Over-expression, purification and biochemical characterisation of trypanosomal heat shock protein
- Authors: Edkins, Adrienne Lesley
- Date: 2003
- Subjects: Heat shock proteins , Heat shock proteins -- Structure activity relationships
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4106 , http://hdl.handle.net/10962/d1011736 , Heat shock proteins , Heat shock proteins -- Structure activity relationships
- Description: The molecular chaperone process of assisted protein folding, characteristic of members of the Heat Shock Protein 70 kDa (Hsp70) and Heat Shock Protein 40kDa (Hsp40) families, is essential for cytoprotection in stressful cellular conditions. Examples of such conditions are heat shock or invasion by pathogens. The Hsp70/Hsp40 process of assisted protein folding is dependent on ATP (governed by the intrinsic ATPase activity of Hsp70) and the ability of molecular chaperones to recognise and bind non-native protein conformations. Here, we analyse and attempt to characterise the molecular chaperone activity of an inducible, cytoplasmic Hsp70 (TcHsp70) from Trypanosoma cruzi and its interactions with its potential partner Hsp40s, Tcj 1, Tcj2, Tcj3 and Tcj4. A bioinformatic analyses of the primary sequences of the trypanosomal proteins revealed that they all contained the canonical domains that define other members of the Hsp70 and Hsp40 family. Tcj2 and Tcj4 showed deviations from the consensus sequence in their substrate binding regions, which may have implications for their substrate binding specificities. TcHsp70, Tcj 1, Tcj2, Tcj3 and Tcj4 were over-expressed recombinantly as 6xHis-tag fusion proteins in Escherichia coli. His-TcHsp70, Tcjl-His and His-Tcj2 were successfully purified by Nickel-affinity chromatography for functional analyses to assess the molecular chaperone activity of His-TcHsp70 in terms of its ATPase activity and substrate binding ability. The basal ATPase activity of His-TcHsp70 was determined as 40 nmol Pi/min/mg, significantly higher than that reported for other Hsp70s. This basal ATPase activity was stimulated to a maximal level of 60 nmol Pi/min/mg in the presence of His-Tcj2 and a model non-native substrate, reduced carboxymethylated αx-lactalbumin (RCMLA). Using native polyacrylamide gel electrophoresis and Western analysis, His-TcHsp70 was shown to form discrete complexes when in the presence of Tcj 1- His, His-Tcj2 and/or RCMLA. These complexes potentially represent His-TcHsp70 - RCMLA or His-TcHsp70 - Tcj interactions, that may be indicative of chaperone activity. In vivo complementation assays showed that Tcj2, but not Tcj3, was able to overcome the temperature sensitivity of the ydjJ mutant Saccharomyces cerevisiae strain JJ160, suggesting that Tcj2 may be functionally equivalent to the yeast Hsp40 Ydj1.
- Full Text:
- Date Issued: 2003
- Authors: Edkins, Adrienne Lesley
- Date: 2003
- Subjects: Heat shock proteins , Heat shock proteins -- Structure activity relationships
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4106 , http://hdl.handle.net/10962/d1011736 , Heat shock proteins , Heat shock proteins -- Structure activity relationships
- Description: The molecular chaperone process of assisted protein folding, characteristic of members of the Heat Shock Protein 70 kDa (Hsp70) and Heat Shock Protein 40kDa (Hsp40) families, is essential for cytoprotection in stressful cellular conditions. Examples of such conditions are heat shock or invasion by pathogens. The Hsp70/Hsp40 process of assisted protein folding is dependent on ATP (governed by the intrinsic ATPase activity of Hsp70) and the ability of molecular chaperones to recognise and bind non-native protein conformations. Here, we analyse and attempt to characterise the molecular chaperone activity of an inducible, cytoplasmic Hsp70 (TcHsp70) from Trypanosoma cruzi and its interactions with its potential partner Hsp40s, Tcj 1, Tcj2, Tcj3 and Tcj4. A bioinformatic analyses of the primary sequences of the trypanosomal proteins revealed that they all contained the canonical domains that define other members of the Hsp70 and Hsp40 family. Tcj2 and Tcj4 showed deviations from the consensus sequence in their substrate binding regions, which may have implications for their substrate binding specificities. TcHsp70, Tcj 1, Tcj2, Tcj3 and Tcj4 were over-expressed recombinantly as 6xHis-tag fusion proteins in Escherichia coli. His-TcHsp70, Tcjl-His and His-Tcj2 were successfully purified by Nickel-affinity chromatography for functional analyses to assess the molecular chaperone activity of His-TcHsp70 in terms of its ATPase activity and substrate binding ability. The basal ATPase activity of His-TcHsp70 was determined as 40 nmol Pi/min/mg, significantly higher than that reported for other Hsp70s. This basal ATPase activity was stimulated to a maximal level of 60 nmol Pi/min/mg in the presence of His-Tcj2 and a model non-native substrate, reduced carboxymethylated αx-lactalbumin (RCMLA). Using native polyacrylamide gel electrophoresis and Western analysis, His-TcHsp70 was shown to form discrete complexes when in the presence of Tcj 1- His, His-Tcj2 and/or RCMLA. These complexes potentially represent His-TcHsp70 - RCMLA or His-TcHsp70 - Tcj interactions, that may be indicative of chaperone activity. In vivo complementation assays showed that Tcj2, but not Tcj3, was able to overcome the temperature sensitivity of the ydjJ mutant Saccharomyces cerevisiae strain JJ160, suggesting that Tcj2 may be functionally equivalent to the yeast Hsp40 Ydj1.
- Full Text:
- Date Issued: 2003
Over-expression, purification and biochemical characterization of DOXP reductoisomerase and the rational design of novel anti-malarial drugs
- Authors: Tanner, Delia Caroline
- Date: 2004
- Subjects: Malaria -- Prevention , Malaria -- Drug therapy , Antimalarials -- Therapeutic use , Proteins -- Purification , Amino acids -- Analysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3931 , http://hdl.handle.net/10962/d1003990 , Malaria -- Prevention , Malaria -- Drug therapy , Antimalarials -- Therapeutic use , Proteins -- Purification , Amino acids -- Analysis
- Description: Malaria poses the greatest threat of all parasites to human life. Current vaccines and efficacious drugs are available however their use is limited due to toxicity, emergence of drug resistance, and cost. The discovery of an alternative pathway of isoprenoid biosynthesis, the non-mevalonate pathway, within the malarial parasite has resulted in development of novel anti-malarial drugs. 1-Deoxy-D-xylulose-5-phosphate (DOXP) reductoisomerase, the second enzyme in this pathway, is responsible for the synthesis of 2-C-methyl-D-erythritol 4-phosphate (MEP) in an intramolecular rearrangement step followed by a reduction process involving NADPH as a hydrogen donor and divalent cations as co-factors. Fosmidomycin and FR900098 have been identified as inhibitors of DOXP reductoisomerase. However, they lack clinical efficacy. In this investigation recombinant DOXP reductoisomerase from Escherichia coli (EcDXR) and Plasmodium falciparum (pfDXR) were biochemically characterized as potential targets for inhibition. (His)6-EcDXR was successfully purified using nickel-chelate affinity chromatography with a specific activity of 1.77 μmoles/min/mg and Km value 282 μM. Utilizing multiple sequence alignment, previous structural data predictions and homology modeling approaches, critical active site amino acid residues were identified and their role in the catalytic activity investigated utilizing site-directed mutagenesis techniques. We have shown evidence that suggests that Trp212 and Met214 interact to maintain the active site architecture and hydrophobic interactions necessary for substrate binding, cofactor binding and enzyme activity. Replacement of Trp212 with Tyr, Phe, and Leu reduced specific activity relative to EcDXR. EcDXR(W212F) and EcDXR(W212Y) had an increased Km relative to EcDXR indicative of loss in affinity toward DOXP, whereas EcDXR(W212L) had a lower Km of ~8 μM indicative of increased affinity for DOXP. The W212L substitution possibly removed contacts necessary for full catalytic activity, but could be considered a non-disruptive substitution in that it maintained active site architecture sufficient for DOXP reductoisomerase activity. EcDXR(M214I) had 36-fold reduced enzyme activity relative to EcDXR, while its Km (~8 μM) was found to be lower than that of EcDXR. This suggested that the M214I substitution had maintained (perhaps improved) substrate and active site architecture, but may have perturbed interactions with NADPH. Rational drug design strategies and docking methods have been utilized in the development of furan derivatives as DOXP reductoisomerase inhibitors, and the synthesis of phosphorylated derivatives (5) and (6) has been achieved. Future inhibitor studies using these novel potential DOXP reductoisomerase inhibitors may lead to the development of effective anti-malarial drug candidates.
- Full Text:
- Date Issued: 2004
- Authors: Tanner, Delia Caroline
- Date: 2004
- Subjects: Malaria -- Prevention , Malaria -- Drug therapy , Antimalarials -- Therapeutic use , Proteins -- Purification , Amino acids -- Analysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3931 , http://hdl.handle.net/10962/d1003990 , Malaria -- Prevention , Malaria -- Drug therapy , Antimalarials -- Therapeutic use , Proteins -- Purification , Amino acids -- Analysis
- Description: Malaria poses the greatest threat of all parasites to human life. Current vaccines and efficacious drugs are available however their use is limited due to toxicity, emergence of drug resistance, and cost. The discovery of an alternative pathway of isoprenoid biosynthesis, the non-mevalonate pathway, within the malarial parasite has resulted in development of novel anti-malarial drugs. 1-Deoxy-D-xylulose-5-phosphate (DOXP) reductoisomerase, the second enzyme in this pathway, is responsible for the synthesis of 2-C-methyl-D-erythritol 4-phosphate (MEP) in an intramolecular rearrangement step followed by a reduction process involving NADPH as a hydrogen donor and divalent cations as co-factors. Fosmidomycin and FR900098 have been identified as inhibitors of DOXP reductoisomerase. However, they lack clinical efficacy. In this investigation recombinant DOXP reductoisomerase from Escherichia coli (EcDXR) and Plasmodium falciparum (pfDXR) were biochemically characterized as potential targets for inhibition. (His)6-EcDXR was successfully purified using nickel-chelate affinity chromatography with a specific activity of 1.77 μmoles/min/mg and Km value 282 μM. Utilizing multiple sequence alignment, previous structural data predictions and homology modeling approaches, critical active site amino acid residues were identified and their role in the catalytic activity investigated utilizing site-directed mutagenesis techniques. We have shown evidence that suggests that Trp212 and Met214 interact to maintain the active site architecture and hydrophobic interactions necessary for substrate binding, cofactor binding and enzyme activity. Replacement of Trp212 with Tyr, Phe, and Leu reduced specific activity relative to EcDXR. EcDXR(W212F) and EcDXR(W212Y) had an increased Km relative to EcDXR indicative of loss in affinity toward DOXP, whereas EcDXR(W212L) had a lower Km of ~8 μM indicative of increased affinity for DOXP. The W212L substitution possibly removed contacts necessary for full catalytic activity, but could be considered a non-disruptive substitution in that it maintained active site architecture sufficient for DOXP reductoisomerase activity. EcDXR(M214I) had 36-fold reduced enzyme activity relative to EcDXR, while its Km (~8 μM) was found to be lower than that of EcDXR. This suggested that the M214I substitution had maintained (perhaps improved) substrate and active site architecture, but may have perturbed interactions with NADPH. Rational drug design strategies and docking methods have been utilized in the development of furan derivatives as DOXP reductoisomerase inhibitors, and the synthesis of phosphorylated derivatives (5) and (6) has been achieved. Future inhibitor studies using these novel potential DOXP reductoisomerase inhibitors may lead to the development of effective anti-malarial drug candidates.
