Physiological signal transduction from the photosynthetic apparatus in the green alga Dunaliella salina
- Logie, Malcolme Ronald Ruxton
- Authors: Logie, Malcolme Ronald Ruxton
- Date: 1995
- Subjects: Cellular signal transduction Photosynthesis -- Research Green algae Dunaliella
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4035 , http://hdl.handle.net/10962/d1004095
- Description: The transduction of stress signals in plants is known to involve complex hysiological responses. In D. salina a range of stresses results in hyperaccumulation of ft-carotene and an understanding of stress responses in this organism has important biotechnological implications. In this thesis an attempt was made to elucidate the physiological components involved and establish a role for pH in response to high light stress. In order to achieve this the effect of high light stress on photosynthesis and cell productivity was measured. Results showed that photosynthetic carbon assimilation, oxygen evolution and cellular productivity was initially inhibited by exposure to high light intensities, but this inhibition was transient and was overcome by a rapid increase in all three parameters. The response of the carbon pool intermediates was also investigated. It was shown that on exposure to high light ft-carotene declined but then showed a rapid increase after about 4 hours of exposure. It was also demonstrated that the initial loss of ft-carotene was due to loss of this pigment from the photosynthetic pigment bed and that the hyper-accumulation of ft-carotene was due to accumulation of ft-carotene in lipoidal globules located in the chloroplast stroma. It was further demonstrated that there was mass movement of carbon in the xanthophyll cycle shortly after exposure to high light. This was characterized by the de-epoxidation of violaxanthin to antheraxanthin with a further de-epoxidation to zeaxanthin, thereby decreasing the epoxidation state of the cycle. Furthermore, it was shown that there was relocation of carbon from violaxanthin to the plant growth regulator abscisic acid. It was also shown for the first time in D. salina that the production of ft-carotene and operation of the epoxidation state of the xanthophyll cycle has a periodicity which is established after exposure to successive cycles of a light regime. Chlorophyll fluorescence was used together with well established ammonia stress responses to acquire a general overview of energy dissipation from the photosynthetic pigment bed. In conjunction with an understanding of xanthophyll cycle operation during exposure to high light stress it has been possible to establish a relationship between chlorophyll florescence, xanthophyll cycle operation and intracellular pH. It was also shown using chlorophyll fluorescence that after 4 hour exposure to high light a maximum fluorescence peak could no longer be induced indicating a transition at about this point from a state of reversibility to commitment of the full stress response. Nuclear magnetic resonance was used to follow intracellular pH fluxes during exposure to high light. A novel technique was developed for studying photosynthetically active organisms in the dark using nuclear magnetic resonance. These results showed that on exposure to high light stress there is rapid acidification of the chloroplast stroma and to a lesser degree of the acidic vacuole. The pH of these compartments is re-established after about 4 hours which is co-incident with the onset of fl-carotene hyper-accumulation and the loss of the induction of the chlorophyll fluorescence peak indicating an intimate relationship for fl-carotene, chlorophyll fluorescence, xanthophyll cycle operation and pH. The results from this study allow for the proposal of a general physiological stress transduction response mechanism for D. salina which is common for a range of different stresses and where intracellular pH plays a central role.
- Full Text:
- Date Issued: 1995
- Authors: Logie, Malcolme Ronald Ruxton
- Date: 1995
- Subjects: Cellular signal transduction Photosynthesis -- Research Green algae Dunaliella
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4035 , http://hdl.handle.net/10962/d1004095
- Description: The transduction of stress signals in plants is known to involve complex hysiological responses. In D. salina a range of stresses results in hyperaccumulation of ft-carotene and an understanding of stress responses in this organism has important biotechnological implications. In this thesis an attempt was made to elucidate the physiological components involved and establish a role for pH in response to high light stress. In order to achieve this the effect of high light stress on photosynthesis and cell productivity was measured. Results showed that photosynthetic carbon assimilation, oxygen evolution and cellular productivity was initially inhibited by exposure to high light intensities, but this inhibition was transient and was overcome by a rapid increase in all three parameters. The response of the carbon pool intermediates was also investigated. It was shown that on exposure to high light ft-carotene declined but then showed a rapid increase after about 4 hours of exposure. It was also demonstrated that the initial loss of ft-carotene was due to loss of this pigment from the photosynthetic pigment bed and that the hyper-accumulation of ft-carotene was due to accumulation of ft-carotene in lipoidal globules located in the chloroplast stroma. It was further demonstrated that there was mass movement of carbon in the xanthophyll cycle shortly after exposure to high light. This was characterized by the de-epoxidation of violaxanthin to antheraxanthin with a further de-epoxidation to zeaxanthin, thereby decreasing the epoxidation state of the cycle. Furthermore, it was shown that there was relocation of carbon from violaxanthin to the plant growth regulator abscisic acid. It was also shown for the first time in D. salina that the production of ft-carotene and operation of the epoxidation state of the xanthophyll cycle has a periodicity which is established after exposure to successive cycles of a light regime. Chlorophyll fluorescence was used together with well established ammonia stress responses to acquire a general overview of energy dissipation from the photosynthetic pigment bed. In conjunction with an understanding of xanthophyll cycle operation during exposure to high light stress it has been possible to establish a relationship between chlorophyll florescence, xanthophyll cycle operation and intracellular pH. It was also shown using chlorophyll fluorescence that after 4 hour exposure to high light a maximum fluorescence peak could no longer be induced indicating a transition at about this point from a state of reversibility to commitment of the full stress response. Nuclear magnetic resonance was used to follow intracellular pH fluxes during exposure to high light. A novel technique was developed for studying photosynthetically active organisms in the dark using nuclear magnetic resonance. These results showed that on exposure to high light stress there is rapid acidification of the chloroplast stroma and to a lesser degree of the acidic vacuole. The pH of these compartments is re-established after about 4 hours which is co-incident with the onset of fl-carotene hyper-accumulation and the loss of the induction of the chlorophyll fluorescence peak indicating an intimate relationship for fl-carotene, chlorophyll fluorescence, xanthophyll cycle operation and pH. The results from this study allow for the proposal of a general physiological stress transduction response mechanism for D. salina which is common for a range of different stresses and where intracellular pH plays a central role.
- Full Text:
- Date Issued: 1995
African mead biotechnology and indigenous knowledge systems in iQhilika process development
- Authors: Cambray, Garth Anton
- Date: 2005
- Subjects: Biotechnology Indigenous peoples -- Africa Ethnoscience -- Africa Mead -- Africa Brewing -- Microbiology Honeybee Honey
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3929 , http://hdl.handle.net/10962/d1003988
- Description: While the production of mead, a fermented honey beverage, has declined in popularity around the world in recent centuries, a substantial mead industry continues to exist in Africa with an estimated annual production of 1 to 1.7 billion litres. This is largely an ‘invisible industry’, and has functioned outside the formal economy due to proscription of indigenous beverages during colonial times. The traditional African mead industry is, however, also now under pressure due to the environmental degradation of scarce natural ingredients, urbanisation and loss of indigenous knowledge systems (IKS) and, with time, the beverage will likely follow the declining trend of mead consumption observed elsewhere. An analysis of early reports of African mead production suggested that the Khoi-San, among the earliest inhabitants of the continent, are the originators of the mead making techniques which use fibrous plant materials derived from specific plant species, to facilitate mead fermentation in some way. The Eastern Cape represents a region with a large body of Khoi-San IKS preserved in their descendants among the Afrikaans and Xhosa populations. A survey to establish a baseline of mead-making technology in the Eastern Cape was undertaken, and involved interviewing traditional mead makers across an area of roughly 100 000 km2, showing that the mead, iQhilika(Xhosa) Kari (Khoi-San/Afrikaans), is produced using a very similar process throughout the region. This involves the roots of a Trichodiadema sp. plant (imoela – Xhosa, karimoer – Afrikaans/Khoi-San), honey, extract of brood and/or pollen and water. Various other fruit sugar sources were also found to be added at times producing seasonal beverages with unique organoleptic properties. A model traditional iQhilika production operation was investigated in order to describe the main features of the process. Biomass immobilised on Trichodiadema root segments was found to be distributed evenly through the profile of the bioreactor resulting in a well mixed fermentation and a productivity of 0.74 g EtOH/l/h. In the initial stages of fermentation, the ethanol yield was highest in the mid-regions of the bioreactor, but with time the regions closer to the surface, which had atmospheric contact had a higher yield. This phenomenon was attributed to aerobic fatty acid synthesis which allowed the yeast close to the surface to function more efficiently despite rising ethanol concentrations. The mead contained 44.25 g/l (7 % volume) ethanol produced in a fermentation time of 43.5 h. Yeast biomass in the traditional process was either immobilised in the form of flocs or attached to the Trichodiadema intonsum support. Electron microscopy revealed that the cells were covered in a layer of extra-cellular polymeric substance apparently assisting the immobilization, and which was populated by a consortium of yeasts and bacteria. Yeasts isolated from iQhilika brewed in two regions separated by 350 km were found to be very closely related Saccharomyces cerevisiae strains as determined by molecular genetic analysis. The traditional beverage was found to contain populations of Lactic acid bacteria (LAB), which are known spoilage organisms in other beverages. Spoilage characteristics of these organisms matched descriptions of spoilage provided by the IKS survey. Other possibly beneficial LAB, which may contribute useful flavour compounds, were also found to be present in the system. The basic functional aspects of the traditional process were used to design a continuous bench-scale tower bioreactor and process development was based on the IKS survey. This consisted of a packed bed bioreactor, consisting of 2 mm3 T. intonsum root segments, immobilising a novel Saccharomyces cerevisiae strain isolated from a traditional batch of iQhilika. The bioreactor performed well with a yield of close to the theoretical maximum and an ethanol productivity of 3.45 g EtOH/l/h. The parameters of the 5.6 l/d bench-scale bioreactor were used to design a full-scale production bioreactor with a planned maximum output of 330 l/d. This bioreactor had a productivity of 0.19 g EtOH/l/h. The organoleptic properties of the product produced were considered by a taste panel to be better than those of the product of the bench-scale tower bioreactor. This research was based on the development of IKS which imposed a number of constraints and obligations on the project to ensure environmental, and social, in addition to financial viability of the scale-up operation. Makana Meadery was established in partnership with Rhodes University as an empowerment company which, in addition to undertaking the commercialisation of the iQhilika process, would also develop methods for the production of scarce ingredients traditionally unsustainably sourced from fragile ecosystems, provide beekeeping training and the manufacture of beehives.
- Full Text:
- Date Issued: 2005
- Authors: Cambray, Garth Anton
- Date: 2005
- Subjects: Biotechnology Indigenous peoples -- Africa Ethnoscience -- Africa Mead -- Africa Brewing -- Microbiology Honeybee Honey
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3929 , http://hdl.handle.net/10962/d1003988
- Description: While the production of mead, a fermented honey beverage, has declined in popularity around the world in recent centuries, a substantial mead industry continues to exist in Africa with an estimated annual production of 1 to 1.7 billion litres. This is largely an ‘invisible industry’, and has functioned outside the formal economy due to proscription of indigenous beverages during colonial times. The traditional African mead industry is, however, also now under pressure due to the environmental degradation of scarce natural ingredients, urbanisation and loss of indigenous knowledge systems (IKS) and, with time, the beverage will likely follow the declining trend of mead consumption observed elsewhere. An analysis of early reports of African mead production suggested that the Khoi-San, among the earliest inhabitants of the continent, are the originators of the mead making techniques which use fibrous plant materials derived from specific plant species, to facilitate mead fermentation in some way. The Eastern Cape represents a region with a large body of Khoi-San IKS preserved in their descendants among the Afrikaans and Xhosa populations. A survey to establish a baseline of mead-making technology in the Eastern Cape was undertaken, and involved interviewing traditional mead makers across an area of roughly 100 000 km2, showing that the mead, iQhilika(Xhosa) Kari (Khoi-San/Afrikaans), is produced using a very similar process throughout the region. This involves the roots of a Trichodiadema sp. plant (imoela – Xhosa, karimoer – Afrikaans/Khoi-San), honey, extract of brood and/or pollen and water. Various other fruit sugar sources were also found to be added at times producing seasonal beverages with unique organoleptic properties. A model traditional iQhilika production operation was investigated in order to describe the main features of the process. Biomass immobilised on Trichodiadema root segments was found to be distributed evenly through the profile of the bioreactor resulting in a well mixed fermentation and a productivity of 0.74 g EtOH/l/h. In the initial stages of fermentation, the ethanol yield was highest in the mid-regions of the bioreactor, but with time the regions closer to the surface, which had atmospheric contact had a higher yield. This phenomenon was attributed to aerobic fatty acid synthesis which allowed the yeast close to the surface to function more efficiently despite rising ethanol concentrations. The mead contained 44.25 g/l (7 % volume) ethanol produced in a fermentation time of 43.5 h. Yeast biomass in the traditional process was either immobilised in the form of flocs or attached to the Trichodiadema intonsum support. Electron microscopy revealed that the cells were covered in a layer of extra-cellular polymeric substance apparently assisting the immobilization, and which was populated by a consortium of yeasts and bacteria. Yeasts isolated from iQhilika brewed in two regions separated by 350 km were found to be very closely related Saccharomyces cerevisiae strains as determined by molecular genetic analysis. The traditional beverage was found to contain populations of Lactic acid bacteria (LAB), which are known spoilage organisms in other beverages. Spoilage characteristics of these organisms matched descriptions of spoilage provided by the IKS survey. Other possibly beneficial LAB, which may contribute useful flavour compounds, were also found to be present in the system. The basic functional aspects of the traditional process were used to design a continuous bench-scale tower bioreactor and process development was based on the IKS survey. This consisted of a packed bed bioreactor, consisting of 2 mm3 T. intonsum root segments, immobilising a novel Saccharomyces cerevisiae strain isolated from a traditional batch of iQhilika. The bioreactor performed well with a yield of close to the theoretical maximum and an ethanol productivity of 3.45 g EtOH/l/h. The parameters of the 5.6 l/d bench-scale bioreactor were used to design a full-scale production bioreactor with a planned maximum output of 330 l/d. This bioreactor had a productivity of 0.19 g EtOH/l/h. The organoleptic properties of the product produced were considered by a taste panel to be better than those of the product of the bench-scale tower bioreactor. This research was based on the development of IKS which imposed a number of constraints and obligations on the project to ensure environmental, and social, in addition to financial viability of the scale-up operation. Makana Meadery was established in partnership with Rhodes University as an empowerment company which, in addition to undertaking the commercialisation of the iQhilika process, would also develop methods for the production of scarce ingredients traditionally unsustainably sourced from fragile ecosystems, provide beekeeping training and the manufacture of beehives.
