The effect of sewage effluent from De Beers marine diamond mining operations on the expression of cytochrome P450 (CYP1A) and vitellogenin (vtg)
- Authors: De Almeida, Louise
- Date: 2013-09-20
- Subjects: De Beers Consolidated Mines , Sewage disposal in rivers, lakes, etc. -- Namibia , Endocrine disrupting chemicals in water -- Namibia , Merluccius capensis -- Namibia , Merluccius -- Namibia , Water -- Pollution -- Namibia
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4096 , http://hdl.handle.net/10962/d1009440 , De Beers Consolidated Mines , Sewage disposal in rivers, lakes, etc. -- Namibia , Endocrine disrupting chemicals in water -- Namibia , Merluccius capensis -- Namibia , Merluccius -- Namibia , Water -- Pollution -- Namibia
- Description: Sewage effluents disposed into the marine environment from De Beers Marine Namibia diamond mining vessels have the potential to cause endocrine disruptive effects in marine organisms. Endocrine disruption refers to the alteration of the normal functioning of the endocrine system and various chemicals have the ability to mimic hormones, effecting endogenous hormone synthesis, transport, receptor interaction and intracellular signaling. The potential endocrine disruptive effects, caused by the release of different types of sewage effluents into the ocean, on fish species is a concern due to the commercial importance of fish species found in the mining area e.g. hake, sole, horse mackerel. Increased awareness of marine environmental degradation due to the presence of chemical contaminants has resulted in research being done on early warning systems, in the form of biomarkers. Cytochrome P450 monooxygenase 1A (CYP1A) and vitellogenin (vtg) are important proteins found in fish liver and blood, that have been used as biomarkers for the detection of pollutants in fish. CYP1A is a subfamily of the P450 superfamily of enzymes and catalyzes the oxidation, hydrolysis and reduction of exogenous and endogenous compounds (phase I reactions) and thus has the capacity to regulate the metabolism of several organic contaminants. CYP1A expression is altered by exposure to planar xenobiotic compounds e.g. polyaromatic hydrocarbons. Vtg is an important precursor for egg yolk proteins and plays a role in the growth and development of an oocyte. Expression of this protein is altered upon exposure to estrogenic compounds. The aim of this project was to isolate CYP1A from fish liver by differential centrifugation and optimize conditions for the CYP1A-mediated ethoxyresorufin-Odeethylase (EROD) assay and western blot analysis (to assess CYP1A expression). Another aim of this study was to evaluate the potential effects of biologically disruptive chemicals from sewage effluents, discharged into the marine environment, on the expression of CYP1A in two species of hake, Merluccius capensis and M. paradoxus (Cape hake). CYP1A in Cape hake is approximately a 60 kDa protein and the highest EROD activity was detected in the microsomal fraction after differential centrifugation. Optimal EROD assay conditions were observed at pH 7.5, a temperature of 25 °C, 10 μl of sample and a reaction time of 30 seconds. Enzyme stability assays indicated a drastic decrease in enzyme activity after 30 seconds. The EROD assay was not NADPH dependent but was limited by NADPH supply, with an increase of 300% in EROD activity being observed with the addition of 0.1 M exogenous NADPH. The addition of dicumarol (40 μM), a phase II enzyme inhibitor, showed a 232% increase in EROD activity. This is because dicumarol inhibited enzymes with the capacity to metabolize the product (resorufin) of the EROD reaction. With regard to western blot analysis, the optimal primary (rabbit antifish CYP1A peptide) and secondary (anti-mouse/rabbit antibody-horseradish peroxidase conjugate (POD)) antibody dilutions were determined to be 1:1000 and 1:5000, respectively. The comparison of CYP1A expression in Cape hake samples from De Beers Marine mining area and reference sites showed higher EROD activity (16.29 ± 0.91 pmol/min) in fish samples from the mining area in comparison to the reference site (10.42 ± 2.65 pmol/min). Western blot analysis was in agreement with the EROD assay results and a higher CYP1A expression was observed in fish from the mining sites. The increased CYP1A expression observed in fish from the mining area is