- Full Text:
- Date Issued: 2004
Phenolic compounds in water and the implications for rapid detection of indicator micro-organisms using ß-D-Galactosidase and ß-D-Glucuronidase
- Authors: Abboo, Sagaran
- Date: 2009
- Subjects: Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3978 , http://hdl.handle.net/10962/d1004037 , Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Description: Faecal contamination in water is detected using appropriate microbial models such as total coliforms, faecal coliforms and E. coli. Βeta-D-Galactosidase (β-GAL) and Beta-D-glucuronidase (β-GUD) are two marker enzymes that are used to test for the presence of total coliforms and E. coli in water samples, respectively. Various assay methods have been developed using chromogenic and fluorogenic substrates. In this study, the chromogenic substrates chlorophenol red β-D-galactopyranoside (CPRG) for β-GAL and p-nitrophenyl-β-D-galactopyranoside (PNPG) for β-GUD were used. Potential problems associated with this approach include interference from other organisms present in the environment (e.g. plants, algae and other bacteria), as well as the presence of certain chemicals, such as phenolic compounds in water. Phenolic compounds are present in the aquatic environment due to their extensive industrial applications. The USA Enviromental Protection Agency (EPA) lists 11 Priority Pollutant Phenols (PPP) due to their high level of toxicity. This study investigated the interfering effects of the eleven PPP found in water on the enzyme activities of both the β-GAL and β-GUD enzyme assays. The presence of these PPP in the β-GAL and β-GUD enzyme assays showed that over and underestimation of activity may occur due to inhibition or activation of these enzymes. Three types of inhibition to enzyme activities were identified from double reciprocal Lineweaver-Burk plots. The inhibition constants (Ki) were determined for all inhibitory phenolic compounds from appropriate secondary plots. Furthermore, this study presented a validated reverse phase high performance liquid chromatography (RP-HPLC) method, developed for the simultaneous detection, separation and determination of all eleven phenolic compounds found in the environment. This method demonstrated good linearity, reproducibility, accuracy and sensitivity. Environmental water samples were collected from rivers, streams, industrial sites and wastewater treatment plant effluent. These samples were extracted and concentrated using a solid phase extraction (SPE) procedure prior to analysis employing the newly developed HPLC method in this study. Seasonal variations on the presence of the PPP in the environment were observed at certain collection sites. The concentrations found were between 0.033 μg/ml for 2,4-dinitrophenol in a running stream to 0.890 mg/ml for pentachlorophenol from an tannery industrial site. These concentrations of phenolic compounds found in these environments were able to interfere with the β-GAL and β-GUD enzyme assays.
- Full Text:
- Date Issued: 2009
- Authors: Abboo, Sagaran
- Date: 2009
- Subjects: Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3978 , http://hdl.handle.net/10962/d1004037 , Water -- Purification -- Biological treatment , Pollutants -- Biodegradation , Phenol , Organic water pollutants , Water quality biological assessment , Water -- Pollution
- Description: Faecal contamination in water is detected using appropriate microbial models such as total coliforms, faecal coliforms and E. coli. Βeta-D-Galactosidase (β-GAL) and Beta-D-glucuronidase (β-GUD) are two marker enzymes that are used to test for the presence of total coliforms and E. coli in water samples, respectively. Various assay methods have been developed using chromogenic and fluorogenic substrates. In this study, the chromogenic substrates chlorophenol red β-D-galactopyranoside (CPRG) for β-GAL and p-nitrophenyl-β-D-galactopyranoside (PNPG) for β-GUD were used. Potential problems associated with this approach include interference from other organisms present in the environment (e.g. plants, algae and other bacteria), as well as the presence of certain chemicals, such as phenolic compounds in water. Phenolic compounds are present in the aquatic environment due to their extensive industrial applications. The USA Enviromental Protection Agency (EPA) lists 11 Priority Pollutant Phenols (PPP) due to their high level of toxicity. This study investigated the interfering effects of the eleven PPP found in water on the enzyme activities of both the β-GAL and β-GUD enzyme assays. The presence of these PPP in the β-GAL and β-GUD enzyme assays showed that over and underestimation of activity may occur due to inhibition or activation of these enzymes. Three types of inhibition to enzyme activities were identified from double reciprocal Lineweaver-Burk plots. The inhibition constants (Ki) were determined for all inhibitory phenolic compounds from appropriate secondary plots. Furthermore, this study presented a validated reverse phase high performance liquid chromatography (RP-HPLC) method, developed for the simultaneous detection, separation and determination of all eleven phenolic compounds found in the environment. This method demonstrated good linearity, reproducibility, accuracy and sensitivity. Environmental water samples were collected from rivers, streams, industrial sites and wastewater treatment plant effluent. These samples were extracted and concentrated using a solid phase extraction (SPE) procedure prior to analysis employing the newly developed HPLC method in this study. Seasonal variations on the presence of the PPP in the environment were observed at certain collection sites. The concentrations found were between 0.033 μg/ml for 2,4-dinitrophenol in a running stream to 0.890 mg/ml for pentachlorophenol from an tannery industrial site. These concentrations of phenolic compounds found in these environments were able to interfere with the β-GAL and β-GUD enzyme assays.
- Full Text:
- Date Issued: 2009
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
Pineal-adrenal gland interactions in search of an anti-stressogenic role for melatonin
- Authors: Van Wyk, Elizabeth Joy
- Date: 1993
- Subjects: Pineal gland -- Secretions , Melatonin , Adrenal glands , Pineal gland -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4054 , http://hdl.handle.net/10962/d1004115 , Pineal gland -- Secretions , Melatonin , Adrenal glands , Pineal gland -- Research
- Description: The multiple functions of the pineal gland have been collectively interpreted as constituting a general anti-stressogenic role. The adrenal glands play a central role in maintaining homeostasis. The major neuroendocrine consequence of long-term stress is elevated circulating glucocorticoid levels. In this study, the effect of chronic, oral hydrocortisone treatment on pineal biochemistry was investigated in male Wi star rats of the albino strain. The results show that seven days of oral hydrocortisone treatment endows the pineal gland with the ability to increase melatonin synthesis in organ culture. The increase is accompanied by a rise in NAT activity, cyclic AMP levels and enhanced specific binding to the pineal B-adrenergic receptors. It appears that hydrocortisone sensitizes the pineal gland to stimulation by B-adrenergic agonists. thus rendering the pineal more responsive to B-adrenergic agonists. Further studies were directed at demonstrating an anti-stressogenic function for the pineal gland by investigating whether the principal pineal indole, melatonin. could protect against the deleterious effects of elevated. circulating drocortisone levels. The results show that chronic, oral hydrocortisone treatment significantly increases liver tryptophan pyrrolase activity. The catabolism of tryptophan by tryptophan pyrrolase is an important determinant of tryptophan availability to the brain, and therefore, brain serotonin levels. The findings show that melatonin inhibits basal and hydrocortisone-stimulated liver tryptophan pyrrolase apoenzyme activity in a dose-dependent manner. This inhibition suggests that melatonin may protect against excessive loss of tryptophan from circulation and against deficiencies in the cerebral serotinergic system which are associated with mood and behavioural disorders. It was shown that another deleterious effect of chronic hydrocortisone treatment is a significant increase in the number of glutamate receptors in the forebrain of male Wistar rats. The increase in receptor number observed in this study is probably due to an increase in the synthesis of glutamate receptors and is associated with a marked reduction in the affinity of the glutamate receptors for glutamate. possible to demonstrate an receptor number or the For practical reasons, it was not effect of melatonin on either glutamate affinity of glutamate receptors for glutamate in rat forebrain membranes. In view of the neurotoxic effect of glutamate in the eNS, the functional significance of recently described glutamate receptors in the pineal gland was investigated. The results show that 10-4 M glutamate significantly inhibits the isoprenaline-stimulated synthesis of N-acetylserotonin and melatonin in organ culture when the pineal glands were pre-incubated with glutamate for 4 hours prior to stimulation with isoprenalin and when glutamate and isoprenaline were administered together in vitro. GABA, a glutamate metabolite could not mimic the decrease in isoprenalinestimulated melatonin, and it is likely that the observed effects were directly attributed to glutamate. Incubation of the pineal gland with 10-4 M glutamate in organ culture did not affect HIOMT activity in pineal homogenates, but significantly elevated both basal and isoprenaline-stimulated NAT activity. It was concluded that glutamate only inhibits melatonin synthesis in intact pineal glands and not in pineal homogenates. The present study has provided further support for an interaction between the pineal and the adrenal glands. There is an ever increasing likelihood that melatonin is an anti-stressogenic hormone and that the pineal gland may have a protective role to play in the pathology of stress-related diseases.
- Full Text:
- Date Issued: 1993
- Authors: Van Wyk, Elizabeth Joy
- Date: 1993
- Subjects: Pineal gland -- Secretions , Melatonin , Adrenal glands , Pineal gland -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4054 , http://hdl.handle.net/10962/d1004115 , Pineal gland -- Secretions , Melatonin , Adrenal glands , Pineal gland -- Research
- Description: The multiple functions of the pineal gland have been collectively interpreted as constituting a general anti-stressogenic role. The adrenal glands play a central role in maintaining homeostasis. The major neuroendocrine consequence of long-term stress is elevated circulating glucocorticoid levels. In this study, the effect of chronic, oral hydrocortisone treatment on pineal biochemistry was investigated in male Wi star rats of the albino strain. The results show that seven days of oral hydrocortisone treatment endows the pineal gland with the ability to increase melatonin synthesis in organ culture. The increase is accompanied by a rise in NAT activity, cyclic AMP levels and enhanced specific binding to the pineal B-adrenergic receptors. It appears that hydrocortisone sensitizes the pineal gland to stimulation by B-adrenergic agonists. thus rendering the pineal more responsive to B-adrenergic agonists. Further studies were directed at demonstrating an anti-stressogenic function for the pineal gland by investigating whether the principal pineal indole, melatonin. could protect against the deleterious effects of elevated. circulating drocortisone levels. The results show that chronic, oral hydrocortisone treatment significantly increases liver tryptophan pyrrolase activity. The catabolism of tryptophan by tryptophan pyrrolase is an important determinant of tryptophan availability to the brain, and therefore, brain serotonin levels. The findings show that melatonin inhibits basal and hydrocortisone-stimulated liver tryptophan pyrrolase apoenzyme activity in a dose-dependent manner. This inhibition suggests that melatonin may protect against excessive loss of tryptophan from circulation and against deficiencies in the cerebral serotinergic system which are associated with mood and behavioural disorders. It was shown that another deleterious effect of chronic hydrocortisone treatment is a significant increase in the number of glutamate receptors in the forebrain of male Wistar rats. The increase in receptor number observed in this study is probably due to an increase in the synthesis of glutamate receptors and is associated with a marked reduction in the affinity of the glutamate receptors for glutamate. possible to demonstrate an receptor number or the For practical reasons, it was not effect of melatonin on either glutamate affinity of glutamate receptors for glutamate in rat forebrain membranes. In view of the neurotoxic effect of glutamate in the eNS, the functional significance of recently described glutamate receptors in the pineal gland was investigated. The results show that 10-4 M glutamate significantly inhibits the isoprenaline-stimulated synthesis of N-acetylserotonin and melatonin in organ culture when the pineal glands were pre-incubated with glutamate for 4 hours prior to stimulation with isoprenalin and when glutamate and isoprenaline were administered together in vitro. GABA, a glutamate metabolite could not mimic the decrease in isoprenalinestimulated melatonin, and it is likely that the observed effects were directly attributed to glutamate. Incubation of the pineal gland with 10-4 M glutamate in organ culture did not affect HIOMT activity in pineal homogenates, but significantly elevated both basal and isoprenaline-stimulated NAT activity. It was concluded that glutamate only inhibits melatonin synthesis in intact pineal glands and not in pineal homogenates. The present study has provided further support for an interaction between the pineal and the adrenal glands. There is an ever increasing likelihood that melatonin is an anti-stressogenic hormone and that the pineal gland may have a protective role to play in the pathology of stress-related diseases.
- Full Text:
- Date Issued: 1993
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
Polymers, catalysts and nanostructures a hybrid approach to biomolecule detection
- Authors: Frith, Kelly-Anne
- Date: 2009
- Subjects: Polymers , Nanostructured materials , Biomolecules , Tryptophan , Melatonin , Electrodes , Electrochemistry , Tryptophan oxygenase
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3980 , http://hdl.handle.net/10962/d1004039 , Polymers , Nanostructured materials , Biomolecules , Tryptophan , Melatonin , Electrodes , Electrochemistry , Tryptophan oxygenase
- Description: The main goals in electroanalytical sensing are towards improved sensitivity and selectivity, or specificity, of an analyte. There are several approaches to achieving these goals with the main approach being modification of an electrode surface with synthetic or natural catalysts (enzymes), polymers and also utilisation of nanostructured materials. At present, there is a strong movement towards hybrid sensing which couple different properties of two or more surface modification approaches. In this thesis, a range of these surface modifications were explored for analysis and detection of two main analytes: the amino acid, tryptophan (Trp); and, the neurotransmitter, dopamine (DA). Specifically, this thesis aimed to utilise these methods to enhance the sensitivity and selectivity for Trp over an interferent, the indoleamine, melatonin (Mel); and, DA over the vitamin, ascorbic acid (AA). For Trp detection, immobilisation of an enzyme, Tryptophanase (Trpase) resulted in poor selectivity for the analyte. However, enhanced sensitivity and selectivity was achieved through pH manipulation of the electrolyte medium at a Nafion®-modified electrode surface for both Trp and Mel. At pH 3.0, the Mel and Trp anodic peak potentials were sufficiently resolved allowing for an LOD of 1.60 and 1.62 nM,respectively, and permitting the accurate analysis of Trp in a dietary supplement containing Mel. Multi-walled carbon nanotubes (MWCNTs) suspended in Nafion® exhibited further increases in the signal responses of these analytes at pH 3.0 and 7.4 with minimal change in the resolution of the anodic peaks. A lower sensitivity was, therefore, observed at the Nafion® and MWCNT modified electrode compared to the Nafion®-modified electrode at pH 3.0 with LODs of 0.59 and 0.80 nM exhibited for Trp and Mel, respectively. Enhanced selectivity for Trp in the presence of Mel can be achieved with MWCNTs in the presence of metallotetrasulphonated phthalocyanines (MTSPcs) particularly at pH 3.0, owing to cation exchange effects. However, the lack of sensitivity towards Trp, and even Mel, at this CoTSPc and MWCNT modified electrode remains a drawback. For DA, detection at the MWCNT and Nafion® surface resulted in improved sensitivity over that of both the bare electrode (613.0 nM) and the Nafion® modified electrode (1045.1 nM) with a calculated LOD of 133.9 nM at this layer. Furthermore, improvements in the selectivity of DA were achieved at the Nafion® and MWCNT modified electrode as exclusion of AA (150 μM) was achieved. At the MWCNT and CoTSPc surface, AA was excluded up to 130 μM with sensitivity for DA extending as low as 14.3 nM, far greater than observed for Trp and Mel. These concentrations are well within physiological concentration ranges and represent the most significant solution yet in terms of AA exclusion and enhanced sensitivity for DA. An examination of the surface layering by impedance spectroscopy and atomic force microscopy indicates that the success of the hybrid sensor utilising CoTSPc and MWCNTs lay in improved dispersion of MWCNTs and improved electron transfer kinetics, facilitated by the net charge of the materials present. This thesis, thus, showed the utility of a judicious selection of synthetic and biological catalysts, polymers and carbon nanomaterials towards a hybrid approach to the electrochemical sensing of Trp, Mel, DA and AA with focus on sensitivity and selectivity of these analytes.