- Full Text:
- Date Issued: 2005
Analysis of the anti-cancer activity of novel indigenous algal compounds in breast cancer: towards the development of a model for screening anti-cancer stem cell activity
- Authors: Lawson, Jessica Clair
- Date: 2010
- Subjects: Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3925 , http://hdl.handle.net/10962/d1003984 , Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Description: Breast cancer, the most common malignancy diagnosed in women, is one of the leading causes of death in women worldwide. In South Africa only 32% of women diagnosed with advanced breast cancer survive more than five years. The search for new chemotherapeutic agents capable of effectively treating breast cancer is therefore essential. Recent evidence supporting the cancer stem cell theory of cancer development for breast cancer challenges the current theories of cancer development and hence treatment. Cancer stem cells are a small subpopulation of tumour cells that possess properties of both cancer cells and stem cells and are believed to be the tumour-initiating population of many cancers. Cancer stem cells are inherently resistant to many chemotherapeutic agents and in this way have been associated with repopulation of tumours after chemotherapy. This phenomenon is proposed as a possible mechanism for cancer relapse after treatment. Cancer stem cells have also been implicated in metastasis, the major cause of mortality in cancer patients. Therefore, any treatment that is capable of targeting and removing breast cancer stem cells may have the theoretical potential to effectively treat breast cancer. However, there are currently no such treatments available for clinical use. We were provided access to a library of novel indigenous small molecules isolated from red and brown algae found off the Eastern Cape of South Africa. The aim of this project was to analyse the anti-cancer and anti-cancer stem cell properties of the compounds in this library and to identify „hit‟ compounds which could form the basis for future development into new anti-cancer drugs. Ten novel compounds of algal origin were tested for cytotoxicity, by determining their ability to inhibit the growth of MCF12A breast epithelial cells and MCF7 breast cancer cells using the colorimetric MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay. All but one of the compounds tested exhibited cytotoxicity towards the MCF7 cancer cell line, with IC50 values (the concentration of the compound that leads to a 50% inhibition in cell growth) of between 3 μM and 90 μM. The chemotherapeutic drug paclitaxel was used as a positive control. Four of the compounds (RUMB-001, RUMB-002, RUMB-007 and RUMB-010/saragaquinoic acid) were significantly more toxic to the MCF7 cancer cell line, than the „normal‟ MCF12A breast cells and were selected as priority compounds for further analyses. In addition, two other compounds were selected as priority compounds, one highly cytotoxic towards both MCF12A and MCF7 cell lines (RUMB-015) and one which was non toxic to either cell line (RUMB-017/018). Preliminary studies into the mechanism of cytotoxicity using Western blot analysis for poly (ADP-ribose) polymerase (PARP) cleavage and Hoechst 33342 immunostaining in MCF-7 cells were largely unsuccessful. The Hoechst 33342 immunostaining assay did provide tentative evidence that selected priority compounds were capable of inducing apoptosis, although these assays will need to be repeated using a less subjective assay to confirm the results. The priority compounds were subsequently investigated for their cytotoxic effect on the cancer stem cell-enriched side population in MCF7 cells. The ability of the priority compounds to selectively target the cancer stem cell containing side population was assessed using two complementary flow cytometry-based techniques – namely the Hoechst 33342-exclusion assay, and fluorescent immunostaining for the expression of the putative cancer stem cell marker, ABCG2+. The ABCG2+ staining assay was a novel technique developed during the course of this study. It remains to be fully validated, but it may provide a new and reliable way to identify and analyse cancer stem cell containing side population cells. The MCF7 cells were treated with the compounds and the proportion of putative cancer stem cells compared with the size of the population in untreated cells was assessed. Three compounds (RUMB-010, RUMB-015 and RUMB-017/018) capable of reducing the proportion of side population cells within the MCF7 cell line were identified. Taking these data together, we identified two potential „hit‟ compounds which should be prioritised for future research. These are compounds RUMB-010/sargaquinoic acid and RUMB-017/018. RUMB-010 is of interest as it was shown to target the putative cancer stem cell population, in addition to the bulk MCF7 tumour line, but was relatively less toxic to the „normal‟ MCF12A cell line. RUMB-017/018 is of interest due to the ability to selectively target the cancer stem cell enriched side population, while having little effect on the normal (MCF12A) or bulk tumour (MCF7) cell lines tested. These compounds will be important as „hit‟ compounds for drug development and as tool compounds to study cancer and cancer stem cell biology.
- Full Text:
- Date Issued: 2010
- Authors: Lawson, Jessica Clair
- Date: 2010
- Subjects: Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3925 , http://hdl.handle.net/10962/d1003984 , Breast -- Cancer , Breast -- Cancer -- Chemotherapy , Breast -- Cancer -- Treatment , Red algae , Brown algae , Algae -- Biotechnology
- Description: Breast cancer, the most common malignancy diagnosed in women, is one of the leading causes of death in women worldwide. In South Africa only 32% of women diagnosed with advanced breast cancer survive more than five years. The search for new chemotherapeutic agents capable of effectively treating breast cancer is therefore essential. Recent evidence supporting the cancer stem cell theory of cancer development for breast cancer challenges the current theories of cancer development and hence treatment. Cancer stem cells are a small subpopulation of tumour cells that possess properties of both cancer cells and stem cells and are believed to be the tumour-initiating population of many cancers. Cancer stem cells are inherently resistant to many chemotherapeutic agents and in this way have been associated with repopulation of tumours after chemotherapy. This phenomenon is proposed as a possible mechanism for cancer relapse after treatment. Cancer stem cells have also been implicated in metastasis, the major cause of mortality in cancer patients. Therefore, any treatment that is capable of targeting and removing breast cancer stem cells may have the theoretical potential to effectively treat breast cancer. However, there are currently no such treatments available for clinical use. We were provided access to a library of novel indigenous small molecules isolated from red and brown algae found off the Eastern Cape of South Africa. The aim of this project was to analyse the anti-cancer and anti-cancer stem cell properties of the compounds in this library and to identify „hit‟ compounds which could form the basis for future development into new anti-cancer drugs. Ten novel compounds of algal origin were tested for cytotoxicity, by determining their ability to inhibit the growth of MCF12A breast epithelial cells and MCF7 breast cancer cells using the colorimetric MTT [(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] cell proliferation assay. All but one of the compounds tested exhibited cytotoxicity towards the MCF7 cancer cell line, with IC50 values (the concentration of the compound that leads to a 50% inhibition in cell growth) of between 3 μM and 90 μM. The chemotherapeutic drug paclitaxel was used as a positive control. Four of the compounds (RUMB-001, RUMB-002, RUMB-007 and RUMB-010/saragaquinoic acid) were significantly more toxic to the MCF7 cancer cell line, than the „normal‟ MCF12A breast cells and were selected as priority compounds for further analyses. In addition, two other compounds were selected as priority compounds, one highly cytotoxic towards both MCF12A and MCF7 cell lines (RUMB-015) and one which was non toxic to either cell line (RUMB-017/018). Preliminary studies into the mechanism of cytotoxicity using Western blot analysis for poly (ADP-ribose) polymerase (PARP) cleavage and Hoechst 33342 immunostaining in MCF-7 cells were largely unsuccessful. The Hoechst 33342 immunostaining assay did provide tentative evidence that selected priority compounds were capable of inducing apoptosis, although these assays will need to be repeated using a less subjective assay to confirm the results. The priority compounds were subsequently investigated for their cytotoxic effect on the cancer stem cell-enriched side population in MCF7 cells. The ability of the priority compounds to selectively target the cancer stem cell containing side population was assessed using two complementary flow cytometry-based techniques – namely the Hoechst 33342-exclusion assay, and fluorescent immunostaining for the expression of the putative cancer stem cell marker, ABCG2+. The ABCG2+ staining assay was a novel technique developed during the course of this study. It remains to be fully validated, but it may provide a new and reliable way to identify and analyse cancer stem cell containing side population cells. The MCF7 cells were treated with the compounds and the proportion of putative cancer stem cells compared with the size of the population in untreated cells was assessed. Three compounds (RUMB-010, RUMB-015 and RUMB-017/018) capable of reducing the proportion of side population cells within the MCF7 cell line were identified. Taking these data together, we identified two potential „hit‟ compounds which should be prioritised for future research. These are compounds RUMB-010/sargaquinoic acid and RUMB-017/018. RUMB-010 is of interest as it was shown to target the putative cancer stem cell population, in addition to the bulk MCF7 tumour line, but was relatively less toxic to the „normal‟ MCF12A cell line. RUMB-017/018 is of interest due to the ability to selectively target the cancer stem cell enriched side population, while having little effect on the normal (MCF12A) or bulk tumour (MCF7) cell lines tested. These compounds will be important as „hit‟ compounds for drug development and as tool compounds to study cancer and cancer stem cell biology.
- Full Text:
- Date Issued: 2010
Cleaning of fouled membranes using enzymes from a sulphidogenic bioreactor
- Authors: Melamane, Xolisa
- Date: 2004
- Subjects: Membrane filters , Membrane filters -- Fouling , Enzymes -- Biotechnology , Enzymes -- Purification , Water -- Purification -- Membrane filtration , Ultrafiltration
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4133 , http://hdl.handle.net/10962/d1015764
- Description: Maintenance of membrane performance requires inevitable cleaning or defouling of fouled membranes. Membrane cleaning using enzymes such as proteases, lipases, α-glucosidases from a sulphidogenic bioreactor was investigated. At first, dilute and concentrated enzyme extract were prepared form the sulphidogenic pellet. Enzyme assays on 0.5 % azocaisen, 1 % triacetin and 1 mg/ml ρ-nitrophenyl-α-D-glucopyranoside were performed using the concentrated enzyme extract (0 – 200 mg/ml). For membrane fouling, an abattoir effluent was obtained from Ostritech Pty (Ltd), Grahamstown, South Africa. The effluent was characterised for presence of potential foulants such as lipids, proteins, amino acids and carbohydrates. Static fouling of polysulphone membranes (0.22 μm, 47 mm) was then performed using the abattoir effluent. Cleaning of the fouled membranes was also performed using at first the dilute and then the concentrated form (200 mg/ml) of enzyme extracts. Qualitative and quantitative biochemical analysis for proteins, lipids and carbohydrates was performed to ascertain the presence of foulants on polysulphone membranes and their removal by dilute or concentrated enzyme extracts. The ability of dilute enzyme extracts to remove proteins lipids, and carbohydrates fouling capillary UF membrane module; their ability to restore permeate fluxes and transmembrane pressure after cleaning/defouling was also investigated. Permeate volumes from this UF membrane module were analysed for protein, amino acids, lipids, and carbohydrates concentrations after fouling and defouling. Fouling was further characterized by standard blocking, cake filtration and pore blocking models using stirred UF cell and polyethersulphone membranes with MWCO of 30 000, 100 000 and 300 000. After characterization of fouling, polyethersulphone membranes with MWCO of 30 000 and 300 000 were defouled using the concentrated enzyme extract (100 mg ml). Enzyme activities at 200 mg/ml of enzyme concentration were 8.071 IU, 86.71 IU and 789.02 IU for proteases, lipases and α-glucosidases. The abattoir effluent contained 553 μg/ml of lipid, 301 μg/ml of protein, 141 μg/ml of total carbohydrate, and 0.63 μg/ml of total reducing sugars. Proteins, lipids and carbohydrates fouling polysulphone membranes after a day were removed by 23.4 %, when a dilute enzyme was used. A concentrated enzyme extract of 200 mg/ml was able to remove proteins, lipids and carbohydrates up to 5 days of fouling by 100 %, 82 %, 71 %, 68 % and 76 % respectively. Defouling of dynamically fouled capillary ultrafiltration membranes using sulphidogenic proteases was successful at pH 10, 37°C, within 1 hour. Sulphidogenic proteases activity was 2.1 U/ml and flux Recovery (FR %) was 64. Characterization of fouling revealed that proteins and lipids were major foulants while low concentration of carbohydrates fouled polyethersulphone membranes. Fouling followed standard blocking for 10 minutes in all the membranes; afterwards fouling adopted cake filtration model for membranes with 30 000 MWCO and pore blocking model for membranes with 300 000 MWCO. A concentration of 100 mg/ml of enzyme extract was able to remove fouling from membranes with MWCO of 30 000. Defouling membranes that followed pore blocking model i.e. 300 000 MWCO was not successful due to a mass transfer problem. From the results of defouling of 30 000 and 300 000 MWCO it was concluded that defouling of cake layer fouling (30 000 MWCO) was successful while defouling of pore blocking fouling was unsuccessful due to a mass transfer problem. The ratio of enzymes present in the enzyme extract when calculated based on enzymatic activity for proteases, lipases and α-glucosidases was 1.1 %, 11 % and 87.9 %. It was hypothesized that apart from proteases, lipases, α and β-glucosidases; phosphatases, sulphatases, amonipeptidases etc. from a sulphidogenic bioreactor clean or defoul cake layer fouling by organic foulants and pore blocking fouling provided the mass transfer problem is solved. However, concentration of enzymes from a sulphidogenic bioreactor has not been optimized yet. Other methods of concentrating the enzyme extract can be investigated for example use of organic solvents.
- Full Text:
- Date Issued: 2004
- Authors: Melamane, Xolisa
- Date: 2004
- Subjects: Membrane filters , Membrane filters -- Fouling , Enzymes -- Biotechnology , Enzymes -- Purification , Water -- Purification -- Membrane filtration , Ultrafiltration
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4133 , http://hdl.handle.net/10962/d1015764
- Description: Maintenance of membrane performance requires inevitable cleaning or defouling of fouled membranes. Membrane cleaning using enzymes such as proteases, lipases, α-glucosidases from a sulphidogenic bioreactor was investigated. At first, dilute and concentrated enzyme extract were prepared form the sulphidogenic pellet. Enzyme assays on 0.5 % azocaisen, 1 % triacetin and 1 mg/ml ρ-nitrophenyl-α-D-glucopyranoside were performed using the concentrated enzyme extract (0 – 200 mg/ml). For membrane fouling, an abattoir effluent was obtained from Ostritech Pty (Ltd), Grahamstown, South Africa. The effluent was characterised for presence of potential foulants such as lipids, proteins, amino acids and carbohydrates. Static fouling of polysulphone membranes (0.22 μm, 47 mm) was then performed using the abattoir effluent. Cleaning of the fouled membranes was also performed using at first the dilute and then the concentrated form (200 mg/ml) of enzyme extracts. Qualitative and quantitative biochemical analysis for proteins, lipids and carbohydrates was performed to ascertain the presence of foulants on polysulphone membranes and their removal by dilute or concentrated enzyme extracts. The ability of dilute enzyme extracts to remove proteins lipids, and carbohydrates fouling capillary UF membrane module; their ability to restore permeate fluxes and transmembrane pressure after cleaning/defouling was also investigated. Permeate volumes from this UF membrane module were analysed for protein, amino acids, lipids, and carbohydrates concentrations after fouling and defouling. Fouling was further characterized by standard blocking, cake filtration and pore blocking models using stirred UF cell and polyethersulphone membranes with MWCO of 30 000, 100 000 and 300 000. After characterization of fouling, polyethersulphone membranes with MWCO of 30 000 and 300 000 were defouled using the concentrated enzyme extract (100 mg ml). Enzyme activities at 200 mg/ml of enzyme concentration were 8.071 IU, 86.71 IU and 789.02 IU for proteases, lipases and α-glucosidases. The abattoir effluent contained 553 μg/ml of lipid, 301 μg/ml of protein, 141 μg/ml of total carbohydrate, and 0.63 μg/ml of total reducing sugars. Proteins, lipids and carbohydrates fouling polysulphone membranes after a day were removed by 23.4 %, when a dilute enzyme was used. A concentrated enzyme extract of 200 mg/ml was able to remove proteins, lipids and carbohydrates up to 5 days of fouling by 100 %, 82 %, 71 %, 68 % and 76 % respectively. Defouling of dynamically fouled capillary ultrafiltration membranes using sulphidogenic proteases was successful at pH 10, 37°C, within 1 hour. Sulphidogenic proteases activity was 2.1 U/ml and flux Recovery (FR %) was 64. Characterization of fouling revealed that proteins and lipids were major foulants while low concentration of carbohydrates fouled polyethersulphone membranes. Fouling followed standard blocking for 10 minutes in all the membranes; afterwards fouling adopted cake filtration model for membranes with 30 000 MWCO and pore blocking model for membranes with 300 000 MWCO. A concentration of 100 mg/ml of enzyme extract was able to remove fouling from membranes with MWCO of 30 000. Defouling membranes that followed pore blocking model i.e. 300 000 MWCO was not successful due to a mass transfer problem. From the results of defouling of 30 000 and 300 000 MWCO it was concluded that defouling of cake layer fouling (30 000 MWCO) was successful while defouling of pore blocking fouling was unsuccessful due to a mass transfer problem. The ratio of enzymes present in the enzyme extract when calculated based on enzymatic activity for proteases, lipases and α-glucosidases was 1.1 %, 11 % and 87.9 %. It was hypothesized that apart from proteases, lipases, α and β-glucosidases; phosphatases, sulphatases, amonipeptidases etc. from a sulphidogenic bioreactor clean or defoul cake layer fouling by organic foulants and pore blocking fouling provided the mass transfer problem is solved. However, concentration of enzymes from a sulphidogenic bioreactor has not been optimized yet. Other methods of concentrating the enzyme extract can be investigated for example use of organic solvents.
