A comparative study on Staff Retention in South Africa’s multinational banking corporations and micro-lenders
- Carolus, Chris Mario Periandros
- Authors: Carolus, Chris Mario Periandros
- Date: 2017-04
- Subjects: Employee retention -- South Africa , Employee motivation -- South Africa , Job satisfaction -- South Africa
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/66434 , vital:75319
- Description: The financial industry has changed rapidly over the past decade with competition being ferocious in the South African market. Traditionally most competition was among the four major banks, being ABSA, Nedbank, First National Bank and Standard Bank; however various small to medium micro-lenders, such as Capitec Bank, have grown more rapidly than the four major banks. These are the banks against which Multinational Corporations compete for products, services as well as employees. Staff employed in sales plays a critical role in any organisation and once that person leaves the organisation, productivity levels are set to be impacted along with margins on which banks are competing. It is thus of great importance for banks to pay close attention to staff retention in their respective organisations. Employees are viewed as the most important resource within any business, as firms are set to ensure a competitive advantage to remain profitable and cope with adversities that the global economy might present. The objective of this research was to establish whether the aspects of staff retention in Multinational Banking Corporations in South Africa differ from Micro- lenders locally. To ensure the objective was achieved, a literature review was conducted to understand the concepts that influence aspects of staff retention. Secondly, based on these aspects, a quantitative questionnaire was designed to obtain staff responses to the aspects of staff retention. The primary findings from the study indicate that the aspects of staff retention do indeed differ between Micro-lenders and Multinational Banking Corporations. Some of the main differences that were uncovered by the study occurred around transparency from leadership, lack of ability to develop talent and working conditions. Overall most respondents from Micro-lenders appear to be happier than respondents in Multinational Banking Corporations. The above factors are set to contribute significantly towards staff retention challenges for Multinational Banking Corporations, where employees are more disengaged, and that influences productivity negatively. , Thesis (MBA) -- Faculty of Business and Economic Sciences, Business School, 2017
- Full Text:
- Date Issued: 2017-04
- Authors: Carolus, Chris Mario Periandros
- Date: 2017-04
- Subjects: Employee retention -- South Africa , Employee motivation -- South Africa , Job satisfaction -- South Africa
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10948/66434 , vital:75319
- Description: The financial industry has changed rapidly over the past decade with competition being ferocious in the South African market. Traditionally most competition was among the four major banks, being ABSA, Nedbank, First National Bank and Standard Bank; however various small to medium micro-lenders, such as Capitec Bank, have grown more rapidly than the four major banks. These are the banks against which Multinational Corporations compete for products, services as well as employees. Staff employed in sales plays a critical role in any organisation and once that person leaves the organisation, productivity levels are set to be impacted along with margins on which banks are competing. It is thus of great importance for banks to pay close attention to staff retention in their respective organisations. Employees are viewed as the most important resource within any business, as firms are set to ensure a competitive advantage to remain profitable and cope with adversities that the global economy might present. The objective of this research was to establish whether the aspects of staff retention in Multinational Banking Corporations in South Africa differ from Micro- lenders locally. To ensure the objective was achieved, a literature review was conducted to understand the concepts that influence aspects of staff retention. Secondly, based on these aspects, a quantitative questionnaire was designed to obtain staff responses to the aspects of staff retention. The primary findings from the study indicate that the aspects of staff retention do indeed differ between Micro-lenders and Multinational Banking Corporations. Some of the main differences that were uncovered by the study occurred around transparency from leadership, lack of ability to develop talent and working conditions. Overall most respondents from Micro-lenders appear to be happier than respondents in Multinational Banking Corporations. The above factors are set to contribute significantly towards staff retention challenges for Multinational Banking Corporations, where employees are more disengaged, and that influences productivity negatively. , Thesis (MBA) -- Faculty of Business and Economic Sciences, Business School, 2017