not definitively an indication of a pollutant effect in the environment, as several environmental and biological factors (e.g. photoperiod and age) must also be considered before reaching this conclusion. Another aim of this study was to purify vtg from Cape hake blood samples. Cape hake vtg was purified from fish plasma by selective precipitation with MgCl2 and EDTA. Precipitated sample was subjected to anion exchange chromatography using fast protein liquid chromatography (FPLC). Vtg eluted as two broad peaks and had a molecular weight above 200 kDa. SDS-PAGE analysis also resolved smaller molecular weight proteins below 70 kDa, which were thought to be vitellogenin cleavage proteins, lipovitellin and phosphovitins. Western blot analysis was performed; however, it did not produce any conclusive results. The purification of vtg enables further studies in characterizing this protein and developing assay aimed at detecting estrogenic pollutants in the marine environment
- Full Text:
- Authors: De Almeida, Louise
- Date: 2013-09-20
- Subjects: De Beers Consolidated Mines , Sewage disposal in rivers, lakes, etc. -- Namibia , Endocrine disrupting chemicals in water -- Namibia , Merluccius capensis -- Namibia , Merluccius -- Namibia , Water -- Pollution -- Namibia
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4096 , http://hdl.handle.net/10962/d1009440 , De Beers Consolidated Mines , Sewage disposal in rivers, lakes, etc. -- Namibia , Endocrine disrupting chemicals in water -- Namibia , Merluccius capensis -- Namibia , Merluccius -- Namibia , Water -- Pollution -- Namibia
- Description: Sewage effluents disposed into the marine environment from De Beers Marine Namibia diamond mining vessels have the potential to cause endocrine disruptive effects in marine organisms. Endocrine disruption refers to the alteration of the normal functioning of the endocrine system and various chemicals have the ability to mimic hormones, effecting endogenous hormone synthesis, transport, receptor interaction and intracellular signaling. The potential endocrine disruptive effects, caused by the release of different types of sewage effluents into the ocean, on fish species is a concern due to the commercial importance of fish species found in the mining area e.g. hake, sole, horse mackerel. Increased awareness of marine environmental degradation due to the presence of chemical contaminants has resulted in research being done on early warning systems, in the form of biomarkers. Cytochrome P450 monooxygenase 1A (CYP1A) and vitellogenin (vtg) are important proteins found in fish liver and blood, that have been used as biomarkers for the detection of pollutants in fish. CYP1A is a subfamily of the P450 superfamily of enzymes and catalyzes the oxidation, hydrolysis and reduction of exogenous and endogenous compounds (phase I reactions) and thus has the capacity to regulate the metabolism of several organic contaminants. CYP1A expression is altered by exposure to planar xenobiotic compounds e.g. polyaromatic hydrocarbons. Vtg is an important precursor for egg yolk proteins and plays a role in the growth and development of an oocyte. Expression of this protein is altered upon exposure to estrogenic compounds. The aim of this project was to isolate CYP1A from fish liver by differential centrifugation and optimize conditions for the CYP1A-mediated ethoxyresorufin-Odeethylase (EROD) assay and western blot analysis (to assess CYP1A expression). Another aim of this study was to evaluate the potential effects of biologically disruptive chemicals from sewage effluents, discharged into the marine environment, on the expression of CYP1A in two species of hake, Merluccius capensis and M. paradoxus (Cape hake). CYP1A in Cape hake is approximately a 60 kDa protein and the highest EROD activity was detected in the microsomal fraction after differential centrifugation. Optimal EROD assay conditions were observed at pH 7.5, a temperature of 25 °C, 10 μl of sample and a reaction time of 30 seconds. Enzyme stability assays indicated a drastic decrease in enzyme activity after 30 seconds. The EROD assay was not NADPH dependent but was limited by NADPH supply, with an increase of 300% in EROD activity being observed with the addition of 0.1 M exogenous NADPH. The addition of dicumarol (40 μM), a phase II enzyme inhibitor, showed a 232% increase in EROD