its clone showed a high similarity to Corynebacterium glutamicum and Corynebacterium acedopltilum. The rapid degradation of vanillin to vanillic acid suggests that extracts from RU-LV1 degrade ferulic acid into vanillin which is immediately oxidized to vanillic acid. Vanillic acid is also considered as a high value chemical. This project has a potential of producing useful chemicals from cheap substrates that can be found in olive effluents. , KMBT_363
- Full Text:
- Date Issued: 2002
- Authors: Notshe, Thandiwe Loretta
- Date: 2002
- Subjects: Phenols , Sewage -- Purification , Effluent quality
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4078 , http://hdl.handle.net/10962/d1007446 , Phenols , Sewage -- Purification , Effluent quality
- Description: Olive mill wastewater is produced in large quantities during the production of olive oil and olive production effluents are produced during the processing of olives. This project was planned to find a use for constituents found in olive production wastewater. The task was carried out by first characterizing the olive effluents, then screening microorganisms for growth in the effluents and reduction of the pollutant properties of the effluents. An investigation into the biotransformation of aromatic compounds present in the effluents into useful chemicals, was carried out. The olive production effluents were collected from different stages in the process for treating olive wastewater, viz, a fermentation tank (FB), the surface of a digester (LV) and an evaporation pond (SO). The three effluents were characterized by investigating their phenolic composition. Protocatechuic acid, vanillic acid, syringic acid, hydroxyphenyl acetic acid, coumaric acid and ferulic acid were identified in an olive effluent, FB, using thin layer chromatography (TLC) and High perfomance liquid chromatography (HPLC). Hydroxyphenyl acetic acid constitutes almost 60% of the organics in olive effluent FB. Five bacteria, namely RU-LV1; RU-FBI and RU-FB2; RU-SOI and RU-S02, were isolated from the olive effluents LV, FB and SO respectively. These isolates were found to be halotolerant and were able to grow over a broad temperature and pH range, with the maximum temperature and pH for growth being 28°C and pH 7 respectively. A range of microorganisms were evaluated for their ability to grow and reduce the total phenolic content of the olive effluents. Among these Neurospora crassa showed the highest potential for the biological reduction of total phenolics in olive effluents. Approximately 70% of the total phenolic content was removed by N. crassa. Trametes verscilor, Pseudomonas putida strains, RU-KMI and RU-KM3s, and the bacteria isolated from olive effluents could also degrade the total phenolic content of olive effluents, but to a lesser extent. The ability of the five bacterial isolates to grow and degrade aromatic compounds was assessed by growing them in medium with standard aromatic compounds. RU-L V1 degraded 96%, 100%, 73% and 100% of caffeic acid, protocatechuic acid, p-coumaric acid and vanillic acid respectively. The other isolates degraded caffeic acid and protocatechuic acid, but their ability to degraded p-coumaric acid and vanillic acid was found to be lesser than the ability of RU-LV1 to degrade the same aromatic compounds. Whole cells of RU-LV1 degraded vanillic acid but no metabolic products were observed on HPLC analysis. Resting cells, French pressed extract, cell free extracts and cell debris from RU-LV1 cells induced with vanillic acid degraded vanillic acid, ferulic acid and vanillin at rates higher than those obtained from non-induced cultures. No products were observed during the degradation of vanillic acid. Ferulic acid was converted into vanillic acid by French pressed extract, cell free extract and cell debris of RU-LV1. The maximum yield of vanillic acid as a product (0 .23 mM, 50 %yield) was obtained when cell free extracts of RU-LVI, grown in glucose and induced by vanillic acid, were used for the degradation of 0.4 mM ferulic acid. Vanillin was rapidly converted into vanillic acid by resting cells, cell free extracts and French pressed extract of RU-LVI. Using molecular techniques, the similarity ranking of the RU-LVI 16S rRNA gene and its clone showed a high similarity to Corynebacterium glutamicum and Corynebacterium acedopltilum. The rapid degradation of vanillin to vanillic acid suggests that extracts from RU-LV1 degrade ferulic acid into vanillin which is immediately oxidized to vanillic acid. Vanillic acid is also considered as a high value chemical. This project has a potential of producing useful chemicals from cheap substrates that can be found in olive effluents. , KMBT_363
- Full Text:
- Date Issued: 2002