- Full Text:
- Date Issued: 2017-04
Mediating teaching and learning in Foundation Phase Grade 3 Life Skills classrooms: role and use of learning and teaching support materials
- Authors: Nqabeni, Pelokazi
- Date: 2017-04
- Subjects: teaching strategies
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/11260/9436 , vital:73535
- Description: The study explored the role and use of learning and teaching support materials (LTSMs) to mediate teaching and learning in Life Skills Grade 3 classrooms of Dutywa District, in the province of the Eastern Cape, South Africa. It was prompted by a deep concern about the role and use of LTSMs to mediate teaching and learning in the Foundation Phase. While studies have been carried out on LTSMs in Foundation Phase, few studies have looked at the role and use of LTSMs to mediate teaching and learning in Life Skills Grade 3 classrooms. The study used a qualitative approach and is a multiple-case study of three purposively sampled schools from the rural areas. Teachers who participated in the study were Grade 3 teachers from the three schools. A pilot study was conducted in three schools with Foundation Phase, which were not used for the main study, for the purpose of validity and reliability. In this study, Cultural Historical Activity Theory (CHAT) was used as a framework to understand that actions are mediated by a complex network of socio-historical activities. I drew on the second generation of the activity theory, which has roots in Leontiev’s work on the Cultural Historical Activity Theory (CHAT), which provided both explanatory and analytical tools. I collected data using documents including journal entries, individual semi-structured interviews, and structured observations. The interviews were voice recorded with participant’s permission in order to present verbatim statements. In structured observations, I observed the role and use of LTSMs to mediate teaching and learning in Life Skills Grade 3 classrooms in the three selected schools. Content analysis guided the data analysis through themes derived from the subsidiary questions and coded for easy referencing. Data reporting took the form of thick description and verbatim quotations in line with the qualitative approach of the study. The study’s findings revealed non-availability of learning and teaching support materials in Life Skills subject. This suggests that intervention is needed by the Department of Education such as providing LTSMs which are essential in order to address the problem of not being used to mediate teaching and learning in classrooms. Language used in the role and use of learning and teaching support materials to mediate teaching and learning, lack of professional development, the effect of the non-examinable nature of Life Skills, and shortage and overcrowded classrooms were some of the aspects affecting the role and use of LTSMs to mediate teaching and learning in Life Skills Grade 3 classrooms. Based on the study findings, then I recommend that further research be conducted on strategies that could be employed to improve the role and use of LTSMs to mediate teaching and learning in Life Skills Grade 3 classrooms in a way that benefits learners. The Departmental officials including subject advisors, principals, teachers and learners should work together to improve the role and use of LTSMs to mediate teaching and learning in classrooms for quality education and to improve results in Foundation Phase. A model is proposed as the new knowledge for the existing literature on the role and use of LTSMs to mediate teaching and learning in classroom based on the theoretical framework and findings of this study. , Thesis (D.Ed) -- Faculty of Education Sciences, 2017
- Full Text:
- Date Issued: 2017-04
- Authors: Nqabeni, Pelokazi
- Date: 2017-04
- Subjects: teaching strategies
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/11260/9436 , vital:73535
- Description: The study explored the role and use of learning and teaching support materials (LTSMs) to mediate teaching and learning in Life Skills Grade 3 classrooms of Dutywa District, in the province of the Eastern Cape, South Africa. It was prompted by a deep concern about the role and use of LTSMs to mediate teaching and learning in the Foundation Phase. While studies have been carried out on LTSMs in Foundation Phase, few studies have looked at the role and use of LTSMs to mediate teaching and learning in Life Skills Grade 3 classrooms. The study used a qualitative approach and is a multiple-case study of three purposively sampled schools from the rural areas. Teachers who participated in the study were Grade 3 teachers from the three schools. A pilot study was conducted in three schools with Foundation Phase, which were not used for the main study, for the purpose of validity and reliability. In this study, Cultural Historical Activity Theory (CHAT) was used as a framework to understand that actions are mediated by a complex network of socio-historical activities. I drew on the