- Full Text:
- Date Issued: 2004
The role of vitamin E succinate in regulation of growth and cyclooxygenase expression in B16 murine melanoma cells
- Van der Merwe, Adele Shanette
- Authors: Van der Merwe, Adele Shanette
- Date: 1999
- Subjects: Melanoma , Vitamin E
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3988 , http://hdl.handle.net/10962/d1004047 , Melanoma , Vitamin E
- Description: This study was undertaken to determine the effects and possible mechanism of action of vitamin E succinate supplementation on B16 murine melanoma cell growth in vitro. Studies revealed that supplementation of 5, 7 and 10µg/ml of this vitamin significantly inhibited growth of B16 cells. Non-malignant LLCMK cells supplemented with the same concentrations of vitamin E succinate resulted in similar inhibition of cell growth. The actual mechanism by which vitamin E succinate inhibits B16 cell growth is unclear, though there has been much speculation about its possible role as an antioxidant. Vitamin E succinate is not a physiological antioxidant and for this ester to behave as an antioxidant, cleavage of the ester bond must occur, releasing the antioxidant vitamin E part of the molecule. To determine whether the observed inhibitory effects on B16 cell growth were due to the intact vitamin E succinate or the vitamin E cleavage product, cleavage studies were undertaken. Results from these studies revealed that in B16 cells vitamin E succinate cleavage did not occur suggesting that the observed inhibitory effects of vitamin E succinate on B16 cells were due to the intact compound. In contrast vitamin E succinate cleavage was shown to occur in LLCMK cells, suggesting that these cells may contain an esterase capable of liberating succinic acid and vitamin E. Further studies focussed on the possible role of vitamin E succinate in regulation of cyclooxygenase activity in B16 cells as vitamin E succinate was found to effect the activity of various enzymes involved in the arachidonic acid cascade, notably cyclooxygenase, the rate-limiting enzyme in prostaglandin synthesis. Time course studies were used to determine when the cyclooxygenase protein was being produced, thus allowing an estimation of when the gene was being 'switched on'. These studies revealed that vitamin E succinate does not significantly effect cyclooxygenase activity in B16 cells over a period of 2 to 12 hours as compared to the OE control cultures. Further studies using RNA techniques investigated whether vitamin E succinate was having an effect on cyclooxygenase activity at a molecular level. These investigations were unsuccessful for the 6 day supplementation for a number of possible reasons, the main reason being RNA stability. Subsequent studies revealed an increase in COX mRNA after 2 hours, suggesting that the gene was 'switched on' soon after supplementation with vitamin E succinate, and further increases in COX mRNA were observed after 8 to 12 hours. The molecular studies were, however, inconclusive. Previous studies suggested that vitamin E succinate was indirectly causing growth inhibition of B16 cells via regulation of cyclooxygenase activity, however, this study does not support these findings and it would seem unlikely that regulation of cyclooxygenase expression in B16 cells by vitamin E succinate has a role to play in the mechanism by which vitamin E succinate inhibits growth in B16 cells.
- Full Text:
- Date Issued: 1999
- Authors: Van der Merwe, Adele Shanette
- Date: 1999
- Subjects: Melanoma , Vitamin E
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3988 , http://hdl.handle.net/10962/d1004047 , Melanoma , Vitamin E
- Description: This study was undertaken to determine the effects and possible mechanism of action of vitamin E succinate supplementation on B16 murine melanoma cell growth in vitro. Studies revealed that supplementation of 5, 7 and 10µg/ml of this vitamin significantly inhibited growth of B16 cells. Non-malignant LLCMK cells supplemented with the same concentrations of vitamin E succinate resulted in similar inhibition of cell growth. The actual mechanism by which vitamin E succinate inhibits B16 cell growth is unclear, though there has been much speculation about its possible role as an antioxidant. Vitamin E succinate is not a physiological antioxidant and for this ester to behave as an antioxidant, cleavage of the ester bond must occur, releasing the antioxidant vitamin E part of the molecule. To determine whether the observed inhibitory effects on B16 cell growth were due to the intact vitamin E succinate or the vitamin E cleavage product, cleavage studies were undertaken. Results from these studies revealed that in B16 cells vitamin E succinate cleavage did not occur suggesting that the observed inhibitory effects of vitamin E succinate on B16 cells were due to the intact compound. In contrast vitamin E succinate cleavage was shown to occur in LLCMK cells, suggesting that these cells may contain an esterase capable of liberating succinic acid and vitamin E. Further studies focussed on the possible role of vitamin E succinate in regulation of cyclooxygenase activity in B16 cells as vitamin E succinate was found to effect the activity of various enzymes involved in the arachidonic acid cascade, notably cyclooxygenase, the rate-limiting enzyme in prostaglandin synthesis. Time course studies were used to determine when the cyclooxygenase protein was being produced, thus allowing an estimation of when the gene was being 'switched on'. These studies revealed that vitamin E succinate does not significantly effect cyclooxygenase activity in B16 cells over a period of 2 to 12 hours as compared to the OE control cultures. Further studies using RNA techniques investigated whether vitamin E succinate was having an effect on cyclooxygenase activity at a molecular level. These investigations were unsuccessful for the 6 day supplementation for a number of possible reasons, the main reason being RNA stability. Subsequent studies revealed an increase in COX mRNA after 2 hours, suggesting that the gene was 'switched on' soon after supplementation with vitamin E succinate, and further increases in COX mRNA were observed after 8 to 12 hours. The molecular studies were, however, inconclusive. Previous studies suggested that vitamin E succinate was indirectly causing growth inhibition of B16 cells via regulation of cyclooxygenase activity, however, this study does not support these findings and it would seem unlikely that regulation of cyclooxygenase expression in B16 cells by vitamin E succinate has a role to play in the mechanism by which vitamin E succinate inhibits growth in B16 cells.
- Full Text:
- Date Issued: 1999
Metabolic responses in melanoma cells to combined nutrient supplementation
- Authors: Midgley, Nicola-Ann
- Date: 1997
- Subjects: Melanoma Tumors -- Growth
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4036 , http://hdl.handle.net/10962/d1004096
- Description: This thesis examined the effect and biochemical mechanism by which combined vitamin E and C supplementation may influence tumour cell growth. The study initially addressed the effect of combined vitamin E succinate and Asc supplementation over a nutritional concentration range (5- 20μg/ml) and (25-50μg/ml) respectively, on the in vitro growth of non-malignant LLCMK and malignant BL6 cells. Supplementation of BL6 and LLCMK cells with combined vitamin E succinate and ascorbic acid, resulted in no significant increasing or decreasing trend in LLCMK cell growth, while in BL6 cells a significant decrease in cell growth was observed at all combined vitamin concentrations. It has been suggested that these vitamins may act synergistically to inhibit tumour cell growth through their antioxidant properties in quenching free radicals and lipid peroxidation and furthermore through their modulation of the activities of various enzymes and metabolites in the eicosanoid pathway. This study consequently investigated the effects of combined vitamin E succinate and ascorbic acid supplementation on these parameters. Throughout this study, emphasis was placed on the BL6 melanoma cells, as combined vitamin E succinate and ascorbic acid supplementation did not significantly affect growth or levels of secondary metabolites in the non-malignant LLCMK cells. Combined vitamin E succinate and ascorbic acid supplementation of BL6 cells resulted in a marked but non significant increase in free radical and a significant increase in lipid peroxidation levels. This prooxidant effect was accompanied by a significant decrease in BL6 cell growth, suggesting that the growth inhibitory effects of combined vitainin E succinate and ascorbic acid on BL6 cells in vitro was not mediated through their synergistic antioxidant properties. Vitamin E succinate is a nonphysiological antioxidant in its esterified form, hence cleavage of the succinate group must occur in order for ascorbic acid to interact with the free alcohol, vitamin E. The inability of combined vitamin E succinate and ascorbic acid to reduce free radicals and lipid peroxidation levels within BL6 cells may not be due to their ineffectiveness as antioxidants but rather the presence of other contributing factors which influence the oxidation state within the BL6 cells. Vitamin E is believed to modulate membrane-bound enzymes through membrane stabilization. Furthermore, the stabilizing effect of vitamin E may be enhanced by the ascorbic acid-sparing effect of vitamin E. Hence, this study investigated the effect of combined vitamin E succinate and ascorbic acid in modulating the activity of various enzymes and secondary messengers in the eicosanoid pathway. Supplementation with combined vitamin E succinate (5-20μg/ml) and ascorbic acid (25-50μg/ml) resulted in significant increases in phospholipase A₂, 5-lipoxygenase, cyclooxygenase and adenyl ate cyclase activity, with a significant decrease in BL6 cell growth. The possible synergistic action of these vitamins in terms of modulating membrane-bound enzymes was further substantiated by uptake and cellular distribution studies. Vitamin E succinate and vitamin E in the membrane fraction increased significantly compared to control cultures, while ascorbic acid levels were significantly higher in the stroma fraction when compared to membrane fractions. Consequently, another factor accounting for increased activities of phospholipase A2, 5-lipoxygenase and adenylate cyclase activities as a result of vitamin supplementation in BL6 cells may be an increased availability of Ca²+. Supplementation of BL6 cells with combined vitamin E succinate and ascorbic acid resulted in significant increases in intracellular Ca²+ levels at all combined vitamin groups. Furthermore, this increase in intracellular Ca²+ was positively correlated with cl1anges of the above-mentioned enzyme activities. Within the eicosanoid pathway, the rate of prostaglandin synthesis is regulated by phospholipase A₂ activity and arachidonic acid release, and the net prostaglandin production is dependent on cyclooxygenase activity, hence the effects of combined vitamin E succinate and ascorbic acid on arachidonic acid composition and prostaglandin production within BL6 cells was determined. The percentage arachidonic acid composition of the BL6 cells was elevated and inversely related to cell growth following combined vitamin E succinate and ascorbic acid supplementation. Prostaglandin E₂ and prostaglandin I₂ levels increased significantly, while those of prostaglandin D2 and prostaglandin F₂α increased markedly following supplementation of combined vitamin E succinate and ascorbic acid. These increases in prostaglandin levels were inversely related to BL6 cell growth, suggesting that the prostaglandins were involved in negative regulation of BL6 cell growth. When comparing the levels of prostaglandins, prostaglandin E2 levels were significantly higher when compared to prostaglandin D₂, prostaglandin F₂α and prostaglandin I₂ suggesting that vitamin E₂ succinate and ascorbic acid effects were mediated primarily through an increase in prostaglandin E2. Hence, prostaglandin E2 levels in combined vitamin E succinate and ascorbic acid appeared to be dependent on the amount of precursor present and the activity of its synthetic enzymes. This was confirmed when BL6 cells were supplemented with arachidonic acid. Arachidonic acid had an inhibitory effect on BL6 cell growth and also stimulated prostaglandin E₂ production. Prostaglandin E₂ levels are in turn believed to modulate adenylate cyclase activity in BL6 cells, hence it is reasonable to conclude that adenylate cyclase activity is dependent on prostaglandin E₂ levels. Combined vitamin E succinate and Asc supplementation to BL6 cells resulted in significant increases in adenyl ate cyclase and cyclic adenosine monophosphate, which again correlated with a significant decrease in cell growth. As cyclic adenosine monophosphate has a regulatory role in the cell cycle this study suggested that the effect of combined vitamin E succinate and ascorbic acid supplementation was mediated through the final effect provided by the second messenger, cyclic adenosine monophosphate. This was confirmed when BL6 cells were supplemented with dexamethasone, a phospholipase A₂ inhibitor. This treatment rsulted in combined vitamin E succinate and ascorbic acid having no inhibitory effect on BL6 cell growth. Cyclooxygenase activity, prostaglandin E₂ levels, adenylate cyclase activity and cyclic adenosine monophosphate levels were significantly lower in dexamethasone-treated cells compared to non-treated dexamethasone cultures. The reason for the increased free radical and lipid peroxidation levels in BL6 cells was further investigated. Cyclooxygenase enzymes are believed to generate free radical species during catalytic activity. Analysis of free radical and lipid peroxidation levels following supplementation with dexamethasone revealed markedly lower free radical and significantly lower lipid peroxidation levels in comparison with control cultures and non dexamethasone-treated cultures. These results suggest that the observed increases in free radical and lipid peroxidation levels in BL6 cells supplemented with combined vitamin E succinate and ascorbic acid were indirectly due to the increase in cyclooxygenase activity in these cells.
- Full Text:
- Date Issued: 1997
- Authors: Midgley, Nicola-Ann
- Date: 1997
- Subjects: Melanoma Tumors -- Growth
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4036 , http://hdl.handle.net/10962/d1004096
- Description: This thesis examined the effect and biochemical mechanism by which combined vitamin E and C supplementation may influence tumour cell growth. The study initially addressed the effect of combined vitamin E succinate and Asc supplementation over a nutritional concentration range (5- 20μg/ml) and (25-50μg/ml) respectively, on the in vitro growth of non-malignant LLCMK and malignant BL6 cells. Supplementation of BL6 and LLCMK cells with combined vitamin E succinate and ascorbic acid, resulted in no significant increasing or decreasing trend in LLCMK cell growth, while in BL6 cells a significant decrease in cell growth was observed at all combined vitamin concentrations. It has been suggested that these vitamins may act synergistically to inhibit tumour cell growth through their antioxidant properties in quenching free radicals and lipid peroxidation and furthermore through their modulation of the activities of various enzymes and metabolites in the eicosanoid pathway. This study consequently investigated the effects of combined vitamin E succinate and ascorbic acid supplementation on these parameters. Throughout this study, emphasis was placed on the BL6 melanoma cells, as combined vitamin E succinate and ascorbic acid supplementation did not significantly affect growth or levels of secondary metabolites in the non-malignant LLCMK cells. Combined vitamin E succinate and ascorbic acid supplementation of BL6 cells resulted in a marked but non significant increase in free radical and a significant increase in lipid peroxidation levels. This prooxidant effect was accompanied by a significant decrease in BL6 cell growth, suggesting that the growth inhibitory effects of combined vitainin E succinate and ascorbic acid on BL6 cells in vitro was not mediated through their synergistic antioxidant properties. Vitamin E succinate is a nonphysiological antioxidant in its esterified form, hence cleavage of the succinate group must occur in order for ascorbic acid to interact with the free alcohol, vitamin E. The inability of combined vitamin E succinate and ascorbic acid to reduce free radicals and lipid peroxidation levels within BL6 cells may not be due to their ineffectiveness as antioxidants but rather the presence of other contributing factors which influence the oxidation state within the BL6 cells. Vitamin E is believed to modulate membrane-bound enzymes through membrane stabilization. Furthermore, the stabilizing effect of vitamin E may be enhanced by the ascorbic acid-sparing effect of vitamin E. Hence, this study investigated the effect of combined vitamin E succinate and ascorbic acid in modulating the activity of various enzymes and secondary messengers in the eicosanoid pathway. Supplementation with combined vitamin E succinate (5-20μg/ml) and ascorbic acid (25-50μg/ml) resulted in significant increases in phospholipase A₂, 5-lipoxygenase, cyclooxygenase and adenyl ate cyclase activity, with a significant decrease in BL6 cell growth. The possible synergistic action of these vitamins in terms of modulating membrane-bound enzymes was further substantiated by uptake and cellular distribution studies. Vitamin E succinate and vitamin E in the membrane fraction increased significantly compared to control cultures, while ascorbic acid levels were significantly higher in the stroma fraction when compared to membrane fractions. Consequently, another factor accounting for increased activities of phospholipase A2, 5-lipoxygenase and adenylate cyclase activities as a result of vitamin supplementation in BL6 cells may be an increased availability of Ca²+. Supplementation of BL6 cells with combined vitamin E succinate and ascorbic acid resulted in significant increases in intracellular Ca²+ levels at all combined vitamin groups. Furthermore, this increase in intracellular Ca²+ was positively correlated with cl1anges of the above-mentioned enzyme activities. Within the eicosanoid pathway, the rate of prostaglandin synthesis is regulated by phospholipase A₂ activity and arachidonic acid release, and the net prostaglandin production is dependent on cyclooxygenase activity, hence the effects of combined vitamin E succinate and ascorbic acid on arachidonic acid composition and prostaglandin production within BL6 cells was determined. The percentage arachidonic acid composition of the BL6 cells was elevated and inversely related to cell growth following combined vitamin E succinate and ascorbic acid supplementation. Prostaglandin E₂ and prostaglandin I₂ levels increased significantly, while those of prostaglandin D2 and prostaglandin F₂α increased markedly following supplementation of combined vitamin E succinate and ascorbic acid. These increases in prostaglandin levels were inversely related to BL6 cell growth, suggesting that the prostaglandins were involved in negative regulation of BL6 cell growth. When comparing the levels of prostaglandins, prostaglandin E2 levels were significantly higher when compared to prostaglandin D₂, prostaglandin F₂α and prostaglandin I₂ suggesting that vitamin E₂ succinate and ascorbic acid effects were mediated primarily through an increase in prostaglandin E2. Hence, prostaglandin E2 levels in combined vitamin E succinate and ascorbic acid appeared to be dependent on the amount of precursor present and the activity of its synthetic enzymes. This was confirmed when BL6 cells were supplemented with arachidonic acid. Arachidonic acid had an inhibitory effect on BL6 cell growth and also stimulated prostaglandin E₂ production. Prostaglandin E₂ levels are in turn believed to modulate adenylate cyclase activity in BL6 cells, hence it is reasonable to conclude that adenylate cyclase activity is dependent on prostaglandin E₂ levels. Combined vitamin E succinate and Asc supplementation to BL6 cells resulted in significant increases in adenyl ate cyclase and cyclic adenosine monophosphate, which again correlated with a significant decrease in cell growth. As cyclic adenosine monophosphate has a regulatory role in the cell cycle this study suggested that the effect of combined vitamin E succinate and ascorbic acid supplementation was mediated through the final effect provided by the second messenger, cyclic adenosine monophosphate. This was confirmed when BL6 cells were supplemented with dexamethasone, a phospholipase A₂ inhibitor. This treatment rsulted in combined vitamin E succinate and ascorbic acid having no inhibitory effect on BL6 cell growth. Cyclooxygenase activity, prostaglandin E₂ levels, adenylate cyclase activity and cyclic adenosine monophosphate levels were significantly lower in dexamethasone-treated cells compared to non-treated dexamethasone cultures. The reason for the increased free radical and lipid peroxidation levels in BL6 cells was further investigated. Cyclooxygenase enzymes are believed to generate free radical species during catalytic activity. Analysis of free radical and lipid peroxidation levels following supplementation with dexamethasone revealed markedly lower free radical and significantly lower lipid peroxidation levels in comparison with control cultures and non dexamethasone-treated cultures. These results suggest that the observed increases in free radical and lipid peroxidation levels in BL6 cells supplemented with combined vitamin E succinate and ascorbic acid were indirectly due to the increase in cyclooxygenase activity in these cells.