- Full Text:
- Date Issued: 2009
- Authors: Frith, Kelly-Anne
- Date: 2009
- Subjects: Polymers , Nanostructured materials , Biomolecules , Tryptophan , Melatonin , Electrodes , Electrochemistry , Tryptophan oxygenase
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3980 , http://hdl.handle.net/10962/d1004039 , Polymers , Nanostructured materials , Biomolecules , Tryptophan , Melatonin , Electrodes , Electrochemistry , Tryptophan oxygenase
- Description: The main goals in electroanalytical sensing are towards improved sensitivity and selectivity, or specificity, of an analyte. There are several approaches to achieving these goals with the main approach being modification of an electrode surface with synthetic or natural catalysts (enzymes), polymers and also utilisation of nanostructured materials. At present, there is a strong movement towards hybrid sensing which couple different properties of two or more surface modification approaches. In this thesis, a range of these surface modifications were explored for analysis and detection of two main analytes: the amino acid, tryptophan (Trp); and, the neurotransmitter, dopamine (DA). Specifically, this thesis aimed to utilise these methods to enhance the sensitivity and selectivity for Trp over an interferent, the indoleamine, melatonin (Mel); and, DA over the vitamin, ascorbic acid (AA). For Trp detection, immobilisation of an enzyme, Tryptophanase (Trpase) resulted in poor selectivity for the analyte. However, enhanced sensitivity and selectivity was achieved through pH manipulation of the electrolyte medium at a Nafion®-modified electrode surface for both Trp and Mel. At pH 3.0, the Mel and Trp anodic peak potentials were sufficiently resolved allowing for an LOD of 1.60 and 1.62 nM,respectively, and permitting the accurate analysis of Trp in a dietary supplement containing Mel. Multi-walled carbon nanotubes (MWCNTs) suspended in Nafion® exhibited further increases in the signal responses of these analytes at pH 3.0 and 7.4 with minimal change in the resolution of the anodic peaks. A lower sensitivity was, therefore, observed at the Nafion® and MWCNT modified electrode compared to the Nafion®-modified electrode at pH 3.0 with LODs of 0.59 and 0.80 nM exhibited for Trp and Mel, respectively. Enhanced selectivity for Trp in the presence of Mel can be achieved with MWCNTs in the presence of metallotetrasulphonated phthalocyanines (MTSPcs) particularly at pH 3.0, owing to cation exchange effects. However, the lack of sensitivity towards Trp, and even Mel, at this CoTSPc and MWCNT modified electrode remains a drawback. For DA, detection at the MWCNT and Nafion® surface resulted in improved sensitivity over that of both the bare electrode (613.0 nM) and the Nafion® modified electrode (1045.1 nM) with a calculated LOD of 133.9 nM at this layer. Furthermore, improvements in the selectivity of DA were achieved at the Nafion® and MWCNT modified electrode as exclusion of AA (150 μM) was achieved. At the MWCNT and CoTSPc surface, AA was excluded up to 130 μM with sensitivity for DA extending as low as 14.3 nM, far greater than observed for Trp and Mel. These concentrations are well within physiological concentration ranges and represent the most significant solution yet in terms of AA exclusion and enhanced sensitivity for DA. An examination of the surface layering by impedance spectroscopy and atomic force microscopy indicates that the success of the hybrid sensor utilising CoTSPc and MWCNTs lay in improved dispersion of MWCNTs and improved electron transfer kinetics, facilitated by the net charge of the materials present. This thesis, thus, showed the utility of a judicious selection of synthetic and biological catalysts, polymers and carbon nanomaterials towards a hybrid approach to the electrochemical sensing of Trp, Mel, DA and AA with focus on sensitivity and selectivity of these analytes.
- Full Text:
- Date Issued: 2009
Polyunsaturated fatty acid metabolism and effects on colon cancer cell biology in vitro.
- Authors: Bulcao, Candice
- Date: 2013
- Subjects: Unsaturated fatty acids , Unsaturated fatty acids in human nutrition , Colon (Anatomy)-- Cancer , Cancer -- Nutritional aspects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4137 , http://hdl.handle.net/10962/d1016128
- Description: Colon cancer is a leading cause of cancer related deaths worldwide. Lifestyle factors such as diet and exercise have been implicated as important agents in colon cancer development and progression. Epidemiological, in vivo and in vitro studies have found that n-3 polyunsaturated fatty acids (PUFAs) reduce colon carcinoma. The role of n-6 PUFAs remains a controversial topic, with studies indicating both promoting and preventing capabilities published. In order to better understand the effects of PUFAs on colon carcinoma, it is important to have an understanding of how they will be broken down in the body. During this study, in silico metabolism of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) predicted the formation of hydroxy-, di-hydroxy- and epoxy-FAs. A gas chromatography-mass spectrometry method was developed and validated for the detection of these PUFAs and their cytochrome P450 (CYP) metabolites. A human liver microsomal system for the in vitro metabolism of EPA, DHA and AA was optimised in terms of microsomal and PUFA concentration. The system resulted in the metabolism of the positive control, lauric acid, to 12-hydroxy-lauric acid but was unable to metabolise the PUFAs of interest. EPA, DHA and AA reduced cell viability in the colon carcinoma cell lines SW480 and SW620 in the micromolar concentration range (25 – 200 μM). The CYP epoxidation metabolite of EPA, 17, 18-epoxyeicosatetraenoic acid (17, 18-EpETE) resulted in a significant reduction in SW480 cell viability relative to the parent compound at lower concentrations (25 and 50 μM). Annexin V apoptosis analysis revealed that EPA and 17, 18- EpETE did not result in apoptosis in SW480 cells at a concentration of 25 μM and over an incubation period of 24 hours. A significant reduction in reactive oxygen species production was seen in SW480 cells after incubation with 25 μM 17, 18-EpETE for 24 hours. EPA and 17, 18-EpETE were implicated in the reduction of colon cancer metastasis since they were able to reduce SW480 migration and anchorage independent cell growth. These results indicate that the dietary intake of EPA, DHA and AA may be beneficial to one’s health due to the negative effects that these PUFAs had on colon carcinoma. Future studies are needed to confirm these benefits and compare the effects of the PUFAs to their CYP-metabolites.
- Full Text:
- Date Issued: 2013
- Authors: Bulcao, Candice
- Date: 2013
- Subjects: Unsaturated fatty acids , Unsaturated fatty acids in human nutrition , Colon (Anatomy)-- Cancer , Cancer -- Nutritional aspects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4137 , http://hdl.handle.net/10962/d1016128
- Description: Colon cancer is a leading cause of cancer related deaths worldwide. Lifestyle factors such as diet and exercise have been implicated as important agents in colon cancer development and progression. Epidemiological, in vivo and in vitro studies have found that n-3 polyunsaturated fatty acids (PUFAs) reduce colon carcinoma. The role of n-6 PUFAs remains a controversial topic, with studies indicating both promoting and preventing capabilities published. In order to better understand the effects of PUFAs on colon carcinoma, it is important to have an understanding of how they will be broken down in the body. During this study, in silico metabolism of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) predicted the formation of hydroxy-, di-hydroxy- and epoxy-FAs. A gas chromatography-mass spectrometry method was developed and validated for the detection of these PUFAs and their cytochrome P450 (CYP) metabolites. A human liver microsomal system for the in vitro metabolism of EPA, DHA and AA was optimised in terms of microsomal and PUFA concentration. The system resulted in the metabolism of the positive control, lauric acid, to 12-hydroxy-lauric acid but was unable to metabolise the PUFAs of interest. EPA, DHA and AA reduced cell viability in the colon carcinoma cell lines SW480 and SW620 in the micromolar concentration range (25 – 200 μM). The CYP epoxidation metabolite of EPA, 17, 18-epoxyeicosatetraenoic acid (17, 18-EpETE) resulted in a significant reduction in SW480 cell viability relative to the parent compound at lower concentrations (25 and 50 μM). Annexin V apoptosis analysis revealed that EPA and 17, 18- EpETE did not result in apoptosis in SW480 cells at a concentration of 25 μM and over an incubation period of 24 hours. A significant reduction in reactive oxygen species production was seen in SW480 cells after incubation with 25 μM 17, 18-EpETE for 24 hours. EPA and 17, 18-EpETE were implicated in the reduction of colon cancer metastasis since they were able to reduce SW480 migration and anchorage independent cell growth. These results indicate that the dietary intake of EPA, DHA and AA may be beneficial to one’s health due to the negative effects that these PUFAs had on colon carcinoma. Future studies are needed to confirm these benefits and compare the effects of the PUFAs to their CYP-metabolites.
- Full Text:
- Date Issued: 2013
Probing the biocompatibility of biomedical interfaces using the Quartz Crystal Microbalance with Dissipation
- Authors: Cromhout, Mary
- Date: 2011
- Subjects: Biomedical materials , Nanostructured materials , Biomedical engineering , Quartz crystal microbalances , Blood proteins , Nanoparticles
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4102 , http://hdl.handle.net/10962/d1010660
- Description: The biomedical application of nanotechnology has come into the spotlight, with the promise of ‘personalised’ therapeutics that couple early diagnosis with targeted therapeutic activity. Due to the rapid growth of the biomedical applications of nanoparticles, along with the lack of understanding concerning their interactions with biomolecules, there is a pressing need for the development of standard methods directed at investigating the effect of introducing these unique particles into the human body. The central aim of this research is to establish a platform directed at assessing the biological fate of pioneering therapeutic particulate agents, such as metallophthalocyanines (MPcs) and multi-walled carbon nanotubes (FMWCNTs). In particular, we proposed, that Quartz Crystal Microbalance with Dissipation (QCM-D) technology may be employed to assess the composition of blood protein corona deposited on the therapeutic surface, and subsequently assess the biocompatibility of such particles. The proposed method of protein detection utilises the nanogram sensitivity of QCM-D technology to monitor highly specific antibody-antigen interactions. In particular those interactions which occur when probe antibodies are used to detect adsorbed blood proteins deposited on target particle-modified sensor surfaces. Protein detection analysis was directed toward identification of surface bound human serum albumin, complement factor C3c, and human plasma fibrinogen. Preliminary analysis of generic biomedical surfaces indicated human serum albumin demonstrates a higher binding affinity towards positively charged surfaces (i.e. cysteamine self-assembled monolayer), followed by hydrophobic surfaces. Detection of complement C3c, corresponded with literature, where lower levels were detected on negatively charged surfaces (i.e. mercapto undecanoic acid self-assembled monolayer), and higher levels of more hydrophobic surfaces (i.e. 11-amino undecane thiol self-assembled monolayer). Human plasma fibrinogen was observed to favour hydrophilic over hydrophobic self-assembled monolayer surfaces, which was in accordance with literature. Application of the proposed protein detection method for biocompatibility analysis of target therapeutic molecules, namely metallophthalocyanines and acid functionalised multi-walled carbon nanotubes, demonstrated a dependence on modified-surface film characteristics, such as surface charge and topography with regards to human serum albumin and human plasma fibrinogen analysis representing new insights into their potential biomolecular interactions The highest levels of detected human serum albumin and complement C3c were detected on the GePcSmix-modified surfaces. AlPcSmix-modified surfaces analysis suggested the highest levels of human plasma fibrinogen. Two methods of acid functionalisation were employed, using both nitric and sulphuric acid, and pure nitric acid. A general increase in detected human serum albumin, corresponding with an increase in functionalisation time, was observed. Complement C3c detection suggested an increase in deposited complement C3c, with increasing functionalisation time, when assessing nitric acid functionalised multi-walled carbon nanotubes, and a decrease, with increasing functionalisation time, when assessing nitric and sulphuric acid functionalised multi-walled carbon nanotubes. Analysis of human plasma fibrinogen was inconclusive, as were cytotoxicity experiments utilising MCF-7 cells in the presence of metallophthalocyanine complexes, raising simultaneously important considerations for their application and study. In the first such detailed examination of its kind it was concluded that the proposed method of protein detection, using QCM-D, allows for the rudimentary but rapid means of analysis of select protein corona deposited on particulate biomedical surfaces.