activity. This is because dicumarol inhibited enzymes with the capacity to metabolize the product (resorufin) of the EROD reaction. With regard to western blot analysis, the optimal primary (rabbit antifish CYP1A peptide) and secondary (anti-mouse/rabbit antibody-horseradish peroxidase conjugate (POD)) antibody dilutions were determined to be 1:1000 and 1:5000, respectively. The comparison of CYP1A expression in Cape hake samples from De Beers Marine mining area and reference sites showed higher EROD activity (16.29 ± 0.91 pmol/min) in fish samples from the mining area in comparison to the reference site (10.42 ± 2.65 pmol/min). Western blot analysis was in agreement with the EROD assay results and a higher CYP1A expression was observed in fish from the mining sites. The increased CYP1A expression observed in fish from the mining area is not definitively an indication of a pollutant effect in the environment, as several environmental and biological factors (e.g. photoperiod and age) must also be considered before reaching this conclusion. Another aim of this study was to purify vtg from Cape hake blood samples. Cape hake vtg was purified from fish plasma by selective precipitation with MgCl2 and EDTA. Precipitated sample was subjected to anion exchange chromatography using fast protein liquid chromatography (FPLC). Vtg eluted as two broad peaks and had a molecular weight above 200 kDa. SDS-PAGE analysis also resolved smaller molecular weight proteins below 70 kDa, which were thought to be vitellogenin cleavage proteins, lipovitellin and phosphovitins. Western blot analysis was performed; however, it did not produce any conclusive results. The purification of vtg enables further studies in characterizing this protein and developing assay aimed at detecting estrogenic pollutants in the marine environment
- Full Text:
An organisation development intervention in an Anglican church theological seminary in Southern Africa
- Authors: Chinganga, Percy
- Date: 2013-08-08
- Subjects: College of the Transfiguration (Grahamstown, South Africa) Organizational change -- Church of England -- Africa, Southern Leadership -- Religious aspects -- Christianity -- Africa, Southern Church management -- Church of England -- Africa, Southern
- Language: English
- Type: Thesis , Masters , MEd
- Identifier: vital:1959 , http://hdl.handle.net/10962/d1008377
- Description: "Organisation development is a planned, systematic process in which applied behavioral science principles and practices are introduced into ongoing organisations toward the goal of increasing individual and organisational effectiveness. " [French and Bell] This study describes and analyses the implementation of Organisation Development (OD) to an Anglican Church theological seminary, The College of the Transfiguration (Cott), in the Anglican Church of the Province of Southern Africa (ACSA). The origins of OD are business related, emerging in the late 1950s and early 1960s. Over the years, and recently in South Africa, OD has been applied in educational change initiatives. Unlike more traditional change strategies, OD promotes collaboration in organisational change processes through the inclusive participation of all stakeholders. This study is unique in the sense that OD is applied to an Anglican Church theological institution in Southern Africa. The goal of theological institutions, particularly Cot!, is to "form, inform and transform" (Cot! Prospectus, 2011) those who feel called to ordained ministry. Personal experience in this practice has confirmed that organisational emphasis is placed more on product than process; on results rather than the leadership and management of the organisation. This study was an attempt to introduce a process of planned change to such an organisational context. OD was introduced to The College of the Transfiguration in the form of action research using the Survey Data Feedback (SDF) strategy. Data gathered was interpreted and analysed, followed by action planning and implementation of agreed plans. The process had a positive impact on both stakeholders and the organisation despite the challenges associated with the unpredictable world of organisations. Ultimately, I propose tentative recommendations which could help Cott and other educational institutions to achieve long-term improvement in organisational leadership and management.