second generation of the activity theory, which has roots in Leontiev’s work on the Cultural Historical Activity Theory (CHAT), which provided both explanatory and analytical tools. I collected data using documents including journal entries, individual semi-structured interviews, and structured observations. The interviews were voice recorded with participant’s permission in order to present verbatim statements. In structured observations, I observed the role and use of LTSMs to mediate teaching and learning in Life Skills Grade 3 classrooms in the three selected schools. Content analysis guided the data analysis through themes derived from the subsidiary questions and coded for easy referencing. Data reporting took the form of thick description and verbatim quotations in line with the qualitative approach of the study. The study’s findings revealed non-availability of learning and teaching support materials in Life Skills subject. This suggests that intervention is needed by the Department of Education such as providing LTSMs which are essential in order to address the problem of not being used to mediate teaching and learning in classrooms. Language used in the role and use of learning and teaching support materials to mediate teaching and learning, lack of professional development, the effect of the non-examinable nature of Life Skills, and shortage and overcrowded classrooms were some of the aspects affecting the role and use of LTSMs to mediate teaching and learning in Life Skills Grade 3 classrooms. Based on the study findings, then I recommend that further research be conducted on strategies that could be employed to improve the role and use of LTSMs to mediate teaching and learning in Life Skills Grade 3 classrooms in a way that benefits learners. The Departmental officials including subject advisors, principals, teachers and learners should work together to improve the role and use of LTSMs to mediate teaching and learning in classrooms for quality education and to improve results in Foundation Phase. A model is proposed as the new knowledge for the existing literature on the role and use of LTSMs to mediate teaching and learning in classroom based on the theoretical framework and findings of this study. , Thesis (D.Ed) -- Faculty of Education Sciences, 2017
- Full Text:
- Date Issued: 2017-04
Synthesis and biolgical screening of potential plasmodium falciparum DXR inhibitors
- Authors: Adeyemi, Christiana Modupe
- Date: 2017-04
- Subjects: Plasmodium falciparum , Enzyme inhibitors , Malaria , Antimalarials , Drug development , Malaria -- Chemotherapy , Isopentenoids -- Synthesis , Fosmidomycin , 1-Deoxy-D-xylulose 5-phosphate
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/61790 , vital:28060
- Description: The non-mevalonate isoprenoid pathway, also known as the 1-deoxy-D-xylulose-5- phosphate DXP pathway, is absent in humans, but present in the anopheles mosquito responsible for the transmission of malaria. DXP reductoisomerase - a key enzyme in the DXP pathway in Plasmodium falciparum (PfDXR) has been identified as a target for the design of novel anti-malarial drugs. Fosmidomycin and its acetyl analogue (FR900098) are known to be inhibitors of PfDXR and, in this study, synthetic variations of the fosmidomycin scaffold have led to four series of novel analogues. Particular attention has been centred on the introduction of various substituted benzyl groups in each of these series in order to occupy a recently discovered vacant pocket in the PfDXR active-site and thus enhance ligand-enzyme binding. In the process 160 ligands and precursors have been prepared, no less than 119 of them novel. Fistly, a series of C-benzylated phosphonate esters and phosphonic acids were synthesised, in which the fosmidomycin hydroxamate Mg2+- coordinating moiety was replaced by an amide funtionality and the number of methylene groups in the “hydrophobic patch” between the phosphonate and the hydroxamate moiety was decreased from two to one. Several approaches were explored for this series, the most successful involving reaction of 3- substituted anilines with a-bromo propanoic acid in the presence of the coupling agent 1,1'- carbonyldiimidazole (CDI), followed by Michaelis-Arbuzov phosphonation using triethyl phosphite. Reaction of the resulting chiral phosphonate esters with bromotrimethylsilane gave the corresponding phosphonic acids in good yields. In order to obviate chirality issues, a second series of potential “reverse” fosmidomycin analogues was synthesised by replacing the methylene group adjacent to the the phosphonate moiety with a nitrogen atom. Deprotonation, alkylation and phosphorylation of various amines gave diethyl #-benzylphosphoramidate ester intermediate. Aza-Michael addition of these intermediates, followed by hydrolysis gave the corresponding carboxylic acids which could be reacted with different hydroxylamine hydrochloride derivatives to afford the novel hydroxamic acid derivatives in good yields. Thirdly, a series of a novel #-benzylated phosphoramidate derivatives were prepared