- Full Text:
- Date Issued: 1997
Bioaccumulation of heavy metals by the yeast S. cerevisiae and the bioremediation of industrial waste water
- Authors: Stoll, Anita
- Date: 1997
- Subjects: Saccharomyces cerevisiae Yeast fungi -- Biotechnology Metal ions Bioremediation Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4015 , http://hdl.handle.net/10962/d1004075
- Description: Water is an essential element in all aspects of life and is vital for both domestic and industrial purposes regarding both the quality and quantity thereof. Similar to many other drought stricken countries, South Africa requires water for the socio-economic growth of the country, yet is faced with the problem of maintaining the quality of its drinking water as well as protecting the dwindling supplies. In an attempt to prevent the deterioration of South African water supplies the treatment, purification and recycling of industrial and mining waste water has recently become of prime importance. Many industrial and mining waste waters contain heavy metals in toxic quantities. The conventional processes that have been used till recently to address this problem, are often expensive or contain chemical agents which compound the environmental problem. As an alternative biological methods of metal accumulation appear to offer an economic and efficient alternative to these methods. An advantage to the South African scenario is the commercial production of the yeast, S. cerevisiae as a readily inexpensive by-product from some fermentation industries, Yeast cells, and in particular S. cerevisiae have proven to be capable of accumulating heavy metals, and therefore exhibit potential application in the bioremediation of waste water. The aim of this project was twofold. The initial part of this work attempted to define the mechanisms of metal accumulation by the yeast cells and cellular components. The information obtained from these initial studies provided a data base required for the development of a bioremediation system. Initial contact with the metal ions occurs at the wall interface of the yeast cell. Metal accumulation appears to be a function of all the cell wall components. The isolated cell wall components are better metal chelators then the intact cell walls. An apparent affinity series of mannan > chitin> glucan > intact cell walls exists. However, these components differ in their affinities for metal ions. Storage of metal ions within the cell occurs predominantly in the vacuole. The present study concluded that metal accumulation by the vacuole could be related to size. Metal accumulation occurred in the order of Cu2+ > Co2+ > Cd2+ with a corresponding decrease in atomic radii of Cd2+ > C02+ > Cu2+. Vacuolar ion deposition occurs at an early stage during the internalization of metal ions within the yeast cells. At the onset of vacuolar saturation, depositions of metal ions as granules within the cytosol occurs. In the presence of heavy metal cations viable yeast cells can be shown to exhibit two types of cellular responses. Uptake of Cu2+ and Cd2+ causes the loss of intracellular physiological cations from within the yeast cell. In comparison, uptake of Co2+ into the cell does not have this effect. All three heavy metal cations initiate plasma cell membrane permeability, thus the Cu2+ and Cd2+ induced loss of the intracellular cations, occurs. ~ a result of ion-exchange mechanisms and not due to cation leakage brought about by membrane permeabilization. Uptake of heavy metals by viable yeasts appears to be generally non-selective though the amount of metals accumulated are largely affected by the ratio of ambient metal concentration to biomass quantity. In addition, the energy dependent nature of internalization necessitates the availability of an external energy source for metal uptake by viable yeast cells. For these reasons metal removal from industrial waste water was investigated using non-viable biomass. By immobilizing the yeast cells additional mechanical integrity and stability was conferred apon the biomass. The three types of biomass preparations developed in this study, viz. polyvinyl alcohol (PV A) Na-alginate, PV A Na-orthophosphate and alkali treated polyethylenimine (PEI):glutaraldehyde (GA) biomass pellets, all fulfilled the necessary physical requirements. However, the superior metal accumulating properties of the PEI:GA biomass determined its selection as a biosorbent for bioremediation purposes. Biosorption of heavy metals by PEI:GA biomass is of a competitive nature, with the amount of metal accumulated influenced by the availability of the metal ions. This availability is largely determined by the solution pH. At low pH values the affinity of the biomass for metals decreases, whilst enhanced metal biosorption occurs at higher pHs, ego pH 4.5 - 6.0. PEI:GA biomass pellets can be implemented -as a biosorbent for the bi9remediaiton of high concentration, low-volume metal containing industrial waste. Several options regarding the bioremediation system are available. Depending on the concentration of the metals in the effluent, the bioremediation process can either be used independently or as part of a biphasic remediation system for the treatment of waste water. Initial phase chemical modification may be required, whilst two types of biological systems can be implemented as 'part of the second phase. The PEI:GA biomass can either be contained within continuous-flow fixed bed tanks or continuous-flow stirred bioreactor tanks. Due to the simplicity of the process and the ease with which scale-up is facilitated, the second type of system shows greater application potential for the treatment of this type of industrial waste water than the fixed-bed systems.
- Full Text:
- Date Issued: 1997
- Authors: Stoll, Anita
- Date: 1997
- Subjects: Saccharomyces cerevisiae Yeast fungi -- Biotechnology Metal ions Bioremediation Water -- Purification -- Biological treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4015 , http://hdl.handle.net/10962/d1004075
- Description: Water is an essential element in all aspects of life and is vital for both domestic and industrial purposes regarding both the quality and quantity thereof. Similar to many other drought stricken countries, South Africa requires water for the socio-economic growth of the country, yet is faced with the problem of maintaining the quality of its drinking water as well as protecting the dwindling supplies. In an attempt to prevent the deterioration of South African water supplies the treatment, purification and recycling of industrial and mining waste water has recently become of prime importance. Many industrial and mining waste waters contain heavy metals in toxic quantities. The conventional processes that have been used till recently to address this problem, are often expensive or contain chemical agents which compound the environmental problem. As an alternative biological methods of metal accumulation appear to offer an economic and efficient alternative to these methods. An advantage to the South African scenario is the commercial production of the yeast, S. cerevisiae as a readily inexpensive by-product from some fermentation industries, Yeast cells, and in particular S. cerevisiae have proven to be capable of accumulating heavy metals, and therefore exhibit potential application in the bioremediation of waste water. The aim of this project was twofold. The initial part of this work attempted to define the mechanisms of metal accumulation by the yeast cells and cellular components. The information obtained from these initial studies provided a data base required for the development of a bioremediation system. Initial contact with the metal ions occurs at the wall interface of the yeast cell. Metal accumulation appears to be a function of all the cell wall components. The isolated cell wall components are better metal chelators then the intact cell walls. An apparent affinity series of mannan > chitin> glucan > intact cell walls exists. However, these components differ in their affinities for metal ions. Storage of metal ions within the cell occurs predominantly in the vacuole. The present study concluded that metal accumulation by the vacuole could be related to size. Metal accumulation occurred in the order of Cu2+ > Co2+ > Cd2+ with a corresponding decrease in atomic radii of Cd2+ > C02+ > Cu2+. Vacuolar ion deposition occurs at an early stage during the internalization of metal ions within the yeast cells. At the onset of vacuolar saturation, depositions of metal ions as granules within the cytosol occurs. In the presence of heavy metal cations viable yeast cells can be shown to exhibit two types of cellular responses. Uptake of Cu2+ and Cd2+ causes the loss of intracellular physiological cations from within the yeast cell. In comparison, uptake of Co2+ into the cell does not have this effect. All three heavy metal cations initiate plasma cell membrane permeability, thus the Cu2+ and Cd2+ induced loss of the intracellular cations, occurs. ~ a result of ion-exchange mechanisms and not due to cation leakage brought about by membrane permeabilization. Uptake of heavy metals by viable yeasts appears to be generally non-selective though the amount of metals accumulated are largely affected by the ratio of ambient metal concentration to biomass quantity. In addition, the energy dependent nature of internalization necessitates the availability of an external energy source for metal uptake by viable yeast cells. For these reasons metal removal from industrial waste water was investigated using non-viable biomass. By immobilizing the yeast cells additional mechanical integrity and stability was conferred apon the biomass. The three types of biomass preparations developed in this study, viz. polyvinyl alcohol (PV A) Na-alginate, PV A Na-orthophosphate and alkali treated polyethylenimine (PEI):glutaraldehyde (GA) biomass pellets, all fulfilled the necessary physical requirements. However, the superior metal accumulating properties of the PEI:GA biomass determined its selection as a biosorbent for bioremediation purposes. Biosorption of heavy metals by PEI:GA biomass is of a competitive nature, with the amount of metal accumulated influenced by the availability of the metal ions. This availability is largely determined by the solution pH. At low pH values the affinity of the biomass for metals decreases, whilst enhanced metal biosorption occurs at higher pHs, ego pH 4.5 - 6.0. PEI:GA biomass pellets can be implemented -as a biosorbent for the bi9remediaiton of high concentration, low-volume metal containing industrial waste. Several options regarding the bioremediation system are available. Depending on the concentration of the metals in the effluent, the bioremediation process can either be used independently or as part of a biphasic remediation system for the treatment of waste water. Initial phase chemical modification may be required, whilst two types of biological systems can be implemented as 'part of the second phase. The PEI:GA biomass can either be contained within continuous-flow fixed bed tanks or continuous-flow stirred bioreactor tanks. Due to the simplicity of the process and the ease with which scale-up is facilitated, the second type of system shows greater application potential for the treatment of this type of industrial waste water than the fixed-bed systems.
- Full Text:
- Date Issued: 1997
A novel adjuvant : polymerised serum albumin beads
- Authors: Dewar, John Barr
- Date: 1985
- Subjects: Antigens , Serum albumin
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4104 , http://hdl.handle.net/10962/d1011146 , Antigens , Serum albumin
- Description: Lee, T. et al (1981) proposed the encapsulation of hormones such as progesterone into serum albumin beads, such that their in vivo proteolysis would allow a gradual release of hormone at low levels, for extended hormone action. It was proposed, in the Department of Microbiology, Rhodes University, to replace the hormone component of the above bead formulation, with virus as antigen, in the development of a vaccine. Beads optimally crosslinked at 1% final glutaraldehyde concentration, containing Nodamura virus, were shown to promote an adjuvant effect in vivo, analogous to the release of antigen from Freund's Complete Adjuvant (FCA), so that extended immunostimulation resulted. It was shown that soluble antigen promoted a short-lived primary immune response, peaking around day 25 following inoculation. Antigen presented in beads, on the other hand, initially elicited a low humoral response, but this response gradually increased up to a peak around day 110 post inoculation, before decreasing. No apparent adverse side-effects were noted following inoculation of antigen-containing serum albumin beads, compared to necrosis following antigen in FCA inoculation, supporting the proposal of using albumin homotypic for the test inoculee animal, so that the beads would themselves be non-immunogenic and would merely act as a vehicle in the vaccine formulation. The indirect enzyme-linked immunosorbent assay (ELISA) was used to monitor the humoral response to antigen following inoculation. Results showed that covalent crosslinking of albumin in the formation of the beads did not promote immunogenicity on the part of the chemically altered albumin. The ELISA test was used to indicate the kinetics of the IgG response to Nodamura virus when presented in formulations such as: Freely soluble virus or its subunit; soluble intact virus inactivated by treatment with glutaraldehyde; intact virus entrapped in serum albumin beads cross; linked at different percentage final glutaraldehyde concentrations and also virus subunit prepared in albumin beads. The presence of virus-neutral ising antibodies was noted in serum obtained from rabbits inoculated with virus entrapped in albumin beads. Virus infectivity, titrated in mice, showed protection against virus challenge after incubation of virus with serum obtained above.
- Full Text:
- Date Issued: 1985
- Authors: Dewar, John Barr
- Date: 1985
- Subjects: Antigens , Serum albumin
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4104 , http://hdl.handle.net/10962/d1011146 , Antigens , Serum albumin
- Description: Lee, T. et al (1981) proposed the encapsulation of hormones such as progesterone into serum albumin beads, such that their in vivo proteolysis would allow a gradual release of hormone at low levels, for extended hormone action. It was proposed, in the Department of Microbiology, Rhodes University, to replace the hormone component of the above bead formulation, with virus as antigen, in the development of a vaccine. Beads optimally crosslinked at 1% final glutaraldehyde concentration, containing Nodamura virus, were shown to promote an adjuvant effect in vivo, analogous to the release of antigen from Freund's Complete Adjuvant (FCA), so that extended immunostimulation resulted. It was shown that soluble antigen promoted a short-lived primary immune response, peaking around day 25 following inoculation. Antigen presented in beads, on the other hand, initially elicited a low humoral response, but this response gradually increased up to a peak around day 110 post inoculation, before decreasing. No apparent adverse side-effects were noted following inoculation of antigen-containing serum albumin beads, compared to necrosis following antigen in FCA inoculation, supporting the proposal of using albumin homotypic for the test inoculee animal, so that the beads would themselves be non-immunogenic and would merely act as a vehicle in the vaccine formulation. The indirect enzyme-linked immunosorbent assay (ELISA) was used to monitor the humoral response to antigen following inoculation. Results showed that covalent crosslinking of albumin in the formation of the beads did not promote immunogenicity on the part of the chemically altered albumin. The ELISA test was used to indicate the kinetics of the IgG response to Nodamura virus when presented in formulations such as: Freely soluble virus or its subunit; soluble intact virus inactivated by treatment with glutaraldehyde; intact virus entrapped in serum albumin beads cross; linked at different percentage final glutaraldehyde concentrations and also virus subunit prepared in albumin beads. The presence of virus-neutral ising antibodies was noted in serum obtained from rabbits inoculated with virus entrapped in albumin beads. Virus infectivity, titrated in mice, showed protection against virus challenge after incubation of virus with serum obtained above.
- Full Text:
- Date Issued: 1985
Polymerized serum albumin beads for use as slow-release adjuvants
- Martin, Michelle Elizabeth Denny
- Authors: Martin, Michelle Elizabeth Denny
- Date: 1988
- Subjects: Serum albumin , Antigens , Vaccines
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3879 , http://hdl.handle.net/10962/d1001613
- Description: Experimental vaccines have been made by covalently bonding virus particles into polymerized rabbit serum albumin beads. Using Nodamura virus as a model antigen, these model vaccines induced specific humoral antibody production, comparable with that achieved using Freund's adjuvants. Virus specific antibodies were also induced when Nodamura virus was covalently attached to the bead surface using different crosslinkers. However, when poliovirus type 2 (Sabin strain) was polymerized into beads, the levels of neutralizing antibodies were insignificant compared with control aqueous vaccines. The synthetic immunostimulator, muramyl dipeptide, was included with bead vaccines in an attempt to potentiate the immune response. Immunostimulation is achieved by a slow release of antigen coinciding with the gradual breakdown of bead structure.