- Full Text:
- Authors: Chinganga, Percy
- Date: 2013-08-08
- Subjects: College of the Transfiguration (Grahamstown, South Africa) Organizational change -- Church of England -- Africa, Southern Leadership -- Religious aspects -- Christianity -- Africa, Southern Church management -- Church of England -- Africa, Southern
- Language: English
- Type: Thesis , Masters , MEd
- Identifier: vital:1959 , http://hdl.handle.net/10962/d1008377
- Description: "Organisation development is a planned, systematic process in which applied behavioral science principles and practices are introduced into ongoing organisations toward the goal of increasing individual and organisational effectiveness. " [French and Bell] This study describes and analyses the implementation of Organisation Development (OD) to an Anglican Church theological seminary, The College of the Transfiguration (Cott), in the Anglican Church of the Province of Southern Africa (ACSA). The origins of OD are business related, emerging in the late 1950s and early 1960s. Over the years, and recently in South Africa, OD has been applied in educational change initiatives. Unlike more traditional change strategies, OD promotes collaboration in organisational change processes through the inclusive participation of all stakeholders. This study is unique in the sense that OD is applied to an Anglican Church theological institution in Southern Africa. The goal of theological institutions, particularly Cot!, is to "form, inform and transform" (Cot! Prospectus, 2011) those who feel called to ordained ministry. Personal experience in this practice has confirmed that organisational emphasis is placed more on product than process; on results rather than the leadership and management of the organisation. This study was an attempt to introduce a process of planned change to such an organisational context. OD was introduced to The College of the Transfiguration in the form of action research using the Survey Data Feedback (SDF) strategy. Data gathered was interpreted and analysed, followed by action planning and implementation of agreed plans. The process had a positive impact on both stakeholders and the organisation despite the challenges associated with the unpredictable world of organisations. Ultimately, I propose tentative recommendations which could help Cott and other educational institutions to achieve long-term improvement in organisational leadership and management.
- Full Text:
A framework for successful SOA adoption in selected South African universities
- Authors: Jacobs, Carmen
- Date: 2013-07-26
- Subjects: System analysis -- Data processing , System design -- Data processing , Systems integration , Service-oriented architecture (Computer science) , Education, Higher -- Data processing
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:1153 , http://hdl.handle.net/10962/d1008366 , System analysis -- Data processing , System design -- Data processing , Systems integration , Service-oriented architecture (Computer science) , Education, Higher -- Data processing
- Description: The demand for systems i·ntegration has become more and more significant in higher education as institutions want systems that provide coherent information with data that is up to date and not redundant and can seamlessly support the end user experience. Institutions have become more reliant on information systems to support both administrative functions and those involved in teaching, learning and research, but because each department supports a diverse array of computing platforms and applications, it becomes very difficult to integrate these systems. SOA is classified as an innovative approach to integrating existing systems involving the use of independent services that can be accessed without knowledge of the underlying platform implementation. Unfortunately, the SOA initiative will not be success if it is not understood and used correctly by various applications and systems throughout the organisation. SOA introduces complexity and challenges in systems integration, acceptance, governance, data, development planning, security and external opportunities. If an organisation does not embrace or enable change in each of these areas, it is not ready for the adoption of SOA. This research investigates the systems integration challenge in selected South African universities and explores factors for SOA adoption. The framework for the adoption of SOA comprises seven factors, of which Systems Integration is the most significant and represents an efficient starting point for institutions considering SOA adoption. Acceptance, Governance, Data, Development Planning, Security and External Opportunities are other factors of SOA adoption that require careful and thorough consideration before an institution can successfully adopt SOA. The results of this research emphasise the importance of being able to embrace change and innovation and modify strategies in order to reflect the constant changes required for the adoption of SOA. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Authors: Jacobs, Carmen