as aza- FR900098 analogues. Alkylation of different amines using bromoacetalde-hyde diethylacetal gave a series of N-benzyl-2,2-diethoxyethylamine compounds, which were then elaborated via a futher six steps to afford novel #-benzylated phosphoramidate derivatives. Finally, in order to ensure syn-orientation of the donor atoms in the Mg - coordinating group and, at the same time, introduce conformational constraints in the ligand, the hydrophobic patch and the hydroxamate moiety were replaced by cyclic systems. Several approaches towards the synthesis of such conformationally constrained phosphoramidate analogues from maleic anhydride led to the unexpected isolation of an unprecedented acyclic furfuryl compound, and 1H NMR and DFT-level theoretical studies have been initiated to explore the reaction sequence. A series of #-benzylated phosphoramidate derivatives containing dihydroxy aromatic rings (as the conformationally constrained groups) to replace the hydroxamate moiety, were successfully obtained in six steps from the starting material, 3,4-dihydroxylbenzaldehyde. While in vitro assays have been conducted on all of the synthesised compounds, and some of the ligands show promising anti-malarial inhibitory activity - most especially the conformationally constrained cyclic #-benzylated phosphoramidate series. Interestingly, a number of these compounds has also shown activity against T.brucei - the causative agent of sleeping sickness. In silico docking studies of selected compounds has revealed the capacity of some of the ligands to bind effectively in the PfDXR active-site with the newly introduced benzyl group occupying the adjacent vacant pocket. The physico-chemical properties of these ligands were also explored in order to predict the oral-bioavailability. Most of the ligands obeyed the Lipinski rule of 5, while QSAR methods have been used in an attempt to correlate structural variations and calculated molecular properties with the bioassay data. , Thesis (PhD) -- Faculty of Science, Chemistry, 2017
- Full Text:
- Date Issued: 2017-04
- Authors: Adeyemi, Christiana Modupe
- Date: 2017-04
- Subjects: Plasmodium falciparum , Enzyme inhibitors , Malaria , Antimalarials , Drug development , Malaria -- Chemotherapy , Isopentenoids -- Synthesis , Fosmidomycin , 1-Deoxy-D-xylulose 5-phosphate
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/61790 , vital:28060
- Description: The non-mevalonate isoprenoid pathway, also known as the 1-deoxy-D-xylulose-5- phosphate DXP pathway, is absent in humans, but present in the anopheles mosquito responsible for the transmission of malaria. DXP reductoisomerase - a key enzyme in the DXP pathway in Plasmodium falciparum (PfDXR) has been identified as a target for the design of novel anti-malarial drugs. Fosmidomycin and its acetyl analogue (FR900098) are known to be inhibitors of PfDXR and, in this study, synthetic variations of the fosmidomycin scaffold have led to four series of novel analogues. Particular attention has been centred on the introduction of various substituted benzyl groups in each of these series in order to occupy a recently discovered vacant pocket in the PfDXR active-site and thus enhance ligand-enzyme binding. In the process 160 ligands and precursors have been prepared, no less than 119 of them novel. Fistly, a series of C-benzylated phosphonate esters and phosphonic acids were synthesised, in which the fosmidomycin hydroxamate Mg2+- coordinating moiety was replaced by an amide funtionality and the number of methylene groups in the “hydrophobic patch” between the phosphonate and the hydroxamate moiety was decreased from two to one. Several approaches were explored for this series, the most successful involving reaction of 3- substituted anilines with a-bromo propanoic acid in the presence of the coupling agent 1,1'- carbonyldiimidazole (CDI), followed by Michaelis-Arbuzov phosphonation using triethyl phosphite. Reaction of the resulting chiral phosphonate esters with bromotrimethylsilane gave the corresponding phosphonic acids in good yields. In order to obviate chirality issues, a second series of potential “reverse” fosmidomycin analogues was synthesised by replacing the methylene group adjacent to the the phosphonate moiety with a nitrogen atom. Deprotonation, alkylation and phosphorylation of various amines gave diethyl #-benzylphosphoramidate ester intermediate. Aza-Michael addition of these intermediates, followed by hydrolysis gave the corresponding carboxylic acids which could be reacted with different hydroxylamine hydrochloride derivatives to afford the novel hydroxamic acid derivatives in good yields. Thirdly, a series of a novel #-benzylated phosphoramidate derivatives were prepared as aza- FR900098 analogues. Alkylation of different amines using bromoacetalde-hyde diethylacetal gave a series of N-benzyl-2,2-diethoxyethylamine compounds, which were then elaborated via a futher six steps to afford novel #-benzylated phosphoramidate derivatives. Finally, in order to ensure syn-orientation of the donor atoms in the Mg - coordinating