- Full Text:
- Date Issued: 1988
- Authors: Martin, Michelle Elizabeth Denny
- Date: 1988
- Subjects: Serum albumin , Antigens , Vaccines
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3879 , http://hdl.handle.net/10962/d1001613
- Description: Experimental vaccines have been made by covalently bonding virus particles into polymerized rabbit serum albumin beads. Using Nodamura virus as a model antigen, these model vaccines induced specific humoral antibody production, comparable with that achieved using Freund's adjuvants. Virus specific antibodies were also induced when Nodamura virus was covalently attached to the bead surface using different crosslinkers. However, when poliovirus type 2 (Sabin strain) was polymerized into beads, the levels of neutralizing antibodies were insignificant compared with control aqueous vaccines. The synthetic immunostimulator, muramyl dipeptide, was included with bead vaccines in an attempt to potentiate the immune response. Immunostimulation is achieved by a slow release of antigen coinciding with the gradual breakdown of bead structure.
- Full Text:
- Date Issued: 1988
The effect of appetite suppressants on pineal function
- Authors: Mchunu, Bongani Isaac
- Date: 1994
- Subjects: Pineal gland -- Research , Pineal gland -- Secretions , Appetite depressants -- Physiological effect
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4038 , http://hdl.handle.net/10962/d1004098 , Pineal gland -- Research , Pineal gland -- Secretions , Appetite depressants -- Physiological effect
- Description: The pineal gland has become the subject of considerable investigation as it provides a productive experimental model for studying circadian rhythms and regulation of end organs. In the rat, the pineal gland provides a convenient model for investigating the noradrenergic receptor system and the effects of various drugs on this system. The effect of appetite suppressants on the rat pineal gland function is described. Appetite suppressants increase melatonin synthesis in organ cultures of rat pineal glands. This effect appears to be mediated by noradrenaline acting on β-adrenoceptors on the pinealocyte membrane. When β-adrenoceptors are blocked, the appetite suppressant-induced rise in melatonin synthesis is prevented. Depletion of noradrenaline in sympathetic nerve terminals also prevented the appetite suppressant-induced rise in melatonin synthesis. Activation of β-adrenoceptors is followed by a rise in N-acetyltransferase activity via a cyclic adenosine monophosphate second messenger system. The effect of appetite suppressants on the activity of liver tryptophan pyrrolase was also investigated. The activity of this enzyme is an important determinant of tryptophan availability to the brain and consequently of brain serotonin levels. The results show that appetite suppressants inhibit both holoenzyme and total enzyme activities of tryptophan pyrrolase. This finding suggests that appetite suppressants may act by inhibiting tryptophan pyrrolase activity thereby increasing brain serotonin, a phenomenon known to be associated with anorexia. There are two possible mechanisms by which appetite suppressants inhibit tryptophan pyrrolase activity. Firstly, these agents, being drugs of dependence, may increase liver NADPH concentrations which inhibit pyrrolase activity. Secondly, appetite suppressants may act on the pineal gland to stimulate melatonin synthesis. Melatonin inhibits pyrrolase activity in a dose-dependent manner. This inhibition will elevate plasma tryptophan levels which result in a rise in brain serotonin synthesis. The present study suggests a possible relationship between the pineal gland and appetite centres in the hypothalamus. Melatonin may have a direct effect on appetite centres since food restriction is associated with an increased melatonin binding in the hypothalamus. If this possible relationship can be extended, melatonin can open new possibilities for the control of food intake and consequently, of pathological obesity.
- Full Text:
- Date Issued: 1994
- Authors: Mchunu, Bongani Isaac
- Date: 1994
- Subjects: Pineal gland -- Research , Pineal gland -- Secretions , Appetite depressants -- Physiological effect
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4038 , http://hdl.handle.net/10962/d1004098 , Pineal gland -- Research , Pineal gland -- Secretions , Appetite depressants -- Physiological effect
- Description: The pineal gland has become the subject of considerable investigation as it provides a productive experimental model for studying circadian rhythms and regulation of end organs. In the rat, the pineal gland provides a convenient model for investigating the noradrenergic receptor system and the effects of various drugs on this system. The effect of appetite suppressants on the rat pineal gland function is described. Appetite suppressants increase melatonin synthesis in organ cultures of rat pineal glands. This effect appears to be mediated by noradrenaline acting on β-adrenoceptors on the pinealocyte membrane. When β-adrenoceptors are blocked, the appetite suppressant-induced rise in melatonin synthesis is prevented. Depletion of noradrenaline in sympathetic nerve terminals also prevented the appetite suppressant-induced rise in melatonin synthesis. Activation of β-adrenoceptors is followed by a rise in N-acetyltransferase activity via a cyclic adenosine monophosphate second messenger system. The effect of appetite suppressants on the activity of liver tryptophan pyrrolase was also investigated. The activity of this enzyme is an important determinant of tryptophan availability to the brain and consequently of brain serotonin levels. The results show that appetite suppressants inhibit both holoenzyme and total enzyme activities of tryptophan pyrrolase. This finding suggests that appetite suppressants may act by inhibiting tryptophan pyrrolase activity thereby increasing brain serotonin, a phenomenon known to be associated with anorexia. There are two possible mechanisms by which appetite suppressants inhibit tryptophan pyrrolase activity. Firstly, these agents, being drugs of dependence, may increase liver NADPH concentrations which inhibit pyrrolase activity. Secondly, appetite suppressants may act on the pineal gland to stimulate melatonin synthesis. Melatonin inhibits pyrrolase activity in a dose-dependent manner. This inhibition will elevate plasma tryptophan levels which result in a rise in brain serotonin synthesis. The present study suggests a possible relationship between the pineal gland and appetite centres in the hypothalamus. Melatonin may have a direct effect on appetite centres since food restriction is associated with an increased melatonin binding in the hypothalamus. If this possible relationship can be extended, melatonin can open new possibilities for the control of food intake and consequently, of pathological obesity.
- Full Text:
- Date Issued: 1994
Removal of copper and nickel from solution by the non-viable biomass of the water fern Azolla filiculoides in an upscaled fixed-bed column system
- Authors: Thompson, Denis Alan
- Date: 2001
- Subjects: Copper , Nickel , Azolla , Heavy metals -- Absorption and adsorption
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3914 , http://hdl.handle.net/10962/d1003973 , Copper , Nickel , Azolla , Heavy metals -- Absorption and adsorption
- Description: The potential of non-viable Azalia filiculaides for the removal of Cu and Ni from aqueous solutions and the possibility of scaling up existing lab scale Azalia column systems was investigated. The effects of factors such as metal starting concentration, pH and two metals in solution on the removal of Ni and Cu from aqueous solution by dried and crushed Azalia biomass were studied in batch systems. Aqueous solutions of Ni with starting concentrations between 1000 and 2000J.lmolll gave the most efficient Ni removal by Azalla biomass. For Cu the optimum starting concentration for adsorption was 50J.lmol/l. The adsorption capacity of both eu and Ni increased as the starting pH of the sorption media increased. The optimum pH for Ni adsorption was found at pH 7 and for Cu, at pH 5. - Awlla biomass had a higher. maximum binding capacity (qrnax) for Cu than for Ni at pH 5. The removal of both Cu allct Ni showed little or no variation with the presence another metal in solution. Kinetic studies show that both Cu and Ni adsorbed rapidly onto the Azalia biomass. The removal of Cu and Ni from aqueous solutions using non-viable Azalia biomass was investigated in a lab scale fixed-bed column and an upscaled 4L column system. The nonviable Azalla filiculaides biomass when dried and used in a column for adsorption of Cu and Ni showed good physical stability under many different conditions. Preparation of the biomass before it could be used in the columns was very simple and did not involve any significant pretreatment steps. Prolonged exposure to UV light decreases Azalia biomass capacity for Ni and Cu adsorption. Column adsorption of Cu and Ni from aqueous solutions was successfully upscaled approximately 100 times. Relative to the lab scale column, the 4L column performed better for the uptake of Cu and Ni per gram of biomass. The larger column was also able to operate at relatively higher flow rates. The biomass showed good reusability with little change in the amount of Ni adsorbed in 10 consecutive cycles. Electron micrographs showecf little or no change in the physical structure and integrity of the Azolla biomass after exposure to mineral acids, Ni solution and high flow rates over 10 consecutive adsorption and desorption cycles. As much as 80% Ni and 70 % Cu was recovered when desorption profiles were generated using O.lMHCI as a desorption agent. The 4L column system was also tested using a highly concen~rat:~ Ni plating bath solution.(Nicrolyte 1). Only 18 % of the Ni could be removed from the expended Nicrolyte 1 pla~Jng solution after treating only 25L, indicating that Azolla biomass is more suited for removal of metals from more dilute industrial effluents.
- Full Text:
- Date Issued: 2001
- Authors: Thompson, Denis Alan
- Date: 2001
- Subjects: Copper , Nickel , Azolla , Heavy metals -- Absorption and adsorption
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3914 , http://hdl.handle.net/10962/d1003973 , Copper , Nickel , Azolla , Heavy metals -- Absorption and adsorption
- Description: The potential of non-viable Azalia filiculaides for the removal of Cu and Ni from aqueous solutions and the possibility of scaling up existing lab scale Azalia column systems was investigated. The effects of factors such as metal starting concentration, pH and two metals in solution on the removal of Ni and Cu from aqueous solution by dried and crushed Azalia biomass were studied in batch systems. Aqueous solutions of Ni with starting concentrations between 1000 and 2000J.lmolll gave the most efficient Ni removal by Azalla biomass. For Cu the optimum starting concentration for adsorption was 50J.lmol/l. The adsorption capacity of both eu and Ni increased as the starting pH of the sorption media increased. The optimum pH for Ni adsorption was found at pH 7 and for Cu, at pH 5. - Awlla biomass had a higher. maximum binding capacity (qrnax) for Cu than for Ni at pH 5. The removal of both Cu allct Ni showed little or no variation with the presence another metal in solution. Kinetic studies show that both Cu and Ni adsorbed rapidly onto the Azalia biomass. The removal of Cu and Ni from aqueous solutions using non-viable Azalia biomass was investigated in a lab scale fixed-bed column and an upscaled 4L column system. The nonviable Azalla filiculaides biomass when dried and used in a column for adsorption of Cu and Ni showed good physical stability under many different conditions. Preparation of the biomass before it could be used in the columns was very simple and did not involve any significant pretreatment steps. Prolonged exposure to UV light decreases Azalia biomass capacity for Ni and Cu adsorption. Column adsorption of Cu and Ni from aqueous solutions was successfully upscaled approximately 100 times. Relative to the lab scale column, the 4L column performed better for the uptake of Cu and Ni per gram of biomass. The larger column was also able to operate at relatively higher flow rates. The biomass showed good reusability with little change in the amount of Ni adsorbed in 10 consecutive cycles. Electron micrographs showecf little or no change in the physical structure and integrity of the Azolla biomass after exposure to mineral acids, Ni solution and high flow rates over 10 consecutive adsorption and desorption cycles. As much as 80% Ni and 70 % Cu was recovered when desorption profiles were generated using O.lMHCI as a desorption agent. The 4L column system was also tested using a highly concen~rat:~ Ni plating bath solution.(Nicrolyte 1). Only 18 % of the Ni could be removed from the expended Nicrolyte 1 pla~Jng solution after treating only 25L, indicating that Azolla biomass is more suited for removal of metals from more dilute industrial effluents.
- Full Text:
- Date Issued: 2001
The removal and recovery of toxic and valuable metals from aqueous solutions by the yeast Saccharomyces cerevisiae
- Authors: Wilhelmi, Brendan Shane
- Date: 1998
- Subjects: Saccharomyces cerevisiae , Yeast fungi -- Biotechnology , Sewage -- Purification -- Heavy metals removal
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4002 , http://hdl.handle.net/10962/d1004062 , Saccharomyces cerevisiae , Yeast fungi -- Biotechnology , Sewage -- Purification -- Heavy metals removal
- Description: This project considered the use of the yeast Saccharomyces cerevisiae as a biosorbent for the removal and recovery of a range of metals from contaminated waters. S. cerevisiae, as a biosorbent, has the potential to provide a cost effective, selective and highly efficient purification system. Initial studies focused on metal accumulation by an immobilized baker's S. cerevisiae biosorbent. The parameters affecting metal uptake were investigated, these included metal concentration, time and solution pH. Metal uptake was rapid. Gold and cobalt reached saturation within 5 min of contact with the biosorbent in batch reactors. Copper, zinc, nickel, cadmium and chromium reached saturation within 30 min of contact. Metal accumulation was pH dependent and was generally unaffected at a solution pH ≥ 4, and was substantially decreased at pH ≤ 2. The exception was gold which was preferentially accumulated at a solution pH of 2. The immobilized baker's yeast accumulated metals with maximum binding capacities in the order of gold > cadmium > cobalt > zinc > copper > chromium > nickel. A rapid method to assess metal recovery was developed. Bioaccumulated metal was efficiently recovered using dilute mineral acids. Copper recovery of ≥ 80 % was achieved by decreasing the solution pH of the reaction mixture to 2 with the addition of nominal quantities of HCl, H₂SO₄ or RNO₃. Adsorption-desorption over 8 cycles had no apparent adverse effect on metal uptake or recovery in batch reactors. Transmission electron microscopy showed no evidence of damage to cells used in copper adsorption-desorption investigations. Biosorption columns were investigated as bioreactors due to their application potential. The metals investigated were effectively removed from solution. At a saturation threshold, metal uptake declined rapidly. Most metals investigated were desorbed from the columns by eluting with 0.1 M HCl. Initially recoveries of copper, cobalt and cadmium were as high as 100%. Desorbed copper, zinc, cadmium, nickel and cobalt were concentrated in 10 to 15 ml of eluent, representing up to a 40 fold decrease in solution volume. Cadmium, nickel and zinc uptake increased with the second application to the columns. Initial accumulation of gold and chromium was 42.2 μmol/g and 28.6 μmol/g, however, due to the low recoveries of these two metals, a second application was not investigated. Copper was applied to a single column for 8 consecutive adsorption-desorption cycles. Uptake increased from an initial 31.3 μmol/g to 47.8 μmol/g at cycle 7. The potential for selective metal recovery was demonstrated using two biosorption columns in series. Copper was accumulated and recovered most efficiently. Zinc, cobalt and cadmium were displaced to the second column. Copper bound preferentially to zinc at a ratio of 6:1. Copper bound preferentially to cobalt at a ratio of 4:1. Cadmium was only displaced at a ratio of 2:1. The successful transfer of the bioremediation technology from the laboratory to an industrial application has yet to be realized. Bioremediation of a Plaatjiesvlei Black Mountain mine effluent, which contained copper, zinc, lead and iron, was investigated in this project. The removal of the metals was most effective at pH 4. A combined strategy of pH adjustment and bioremediation using immobilized S. cerevisiae decreased the copper concentration by 92.5%, lead was decreased by 90% and zinc was decreased by 60%. Iron was mostly precipitated from solution at pH ≥ 4. An ageing pond at the mine with conditions such as; pH, water volume and metal concentration, which were more conducive to biological treatment was subsequently identified. The investigation indicated a possible application of the biomass as a supplement to chemical remediation. The metal removal capability of a waste brewer's yeast was subsequently investigated. A yeast conditioning step increased metal uptake up to 100% and enhanced reproducibility. Metal removal from solution was rapid and pH dependent. The metals were efficiently removed from solution at pH ≥ 4. Uptake was substantially inhibited at pH ≤ 3. The waste brewer's yeast accumulated metals with maximum binding capacities in the order of copper (25.4 μmol/g) > lead (19.4 μmol/g) > iron (15.6 μmol/g) > zinc (12.5 μmol/g). No correlation between cell physiology and metal uptake was observed. Uptake of the four metals was confirmed by energy dispersive X-ray microanalysis. The interference of lead, zinc and iron on copper uptake by the waste brewer's yeast, and the interference of copper on the uptake of lead, zinc and iron was investigated. Maximum copper uptake was not decreased in the presence of lead. The Bmax remained constant at approximately 25 μmol/g. The dissociation constants increased with increasing lead concentrations. Lead bioaccumulation was significantly decreased in the presence of copper. The type of inhibition was dependent on the initial copper concentrations. Zinc had a slight synergistic effect on copper uptake. The copper Bmax increased from 30.8 μmol/g in a single-ion system to 34.5 μmol/g in the presence of 200 μmol/l of zinc. Zinc uptake was severely inhibited in the presence of copper. The maximum uptake and dissociation constant values were decreased in the presence of copper, which suggested an uncompetitive inhibition. The affinity of copper was substantially higher than zinc. The presence of higher levels of copper than zinc in the yeast cells was confirmed by energy dispersive microanalysis. Copper uptake was decreased in the presence of iron, with the copper Bmax being decreased from 25.4 μmol/g in a single-ion system to 20.1 μmol/g in the presence of 200 μmol/l iron. Iron Bmax values remained constant at 16.0 μmol/g. Combined biosorption and EDXA results suggested the iron bound at a higher affinity than copper to the cell wall. Total copper removal was higher as larger quantities of copper were deposited in the cell cytoplasm. Metal removal from the Plaatjiesvlei effluent by free cell suspensions of the waste brewer's yeast was satisfactory. Copper levels were decreased by 96%, iron by 42%, lead 25% and zinc 2%. Waste brewer's yeast is a cheap source of biomass in South Africa, and could potentially provide the basis for the development of an innovative purification system for metal-contaminated waters.