- Date: 2013-07-26
- Subjects: System analysis -- Data processing , System design -- Data processing , Systems integration , Service-oriented architecture (Computer science) , Education, Higher -- Data processing
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:1153 , http://hdl.handle.net/10962/d1008366 , System analysis -- Data processing , System design -- Data processing , Systems integration , Service-oriented architecture (Computer science) , Education, Higher -- Data processing
- Description: The demand for systems i·ntegration has become more and more significant in higher education as institutions want systems that provide coherent information with data that is up to date and not redundant and can seamlessly support the end user experience. Institutions have become more reliant on information systems to support both administrative functions and those involved in teaching, learning and research, but because each department supports a diverse array of computing platforms and applications, it becomes very difficult to integrate these systems. SOA is classified as an innovative approach to integrating existing systems involving the use of independent services that can be accessed without knowledge of the underlying platform implementation. Unfortunately, the SOA initiative will not be success if it is not understood and used correctly by various applications and systems throughout the organisation. SOA introduces complexity and challenges in systems integration, acceptance, governance, data, development planning, security and external opportunities. If an organisation does not embrace or enable change in each of these areas, it is not ready for the adoption of SOA. This research investigates the systems integration challenge in selected South African universities and explores factors for SOA adoption. The framework for the adoption of SOA comprises seven factors, of which Systems Integration is the most significant and represents an efficient starting point for institutions considering SOA adoption. Acceptance, Governance, Data, Development Planning, Security and External Opportunities are other factors of SOA adoption that require careful and thorough consideration before an institution can successfully adopt SOA. The results of this research emphasise the importance of being able to embrace change and innovation and modify strategies in order to reflect the constant changes required for the adoption of SOA. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
Synthesis and evaluation of novel inhibitors of 1-Deoxy-D-xylolose-5-phosphate reductoisomerase as potential antimalarials
- Authors: Conibear, Anne Claire
- Date: 2013-07-19
- Subjects: Antimalarials -- Development , Malaria -- Chemotherapy , Drug development , Enzyme kinetics , Phosphate esters
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4451 , http://hdl.handle.net/10962/d1008282 , Antimalarials -- Development , Malaria -- Chemotherapy , Drug development , Enzyme kinetics , Phosphate esters
- Description: Malaria continues to be an enormous health-threat in the developing world and the emergence of drug resistance has further compounded the problem. The parasite-specific enzyme, 1-deoxY-D-xylulose-S-phosphate reductoisomerase (DXR), has recently been validated as a promising antimalarial drug target. The present study comprises a combination of synthetic, physical organic, computer modelling and bioassay techniques directed towards the development of novel DXR inhibitors. A range of 2-heteroarylamino-2-oxoethyl- and 2- heteroarylamino-2-oxopropyl phosphonate esters and their corresponding phosphonic acid salts have been synthesised as analogues of the highly active DXR inhibitor, fosmidomycin. Treatment of the heteroarylamino precursors with chloroacetyl chloride or chloropropionyl chloride afforded chloroamide intermediates, Arbuzov reactions of which led to the corresponding diethyl phosphonate esters. Hydrolysis of the esters has been effected using bromotrimethylsilane. Twenty-four new compounds have been prepared and fully characterised using elemental (HRMS or combustion) and spectroscopic (1- and 2-D NMR and IR) analysis. A 31p NMR kinetic study has been carried out on the two-step silylation reaction involved in the hydrolysis of the phosphonate esters and has provided activation parameters for the reaction. The kinetic analysis was refined using a computational method to give an improved fit with the experimental data. Saturation transfer difference (STD) NMR analysis, computer-simulated docking and enzyme inhibition assays have been used to evaluate the enzyme-binding and -inhibition potential of the synthesised ligands. Minimal to moderate inhibitory activity has been observed and several structure-activity relationships have been identified. In silica exploration of the DXR active site has revealed an additional binding pocket and information on the topology of the active site has led to the de novo design of a new series of potential ligands. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Authors: Conibear, Anne Claire