group and, at the same time, introduce conformational constraints in the ligand, the hydrophobic patch and the hydroxamate moiety were replaced by cyclic systems. Several approaches towards the synthesis of such conformationally constrained phosphoramidate analogues from maleic anhydride led to the unexpected isolation of an unprecedented acyclic furfuryl compound, and 1H NMR and DFT-level theoretical studies have been initiated to explore the reaction sequence. A series of #-benzylated phosphoramidate derivatives containing dihydroxy aromatic rings (as the conformationally constrained groups) to replace the hydroxamate moiety, were successfully obtained in six steps from the starting material, 3,4-dihydroxylbenzaldehyde. While in vitro assays have been conducted on all of the synthesised compounds, and some of the ligands show promising anti-malarial inhibitory activity - most especially the conformationally constrained cyclic #-benzylated phosphoramidate series. Interestingly, a number of these compounds has also shown activity against T.brucei - the causative agent of sleeping sickness. In silico docking studies of selected compounds has revealed the capacity of some of the ligands to bind effectively in the PfDXR active-site with the newly introduced benzyl group occupying the adjacent vacant pocket. The physico-chemical properties of these ligands were also explored in order to predict the oral-bioavailability. Most of the ligands obeyed the Lipinski rule of 5, while QSAR methods have been used in an attempt to correlate structural variations and calculated molecular properties with the bioassay data. , Thesis (PhD) -- Faculty of Science, Chemistry, 2017
- Full Text:
- Date Issued: 2017-04
Incidence of unnatural deaths in Transkei subregion of South Africa (1996 –2015)
- Date: 2017
- Subjects: South Africa Death 2078-6204 Journal article
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/11260/6172 , vital:45258 , xlink:href=": https://doi.org/10.1080/20786190.2017.1292697"
- Description: Background: Unnatural deaths are a serious and preventable public health problem in South Africa. Such an event is more than the death of an individual who has died in an unnatural way. It has a negative and long-lasting impact on family members as well as on society as a whole. Objective: To study the pattern of unnatural deaths in the Transkei sub-region of South Africa. Method: A record review was undertaken from 1996 to 2015 of 24 693 medico-legal autopsies performed at Mthatha Forensic Pathology Laboratory. Results: At the Mthatha Forensic Pathology Laboratory, 24 693 autopsies were performed between 1996 and 2015 on people who had died unnatural deaths. The average rate of unnatural death is 205 per 100 000 per year in this region. The figure is 160 per 100 000 among males and 44 per 100 000 among females. The rate of unnatural death has increased from 153 per 100 000 in 1996 to 260 per 100 000 in 2015. In two-thirds (69%) of cases, the cause of death is related to trauma. Slightly less than half (45%) of the victims in this study were murdered. Conclusion: There is an increasing trend of unnatural deaths in the Mthatha region of South Africa. The male-to-female ratio is 3.8:1, and about half (48%) of the victims were between 11 and 30 years old. This situation needs the urgent attention of the law enforcement agencies. Keywords: unnatural deaths, traumatic deaths, violent death
- Full Text:
- Date Issued: 2017
- Date: 2017
- Subjects: South Africa Death 2078-6204 Journal article
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/11260/6172 , vital:45258 , xlink:href=": https://doi.org/10.1080/20786190.2017.1292697"
- Description: Background: Unnatural deaths are a serious and preventable public health problem in South Africa. Such an event is more than the death of an individual who has died in an unnatural way. It has a negative and long-lasting impact on family members as well as on society as a whole. Objective: To study the pattern of unnatural deaths in the Transkei sub-region of South Africa. Method: A record review was undertaken from 1996 to 2015 of 24 693 medico-legal autopsies performed at Mthatha Forensic Pathology Laboratory. Results: At the Mthatha Forensic Pathology Laboratory, 24 693 autopsies were performed between 1996 and 2015 on people who had died unnatural deaths. The average rate of unnatural death is 205 per 100 000 per year in this region. The figure is 160 per 100 000 among males and 44 per 100 000 among females. The rate of unnatural death has increased from 153 per 100 000 in 1996 to 260 per 100 000 in 2015. In two-thirds (69%) of cases, the cause of death is related to trauma. Slightly less than half (45%) of the victims in this study were murdered. Conclusion: There is an increasing trend of unnatural deaths in the Mthatha region of South Africa. The male-to-female ratio is 3.8:1, and about half (48%) of the victims were between 11 and 30 years old. This situation needs the urgent attention of the law enforcement agencies. Keywords: unnatural deaths, traumatic deaths, violent death