- Full Text:
- Date Issued: 2011
- Authors: Cromhout, Mary
- Date: 2011
- Subjects: Biomedical materials , Nanostructured materials , Biomedical engineering , Quartz crystal microbalances , Blood proteins , Nanoparticles
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4102 , http://hdl.handle.net/10962/d1010660
- Description: The biomedical application of nanotechnology has come into the spotlight, with the promise of ‘personalised’ therapeutics that couple early diagnosis with targeted therapeutic activity. Due to the rapid growth of the biomedical applications of nanoparticles, along with the lack of understanding concerning their interactions with biomolecules, there is a pressing need for the development of standard methods directed at investigating the effect of introducing these unique particles into the human body. The central aim of this research is to establish a platform directed at assessing the biological fate of pioneering therapeutic particulate agents, such as metallophthalocyanines (MPcs) and multi-walled carbon nanotubes (FMWCNTs). In particular, we proposed, that Quartz Crystal Microbalance with Dissipation (QCM-D) technology may be employed to assess the composition of blood protein corona deposited on the therapeutic surface, and subsequently assess the biocompatibility of such particles. The proposed method of protein detection utilises the nanogram sensitivity of QCM-D technology to monitor highly specific antibody-antigen interactions. In particular those interactions which occur when probe antibodies are used to detect adsorbed blood proteins deposited on target particle-modified sensor surfaces. Protein detection analysis was directed toward identification of surface bound human serum albumin, complement factor C3c, and human plasma fibrinogen. Preliminary analysis of generic biomedical surfaces indicated human serum albumin demonstrates a higher binding affinity towards positively charged surfaces (i.e. cysteamine self-assembled monolayer), followed by hydrophobic surfaces. Detection of complement C3c, corresponded with literature, where lower levels were detected on negatively charged surfaces (i.e. mercapto undecanoic acid self-assembled monolayer), and higher levels of more hydrophobic surfaces (i.e. 11-amino undecane thiol self-assembled monolayer). Human plasma fibrinogen was observed to favour hydrophilic over hydrophobic self-assembled monolayer surfaces, which was in accordance with literature. Application of the proposed protein detection method for biocompatibility analysis of target therapeutic molecules, namely metallophthalocyanines and acid functionalised multi-walled carbon nanotubes, demonstrated a dependence on modified-surface film characteristics, such as surface charge and topography with regards to human serum albumin and human plasma fibrinogen analysis representing new insights into their potential biomolecular interactions The highest levels of detected human serum albumin and complement C3c were detected on the GePcSmix-modified surfaces. AlPcSmix-modified surfaces analysis suggested the highest levels of human plasma fibrinogen. Two methods of acid functionalisation were employed, using both nitric and sulphuric acid, and pure nitric acid. A general increase in detected human serum albumin, corresponding with an increase in functionalisation time, was observed. Complement C3c detection suggested an increase in deposited complement C3c, with increasing functionalisation time, when assessing nitric acid functionalised multi-walled carbon nanotubes, and a decrease, with increasing functionalisation time, when assessing nitric and sulphuric acid functionalised multi-walled carbon nanotubes. Analysis of human plasma fibrinogen was inconclusive, as were cytotoxicity experiments utilising MCF-7 cells in the presence of metallophthalocyanine complexes, raising simultaneously important considerations for their application and study. In the first such detailed examination of its kind it was concluded that the proposed method of protein detection, using QCM-D, allows for the rudimentary but rapid means of analysis of select protein corona deposited on particulate biomedical surfaces.
- Full Text:
- Date Issued: 2011
Progestin receptor heterogeneity in a breast cancer cell line
- Authors: Levy, Anita Rochelle
- Date: 1995
- Subjects: Breast -- Cancer , Hormone receptors , Cancer cells -- Growth -- Regulation , Progesterone -- Receptors , Cellular control mechanisms
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4039 , http://hdl.handle.net/10962/d1004100 , Breast -- Cancer , Hormone receptors , Cancer cells -- Growth -- Regulation , Progesterone -- Receptors , Cellular control mechanisms
- Description: Anti-oestrogens act via the oestrogen receptor whether they compete with the hormone for binding to the receptor and therefore interfere with DNA binding or inhibit transcriptional activity. These receptors exist as a large 85 complex and/or a small 45 form on sucrose density gradients. High performance ion-exchange chromatography has confirmed that the oestrogen and progestin complex is present in various isoforms. Progestin receptor heterogeneity could be influenced by the presence of oestrogens and anti-oestrogens in the culture media of hormone-dependent neoplastic cells. Cell culture methods offer the opportunity to test effects of specified components in repeated experiments on a homogeneous population of cells. MCF-7 and T47-D human breast cancer cell lines were conditioned to grow in a serum-free environment. There was no difference in cell proliferation rates, nor in their oestrogen or progestin receptor levels when compared to the same cells grown in conventional media. Receptors were present mainly in the large molecular 85 form. Both the MCF-7 and T47-D breast cancer cells showed an increase in proliferation rate with the addition of oestrogen or diethylstilbestrol. There was a corresponding loss of progestin receptor levels and an alteration in the high performance ion-exchange isoforms. Flow cytometry confirmed differences in the S-phase components of the cells following exposure to oestrogens. The proliferation rates of the cell lines as well as their progestin receptor levels decreased when treated with tamoxifen or the hydroxylated tamoxifen. There were marked changes on high performance ion-exchange chromatography profiles. DNA ploidy and S-phase showed signs of toxicity and there was an increase in cellular debris. The MCF-7 and T47-D human breast cancer cell line retained response to antioestrogen saturation.
- Full Text:
- Date Issued: 1995
- Authors: Levy, Anita Rochelle
- Date: 1995
- Subjects: Breast -- Cancer , Hormone receptors , Cancer cells -- Growth -- Regulation , Progesterone -- Receptors , Cellular control mechanisms
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4039 , http://hdl.handle.net/10962/d1004100 , Breast -- Cancer , Hormone receptors , Cancer cells -- Growth -- Regulation , Progesterone -- Receptors , Cellular control mechanisms
- Description: Anti-oestrogens act via the oestrogen receptor whether they compete with the hormone for binding to the receptor and therefore interfere with DNA binding or inhibit transcriptional activity. These receptors exist as a large 85 complex and/or a small 45 form on sucrose density gradients. High performance ion-exchange chromatography has confirmed that the oestrogen and progestin complex is present in various isoforms. Progestin receptor heterogeneity could be influenced by the presence of oestrogens and anti-oestrogens in the culture media of hormone-dependent neoplastic cells. Cell culture methods offer the opportunity to test effects of specified components in repeated experiments on a homogeneous population of cells. MCF-7 and T47-D human breast cancer cell lines were conditioned to grow in a serum-free environment. There was no difference in cell proliferation rates, nor in their oestrogen or progestin receptor levels when compared to the same cells grown in conventional media. Receptors were present mainly in the large molecular 85 form. Both the MCF-7 and T47-D breast cancer cells showed an increase in proliferation rate with the addition of oestrogen or diethylstilbestrol. There was a corresponding loss of progestin receptor levels and an alteration in the high performance ion-exchange isoforms. Flow cytometry confirmed differences in the S-phase components of the cells following exposure to oestrogens. The proliferation rates of the cell lines as well as their progestin receptor levels decreased when treated with tamoxifen or the hydroxylated tamoxifen. There were marked changes on high performance ion-exchange chromatography profiles. DNA ploidy and S-phase showed signs of toxicity and there was an increase in cellular debris. The MCF-7 and T47-D human breast cancer cell line retained response to antioestrogen saturation.
- Full Text:
- Date Issued: 1995
Proportional distribution of predominant rumen bacteria between the solid and the liquid portions of ruminal ingesta
- Authors: Brinkman, Paul A
- Date: 1966
- Subjects: Rumen -- Microbiology , Bacteria -- Rumen
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4100 , http://hdl.handle.net/10962/d1009715 , Rumen -- Microbiology , Bacteria -- Rumen
- Description: That certain bacteria in the rumen of sheep and cattle are attached to solid particles in the ruminal ingesta has been known for many years. In 1942 Baker published direct microscopical evidence that bacteria were attached to cellulose food particles and to starch granules in the rumen. The sites of attachment of these bacteria corresponded to sites of disintegration of the particles when viewed by polarised light. This indicated that at least bacteria attacking solid substrates such as cellulose and starch were attached to particles of ruminal ingesta. Van der Wath (1942) found rumen bacteria attached to particles of chemically pure cellulose and of crushed maize which he suspended in separate compartments of a pure silk bag inside the rumen of sheep. The bacteria associated with the particles of cellulose were mainly Gram negative rods , while clusters of iodophilic cocci were observed in most instances around the maize kernels . The latter organisms were isolated in pure culture and found to be heat-tolerant, short-chain, Gram positive cocci fermenting glucose, maltose, and other soluble sugars as well as starch. It was thus not surprising that many years later Schwartz et al (1964) obtained evidence which suggested that bacteria metabolising soluble substrates such as glucose also showed marked attachment to solid particles of ingesta.
- Full Text:
- Date Issued: 1966
- Authors: Brinkman, Paul A
- Date: 1966
- Subjects: Rumen -- Microbiology , Bacteria -- Rumen
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4100 , http://hdl.handle.net/10962/d1009715 , Rumen -- Microbiology , Bacteria -- Rumen
- Description: That certain bacteria in the rumen of sheep and cattle are attached to solid particles in the ruminal ingesta has been known for many years. In 1942 Baker published direct microscopical evidence that bacteria were attached to cellulose food particles and to starch granules in the rumen. The sites of attachment of these bacteria corresponded to sites of disintegration of the particles when viewed by polarised light. This indicated that at least bacteria attacking solid substrates such as cellulose and starch were attached to particles of ruminal ingesta. Van der Wath (1942) found rumen bacteria attached to particles of chemically pure cellulose and of crushed maize which he suspended in separate compartments of a pure silk bag inside the rumen of sheep. The bacteria associated with the particles of cellulose were mainly Gram negative rods , while clusters of iodophilic cocci were observed in most instances around the maize kernels . The latter organisms were isolated in pure culture and found to be heat-tolerant, short-chain, Gram positive cocci fermenting glucose, maltose, and other soluble sugars as well as starch. It was thus not surprising that many years later Schwartz et al (1964) obtained evidence which suggested that bacteria metabolising soluble substrates such as glucose also showed marked attachment to solid particles of ingesta.
- Full Text:
- Date Issued: 1966
Purification and characterization of fructosyltransferase for the synthesis of short-chain fructo-oligosaccharides and investigation into thier anti-carcinogenic properties
- Authors: Nemukula, Aluwani
- Date: 2009
- Subjects: Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3927 , http://hdl.handle.net/10962/d1003986 , Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Description: There is a growing attention in the synthesis of fructo-oligosaccharides (FOS) due to their excellent bio-functional and health-promoting properties. The current production processes are limited to chemical hydrolysis reactions of plant extracts, which are often associated with several drawbacks. In this study, fructosyltransferase (FTase) and polygalacturonase (PGase) activities, present in a commercial enzyme preparation (Pectinex® Ultra SP-L) sourced from Aspergillus aculeatus, have been separated and fully purified by anion-exchange and sizeexclusion chromatography. The FTase possesses fructosyl transfer activity for FOS synthesis and the PGase has pectin hydrolytic activity. Fructosyltransferase is a single-band protein with a molecular weight of 85 kDa, whereas PGase is a distinct protein of 40 kDa. The temperature and pH optima of FTase were 60 ºC and 6.0, with a half-life of 8 h; while that for PGase were 40 ºC and 6.0, respectively. FTase was slightly inhibited in the presence of Ni²⁺, Mg²⁺ and urea; but PGase was more susceptible to divalent ions such as Ca²⁺, Mg²⁺ and Mn²⁺. The kinetic parameters (Km and Vmax) of FTase for the hydrolysis of β-(2→1) linkages from sucrose were 752.3 mM and 120.5 μmol.min⁻¹.mL⁻¹, respectively; whereas the same parameters for pectin hydrolysis by PGase were 13.0 mg.mL⁻¹ and 263 μmol.min-1.mL⁻¹, respectively. The purified FTase was able to transfer fructosyl residues from sucrose, synthesizing the corresponding chains of FOS. PGase was relatively stable at 40 ºC (t½ > 3 h), depolymerizing the pectin backbone while releasing the inulins from within the chicory roots. Analysis of various mixtures of FOS by mass spectrometry, HPLC and ¹H-NMR was undertaken. Results indicated that MS with electrospray ionization and ¹H-NMR are capable of providing relative quantitative data of the FOS present in the mixtures. The pharmaceutical effects of various sc-FOS (0.5%, v/v) and SCFA (0.3%, v/v) on certain bacterial enzymes (β-glucuronidase, urease and β-glucosidase) associated with the formation of carcinogens were also studied. These enzyme activities were not directly influenced by the sc-FOS, but were found to be remarkably decreased by SCFA, pointing toward the prebiotic effect of FOS in intestinal microflora modulation.