- Full Text:
- Date Issued: 1998
- Authors: Wilhelmi, Brendan Shane
- Date: 1998
- Subjects: Saccharomyces cerevisiae , Yeast fungi -- Biotechnology , Sewage -- Purification -- Heavy metals removal
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4002 , http://hdl.handle.net/10962/d1004062 , Saccharomyces cerevisiae , Yeast fungi -- Biotechnology , Sewage -- Purification -- Heavy metals removal
- Description: This project considered the use of the yeast Saccharomyces cerevisiae as a biosorbent for the removal and recovery of a range of metals from contaminated waters. S. cerevisiae, as a biosorbent, has the potential to provide a cost effective, selective and highly efficient purification system. Initial studies focused on metal accumulation by an immobilized baker's S. cerevisiae biosorbent. The parameters affecting metal uptake were investigated, these included metal concentration, time and solution pH. Metal uptake was rapid. Gold and cobalt reached saturation within 5 min of contact with the biosorbent in batch reactors. Copper, zinc, nickel, cadmium and chromium reached saturation within 30 min of contact. Metal accumulation was pH dependent and was generally unaffected at a solution pH ≥ 4, and was substantially decreased at pH ≤ 2. The exception was gold which was preferentially accumulated at a solution pH of 2. The immobilized baker's yeast accumulated metals with maximum binding capacities in the order of gold > cadmium > cobalt > zinc > copper > chromium > nickel. A rapid method to assess metal recovery was developed. Bioaccumulated metal was efficiently recovered using dilute mineral acids. Copper recovery of ≥ 80 % was achieved by decreasing the solution pH of the reaction mixture to 2 with the addition of nominal quantities of HCl, H₂SO₄ or RNO₃. Adsorption-desorption over 8 cycles had no apparent adverse effect on metal uptake or recovery in batch reactors. Transmission electron microscopy showed no evidence of damage to cells used in copper adsorption-desorption investigations. Biosorption columns were investigated as bioreactors due to their application potential. The metals investigated were effectively removed from solution. At a saturation threshold, metal uptake declined rapidly. Most metals investigated were desorbed from the columns by eluting with 0.1 M HCl. Initially recoveries of copper, cobalt and cadmium were as high as 100%. Desorbed copper, zinc, cadmium, nickel and cobalt were concentrated in 10 to 15 ml of eluent, representing up to a 40 fold decrease in solution volume. Cadmium, nickel and zinc uptake increased with the second application to the columns. Initial accumulation of gold and chromium was 42.2 μmol/g and 28.6 μmol/g, however, due to the low recoveries of these two metals, a second application was not investigated. Copper was applied to a single column for 8 consecutive adsorption-desorption cycles. Uptake increased from an initial 31.3 μmol/g to 47.8 μmol/g at cycle 7. The potential for selective metal recovery was demonstrated using two biosorption columns in series. Copper was accumulated and recovered most efficiently. Zinc, cobalt and cadmium were displaced to the second column. Copper bound preferentially to zinc at a ratio of 6:1. Copper bound preferentially to cobalt at a ratio of 4:1. Cadmium was only displaced at a ratio of 2:1. The successful transfer of the bioremediation technology from the laboratory to an industrial application has yet to be realized. Bioremediation of a Plaatjiesvlei Black Mountain mine effluent, which contained copper, zinc, lead and iron, was investigated in this project. The removal of the metals was most effective at pH 4. A combined strategy of pH adjustment and bioremediation using immobilized S. cerevisiae decreased the copper concentration by 92.5%, lead was decreased by 90% and zinc was decreased by 60%. Iron was mostly precipitated from solution at pH ≥ 4. An ageing pond at the mine with conditions such as; pH, water volume and metal concentration, which were more conducive to biological treatment was subsequently identified. The investigation indicated a possible application of the biomass as a supplement to chemical remediation. The metal removal capability of a waste brewer's yeast was subsequently investigated. A yeast conditioning step increased metal uptake up to 100% and enhanced reproducibility. Metal removal from solution was rapid and pH dependent. The metals were efficiently removed from solution at pH ≥ 4. Uptake was substantially inhibited at pH ≤ 3. The waste brewer's yeast accumulated metals with maximum binding capacities in the order of copper (25.4 μmol/g) > lead (19.4 μmol/g) > iron (15.6 μmol/g) > zinc (12.5 μmol/g). No correlation between cell physiology and metal uptake was observed. Uptake of the four metals was confirmed by energy dispersive X-ray microanalysis. The interference of lead, zinc and iron on copper uptake by the waste brewer's yeast, and the interference of copper on the uptake of lead, zinc and iron was investigated. Maximum copper uptake was not decreased in the presence of lead. The Bmax remained constant at approximately 25 μmol/g. The dissociation constants increased with increasing lead concentrations. Lead bioaccumulation was significantly decreased in the presence of copper. The type of inhibition was dependent on the initial copper concentrations. Zinc had a slight synergistic effect on copper uptake. The copper Bmax increased from 30.8 μmol/g in a single-ion system to 34.5 μmol/g in the presence of 200 μmol/l of zinc. Zinc uptake was severely inhibited in the presence of copper. The maximum uptake and dissociation constant values were decreased in the presence of copper, which suggested an uncompetitive inhibition. The affinity of copper was substantially higher than zinc. The presence of higher levels of copper than zinc in the yeast cells was confirmed by energy dispersive microanalysis. Copper uptake was decreased in the presence of iron, with the copper Bmax being decreased from 25.4 μmol/g in a single-ion system to 20.1 μmol/g in the presence of 200 μmol/l iron. Iron Bmax values remained constant at 16.0 μmol/g. Combined biosorption and EDXA results suggested the iron bound at a higher affinity than copper to the cell wall. Total copper removal was higher as larger quantities of copper were deposited in the cell cytoplasm. Metal removal from the Plaatjiesvlei effluent by free cell suspensions of the waste brewer's yeast was satisfactory. Copper levels were decreased by 96%, iron by 42%, lead 25% and zinc 2%. Waste brewer's yeast is a cheap source of biomass in South Africa, and could potentially provide the basis for the development of an innovative purification system for metal-contaminated waters.
- Full Text:
- Date Issued: 1998
Ectomycorrhizal characterisation, species diversity and community dynamics in Pinus patula Schelcht. et Cham. plantations
- Authors: Hawley, Greer Leigh
- Date: 2006
- Subjects: Mycorrhizas Ectomycorrhizal fungi Pinus patula -- Irrigation -- South Africa Forests and forestry -- South Africa Forest ecology -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3951 , http://hdl.handle.net/10962/d1004010
- Description: Ectomycorrhizal (ECM) associations are important elements of forest biomes, connecting and transferring nutrients through an intricate and complex system of hyphal networks, ensuring plants of the nutrients they require, in nutrient poor soil. ECM research and particularly investigations into the diversity of the fungal partners has not received much attention in South Africa, hindering the advance of research in this field. This has been attributed to the difficulty of identifying the mycobionts involved in the symbiosis. The objectives of this study were to examine the ECM fungal diversity associating with Pinus patula, in selected forest plantations in Mpumalanga, South Africa. Both morphological and molecular techniques were used to identify specimens of both sporocarp collections and ECM root tip morphotypes. Morphological analysis of the ECM root tips involved characterisation of root morphology such as colour, branching and texture, and anatomical analysis examined hyphal arrangement in the root mantle and rhizomorphs. Molecular analysis involved sequencing of the Internal Transcribed Spacer (ITS) region and comparative BLAST analysis. Twenty-four sporocarp species were identified from 13 genera, namely: Amanita, Boletus, Clavulina, Inocybe, Lactarius, Rhizopogon, Russula, Scleroderma, Suillus, Tricholoma, Thelephora, Tomentella and Xerocomus. ECM root tip analysis led to the characterisation of 7 wild-type morphotypes identified as an Albatrellus sp., 2 Amanita species, a Rhizopogon sp., Thelephora terrestris, a Tomentella sp. and Scleroderma citrinum. A secondary objective was to determine whether fertilisation treatments within the study sites were responsible for differences in fungal species community structure. No evidence of a change in species diversity or shift in species composition was encountered. It is envisaged that these comprehensive ECM descriptions will be used as reference material to stimulate continued research in this field in South Africa.
- Full Text:
- Date Issued: 2006
- Authors: Hawley, Greer Leigh
- Date: 2006
- Subjects: Mycorrhizas Ectomycorrhizal fungi Pinus patula -- Irrigation -- South Africa Forests and forestry -- South Africa Forest ecology -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3951 , http://hdl.handle.net/10962/d1004010
- Description: Ectomycorrhizal (ECM) associations are important elements of forest biomes, connecting and transferring nutrients through an intricate and complex system of hyphal networks, ensuring plants of the nutrients they require, in nutrient poor soil. ECM research and particularly investigations into the diversity of the fungal partners has not received much attention in South Africa, hindering the advance of research in this field. This has been attributed to the difficulty of identifying the mycobionts involved in the symbiosis. The objectives of this study were to examine the ECM fungal diversity associating with Pinus patula, in selected forest plantations in Mpumalanga, South Africa. Both morphological and molecular techniques were used to identify specimens of both sporocarp collections and ECM root tip morphotypes. Morphological analysis of the ECM root tips involved characterisation of root morphology such as colour, branching and texture, and anatomical analysis examined hyphal arrangement in the root mantle and rhizomorphs. Molecular analysis involved sequencing of the Internal Transcribed Spacer (ITS) region and comparative BLAST analysis. Twenty-four sporocarp species were identified from 13 genera, namely: Amanita, Boletus, Clavulina, Inocybe, Lactarius, Rhizopogon, Russula, Scleroderma, Suillus, Tricholoma, Thelephora, Tomentella and Xerocomus. ECM root tip analysis led to the characterisation of 7 wild-type morphotypes identified as an Albatrellus sp., 2 Amanita species, a Rhizopogon sp., Thelephora terrestris, a Tomentella sp. and Scleroderma citrinum. A secondary objective was to determine whether fertilisation treatments within the study sites were responsible for differences in fungal species community structure. No evidence of a change in species diversity or shift in species composition was encountered. It is envisaged that these comprehensive ECM descriptions will be used as reference material to stimulate continued research in this field in South Africa.
- Full Text:
- Date Issued: 2006
Wheat stress responses during Russian wheat aphid and Bird Cherry Oat aphid infestation: an analysis of differential protein regulation during plant biotic stress responses
- Louw, Cassandra Alexandrovna
- Authors: Louw, Cassandra Alexandrovna
- Date: 2007
- Subjects: Russian wheat aphid , Plants, Effect of stress on , Wheat -- Diseases and pests , Rhopalosiphum , Plant proteins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3995 , http://hdl.handle.net/10962/d1004055 , Russian wheat aphid , Plants, Effect of stress on , Wheat -- Diseases and pests , Rhopalosiphum , Plant proteins
- Description: Plants possess a complex and poorly understood network of defence mechanisms that enable them to counteract the effects of abiotic and biotic stress. Aphid phloem feeding is source of biotic stress in plants. Russian wheat aphid and Bird Cherry-Oat aphid feeding cause significant losses in the annual wheat crop, and control by conventional methods such as pesticide application, has proved to be ineffective. Infestation by the Russian wheat aphid has a particularly devastating effect in South Africa. Aphid-resistant wheat cultivars have been identified but an incomplete understanding of the mechanism of the plant’s resistance thwarts the development of improved cultivars. A two-dimensional gel electrophoresis method was developed, partially optimised and validated in order to determine the effect of Russian wheat aphid and Bird Cherry-Oat aphid phloem feeding on the Betta and Betta DN wheat proteome. Differentially expressed proteins that were up or down regulated more than two fold were identified using PDQuest™ Basic software and matched to known wheat proteins stored in the SwissProt protein database on the basis of their molecular mass and isolectric point. Initial analysis of the differential protein expression of Betta and Betta DN wheat in response to Russian wheat aphid and Bird Cherry-Oat aphid phloem feeding at different growth stages revealed that younger plants display higher levels of resistance than older plants. Feeding by the Bird-Cherry Oat aphid does not result in the upregulation of proteins implicated in a defence response, which indicates that the damage incurred by the plant due to feeding by this aphid is not enough to trigger a classic defence response. Feeding by the more damaging Russian wheat aphid resulted in a stress response in susceptible wheat cultivar Betta, and a defence response in resistant wheat cultivar Betta DN. The infestation of Betta DN resulted in the upregulation of putative thaumatins and amylase trypsin inhibitors, indicating that the Betta DN resistance response could be due to the combined effect of protease inhibitors that discourage aphid phloem feeding and the activation of the salicylic acid and jasmonic acid plant defence pathways.
- Full Text:
- Date Issued: 2007
- Authors: Louw, Cassandra Alexandrovna
- Date: 2007
- Subjects: Russian wheat aphid , Plants, Effect of stress on , Wheat -- Diseases and pests , Rhopalosiphum , Plant proteins
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3995 , http://hdl.handle.net/10962/d1004055 , Russian wheat aphid , Plants, Effect of stress on , Wheat -- Diseases and pests , Rhopalosiphum , Plant proteins
- Description: Plants possess a complex and poorly understood network of defence mechanisms that enable them to counteract the effects of abiotic and biotic stress. Aphid phloem feeding is source of biotic stress in plants. Russian wheat aphid and Bird Cherry-Oat aphid feeding cause significant losses in the annual wheat crop, and control by conventional methods such as pesticide application, has proved to be ineffective. Infestation by the Russian wheat aphid has a particularly devastating effect in South Africa. Aphid-resistant wheat cultivars have been identified but an incomplete understanding of the mechanism of the plant’s resistance thwarts the development of improved cultivars. A two-dimensional gel electrophoresis method was developed, partially optimised and validated in order to determine the effect of Russian wheat aphid and Bird Cherry-Oat aphid phloem feeding on the Betta and Betta DN wheat proteome. Differentially expressed proteins that were up or down regulated more than two fold were identified using PDQuest™ Basic software and matched to known wheat proteins stored in the SwissProt protein database on the basis of their molecular mass and isolectric point. Initial analysis of the differential protein expression of Betta and Betta DN wheat in response to Russian wheat aphid and Bird Cherry-Oat aphid phloem feeding at different growth stages revealed that younger plants display higher levels of resistance than older plants. Feeding by the Bird-Cherry Oat aphid does not result in the upregulation of proteins implicated in a defence response, which indicates that the damage incurred by the plant due to feeding by this aphid is not enough to trigger a classic defence response. Feeding by the more damaging Russian wheat aphid resulted in a stress response in susceptible wheat cultivar Betta, and a defence response in resistant wheat cultivar Betta DN. The infestation of Betta DN resulted in the upregulation of putative thaumatins and amylase trypsin inhibitors, indicating that the Betta DN resistance response could be due to the combined effect of protease inhibitors that discourage aphid phloem feeding and the activation of the salicylic acid and jasmonic acid plant defence pathways.