- Full Text:
- Date Issued: 2017
The creation and validation of aptamers binding to murine 3T3-L1 Preadipocytes: preliminary implications for controlled cellular attachment, differentiation and cell fate
- Authors: Rubidge, Mark Lourens
- Date: 2017
- Subjects: Oligonucleotides , Fat cells , Stem cells , Ligand binding (Biochemistry) , Fluorimetry
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/65247 , vital:28714
- Description: The controlled seeding of a variety of stem cells in vitro has been reported to alter the patterns of their subsequent differentiation. This has been attributed to the control of the surface microenvironment onto which adherent stem cells are cultured, especially control of the proximal density of neighbouring cells. Simultaneously, advances in the generation of aptamers - synthetic ligand molecules developed using in vitro selection techniques targeting complex molecules - have aided in the production of molecules capable of selectively binding to a variety of commercial stem cell lines. Combining the aforementioned research fields, the project reported in this thesis aimed to generate DNA-based aptamers capable of assisting with the selective binding of murine 3T3- L1 preadipocytes to a solid surface. This was performed with a view to, eventually, control the seeding densities of the adherent preadipocytes on the surface of the tissue culture dish in subsequent researchers. In the process of meeting this goal, several optimisations of the in vitro process by which aptamers binding to cells are generated (Cell-SELEX) were performed: an analysis into a variety of methods used for the removal of the single stranded aptamer candidate sequences attached to the surface of 3T3-L1 preadipocytes, a comparison of methods for the generation of single-stranded aptamer sequences from double-stranded DNA template molecules and a method for quantifying the removed ssDNA from the cell surface. Their use is further reported in this work. Initially, it was determined that a fluorimetric evaluation of the unbound single stranded DNA was the optimum technique to use to evaluate the relative amounts of aptamer DNA binding to target cells during cell-SELEX; this arose from the release of DNA, and other cell lysate contaminates, which interfered UV/ Vis quantification. The evaluation into different methods of ssDNA removal from the cell surface showed that although trypsinisation of the cells demonstrated the highest level of aptamer detachment (quantified by fluorimetry), there is a decrease the number of potential targets that aptamers could attach to. The most common method for detaching bound DNA aptamer molecules from cellular targets reported in literature, the use of high temperatures, was selected for cell-SELEX to increase the variability in potential target sites on the cell surface. Using techniques optimised in this work, fluorescently-tagged single-stranded oligonucleotide aptamers were later generated with a positive selection pressure to bind to the surface of the 3T3-L1 preadipocytes, but not to their differentiated adipocyte counterparts. After eight cycles of cell-SELEX, fluorescent spectroscopic analysis depicted a 74 % binding retention of the selection pool in the positive preadipocyte selection pool, as opposed to a 0.69 % binding of sequences to the negative differentiated preadipocytes. Following the isolation and identification of candidate sequences, seven separate sequences were identified as being successfully generated from the selection process. Bioinformatic characterisation of these placed sequenced aptamer candidates into two separate families, that were then analysed in opposition to each for their binding affinity toward each other. Using fluorescently-tagged sequences, the binding selectivity of the generated aptamers was validated using both epifluorescent microscopy and confocal microscopy. At this stage, an aptamer sequence selected from prior in-house research to serve as a negative control also demonstrated significant binding to the extracellular matrix of both preadipocytes and mature adipocytes. 5’-thiolated aptamer sequences were used to form self-assembled monolayers on the electrode surfaces of the impedimetric Roche xCELLigence Real-Time Cell Analysis. The use of aptamer sequences to capture the seeded preadipocytes, demonstrated a slight increase in the extent of binding of the preadipocytes to the gold electrode surface and produced some preliminary indications of alterations to the pattern and rate of subsequent differentiation in the preadipocytes. This provides preliminary evidence that aptamers developed to bind specifically to a stem cell line in vitro show potential to be used as to capture said cell when cast in a self- assembled monolayer assembly. This provides a future opportunity to control the seeding densities of the cells in vitro. The effects of cellular differentiation at a set of predefined cellular densities can be demonstrated on a desired stem cell line. , Thesis (MSc) -- Faculty of Faculty of Science, Biotechnology Innovation Centre, 2017