- Full Text:
- Date Issued: 2009
- Authors: Nemukula, Aluwani
- Date: 2009
- Subjects: Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3927 , http://hdl.handle.net/10962/d1003986 , Oligosaccharides , Polygalacturonase , Aspergillus , Fructose , Inulin , Cancer -- Prevention , Cancer -- Research , Carcinogens , High performance liquid chromatography
- Description: There is a growing attention in the synthesis of fructo-oligosaccharides (FOS) due to their excellent bio-functional and health-promoting properties. The current production processes are limited to chemical hydrolysis reactions of plant extracts, which are often associated with several drawbacks. In this study, fructosyltransferase (FTase) and polygalacturonase (PGase) activities, present in a commercial enzyme preparation (Pectinex® Ultra SP-L) sourced from Aspergillus aculeatus, have been separated and fully purified by anion-exchange and sizeexclusion chromatography. The FTase possesses fructosyl transfer activity for FOS synthesis and the PGase has pectin hydrolytic activity. Fructosyltransferase is a single-band protein with a molecular weight of 85 kDa, whereas PGase is a distinct protein of 40 kDa. The temperature and pH optima of FTase were 60 ºC and 6.0, with a half-life of 8 h; while that for PGase were 40 ºC and 6.0, respectively. FTase was slightly inhibited in the presence of Ni²⁺, Mg²⁺ and urea; but PGase was more susceptible to divalent ions such as Ca²⁺, Mg²⁺ and Mn²⁺. The kinetic parameters (Km and Vmax) of FTase for the hydrolysis of β-(2→1) linkages from sucrose were 752.3 mM and 120.5 μmol.min⁻¹.mL⁻¹, respectively; whereas the same parameters for pectin hydrolysis by PGase were 13.0 mg.mL⁻¹ and 263 μmol.min-1.mL⁻¹, respectively. The purified FTase was able to transfer fructosyl residues from sucrose, synthesizing the corresponding chains of FOS. PGase was relatively stable at 40 ºC (t½ > 3 h), depolymerizing the pectin backbone while releasing the inulins from within the chicory roots. Analysis of various mixtures of FOS by mass spectrometry, HPLC and ¹H-NMR was undertaken. Results indicated that MS with electrospray ionization and ¹H-NMR are capable of providing relative quantitative data of the FOS present in the mixtures. The pharmaceutical effects of various sc-FOS (0.5%, v/v) and SCFA (0.3%, v/v) on certain bacterial enzymes (β-glucuronidase, urease and β-glucosidase) associated with the formation of carcinogens were also studied. These enzyme activities were not directly influenced by the sc-FOS, but were found to be remarkably decreased by SCFA, pointing toward the prebiotic effect of FOS in intestinal microflora modulation.
- Full Text:
- Date Issued: 2009
Purification and characterization of TbHsp70.c, a novel Hsp70 from Trypanosoma brucei
- Authors: Burger, Adélle
- Date: 2014
- Subjects: African trypanosomiasis -- Research Heat shock proteins -- Research Trypanosoma brucei -- Research Mycobacterial diseases -- Research -- Africa Parasitic diseases -- Africa -- Prevention Parasites -- Physiology Developing countries -- Economic conditions
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4105 , http://hdl.handle.net/10962/d1011618
- Description: One of Africa’s neglected tropical diseases, African Trypanosomiasis, is not only fatal but also has a crippling impact on economic development. Heat shock proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. The expression of heat shock proteins increases during these heat shock conditions, and this is considered to play a role in differentiation of these vector-borne parasites. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is involved in protein homeostasis, Hsp40 acts as a co-chaperone and stimulates its intrinsically weak ATPase activity. In silico analysis of the T. brucei genome has revealed the existence of 12 Hsp70 proteins and 65 Hsp40 proteins to date. A novel Hsp70, TbHsp70.c, was recently identified in T. brucei. Different from the prototypical Hsp70, TbHsp70.c contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. By implication the substrate range and mechanism by which the substrates are recognized may be novel. The ability of a Type I Hsp40, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective of this study was to biochemically characterize TbHsp70.c and its partnership with Tbj2 to further enhance our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed chaperone activities in their ability to suppress aggregation of thermolabile MDH. TbHsp70.c also suppressed aggregation of rhodanese. ATPase assays revealed that the ATPase activity of TbHsp70.c was stimulated by Tbj2. The targeted inhibition of the function of heat shock proteins is emerging as a tool to combat disease. The small molecule modulators quercetin and methylene blue are known to inhibit the ATPase activity of Hsp70. However, methylene blue did not significantly inhibit the ATPase activity of TbHsp70.c; while quercetin, did inhibit the ATPase activity. In vivo heat stress experiments indicated an up-regulation of the expression levels of TbHsp70.c. RNA interference studies showed partial knockdown of TbHsp70.c with no detrimental effect on the parasite. Fluorescence microscopy studies of TbHsp70.c showed a probable cytoplasmic subcellular localization. In this study both TbHsp70.c and Tbj2 demonstrated chaperone activity and Tbj2 possibly functions as a co-chaperone of TbHsp70.c.
- Full Text:
- Date Issued: 2014
- Authors: Burger, Adélle
- Date: 2014
- Subjects: African trypanosomiasis -- Research Heat shock proteins -- Research Trypanosoma brucei -- Research Mycobacterial diseases -- Research -- Africa Parasitic diseases -- Africa -- Prevention Parasites -- Physiology Developing countries -- Economic conditions
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4105 , http://hdl.handle.net/10962/d1011618
- Description: One of Africa’s neglected tropical diseases, African Trypanosomiasis, is not only fatal but also has a crippling impact on economic development. Heat shock proteins play a wide range of roles in the cell and they are required to assist the parasite as it moves from a cold blooded insect vector to a warm blooded mammalian host. The expression of heat shock proteins increases during these heat shock conditions, and this is considered to play a role in differentiation of these vector-borne parasites. Heat shock protein 70 (Hsp70) is an important molecular chaperone that is involved in protein homeostasis, Hsp40 acts as a co-chaperone and stimulates its intrinsically weak ATPase activity. In silico analysis of the T. brucei genome has revealed the existence of 12 Hsp70 proteins and 65 Hsp40 proteins to date. A novel Hsp70, TbHsp70.c, was recently identified in T. brucei. Different from the prototypical Hsp70, TbHsp70.c contains an acidic substrate binding domain and lacks the C-terminal EEVD motif. By implication the substrate range and mechanism by which the substrates are recognized may be novel. The ability of a Type I Hsp40, Tbj2, to function as a co-chaperone of TbHsp70.c was investigated. The main objective of this study was to biochemically characterize TbHsp70.c and its partnership with Tbj2 to further enhance our knowledge of parasite biology. TbHsp70.c and Tbj2 were heterologously expressed and purified and both proteins displayed chaperone activities in their ability to suppress aggregation of thermolabile MDH. TbHsp70.c also suppressed aggregation of rhodanese. ATPase assays revealed that the ATPase activity of TbHsp70.c was stimulated by Tbj2. The targeted inhibition of the function of heat shock proteins is emerging as a tool to combat disease. The small molecule modulators quercetin and methylene blue are known to inhibit the ATPase activity of Hsp70. However, methylene blue did not significantly inhibit the ATPase activity of TbHsp70.c; while quercetin, did inhibit the ATPase activity. In vivo heat stress experiments indicated an up-regulation of the expression levels of TbHsp70.c. RNA interference studies showed partial knockdown of TbHsp70.c with no detrimental effect on the parasite. Fluorescence microscopy studies of TbHsp70.c showed a probable cytoplasmic subcellular localization. In this study both TbHsp70.c and Tbj2 demonstrated chaperone activity and Tbj2 possibly functions as a co-chaperone of TbHsp70.c.
- Full Text:
- Date Issued: 2014
Rapid enzymatic detection of organophosphorous and carbamate pesticides in water
- Authors: Mwila, Katayi
- Date: 2012
- Subjects: Organophosphorus compounds , Carbamates , Water -- Pesticide content -- South Africa -- Eastern Cape , Water quality biological assessment -- South Africa -- Eastern Cape , Water quality management -- South Africa -- Eastern Cape , Pesticides -- Toxicology -- South Africa -- Eastern Cape , Biological assay , Acetylcholinesterase , Parathion , Aldicarb , Carbaryl , Carbofuran , Nitrophenols
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4024 , http://hdl.handle.net/10962/d1004084 , Organophosphorus compounds , Carbamates , Water -- Pesticide content -- South Africa -- Eastern Cape , Water quality biological assessment -- South Africa -- Eastern Cape , Water quality management -- South Africa -- Eastern Cape , Pesticides -- Toxicology -- South Africa -- Eastern Cape , Biological assay , Acetylcholinesterase , Parathion , Aldicarb , Carbaryl , Carbofuran , Nitrophenols
- Description: The increased use of pesticides has resulted in a corresponding increase in concern for the effect they may have on the health of humans and other non-target organisms. The two main areas of concern are the toxicological effects that mixtures of pesticides may have as well as the endocrine disrupting effects. Although the individual pesticides may be present at concentrations below the levels deemed to be detrimental to health, it has been argued that their combined effect may still result in elevated health risks. Another important aspect of pesticide risk assessment requires a consideration of the breakdown products of pesticides and their effect on human health. There has been very little research into the effects of degradation products and this issue should be addressed as these could potentially pose a higher risk than their parent compounds. One of the most important bio-markers available for use is the ubiquitous enzyme acetylcholinesterase (AChE). This enzyme is responsible for one of the most important functions in the body; namely nerve impulse transmission, upon which all life depends. The inhibition of this enzyme indicates toxicity and as a subsequence, a threat to the organism’s well-being. Bioassays have also recently been developed to test chemicals for endocrine disrupting effects. These tests rely on a dose response equivalent to that of the most potent well known estrogen 17-β estradiol. Any chemical that has a measurable response is deemed to display endocrine disrupting effects. This first aim of this study was to investigate the toxicological and endocrine disrupting effects of three organophosphorus pesticides; aldicarb, parathion and demeton-S-methyl, in addition to two breakdown products; aminophenol and p-nitrophenol. Two carbamate pesticides; carbaryl and carbofuran were also analysed. The toxicological effects of mixtures of the parent pesticide compounds were tested to assess if any antagonistic, additive or synergistic effects were observed. This data was then used in conjunction with an artificial neural network to assess if individual pesticides could be distinguished from mixtures of pesticides. A final objective was to sample various Eastern Cape water sources, utilising the enzymatic assay to determine the presence of any of these pesticides in these samples. There were several conclusions drawn from this study. AChE was successfully used as an assay to test the toxicity of the pesticides under investigation, based on their inhibition of this enzyme. An important factor for consideration throughout the study was the need to establish basal and monitor AChE activity (i.e. the need to monitor AChE activity in the absence of any pesticide). This ensured accurate comparison of the results obtained. It was found that demeton-S-methyl was the most potent of these pesticides followed by carbaryl, parathion, aldicarb and finally carbofuran, and that carbofuran could potentiate AChE. The results indicated that pesticide mixtures generally exhibited an additive inhibitory effect on AChE, although at some concentrations of pesticides, synergistic and antagonistic effects were noted. From the data using mixtures of pesticides, a feed forward neural network was created that was successfully able to distinguish individual pesticides from mixtures within its training parameters. None of the pesticides tested displayed endocrine disrupting properties in the Yeast Estrogen Screen (YES), T47D-KBluc and MDA-kb2 bio-assays. Other studies reported mixed results in this regard and thus no final conclusions could be drawn. The Blaauwkrantz River, Kariega River, Sundays River, Swartkops River and Kowie River were all tested for pesticides and although positive results were recorded, conventional methods indicated that there were no pesticides in the rivers. There were, however, trace metals present which are known to inhibit AChE, thus causing a false positive result. These results indicated that AChE can be used as a high throughput initial pre-screening tool, but that it cannot serve as a substitute for more accurate conventional testing methods.