- Date: 2013-07-19
- Subjects: Antimalarials -- Development , Malaria -- Chemotherapy , Drug development , Enzyme kinetics , Phosphate esters
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4451 , http://hdl.handle.net/10962/d1008282 , Antimalarials -- Development , Malaria -- Chemotherapy , Drug development , Enzyme kinetics , Phosphate esters
- Description: Malaria continues to be an enormous health-threat in the developing world and the emergence of drug resistance has further compounded the problem. The parasite-specific enzyme, 1-deoxY-D-xylulose-S-phosphate reductoisomerase (DXR), has recently been validated as a promising antimalarial drug target. The present study comprises a combination of synthetic, physical organic, computer modelling and bioassay techniques directed towards the development of novel DXR inhibitors. A range of 2-heteroarylamino-2-oxoethyl- and 2- heteroarylamino-2-oxopropyl phosphonate esters and their corresponding phosphonic acid salts have been synthesised as analogues of the highly active DXR inhibitor, fosmidomycin. Treatment of the heteroarylamino precursors with chloroacetyl chloride or chloropropionyl chloride afforded chloroamide intermediates, Arbuzov reactions of which led to the corresponding diethyl phosphonate esters. Hydrolysis of the esters has been effected using bromotrimethylsilane. Twenty-four new compounds have been prepared and fully characterised using elemental (HRMS or combustion) and spectroscopic (1- and 2-D NMR and IR) analysis. A 31p NMR kinetic study has been carried out on the two-step silylation reaction involved in the hydrolysis of the phosphonate esters and has provided activation parameters for the reaction. The kinetic analysis was refined using a computational method to give an improved fit with the experimental data. Saturation transfer difference (STD) NMR analysis, computer-simulated docking and enzyme inhibition assays have been used to evaluate the enzyme-binding and -inhibition potential of the synthesised ligands. Minimal to moderate inhibitory activity has been observed and several structure-activity relationships have been identified. In silica exploration of the DXR active site has revealed an additional binding pocket and information on the topology of the active site has led to the de novo design of a new series of potential ligands. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
The nature of geometry instruction and observed learning-outcomes opportunities in Nigerian and South African high schools:
- Atebe, Humphrey U, Schäfer, Marc
- Authors: Atebe, Humphrey U , Schäfer, Marc
- Date: 2013
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/140919 , vital:37929 , DOI: 10.1080/10288457.2011.10740712
- Description: The purpose of this qualitative case study involving six secondary school teachers was to obtain insight into how geometry is taught in selected Nigerian and South African high schools. It also aimed, by making use of the van Hiele model of geometry instruction, to elucidate what possible learning opportunities observed instructional methods could offer learners in the subject. The sample comprised three mathematics teachers from Nigeria and three mathematics teachers from South Africa, all of whom were selected using purposive sampling techniques. Instructional activities in six geometry classrooms were recorded on videotape. The van Hiele learning phases provided the framework for data analysis. The findings of this study indicate that observed teaching methods in geometry classrooms in the participating schools offer learners scant opportunity to learn geometry.
- Full Text:
- Date Issued: 2013
- Authors: Atebe, Humphrey U , Schäfer, Marc
- Date: 2013
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/10962/140919 , vital:37929 , DOI: 10.1080/10288457.2011.10740712
- Description: The purpose of this qualitative case study involving six secondary school teachers was to obtain insight into how geometry is taught in selected Nigerian and South African high schools. It also aimed, by making use of the van Hiele model of geometry instruction, to elucidate what possible learning opportunities observed instructional methods could offer learners in the subject. The sample comprised three mathematics teachers from Nigeria and three mathematics teachers from South Africa, all of whom were selected using purposive sampling techniques. Instructional activities in six geometry classrooms were recorded on videotape. The van Hiele learning phases provided the framework for data analysis. The findings of this study indicate that observed teaching methods in geometry classrooms in the participating schools offer learners scant opportunity to learn geometry.
- Full Text:
- Date Issued: 2013
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