- Full Text:
- Date Issued: 2007
Synthesis and interaction of secondary N-nitrosamines with acetylcholinesterase
- Authors: Mmutle, Tsietso Bernard
- Date: 1991
- Subjects: Chemistry, Physical and theoretical , Enzyme kinetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4058 , http://hdl.handle.net/10962/d1004119 , Chemistry, Physical and theoretical , Enzyme kinetics
- Description: Secondary N-nitrosamines: diphenylnitrosamine (DPhNA), dimethylnitrosamine (DMNA), diethylnitrosamine (DENA), dipropylnitrosamine (DPNA), dibutylnitrosamine (DBNA), diethanolnitrosamine (DEtNA), methylnitrosoglycine (MNGly), nitrosopyrrolidine (NPyr), nitrosomorpholine (NMor) and nitrosopiperidine (NPip) were synthesised and their interaction with acetylcholinesterase (AChE) was investigated. Analyses of kinetic results show that DMNA (Ki=34.78 μM); DENA (Ki=54.24 μM); DPNA(Ki=60.36 μM); DBNA(Ki=95.54 μM); DEtNA(Ki=43.68 μM)MNGly (Ki=30.18 μM); NPip (Ki=123 μM); NPyr (Ki=66.07 μM), NMor (Ki=73.93 μM) and DPhNA (Ki=20.32 μM) are competitive and reversible inhibitors of acetylcholinesterase, with respect to the substrate, acetylthiocholine chloride, ATChCl. With time they act as irreversible covalent inhibitors with dipropy1nitrosamine producing 72% inactivation after 60 minutes. Scatchard analyses of f1uorometric titrations, (Kd=0.75mM-4.09mM); gel chromatography (Kd=O. 80mM-4. 60mM) and equilibrium dia1ysis (Kd=O. 71mM- 4.21mM) for MNG1y, DMNA, DEtNA, DENA, DPNA, NPyr, DSNA, NMor and NPip show that these compounds have weaker affinity for the enzyme, as compared to the much tightly binding aromatic DPhNA, Kd values (0.65mM, 0.68mM and 0.68mM) for fluorometric experiments, gel chromatography and equilibrium dialysis respectively. In all cases, the number of binding sites of acetylcholinesterase averaged to four.
- Full Text:
- Date Issued: 1991
- Authors: Mmutle, Tsietso Bernard
- Date: 1991
- Subjects: Chemistry, Physical and theoretical , Enzyme kinetics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4058 , http://hdl.handle.net/10962/d1004119 , Chemistry, Physical and theoretical , Enzyme kinetics
- Description: Secondary N-nitrosamines: diphenylnitrosamine (DPhNA), dimethylnitrosamine (DMNA), diethylnitrosamine (DENA), dipropylnitrosamine (DPNA), dibutylnitrosamine (DBNA), diethanolnitrosamine (DEtNA), methylnitrosoglycine (MNGly), nitrosopyrrolidine (NPyr), nitrosomorpholine (NMor) and nitrosopiperidine (NPip) were synthesised and their interaction with acetylcholinesterase (AChE) was investigated. Analyses of kinetic results show that DMNA (Ki=34.78 μM); DENA (Ki=54.24 μM); DPNA(Ki=60.36 μM); DBNA(Ki=95.54 μM); DEtNA(Ki=43.68 μM)MNGly (Ki=30.18 μM); NPip (Ki=123 μM); NPyr (Ki=66.07 μM), NMor (Ki=73.93 μM) and DPhNA (Ki=20.32 μM) are competitive and reversible inhibitors of acetylcholinesterase, with respect to the substrate, acetylthiocholine chloride, ATChCl. With time they act as irreversible covalent inhibitors with dipropy1nitrosamine producing 72% inactivation after 60 minutes. Scatchard analyses of f1uorometric titrations, (Kd=0.75mM-4.09mM); gel chromatography (Kd=O. 80mM-4. 60mM) and equilibrium dia1ysis (Kd=O. 71mM- 4.21mM) for MNG1y, DMNA, DEtNA, DENA, DPNA, NPyr, DSNA, NMor and NPip show that these compounds have weaker affinity for the enzyme, as compared to the much tightly binding aromatic DPhNA, Kd values (0.65mM, 0.68mM and 0.68mM) for fluorometric experiments, gel chromatography and equilibrium dialysis respectively. In all cases, the number of binding sites of acetylcholinesterase averaged to four.
- Full Text:
- Date Issued: 1991
Investigation into the biological removal of sulphate from ethanol distillery wastewater using sulphate-reducing prokaryotes
- Authors: Smuts, Lizl
- Date: 2005
- Subjects: Sewage -- Purification -- Biological treatment , Prokaryotes , Sulfates , Distilleries -- Waste disposal
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3941 , http://hdl.handle.net/10962/d1004000 , Sewage -- Purification -- Biological treatment , Prokaryotes , Sulfates , Distilleries -- Waste disposal
- Description: Ethanol production wastewater is known to be toxic, and is not easily biodegradable. It also consists of a variety of coloured components adding to the complex composition of this wastewater. Disposal of this wastewater into water courses is not recommended and yet is performed all over the world. Investigation of this wastewater found that there was a high concentration of sulphate which, in the presence of sulphate-reducing prokaryotes can cause sulphide corrosion of cement. The concentration of sulphate in the wastewater was approximately 2770 mg/L. It was also found that the wastewater pH was very low and discharge of the wastewater into the wastewater treatment works caused a negative impact on the overall quality of the final wastewater discharged to sea. It was found using FISH techniques that there were no sulphate-reducing prokaryotes present in the wastewaters but that a sulphate-reducing population existed on the sewer wall. An anaerobic contact process was designed to treat this wastewater targeting sulphate reduction to sulphide, to be converted into elemental sulphur and to increase the wastewater pH. The process did not achieve this aim and only approximately 20-30 % reduction in sulphate from the wastewater was achieved with little to no change in the pH. A 95 % reduction in sulphate concentration was needed in order to reach acceptable discharge limits. Sulphate reduction could not be carried out, even under ideal laboratory conditions. It was found that the barrier causing the digester failure was the high concentration of phenols present in the wastewater (3.3 g/L) together with the production of high concentrations of volatile fatty acids (on average 13 g acetic/L). These two components are known to cause digester failure, especially phenols, and phenols are usually only degraded by fungal species. It was concluded that the wastewater itself was not amenable to this method of biological treatment.
- Full Text:
- Date Issued: 2005
- Authors: Smuts, Lizl
- Date: 2005
- Subjects: Sewage -- Purification -- Biological treatment , Prokaryotes , Sulfates , Distilleries -- Waste disposal
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3941 , http://hdl.handle.net/10962/d1004000 , Sewage -- Purification -- Biological treatment , Prokaryotes , Sulfates , Distilleries -- Waste disposal
- Description: Ethanol production wastewater is known to be toxic, and is not easily biodegradable. It also consists of a variety of coloured components adding to the complex composition of this wastewater. Disposal of this wastewater into water courses is not recommended and yet is performed all over the world. Investigation of this wastewater found that there was a high concentration of sulphate which, in the presence of sulphate-reducing prokaryotes can cause sulphide corrosion of cement. The concentration of sulphate in the wastewater was approximately 2770 mg/L. It was also found that the wastewater pH was very low and discharge of the wastewater into the wastewater treatment works caused a negative impact on the overall quality of the final wastewater discharged to sea. It was found using FISH techniques that there were no sulphate-reducing prokaryotes present in the wastewaters but that a sulphate-reducing population existed on the sewer wall. An anaerobic contact process was designed to treat this wastewater targeting sulphate reduction to sulphide, to be converted into elemental sulphur and to increase the wastewater pH. The process did not achieve this aim and only approximately 20-30 % reduction in sulphate from the wastewater was achieved with little to no change in the pH. A 95 % reduction in sulphate concentration was needed in order to reach acceptable discharge limits. Sulphate reduction could not be carried out, even under ideal laboratory conditions. It was found that the barrier causing the digester failure was the high concentration of phenols present in the wastewater (3.3 g/L) together with the production of high concentrations of volatile fatty acids (on average 13 g acetic/L). These two components are known to cause digester failure, especially phenols, and phenols are usually only degraded by fungal species. It was concluded that the wastewater itself was not amenable to this method of biological treatment.
- Full Text:
- Date Issued: 2005
Towards understanding the mechanism of dimerisation of Saccharomyces cerevisiae eukaryotic translation initiation factor 5A
- Authors: Gentz, Petra Monika
- Date: 2008
- Subjects: Cytology Molecular biology Biochemistry Proteins -- Analysis Proteomics Polypeptides Amino acids -- Synthesis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3992 , http://hdl.handle.net/10962/d1004052
- Description: Eukaryotic translation initiation factor 5A (eIF5A) is the only known protein to contain hypusine, formed by post-translational modification of a highly conserved lysine residue. Hypusination is essential for eIF5A function, being required for binding of a specific subset of mRNAs necessary for progression of eukaryotic cells through the G1-S checkpoint. Little structural information is available for eIF5A other than that derived from archaeal homologues. The aim of this study was to conduct structure-function studies on Saccharomyces cerevisiae (yeast) eIF5A, encoded by TIF51A. Homology models of eIF5A were generated from the Methanococcus jannaschii archaeal homologue (aIF5A) and two Leishmania eIF5As. The models, along with secondary structure predictions identified an a-helix on the C-terminal domain, unique to eukaryote eIF5A. The Neurospora crassa structural analogue, HEX-1, which dimerises in three configurations, was used to generate similar dimeric model configurations of eIF5A. A biochemical and functional analysis was used to validate the homology models of eIF5A.Since the crystal structures of aIF5A and eIF5A were solved from unhypusinated protein produced in Escherichia coli, 6 x His-tagged eIF5A (His-eIF5A) was used for biochemical analysis. This analysis revealed that eIF5A existed as a dimer in solution, dependent on the presence of the highly conserved Cys 39 residue. A yeast TIF51A/TIF51B null yeast strain, with a chromosomal copy of TIF51A under control of PGAL1, was used to confirm that HiseIF5A and selected eIF5A mutants were functional in vivo. Biochemical analysis showed that hypusinated His-eIF5A also exists as a dimer, but neither the dimerisation, nor the function of eIF5A are dependent on the presence of Cys 39. Rather they depend on the presence of hypusine (Hpu) 51 and the presence of RNA leading to the conclusion that RNA and hypusine are required for dimerisation and hence function, of native eIF5A in vivo. In contrast, a Lys 51 to Arg 51 substitution or RNase treatment of His-eIF5A produced in E. coli did not destabilize the dimeric form, suggesting different folding/dimerisation mechanisms in E. coli and yeast cells. The information obtained from the initial homology models, together with the results of the biochemical analysis was used to propose a mechanism for dimerisation of yeast eIF5A involving both hypusine and RNA.
- Full Text:
- Date Issued: 2008
- Authors: Gentz, Petra Monika
- Date: 2008
- Subjects: Cytology Molecular biology Biochemistry Proteins -- Analysis Proteomics Polypeptides Amino acids -- Synthesis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3992 , http://hdl.handle.net/10962/d1004052
- Description: Eukaryotic translation initiation factor 5A (eIF5A) is the only known protein to contain hypusine, formed by post-translational modification of a highly conserved lysine residue. Hypusination is essential for eIF5A function, being required for binding of a specific subset of mRNAs necessary for progression of eukaryotic cells through the G1-S checkpoint. Little structural information is available for eIF5A other than that derived from archaeal homologues. The aim of this study was to conduct structure-function studies on Saccharomyces cerevisiae (yeast) eIF5A, encoded by TIF51A. Homology models of eIF5A were generated from the Methanococcus jannaschii archaeal homologue (aIF5A) and two Leishmania eIF5As. The models, along with secondary structure predictions identified an a-helix on the C-terminal domain, unique to eukaryote eIF5A. The Neurospora crassa structural analogue, HEX-1, which dimerises in three configurations, was used to generate similar dimeric model configurations of eIF5A. A biochemical and functional analysis was used to validate the homology models of eIF5A.Since the crystal structures of aIF5A and eIF5A were solved from unhypusinated protein produced in Escherichia coli, 6 x His-tagged eIF5A (His-eIF5A) was used for biochemical analysis. This analysis revealed that eIF5A existed as a dimer in solution, dependent on the presence of the highly conserved Cys 39 residue. A yeast TIF51A/TIF51B null yeast strain, with a chromosomal copy of TIF51A under control of PGAL1, was used to confirm that HiseIF5A and selected eIF5A mutants were functional in vivo. Biochemical analysis showed that hypusinated His-eIF5A also exists as a dimer, but neither the dimerisation, nor the function of eIF5A are dependent on the presence of Cys 39. Rather they depend on the presence of hypusine (Hpu) 51 and the presence of RNA leading to the conclusion that RNA and hypusine are required for dimerisation and hence function, of native eIF5A in vivo. In contrast, a Lys 51 to Arg 51 substitution or RNase treatment of His-eIF5A produced in E. coli did not destabilize the dimeric form, suggesting different folding/dimerisation mechanisms in E. coli and yeast cells. The information obtained from the initial homology models, together with the results of the biochemical analysis was used to propose a mechanism for dimerisation of yeast eIF5A involving both hypusine and RNA.
- Full Text:
- Date Issued: 2008
An investigation into dopamine-melatonin interactions in the rat Corpus striatum and pineal gland: a possible pineal-striatal axis
- Authors: Boyd, Clinton Shane
- Date: 2000
- Subjects: Pineal gland -- Research Melatonin Dopamine -- Physiological effect Dopamine Brain chemistry Rats -- Physiology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3906 , http://hdl.handle.net/10962/d1003965
- Description: Dysfunction of central dopaminergic systems has been implicated in neuroendocrine, neurodegenerative and psychiatric disorders. Monoamine oxidase and catechol-Omethyltransferase represent the key catabolic enzymes of dopamine, terminating neurotransmission following synaptic release of this catecholamine. Thus, both enzymes have been associated with the pathology of dopaminergic systems and represent therapeutic targets elf enormous clinical importance. Some neuroendocrine and circadian effects of melatonin have been attributed to an antidopamimetic effect of this pineal hormone in the hypothalamus and pituitary. Furthermore, both melatonin and dopamine modulate the behavioural output of the mesencephalic dopaminergic pathways of the basal ganglia, including movement disorders. However, the biochemical basis for the tonic inhibitory effect of melatonin in the nigro-striatal pathway has been poorly delineated. Thus, this study determined whether melatonin influences dopaminergic function in the corpus striatum of the Wistar rat by modulating monoamine oxidase and catecholO- methyltransferase activity. Reciprocally, the putative existence of an intrapineal dopaminergic system was investigated by determining the effect of selective dopaminergic agents, R-( -)apomorphine, haloperidol and dopamine, on indole metabolism of the pineal gland. The akinetic state of drug-induced catalepsy was employed as an animal model of Parkinson's disease to probe the neurotransmitter systems involved in the behavioural effects of melatonin. Indole metabolism was a reliable indicator of state-dependent metabolic fluxes in pineal gland function. These included a robust diurnal and seasonal variation in N-acetylserotonin and melatonin biosynthesis, and photoperiod- and drug-induced alterations of Inftabolism. The predominant changes could be attributed to an effect on serotonin N-acetyltransferase activity and/or the melatoninl5-methoxytryptophol ratio. Pineal 5-methoxyindole biosynthesis was determined primarily by the bioavailability of the corresponding 5-hydroxyindole and its affinity for hydroxyindole-O-methyltransferase. Evidence was found for the negative feedback or paracrine control of pineal indole metabolism by melatonin. A high inter-individual variability was observed in the biosynthesis of N-acetylserotonin and melatonin biosynthesis, and the weight of the pineal glands. Accordingly, the rats could be classified as either high or low capacity producers of these two indoles. R-(-)-apomorphine and dopamine in vitro, but not acute haloperidol in vivo, had dose- and phase-dependent effects on pineal indole metabolism. The predominant effect was a suppression of the scotophase-dependent induction ofN-acetylserotonin and melatonin biosynthesis by dopamine and R-( -)-apomorphine. It is postulated that these agonists inhibited nocturnal N-acetyltransferase activity via postsynaptic pineal D2 or D2-like receptors. The observed modulatory nature of the intrapineal dopaminergic system suggests that dopamine may be involved in the long-term regulation of pineal indole biosynthesis. Several lines of evidence are presented that the activity of striatal monoamine oxidase A and catechol-O-methyltransferase, represented predominantly by the soluble isoform, is statedependent and regulated in vivo by endogenous melatonin. Firstly, both enzymes showed a daynight variation in activity. Secondly, acute and subchronic administration and photoperiod manipulation studies indicated that both exogenous and endogenous melatonin inhibited each enzyme in a chronotypic fashion, with a more robust effect against catechol- -methyltransferase. The intensity of the in vivo effects was critically dependent on the dose, duration, route and the phase-timing of administration during the light dark cycle, and the length of the exposure to constant light. Melatonin in vitro had no effect on basal or Mg2+ -induced catechol-Omethyltransferase activity. Thus, it is proposed that the in vivo effects of the hormone can be attributed to a time-dependent change in the amount of active molecules of this enzyme. In contrast, melatonin and numerous other endogenous indolic compounds were found to be reversible inhibitors of striatal monoamine oxidase A in vitro. Structure-activity modeling revealed that the 5-methoxy moiety on the indole nucleus and substitution of the free primary amine of these compounds were the principal determinants of the potency and time-dependency of inhibition. Thus melatonin most likely has a direct inhibitory effect in vivo at the level of the active site of monoamine oxidase A. Exogenous melatonin alone had no cataleptogenic potential whereas a variety of behavioural responses were observed following intraperitoneal administration of y-hydroxybutyrate. The latter responses were state-dependent with day-night variations in intensity. Furthermore, yhydroxybutyrate stimulated melatonin biosynthesis during the photophase both in vitro and in vivo. These results point to a possible involvement of melatonin in the behavioural and neurochemical effects of y-hydroxybutyrate. Thus the general conclusion is that dopamine and melatonin display functional antagonism at the level of the pineal gland and corpus striatum of the Wistar rats. Therefore melatonin may be an important homeostatic modulator of dopaminergic neurotransmission throu~out the central nervous system. Furthermore, the putative existence of a functional pineal-striatal axis would greatly strengthen the argument for a holistic concept of brain homeostasis. The ability of endogenous melatonin to regulate monoamine oxidase A and catechol-O-methyltransferase may represent an alternative strategy for the treatment of disorders associated with these enzymes.