- Full Text:
- Date Issued: 2012
- Authors: Mwila, Katayi
- Date: 2012
- Subjects: Organophosphorus compounds , Carbamates , Water -- Pesticide content -- South Africa -- Eastern Cape , Water quality biological assessment -- South Africa -- Eastern Cape , Water quality management -- South Africa -- Eastern Cape , Pesticides -- Toxicology -- South Africa -- Eastern Cape , Biological assay , Acetylcholinesterase , Parathion , Aldicarb , Carbaryl , Carbofuran , Nitrophenols
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4024 , http://hdl.handle.net/10962/d1004084 , Organophosphorus compounds , Carbamates , Water -- Pesticide content -- South Africa -- Eastern Cape , Water quality biological assessment -- South Africa -- Eastern Cape , Water quality management -- South Africa -- Eastern Cape , Pesticides -- Toxicology -- South Africa -- Eastern Cape , Biological assay , Acetylcholinesterase , Parathion , Aldicarb , Carbaryl , Carbofuran , Nitrophenols
- Description: The increased use of pesticides has resulted in a corresponding increase in concern for the effect they may have on the health of humans and other non-target organisms. The two main areas of concern are the toxicological effects that mixtures of pesticides may have as well as the endocrine disrupting effects. Although the individual pesticides may be present at concentrations below the levels deemed to be detrimental to health, it has been argued that their combined effect may still result in elevated health risks. Another important aspect of pesticide risk assessment requires a consideration of the breakdown products of pesticides and their effect on human health. There has been very little research into the effects of degradation products and this issue should be addressed as these could potentially pose a higher risk than their parent compounds. One of the most important bio-markers available for use is the ubiquitous enzyme acetylcholinesterase (AChE). This enzyme is responsible for one of the most important functions in the body; namely nerve impulse transmission, upon which all life depends. The inhibition of this enzyme indicates toxicity and as a subsequence, a threat to the organism’s well-being. Bioassays have also recently been developed to test chemicals for endocrine disrupting effects. These tests rely on a dose response equivalent to that of the most potent well known estrogen 17-β estradiol. Any chemical that has a measurable response is deemed to display endocrine disrupting effects. This first aim of this study was to investigate the toxicological and endocrine disrupting effects of three organophosphorus pesticides; aldicarb, parathion and demeton-S-methyl, in addition to two breakdown products; aminophenol and p-nitrophenol. Two carbamate pesticides; carbaryl and carbofuran were also analysed. The toxicological effects of mixtures of the parent pesticide compounds were tested to assess if any antagonistic, additive or synergistic effects were observed. This data was then used in conjunction with an artificial neural network to assess if individual pesticides could be distinguished from mixtures of pesticides. A final objective was to sample various Eastern Cape water sources, utilising the enzymatic assay to determine the presence of any of these pesticides in these samples. There were several conclusions drawn from this study. AChE was successfully used as an assay to test the toxicity of the pesticides under investigation, based on their inhibition of this enzyme. An important factor for consideration throughout the study was the need to establish basal and monitor AChE activity (i.e. the need to monitor AChE activity in the absence of any pesticide). This ensured accurate comparison of the results obtained. It was found that demeton-S-methyl was the most potent of these pesticides followed by carbaryl, parathion, aldicarb and finally carbofuran, and that carbofuran could potentiate AChE. The results indicated that pesticide mixtures generally exhibited an additive inhibitory effect on AChE, although at some concentrations of pesticides, synergistic and antagonistic effects were noted. From the data using mixtures of pesticides, a feed forward neural network was created that was successfully able to distinguish individual pesticides from mixtures within its training parameters. None of the pesticides tested displayed endocrine disrupting properties in the Yeast Estrogen Screen (YES), T47D-KBluc and MDA-kb2 bio-assays. Other studies reported mixed results in this regard and thus no final conclusions could be drawn. The Blaauwkrantz River, Kariega River, Sundays River, Swartkops River and Kowie River were all tested for pesticides and although positive results were recorded, conventional methods indicated that there were no pesticides in the rivers. There were, however, trace metals present which are known to inhibit AChE, thus causing a false positive result. These results indicated that AChE can be used as a high throughput initial pre-screening tool, but that it cannot serve as a substitute for more accurate conventional testing methods.
- Full Text:
- Date Issued: 2012
Reactor development and process optimisation for the bioremediation of phenolic wastewaters by trametes species
- Authors: Ryan, Daniel Reginald
- Date: 2004-04
- Subjects: Uncatalogued
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/191487 , vital:45103
- Description: In many service industries, the source of a company’s value has shifted from capital to knowledge and ideas, the quality of which is dependent on its employees (Wooldridge, 2006). In fact, human resources can be considered part of factor conditions which can positively impact on a firm’s competitive context. This impact can ultimately translate into improved financial results (Porter and Kramer, 2002). There is therefore a growing interest in ways to attract and retain talent. According to the managers of many big companies, well communicated corporate responsibility practices can improve staff attraction as well as retention rates by improving morale (CSRwire, 2002). To explore this, a small, creative company in Johannesburg which engages in charity work was selected as a case study, with the goal being to understand whether their culture of good deeds has a positive impact on staff wellbeing. While the owner of the company actively attempts to make the company an enjoyable place to work at, he appears to have initiated the philanthropic activities in a true spirit of giving, rather than with the motive of engaging staff in order to make more money. Nevertheless, the researcher’s investigative stance is that of an enlightened egoist, and the study focuses on the business case of giving being beneficial to the giver (ultimately the company) in the long term, as well as to the recipient. While the danger of suggesting that philanthropy could be instrumentalised is acknowledged (Morton, 2004), the investigation explores the possibility because such evidence could persuade other companies to become more socially concerned. Through a qualitative approach involving interviews, observation and analysis of video footage, it becomes apparent that there is clearly value for the staff in the charity work they do. Unfortunately the multiple initiatives undertaken to keep staff morale high at the company make it impossible to establish a clear link between the philanthropy and overall wellbeing, but as the study was conducted in the phenomenological paradigm the main concern was with understanding the experience of participants. However, an unexpected finding was that the employees derive great satisfaction from using their professional skills for charity work rather than just donating money to the charity. They feel that their skills uniquely position them to make significant changes to the lives of others, which gives them a sense of pride and achievement that they don’t necessarily experience in their ordinary activities at work. On the basis of this, it is recommended that companies look to involve staff with projects that require their specific expertise when evaluating philanthropic initiatives. , Thesis (PhD) -- Faculty of Science, Biochemistry, Microbiology and Biotechnology, 2004
- Full Text:
- Date Issued: 2004-04
- Authors: Ryan, Daniel Reginald
- Date: 2004-04
- Subjects: Uncatalogued
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/191487 , vital:45103
- Description: In many service industries, the source of a company’s value has shifted from capital to knowledge and ideas, the quality of which is dependent on its employees (Wooldridge, 2006). In fact, human resources can be considered part of factor conditions which can positively impact on a firm’s competitive context. This impact can ultimately translate into improved financial results (Porter and Kramer, 2002). There is therefore a growing interest in ways to attract and retain talent. According to the managers of many big companies, well communicated corporate responsibility practices can improve staff attraction as well as retention rates by improving morale (CSRwire, 2002). To explore this, a small, creative company in Johannesburg which engages in charity work was selected as a case study, with the goal being to understand whether their culture of good deeds has a positive impact on staff wellbeing. While the owner of the company actively attempts to make the company an enjoyable place to work at, he appears to have initiated the philanthropic activities in a true spirit of giving, rather than with the motive of engaging staff in order to make more money. Nevertheless, the researcher’s investigative stance is that of an enlightened egoist, and the study focuses on the business case of giving being beneficial to the giver (ultimately the company) in the long term, as well as to the recipient. While the danger of suggesting that philanthropy could be instrumentalised is acknowledged (Morton, 2004), the investigation explores the possibility because such evidence could persuade other companies to become more socially concerned. Through a qualitative approach involving interviews, observation and analysis of video footage, it becomes apparent that there is clearly value for the staff in the charity work they do. Unfortunately the multiple initiatives undertaken to keep staff morale high at the company make it impossible to establish a clear link between the philanthropy and overall wellbeing, but as the study was conducted in the phenomenological paradigm the main concern was with understanding the experience of participants. However, an unexpected finding was that the employees derive great satisfaction from using their professional skills for charity work rather than just donating money to the charity. They feel that their skills uniquely position them to make significant changes to the lives of others, which gives them a sense of pride and achievement that they don’t necessarily experience in their ordinary activities at work. On the basis of this, it is recommended that companies look to involve staff with projects that require their specific expertise when evaluating philanthropic initiatives. , Thesis (PhD) -- Faculty of Science, Biochemistry, Microbiology and Biotechnology, 2004
- Full Text:
- Date Issued: 2004-04
Recombinant expression, purification and in vitro interaction analysis of HOP and RhoC
- Vaaltyn, Michaelone Chantelle
- Authors: Vaaltyn, Michaelone Chantelle
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64523 , vital:28555
- Description: Expected release date-May 2019
- Full Text:
- Date Issued: 2016
- Authors: Vaaltyn, Michaelone Chantelle
- Date: 2016
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/64523 , vital:28555
- Description: Expected release date-May 2019
- Full Text:
- Date Issued: 2016
Regulation of hyu gene expression in Agrobacterium tumefaciens strains RU-AE01 and RU-OR
- Authors: Jiwaji, Meesbah
- Date: 2007
- Subjects: Agrobacterium tumefaciens Amino acids Gene expression Hydrolysis Hydantoin Enzymes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3983 , http://hdl.handle.net/10962/d1004042
- Description: Several Agrobacterium tumefaciens strains have been isolated for their ability to produce D-amino acids from D, L-substituted hydantoins. The optically pure D-amino acids are used in the synthesis of pharmaceuticals, as food additives and as insecticides. This hydrolysis of D, L-substituted hydantoins is catalysed by two hydantoin-hydrolyzing enzymes, an hydantoinase and an N-carbamyl amino acid amidohydrolase. While the hydantoin-hydrolyzing enzymes have been studied in detail, the mechanisms that control expression of the hyu genes have not. The research reported in this work elucidates some of the mechanisms involved in the regulation of the hyu genes in A. tumefaciens strains. The hydantoin-hydrolyzing enzyme activity from the environmental isolate A. tumefaciens RU-AE01 was characterized. A broad host range vector for the simultaneous analysis of divergent promoters was constructed. The promoter regions responsible for the activation of transcription of hyuH and hyuC were identified by deletion analysis. It was proposed that transcription of hyuH was activated by a putative σ[superscript 54]-dependent promoter or a putative σ[superscript 70]-dependent promoter identified upstream of the hyuH gene. The hyuC gene was activated by a putative σ[superscript 70]-dependent promoter identified upstream of the hyuC gene. The regulation of hydantoinase and N-carbamyl amino acid amidohydrolase enzyme activity was compared to the regulation of transcription from the RU-AE01 hyuH-hyuC region. Expression of the hydantoin-hydrolyzing enzymes was regulated by induction which correlated with reporter enzyme expression from the hyuH and hyuC promoter regions. However, the expression of the hydantoin-hydrolyzing enzymes was also regulated by nitrogen catabolite repression (NCR). This did not correlate to the reporter gene expression of the hyuH promoter region but did compare to the reporter gene expression of the hyuC promoter region. This suggested that NCR of hyuH was at the post-translational level whereas NCR of the hyuC promoter was at the transcriptional level. Pathways involved in the regulation of the hyu genes were characterized. The production of the hydantoin-hydrolyzing enzymes in both A. tumefaciens strains RU-AE01 and RU-OR were regulated by proteins involved in the global ntr pathway. The levels of the hydantoin-hydrolyzing enzymes in strain RU-AE01 were elevated in the presence of increased levels of NtrB and NtrC illustrating the importance of the ntr pathway in the regulation of the levels of the hydantoin-hydrolyzing enzymes. Similarly, in RU-OR the presence of exogenous NtrB and NtrC elevated levels of N-carbamyl amino acid amidohydrolase activity. However, the levels of hydantoinase enzyme activity in strain RU-OR were elevated in the presence of NtrC alone. In addition, the presence of a His6-tagged NtrC molecule abolished the elevation in the levels of the hydantoinase but not the N-carbamyl amino acid amidohydrolase enzyme activity in strain RU-OR. This suggests that NtrC has a direct role in the regulation of the expression of hyuH in RU-OR. In addition, it indicates that the hyu genes in the two A. tumefaciens strains RU-AE01 and RU-OR are different. The presence of the RU-AE01 hyuH-hyuC fragment caused a dramatic increase in the hydantoin-hydrolyzing enzyme activity in strain RU-OR but not strain RU-AE01. This implied the incidence of a possible repressor protein in RU-OR, which is titrated out by the presence of the RU-AE01 hyuH-hyuC fragment. Protein-DNA binding assays suggest that this putative repressor may be 38 kDa in RU-OR cells.
- Full Text:
- Date Issued: 2007
- Authors: Jiwaji, Meesbah
- Date: 2007
- Subjects: Agrobacterium tumefaciens Amino acids Gene expression Hydrolysis Hydantoin Enzymes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3983 , http://hdl.handle.net/10962/d1004042
- Description: Several Agrobacterium tumefaciens strains have been isolated for their ability to produce D-amino acids from D, L-substituted hydantoins. The optically pure D-amino acids are used in the synthesis of pharmaceuticals, as food additives and as insecticides. This hydrolysis of D, L-substituted hydantoins is catalysed by two hydantoin-hydrolyzing enzymes, an hydantoinase and an N-carbamyl amino acid amidohydrolase. While the hydantoin-hydrolyzing enzymes have been studied in detail, the mechanisms that control expression of the hyu genes have not. The research reported in this work elucidates some of the mechanisms involved in the regulation of the hyu genes in A. tumefaciens strains. The hydantoin-hydrolyzing enzyme activity from the environmental isolate A. tumefaciens RU-AE01 was characterized. A broad host range vector for the simultaneous analysis of divergent promoters was constructed. The promoter regions responsible for the activation of transcription of hyuH and hyuC were identified by deletion analysis. It was proposed that transcription of hyuH was activated by a putative σ[superscript 54]-dependent promoter or a putative σ[superscript 70]-dependent promoter identified upstream of the hyuH gene. The hyuC gene was activated by a putative σ[superscript 70]-dependent promoter identified upstream of the hyuC gene. The regulation of hydantoinase and N-carbamyl amino acid amidohydrolase enzyme activity was compared to the regulation of transcription from the RU-AE01 hyuH-hyuC region. Expression of the hydantoin-hydrolyzing enzymes was regulated by induction which correlated with reporter enzyme expression from the hyuH and hyuC promoter regions. However, the expression of the hydantoin-hydrolyzing enzymes was also regulated by nitrogen catabolite repression (NCR). This did not correlate to the reporter gene expression of the hyuH promoter region but did compare to the reporter gene expression of the hyuC promoter region. This suggested that NCR of hyuH was at the post-translational level whereas NCR of the hyuC promoter was at the transcriptional level. Pathways involved in the regulation of the hyu genes were characterized. The production of the hydantoin-hydrolyzing enzymes in both A. tumefaciens strains RU-AE01 and RU-OR were regulated by proteins involved in the global ntr pathway. The levels of the hydantoin-hydrolyzing enzymes in strain RU-AE01 were elevated in the presence of increased levels of NtrB and NtrC illustrating the importance of the ntr pathway in the regulation of the levels of the hydantoin-hydrolyzing enzymes. Similarly, in RU-OR the presence of exogenous NtrB and NtrC elevated levels of N-carbamyl amino acid amidohydrolase activity. However, the levels of hydantoinase enzyme activity in strain RU-OR were elevated in the presence of NtrC alone. In addition, the presence of a His6-tagged NtrC molecule abolished the elevation in the levels of the hydantoinase but not the N-carbamyl amino acid amidohydrolase enzyme activity in strain RU-OR. This suggests that NtrC has a direct role in the regulation of the expression of hyuH in RU-OR. In addition, it indicates that the hyu genes in the two A. tumefaciens strains RU-AE01 and RU-OR are different. The presence of the RU-AE01 hyuH-hyuC fragment caused a dramatic increase in the hydantoin-hydrolyzing enzyme activity in strain RU-OR but not strain RU-AE01. This implied the incidence of a possible repressor protein in RU-OR, which is titrated out by the presence of the RU-AE01 hyuH-hyuC fragment. Protein-DNA binding assays suggest that this putative repressor may be 38 kDa in RU-OR cells.
- Full Text:
- Date Issued: 2007
Regulation of the indoleamines by sex steroids
- Authors: Awah, Edmund Kpabi
- Date: 1992
- Subjects: Steroids -- Research , Steroid drugs -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4053 , http://hdl.handle.net/10962/d1004114 , Steroids -- Research , Steroid drugs -- Research
- Description: Alteration of serum tryptophan leads to parallel alterations in brain tryptophan levels. Such changes in brain tryptophan levels has been shown to lead to mood disturbances. The primary enzyme responsible for altering serum tryptophan levels is the liver cytosolic enzyme, tryptophan pyrrolase. Activation of this enzyme is responsible for the enhanced catabolism of circulating tryptophan. The purpose of the present study was firstly to establish whether there is a link between sex steroids and tryptophan pyrrolase activity especially since sex steroids are also known to cause mood disturbances and secondly to determine the effects of sex steroids on brain indolamine metabolism. The results show that all three sex steroids induce the activity of tryptophan pyrrolase implying that they decrease serum tryptophan levels by the activation of tryptophan pyrrolase, thus making less tryptophan available for uptake by the brain. It was also shown that the sex steroids enhance the uptake of ¹⁴C-tryptophan by brain synatopsomes. In addition, the sex steroids influenced the pattern of metabolism of serotonin by organ cultures of rat pineal glands. It is possible that the sex steroids regulate the availability and uptake of indoleamines in the brain.
- Full Text:
- Date Issued: 1992
- Authors: Awah, Edmund Kpabi
- Date: 1992
- Subjects: Steroids -- Research , Steroid drugs -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4053 , http://hdl.handle.net/10962/d1004114 , Steroids -- Research , Steroid drugs -- Research
- Description: Alteration of serum tryptophan leads to parallel alterations in brain tryptophan levels. Such changes in brain tryptophan levels has been shown to lead to mood disturbances. The primary enzyme responsible for altering serum tryptophan levels is the liver cytosolic enzyme, tryptophan pyrrolase. Activation of this enzyme is responsible for the enhanced catabolism of circulating tryptophan. The purpose of the present study was firstly to establish whether there is a link between sex steroids and tryptophan pyrrolase activity especially since sex steroids are also known to cause mood disturbances and secondly to determine the effects of sex steroids on brain indolamine metabolism. The results show that all three sex steroids induce the activity of tryptophan pyrrolase implying that they decrease serum tryptophan levels by the activation of tryptophan pyrrolase, thus making less tryptophan available for uptake by the brain. It was also shown that the sex steroids enhance the uptake of ¹⁴C-tryptophan by brain synatopsomes. In addition, the sex steroids influenced the pattern of metabolism of serotonin by organ cultures of rat pineal glands. It is possible that the sex steroids regulate the availability and uptake of indoleamines in the brain.
- Full Text:
- Date Issued: 1992
Regulation of tryptophan-2,3-dioxygenase and pineal indoleamines by selected tryptophan derivatives and antidepressants
- Authors: Walsh, Harold Archibold
- Date: 1997
- Subjects: Antidepressants Tryptophan -- Physiological effect Tryptophan -- Therapeutic use Depression, Mental -- Treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4017 , http://hdl.handle.net/10962/d1004077
- Description: The regulation of tryptophan-2,3-dioxygenase (TDO) (EC 1.13.1.12) and, to a lesser extent, pineal indoleamines, both in vitro and in vivo, is examined in this study. Rat liver TDO is a cytosolic enzyme which plays a crucial role in the regulation of circulating tryptophan (TRP) levels. Stimulation of this enzyme by heme enhances the catabolism of TRP, making less TRP available for uptake into the brain and other tissues, and for protein synthesis. At pH 7, the enzyme has an approximate Km of 100μM, is subject to substrate inhibition immediately beyond Sopt([S] at Vmax), and response of the enzyme is cooperative in both uninhibited and inhibited regions. Hill analysis of the uninhibited region reveals a biphasic plot and two classes of binding sites. Negative cooperativity is brought about through deprotonation of the enzyme. Substrate iphibition also occurs at both acidic and basic pH values with concomitant shifts in Sopt. The results obtained indicate that substrate inhibition could be an additional mechanism whereby the flux through the TRP-kynurenine pathway is regulated. TDO is subject to a diurnal rhythm, with peak activity during the pre-dark period and the loweSt activity towards the end of the dark period. It is possible that the enzyme controls the synthesis of the neurotransmitter serotonin (5-HT), and that the circadian rhythm in TDO activity is due to the endogenous rhythm of melatonin (aMT) production by the pineal gland. In the present study, aMT displaces TRP from bovine serum albumin (BSA) in vitro, and it is therefore possible for the indoleamine to regulate the availability of TRP for uptake into the brain for conversion to its derivatives. Chronic intraperitoneal administration of aMT affects physiological hepatic parameters in rats, such as TDO activity and stromal fatty acid composition, whilst no observable effect is demonstrable with respect to protein synthesis, nucleic acid metabolism, membrane fatty acid composition and pineal indole biosynthesis. On the other hand, chronic treatment of rats with antidepressants, the tricyclic desmethylimipramine (DMI) and the selective serotonin reuptake inhibitor (SSRI), fluoxetine, reveals significant negative alterations in TDO concentrations and pineal indole amine synthesis. Combining aMT with any of these two drugs normalises the activity of the hepatic enzyme. DMI is found to be an effective inhibitor of TDO in the micromolar range in vitro, and also affects total enzyme concentrations in vivo. Fluoxetine has no effect on TDO in vitro, but in vivo also reduces total enzyme levels in the liver. However, the SSRI does not affect conjugation between apo- and holoenzyme. Instead, it decreases extant holoenzyme levels. Indoleamine synthesis by the pineal gland, in organ culture, is altered by both antidepressants, although in different ways. DMI increases N-acetylserotonin levels and reduces the output of methoxyindole acetic acid and meth6xytryptophol. Fluoxetine treatment markedly reduces aMT concentrations and also brings about high levels of the 5-HT catabolites, 5-hydroxytryptophol and 5-hydroxyindole acetic acid. Insulin also lowers aMT synthesis significantly in pineal organ cultures, via a mechamsm that involves inhibition of the enzyme, N-acetyl transferase, that regulates aMT synthesis. The effects of insulin on pineal indole metabolism are due to the observation that a carbohydrate rich diet which induces insulin release elevates plasma TRP and brain 5-HT, but has no effect on pineal TRP and indole amine synthesis. It could thus be possible for insulin to have an effect on the pineal, since the latter is outside the blood brain barrier. The finilings of this study support the biogenic amine deficiency hypothesis, implicating some of the major biogenic amines such as noradrenaline (NA), 5-HT and aMT in depression. There is believed to be a deficiency of NA and 5-HT at their respective synapses in the depressed state. The drug DMI could act, firstly, by inhibiting TDO and thus increasing plasma TRP levels, and could, secondly, stimulate NA release and inhibit NA reuptake at the pineal membrane. The combined effect would be to enhance aMT synthesis, with eventual remission. Fluoxetine, on the other hand, appears to utilize a slightly different mode of action to DMI, which seems to focus on the preservation of 5-HT. The fact that aMT counteracts the effects of both antidepressants, and restores the activity of TDO to that of the controls, is also consistent with the observation that the therapeutic action of drugs such as these coincides willi the restoration of normal plasma levels of the neurohormone in depressives. In view of the biogenic amine deficiency hypothesis of depression and the contentious claim that TDO is the major peripheral determinant of brain TRP, brain 5-HT and ultimately aMT, the regulation of TDO is investigated and discussed. The study concludes that TDO activity is regulated by a number of endogenous compounds which are mainly derivatives of TRP, such as aMT and oxidized nicotinamide adenine dinucleotide and exogenous substances, of which DMI and fluoxetine are but two. In addition, modulation of IDO activity in depression appears to be an important aspect of antidepressant action. aMT, the product of the pineal gland, also has the potential to increase plasma TRP and hence forebrain TRP levels, and ultimately 5-HT concentrations, firstly by displacing TRP from serum albumin and secondly by inhibiting TDO.