- Full Text:
- Date Issued: 2000
- Authors: Boyd, Clinton Shane
- Date: 2000
- Subjects: Pineal gland -- Research Melatonin Dopamine -- Physiological effect Dopamine Brain chemistry Rats -- Physiology
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3906 , http://hdl.handle.net/10962/d1003965
- Description: Dysfunction of central dopaminergic systems has been implicated in neuroendocrine, neurodegenerative and psychiatric disorders. Monoamine oxidase and catechol-Omethyltransferase represent the key catabolic enzymes of dopamine, terminating neurotransmission following synaptic release of this catecholamine. Thus, both enzymes have been associated with the pathology of dopaminergic systems and represent therapeutic targets elf enormous clinical importance. Some neuroendocrine and circadian effects of melatonin have been attributed to an antidopamimetic effect of this pineal hormone in the hypothalamus and pituitary. Furthermore, both melatonin and dopamine modulate the behavioural output of the mesencephalic dopaminergic pathways of the basal ganglia, including movement disorders. However, the biochemical basis for the tonic inhibitory effect of melatonin in the nigro-striatal pathway has been poorly delineated. Thus, this study determined whether melatonin influences dopaminergic function in the corpus striatum of the Wistar rat by modulating monoamine oxidase and catecholO- methyltransferase activity. Reciprocally, the putative existence of an intrapineal dopaminergic system was investigated by determining the effect of selective dopaminergic agents, R-( -)apomorphine, haloperidol and dopamine, on indole metabolism of the pineal gland. The akinetic state of drug-induced catalepsy was employed as an animal model of Parkinson's disease to probe the neurotransmitter systems involved in the behavioural effects of melatonin. Indole metabolism was a reliable indicator of state-dependent metabolic fluxes in pineal gland function. These included a robust diurnal and seasonal variation in N-acetylserotonin and melatonin biosynthesis, and photoperiod- and drug-induced alterations of Inftabolism. The predominant changes could be attributed to an effect on serotonin N-acetyltransferase activity and/or the melatoninl5-methoxytryptophol ratio. Pineal 5-methoxyindole biosynthesis was determined primarily by the bioavailability of the corresponding 5-hydroxyindole and its affinity for hydroxyindole-O-methyltransferase. Evidence was found for the negative feedback or paracrine control of pineal indole metabolism by melatonin. A high inter-individual variability was observed in the biosynthesis of N-acetylserotonin and melatonin biosynthesis, and the weight of the pineal glands. Accordingly, the rats could be classified as either high or low capacity producers of these two indoles. R-(-)-apomorphine and dopamine in vitro, but not acute haloperidol in vivo, had dose- and phase-dependent effects on pineal indole metabolism. The predominant effect was a suppression of the scotophase-dependent induction ofN-acetylserotonin and melatonin biosynthesis by dopamine and R-( -)-apomorphine. It is postulated that these agonists inhibited nocturnal N-acetyltransferase activity via postsynaptic pineal D2 or D2-like receptors. The observed modulatory nature of the intrapineal dopaminergic system suggests that dopamine may be involved in the long-term regulation of pineal indole biosynthesis. Several lines of evidence are presented that the activity of striatal monoamine oxidase A and catechol-O-methyltransferase, represented predominantly by the soluble isoform, is statedependent and regulated in vivo by endogenous melatonin. Firstly, both enzymes showed a daynight variation in activity. Secondly, acute and subchronic administration and photoperiod manipulation studies indicated that both exogenous and endogenous melatonin inhibited each enzyme in a chronotypic fashion, with a more robust effect against catechol- -methyltransferase. The intensity of the in vivo effects was critically dependent on the dose, duration, route and the phase-timing of administration during the light dark cycle, and the length of the exposure to constant light. Melatonin in vitro had no effect on basal or Mg2+ -induced catechol-Omethyltransferase activity. Thus, it is proposed that the in vivo effects of the hormone can be attributed to a time-dependent change in the amount of active molecules of this enzyme. In contrast, melatonin and numerous other endogenous indolic compounds were found to be reversible inhibitors of striatal monoamine oxidase A in vitro. Structure-activity modeling revealed that the 5-methoxy moiety on the indole nucleus and substitution of the free primary amine of these compounds were the principal determinants of the potency and time-dependency of inhibition. Thus melatonin most likely has a direct inhibitory effect in vivo at the level of the active site of monoamine oxidase A. Exogenous melatonin alone had no cataleptogenic potential whereas a variety of behavioural responses were observed following intraperitoneal administration of y-hydroxybutyrate. The latter responses were state-dependent with day-night variations in intensity. Furthermore, yhydroxybutyrate stimulated melatonin biosynthesis during the photophase both in vitro and in vivo. These results point to a possible involvement of melatonin in the behavioural and neurochemical effects of y-hydroxybutyrate. Thus the general conclusion is that dopamine and melatonin display functional antagonism at the level of the pineal gland and corpus striatum of the Wistar rats. Therefore melatonin may be an important homeostatic modulator of dopaminergic neurotransmission throu~out the central nervous system. Furthermore, the putative existence of a functional pineal-striatal axis would greatly strengthen the argument for a holistic concept of brain homeostasis. The ability of endogenous melatonin to regulate monoamine oxidase A and catechol-O-methyltransferase may represent an alternative strategy for the treatment of disorders associated with these enzymes.
- Full Text:
- Date Issued: 2000
Understanding the underlying resistance mechanism of Mycobacterium tuberculosis against Rifampicin by analyzing mutant DNA - directed RNA polymerase proteins via bioinformatics approaches
- Authors: Monama, Mokgerwa Zacharia
- Date: 2020
- Subjects: Mycobacterium tuberculosis , Rifampin , Drug resistance , Homology (Biology) , Tuberculosis -- Chemotherapy
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167508 , vital:41487
- Description: Tuberculosis or TB is an airborne disease caused by the non-motile bacilli, Mycobacterium tuberculosis (MTB). There are two main forms of TB, namely, latent TB or LTB, asymptomatic and non-contagious version which according to the World Health Organization (WHO) is estimated to afflict over a third of the world’s population; and active TB or ATB, a symptomatic and contagious version which continues to spread, affecting millions worldwide. With the already high reported prevalence of TB, the emergence of drug-resistant strains has prompted the development of novel approaches to enhance the efficacy of known drugs and a desperate search for novel compounds to combat MTB infections. It was for this very purpose that this study was conducted. A look into the resistance mechanism of Rifampicin (Rifampin or RIF), one of the more potent first-line drugs, might prove beneficial in predicting the consequence of an introduced mutation (which usually occur as single nucleotide polymorphisms or SNPs) and perhaps even overcome it using appropriate therapeutic interventions that improve RIF’s efficacy. To accomplish this task, models of acceptable quality were generated for the WT and clinically relevant, RIF resistance conferring, SNPs occurring at codon positions D516, H526 and S531 (E .coli numbering system) using MODELLER. The models were accordingly ranked using GA341 and z-DOPE score, and subsequently validated with QMEAN, PROCHECK and VERIFY3D. MD simulations spanning 100 ns were run for RIF-bound (complex) and RIF-free (holo) DNA-directed RNA polymerase (DDRP) protein systems for the WT and SNP mutants using GROMACS. The MD frames were analyzed using RMSD, Rg and RMSF. For further analysis, MD-TASK was used to analyze the calculated dynamic residue networks (DRNs) from the generated MD frames, determining both change in average shortest path (ΔL) and betweenness centrality (ΔBC). The RMSD analysis revealed that all of the SNP complex models displayed a level instability higher than that of the WT complex. A majority of the SNP complex models were also observed to have similar compactness to the WT holo when looking at the calculated Rg. The RMSF results also hinted towards possible physiological consequences of the mutations (generally referred to as a fitness cost) highlighted by the increased fluctuations of the zinc-binding domain and the MTB SI α helical coiled coil. For the first time, to the knowledge of the authors, DRN analysis was employed for the DDRP protein for both holo and complex systems, revealing insightful information about the residues that play a key role in the change in distance between residue pairs along with residues that play an essential role in protein communication within the calculated RIN. Overall, the data supported the conclusions drawn by a recent study that only concentrated on RIF-resistance in rpoB models which suggested that the binding pocket for the SNP models may result in the changed coordination of RIF which may be the main contributor to its impaired efficacy.
- Full Text:
- Date Issued: 2020
- Authors: Monama, Mokgerwa Zacharia
- Date: 2020
- Subjects: Mycobacterium tuberculosis , Rifampin , Drug resistance , Homology (Biology) , Tuberculosis -- Chemotherapy
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/167508 , vital:41487
- Description: Tuberculosis or TB is an airborne disease caused by the non-motile bacilli, Mycobacterium tuberculosis (MTB). There are two main forms of TB, namely, latent TB or LTB, asymptomatic and non-contagious version which according to the World Health Organization (WHO) is estimated to afflict over a third of the world’s population; and active TB or ATB, a symptomatic and contagious version which continues to spread, affecting millions worldwide. With the already high reported prevalence of TB, the emergence of drug-resistant strains has prompted the development of novel approaches to enhance the efficacy of known drugs and a desperate search for novel compounds to combat MTB infections. It was for this very purpose that this study was conducted. A look into the resistance mechanism of Rifampicin (Rifampin or RIF), one of the more potent first-line drugs, might prove beneficial in predicting the consequence of an introduced mutation (which usually occur as single nucleotide polymorphisms or SNPs) and perhaps even overcome it using appropriate therapeutic interventions that improve RIF’s efficacy. To accomplish this task, models of acceptable quality were generated for the WT and clinically relevant, RIF resistance conferring, SNPs occurring at codon positions D516, H526 and S531 (E .coli numbering system) using MODELLER. The models were accordingly ranked using GA341 and z-DOPE score, and subsequently validated with QMEAN, PROCHECK and VERIFY3D. MD simulations spanning 100 ns were run for RIF-bound (complex) and RIF-free (holo) DNA-directed RNA polymerase (DDRP) protein systems for the WT and SNP mutants using GROMACS. The MD frames were analyzed using RMSD, Rg and RMSF. For further analysis, MD-TASK was used to analyze the calculated dynamic residue networks (DRNs) from the generated MD frames, determining both change in average shortest path (ΔL) and betweenness centrality (ΔBC). The RMSD analysis revealed that all of the SNP complex models displayed a level instability higher than that of the WT complex. A majority of the SNP complex models were also observed to have similar compactness to the WT holo when looking at the calculated Rg. The RMSF results also hinted towards possible physiological consequences of the mutations (generally referred to as a fitness cost) highlighted by the increased fluctuations of the zinc-binding domain and the MTB SI α helical coiled coil. For the first time, to the knowledge of the authors, DRN analysis was employed for the DDRP protein for both holo and complex systems, revealing insightful information about the residues that play a key role in the change in distance between residue pairs along with residues that play an essential role in protein communication within the calculated RIN. Overall, the data supported the conclusions drawn by a recent study that only concentrated on RIF-resistance in rpoB models which suggested that the binding pocket for the SNP models may result in the changed coordination of RIF which may be the main contributor to its impaired efficacy.
- Full Text:
- Date Issued: 2020
An investigation of the role of mitochondrial STAT3 and modulation of Reactive Oxygen Species in adipocyte differentiation
- Authors: Kramer, Adam Hildyard
- Date: 2014
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54632 , vital:26595
- Description: Stem cells have the ability to differentiate into a myriad of different cell types. The understanding of the differentiation process is of paramount importance if we are to use these cells in the lab as well as in therapeutics. Here, the levels and localization of the signal transducer and activator of transcription 3 (STAT3), with particular attention focused on the mitochondrial serine 727 phosphorylated form of STAT3 (pSTAT3S727) during differentiation, was investigated. Using the murine preadipocyte progenitor cell line 3T3-L1, as well as adipose derived human mesenchymal stem cells (HMSC-ad) as differentiation models, the relative levels of Reactive Oxygen Species (ROS) and the levels and localization of STAT3 were investigated during the differentiation process. ROS is known to play an important signalling role during differentiation and is well reported during the events of adipogenesis. ROS are generated as a by-product in the Electron Transport Chain (ETC), and it has recently been reported that pSTAT3S727 plays an important role at complex I of the ETC. Various techniques including fluorescence confocal microscopy, flow cytometry and Western blots were utilized to investigate the non-canonical role STAT3 plays during adipogenesis. Mitochondrial isolations were performed to investigate the levels of STAT3 in the mitochondria during differentiation. Further to this, an impedance based real time differentiation assay was developed using the xCELLigence Real Time Cell Analyser to monitor differentiation and the affects various compounds, including a STAT3 inhibitor, have on differentiation. Results indicate that upon induction of differentiation, levels of mitochondrial pSTAT3S727 dramatically decrease and leave the mitochondria. This corresponds to increasing levels of ROS. The canonical active form of STAT3 following phosphorylation on tyrosine 705 (pSTAT3Y705) was found to decrease and lose its nuclear localization. These initial results indicate that STAT3 plays an important non-canonical role in the mitochondria during differentiation.
- Full Text:
- Date Issued: 2014
- Authors: Kramer, Adam Hildyard
- Date: 2014
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/54632 , vital:26595
- Description: Stem cells have the ability to differentiate into a myriad of different cell types. The understanding of the differentiation process is of paramount importance if we are to use these cells in the lab as well as in therapeutics. Here, the levels and localization of the signal transducer and activator of transcription 3 (STAT3), with particular attention focused on the mitochondrial serine 727 phosphorylated form of STAT3 (pSTAT3S727) during differentiation, was investigated. Using the murine preadipocyte progenitor cell line 3T3-L1, as well as adipose derived human mesenchymal stem cells (HMSC-ad) as differentiation models, the relative levels of Reactive Oxygen Species (ROS) and the levels and localization of STAT3 were investigated during the differentiation process. ROS is known to play an important signalling role during differentiation and is well reported during the events of adipogenesis. ROS are generated as a by-product in the Electron Transport Chain (ETC), and it has recently been reported that pSTAT3S727 plays an important role at complex I of the ETC. Various techniques including fluorescence confocal microscopy, flow cytometry and Western blots were utilized to investigate the non-canonical role STAT3 plays during adipogenesis. Mitochondrial isolations were performed to investigate the levels of STAT3 in the mitochondria during differentiation. Further to this, an impedance based real time differentiation assay was developed using the xCELLigence Real Time Cell Analyser to monitor differentiation and the affects various compounds, including a STAT3 inhibitor, have on differentiation. Results indicate that upon induction of differentiation, levels of mitochondrial pSTAT3S727 dramatically decrease and leave the mitochondria. This corresponds to increasing levels of ROS. The canonical active form of STAT3 following phosphorylation on tyrosine 705 (pSTAT3Y705) was found to decrease and lose its nuclear localization. These initial results indicate that STAT3 plays an important non-canonical role in the mitochondria during differentiation.
- Full Text:
- Date Issued: 2014