- Full Text:
- Date Issued: 1997
- Authors: Walsh, Harold Archibold
- Date: 1997
- Subjects: Antidepressants Tryptophan -- Physiological effect Tryptophan -- Therapeutic use Depression, Mental -- Treatment
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4017 , http://hdl.handle.net/10962/d1004077
- Description: The regulation of tryptophan-2,3-dioxygenase (TDO) (EC 1.13.1.12) and, to a lesser extent, pineal indoleamines, both in vitro and in vivo, is examined in this study. Rat liver TDO is a cytosolic enzyme which plays a crucial role in the regulation of circulating tryptophan (TRP) levels. Stimulation of this enzyme by heme enhances the catabolism of TRP, making less TRP available for uptake into the brain and other tissues, and for protein synthesis. At pH 7, the enzyme has an approximate Km of 100μM, is subject to substrate inhibition immediately beyond Sopt([S] at Vmax), and response of the enzyme is cooperative in both uninhibited and inhibited regions. Hill analysis of the uninhibited region reveals a biphasic plot and two classes of binding sites. Negative cooperativity is brought about through deprotonation of the enzyme. Substrate iphibition also occurs at both acidic and basic pH values with concomitant shifts in Sopt. The results obtained indicate that substrate inhibition could be an additional mechanism whereby the flux through the TRP-kynurenine pathway is regulated. TDO is subject to a diurnal rhythm, with peak activity during the pre-dark period and the loweSt activity towards the end of the dark period. It is possible that the enzyme controls the synthesis of the neurotransmitter serotonin (5-HT), and that the circadian rhythm in TDO activity is due to the endogenous rhythm of melatonin (aMT) production by the pineal gland. In the present study, aMT displaces TRP from bovine serum albumin (BSA) in vitro, and it is therefore possible for the indoleamine to regulate the availability of TRP for uptake into the brain for conversion to its derivatives. Chronic intraperitoneal administration of aMT affects physiological hepatic parameters in rats, such as TDO activity and stromal fatty acid composition, whilst no observable effect is demonstrable with respect to protein synthesis, nucleic acid metabolism, membrane fatty acid composition and pineal indole biosynthesis. On the other hand, chronic treatment of rats with antidepressants, the tricyclic desmethylimipramine (DMI) and the selective serotonin reuptake inhibitor (SSRI), fluoxetine, reveals significant negative alterations in TDO concentrations and pineal indole amine synthesis. Combining aMT with any of these two drugs normalises the activity of the hepatic enzyme. DMI is found to be an effective inhibitor of TDO in the micromolar range in vitro, and also affects total enzyme concentrations in vivo. Fluoxetine has no effect on TDO in vitro, but in vivo also reduces total enzyme levels in the liver. However, the SSRI does not affect conjugation between apo- and holoenzyme. Instead, it decreases extant holoenzyme levels. Indoleamine synthesis by the pineal gland, in organ culture, is altered by both antidepressants, although in different ways. DMI increases N-acetylserotonin levels and reduces the output of methoxyindole acetic acid and meth6xytryptophol. Fluoxetine treatment markedly reduces aMT concentrations and also brings about high levels of the 5-HT catabolites, 5-hydroxytryptophol and 5-hydroxyindole acetic acid. Insulin also lowers aMT synthesis significantly in pineal organ cultures, via a mechamsm that involves inhibition of the enzyme, N-acetyl transferase, that regulates aMT synthesis. The effects of insulin on pineal indole metabolism are due to the observation that a carbohydrate rich diet which induces insulin release elevates plasma TRP and brain 5-HT, but has no effect on pineal TRP and indole amine synthesis. It could thus be possible for insulin to have an effect on the pineal, since the latter is outside the blood brain barrier. The finilings of this study support the biogenic amine deficiency hypothesis, implicating some of the major biogenic amines such as noradrenaline (NA), 5-HT and aMT in depression. There is believed to be a deficiency of NA and 5-HT at their respective synapses in the depressed state. The drug DMI could act, firstly, by inhibiting TDO and thus increasing plasma TRP levels, and could, secondly, stimulate NA release and inhibit NA reuptake at the pineal membrane. The combined effect would be to enhance aMT synthesis, with eventual remission. Fluoxetine, on the other hand, appears to utilize a slightly different mode of action to DMI, which seems to focus on the preservation of 5-HT. The fact that aMT counteracts the effects of both antidepressants, and restores the activity of TDO to that of the controls, is also consistent with the observation that the therapeutic action of drugs such as these coincides willi the restoration of normal plasma levels of the neurohormone in depressives. In view of the biogenic amine deficiency hypothesis of depression and the contentious claim that TDO is the major peripheral determinant of brain TRP, brain 5-HT and ultimately aMT, the regulation of TDO is investigated and discussed. The study concludes that TDO activity is regulated by a number of endogenous compounds which are mainly derivatives of TRP, such as aMT and oxidized nicotinamide adenine dinucleotide and exogenous substances, of which DMI and fluoxetine are but two. In addition, modulation of IDO activity in depression appears to be an important aspect of antidepressant action. aMT, the product of the pineal gland, also has the potential to increase plasma TRP and hence forebrain TRP levels, and ultimately 5-HT concentrations, firstly by displacing TRP from serum albumin and secondly by inhibiting TDO.
- Full Text:
- Date Issued: 1997
Removal and recovery of gold and platinum from aqueous solutions utilising the non-viable biomass Asolla filiculoides
- Authors: Antunes, Ana Paula Martins
- Date: 2002
- Subjects: Azolla filiculoides Metal wastes -- Recycling Gold -- Recycling Platinum -- Recycling
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3894 , http://hdl.handle.net/10962/d1003726
- Description: Waste water from the mining industry is generally extremely complex and contains numerous species which influence the adsorption of the metals to any biomass. A variety of factors need to be addressed before treatment is considered viable. It is also beneficial to establish the binding characteristics of the metal of interest to maximise its interaction with the biomass to be utilised. Azalia filiculaides was investigated in the adsorption of gold(III), lead(II), iron(ID), copper(II) and platinum (IV). In batch studies, the optimum biomass and initial gold(III) concentrations were found to be 5 gIL and 8 mgIL respectively. The adsorption of gold(ID) is principally pH-dependent with optimal removal at pH 2. Lead(II), iron(III) and copper(II) did not compete with gold(III) adsorption under equimolar and simulated effluent conditions. Halides, with increasing affinity for gold (chloride < bromide < iodide), can affect gold uptake with the soft base, iodide, exhibiting the most inhibition (25%) and the hard base, chloride, O%. Mercaptoethanol (soft base) showed no interference in gold(III) adsorption while the presence of sulphate (hard base) and sulphite (borderline base) showed that concentrations in excess of 1 0 mM may adversely affect gold(ill) uptake, most likely due to competition for cationic sites on the biomass. Column studies, better suited to high volume treatment, indicated that a flow-rate of 5 mL/min and an initial gold(ill) concentration of 5 mgIL was optimal. Competitive effects between lead, iron, copper and gold again showed little or no interference. The halides, chloride, bromide and iodide, affect gold(ill) uptake similarly to the batch studies, while the bases mercaptoethanol and sulphate minimally affect gold(III) binding with sulphite severely hampering adsorption (70% inhibition). To optimise gold desorption, preliminary batch studies indicated that a ratio of 1:1 of adsorbentdesorbent was optimal, whilst gas purging of thiourea with oxygen, air and nitrogen decreased gold elution in proportion to decreased amounts of oxygen. A series of desorbents were utilised, in column studies, to optimise and determine the speciation of bound gold. The presence of an oxidant with thiourea enhanced desorption greater than 3 fold when compared with thiourea alone. Thiourea desorption studies, aided by the oxidant, suggest that gold is present in the + I and 0 oxidation states. Ultimately thiourea, perchloric acid and hydrochloric acid was found to be the most optimal elutant for gold (J 00% recovery). For selective metal recovery oflead and copper, pre-washing the plant material with water, utilising an acid (0.3 M nitric acid), pumping in an up-flow mode, and recycling the desorbent six times was found to be optimal elutant for gold (J 00% recovery). Cost analysis of utilising elutant versus incinerating the biomass for gold recovery indicated the latter as the most economical. Over a 5 cycle adsorption and desorption series, acid desorption before each adsorption cycle was found to result in greater than 92% desorption for lead and 96% for copper. Gold recovery was 97% with incineration. A preliminary study with gold effluent (Mine C) indicated that nickel and sulphate was removed in batch and column studies. Gold removal was found to be 100% and 4% in batch and column studies respectively. Adsorption of gold in the effluent study was accompanied by the release ofHt. Modifying the plant material with various reagents failed to identify the primary binding sites and the role of polysaccharides, proteins and lipids in gold(ill) uptake. The mode of gold binding is suggested as being initially ionic, this is very rapid, with the interaction of the anionic complex, [AuCI₄]". with the cationic biomass (PH 2). This eventually leads to the displacement of the chloride ligand(s) initiating covalent binding. Spectral studies of the chemical interaction between gold and the representative tannins indicated the protonated hydroxy groups to be responsible. All evidence suggests that the binding mechanisms of gold are not simple. Preliminary adsorption studies of platinum by Azalia filiculaides were conducted. Batch studies indicated that J gIL biomass concentration, initial platinum concentration of 20 mgIL and pH 2 are optimal, while the column studies indicated a flow-rate of! 0 rnL/min and initial platinum concentration of 20 mgIL as optimal. In the platinum effluent study, platinum showed a removal of 23 % and 2 J % for the batch and column studies respectively. Again adsorption was accompanied by //' release. Azalia filiculaides demonstrated its feasibility in the removal of gold and platinum from simulated as well as waste water solutions. Its potential viability as a biosorbent was demonstrated by the high recovery from synthetic solutions of greater than 99% for gold (2-10 mgIL), and greater than 89% for platinum (20 mgIL).
- Full Text:
- Date Issued: 2002
- Authors: Antunes, Ana Paula Martins
- Date: 2002
- Subjects: Azolla filiculoides Metal wastes -- Recycling Gold -- Recycling Platinum -- Recycling
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3894 , http://hdl.handle.net/10962/d1003726
- Description: Waste water from the mining industry is generally extremely complex and contains numerous species which influence the adsorption of the metals to any biomass. A variety of factors need to be addressed before treatment is considered viable. It is also beneficial to establish the binding characteristics of the metal of interest to maximise its interaction with the biomass to be utilised. Azalia filiculaides was investigated in the adsorption of gold(III), lead(II), iron(ID), copper(II) and platinum (IV). In batch studies, the optimum biomass and initial gold(III) concentrations were found to be 5 gIL and 8 mgIL respectively. The adsorption of gold(ID) is principally pH-dependent with optimal removal at pH 2. Lead(II), iron(III) and copper(II) did not compete with gold(III) adsorption under equimolar and simulated effluent conditions. Halides, with increasing affinity for gold (chloride < bromide < iodide), can affect gold uptake with the soft base, iodide, exhibiting the most inhibition (25%) and the hard base, chloride, O%. Mercaptoethanol (soft base) showed no interference in gold(III) adsorption while the presence of sulphate (hard base) and sulphite (borderline base) showed that concentrations in excess of 1 0 mM may adversely affect gold(ill) uptake, most likely due to competition for cationic sites on the biomass. Column studies, better suited to high volume treatment, indicated that a flow-rate of 5 mL/min and an initial gold(ill) concentration of 5 mgIL was optimal. Competitive effects between lead, iron, copper and gold again showed little or no interference. The halides, chloride, bromide and iodide, affect gold(ill) uptake similarly to the batch studies, while the bases mercaptoethanol and sulphate minimally affect gold(III) binding with sulphite severely hampering adsorption (70% inhibition). To optimise gold desorption, preliminary batch studies indicated that a ratio of 1:1 of adsorbentdesorbent was optimal, whilst gas purging of thiourea with oxygen, air and nitrogen decreased gold elution in proportion to decreased amounts of oxygen. A series of desorbents were utilised, in column studies, to optimise and determine the speciation of bound gold. The presence of an oxidant with thiourea enhanced desorption greater than 3 fold when compared with thiourea alone. Thiourea desorption studies, aided by the oxidant, suggest that gold is present in the + I and 0 oxidation states. Ultimately thiourea, perchloric acid and hydrochloric acid was found to be the most optimal elutant for gold (J 00% recovery). For selective metal recovery oflead and copper, pre-washing the plant material with water, utilising an acid (0.3 M nitric acid), pumping in an up-flow mode, and recycling the desorbent six times was found to be optimal elutant for gold (J 00% recovery). Cost analysis of utilising elutant versus incinerating the biomass for gold recovery indicated the latter as the most economical. Over a 5 cycle adsorption and desorption series, acid desorption before each adsorption cycle was found to result in greater than 92% desorption for lead and 96% for copper. Gold recovery was 97% with incineration. A preliminary study with gold effluent (Mine C) indicated that nickel and sulphate was removed in batch and column studies. Gold removal was found to be 100% and 4% in batch and column studies respectively. Adsorption of gold in the effluent study was accompanied by the release ofHt. Modifying the plant material with various reagents failed to identify the primary binding sites and the role of polysaccharides, proteins and lipids in gold(ill) uptake. The mode of gold binding is suggested as being initially ionic, this is very rapid, with the interaction of the anionic complex, [AuCI₄]". with the cationic biomass (PH 2). This eventually leads to the displacement of the chloride ligand(s) initiating covalent binding. Spectral studies of the chemical interaction between gold and the representative tannins indicated the protonated hydroxy groups to be responsible. All evidence suggests that the binding mechanisms of gold are not simple. Preliminary adsorption studies of platinum by Azalia filiculaides were conducted. Batch studies indicated that J gIL biomass concentration, initial platinum concentration of 20 mgIL and pH 2 are optimal, while the column studies indicated a flow-rate of! 0 rnL/min and initial platinum concentration of 20 mgIL as optimal. In the platinum effluent study, platinum showed a removal of 23 % and 2 J % for the batch and column studies respectively. Again adsorption was accompanied by //' release. Azalia filiculaides demonstrated its feasibility in the removal of gold and platinum from simulated as well as waste water solutions. Its potential viability as a biosorbent was demonstrated by the high recovery from synthetic solutions of greater than 99% for gold (2-10 mgIL), and greater than 89% for platinum (20 mgIL).
- Full Text:
- Date Issued: 2002