- Full Text:
- Date Issued: 2017
- Authors: Rubidge, Mark Lourens
- Date: 2017
- Subjects: Oligonucleotides , Fat cells , Stem cells , Ligand binding (Biochemistry) , Fluorimetry
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/65247 , vital:28714
- Description: The controlled seeding of a variety of stem cells in vitro has been reported to alter the patterns of their subsequent differentiation. This has been attributed to the control of the surface microenvironment onto which adherent stem cells are cultured, especially control of the proximal density of neighbouring cells. Simultaneously, advances in the generation of aptamers - synthetic ligand molecules developed using in vitro selection techniques targeting complex molecules - have aided in the production of molecules capable of selectively binding to a variety of commercial stem cell lines. Combining the aforementioned research fields, the project reported in this thesis aimed to generate DNA-based aptamers capable of assisting with the selective binding of murine 3T3- L1 preadipocytes to a solid surface. This was performed with a view to, eventually, control the seeding densities of the adherent preadipocytes on the surface of the tissue culture dish in subsequent researchers. In the process of meeting this goal, several optimisations of the in vitro process by which aptamers binding to cells are generated (Cell-SELEX) were performed: an analysis into a variety of methods used for the removal of the single stranded aptamer candidate sequences attached to the surface of 3T3-L1 preadipocytes, a comparison of methods for the generation of single-stranded aptamer sequences from double-stranded DNA template molecules and a method for quantifying the removed ssDNA from the cell surface. Their use is further reported in this work. Initially, it was determined that a fluorimetric evaluation of the unbound single stranded DNA was the optimum technique to use to evaluate the relative amounts of aptamer DNA binding to target cells during cell-SELEX; this arose from the release of DNA, and other cell lysate contaminates, which interfered UV/ Vis quantification. The evaluation into different methods of ssDNA removal from the cell surface showed that although trypsinisation of the cells demonstrated the highest level of aptamer detachment (quantified by fluorimetry), there is a decrease the number of potential targets that aptamers could attach to. The most common method for detaching bound DNA aptamer molecules from cellular targets reported in literature, the use of high temperatures, was selected for cell-SELEX to increase the variability in potential target sites on the cell surface. Using techniques optimised in this work, fluorescently-tagged single-stranded oligonucleotide aptamers were later generated with a positive selection pressure to bind to the surface of the 3T3-L1 preadipocytes, but not to their differentiated adipocyte counterparts. After eight cycles of cell-SELEX, fluorescent spectroscopic analysis depicted a 74 % binding retention of the selection pool in the positive preadipocyte selection pool, as opposed to a 0.69 % binding of sequences to the negative differentiated preadipocytes. Following the isolation and identification of candidate sequences, seven separate sequences were identified as being successfully generated from the selection process. Bioinformatic characterisation of these placed sequenced aptamer candidates into two separate families, that were then analysed in opposition to each for their binding affinity toward each other. Using fluorescently-tagged sequences, the binding selectivity of the generated aptamers was validated using both epifluorescent microscopy and confocal microscopy. At this stage, an aptamer sequence selected from prior in-house research to serve as a negative control also demonstrated significant binding to the extracellular matrix of both preadipocytes and mature adipocytes. 5’-thiolated aptamer sequences were used to form self-assembled monolayers on the electrode surfaces of the impedimetric Roche xCELLigence Real-Time Cell Analysis. The use of aptamer sequences to capture the seeded preadipocytes, demonstrated a slight increase in the extent of binding of the preadipocytes to the gold electrode surface and produced some preliminary indications of alterations to the pattern and rate of subsequent differentiation in the preadipocytes. This provides preliminary evidence that aptamers developed to bind specifically to a stem cell line in vitro show potential to be used as to capture said cell when cast in a self- assembled monolayer assembly. This provides a future opportunity to control the seeding densities of the cells in vitro. The effects of cellular differentiation at a set of predefined cellular densities can be demonstrated on a desired stem cell line. , Thesis (MSc) -- Faculty of Faculty of Science, Biotechnology Innovation Centre, 2017
- Full Text:
- Date Issued: 2017
The Role of HOP in Emerin-Mediated Nuclear Structure
- Authors: Kituyi, Sarah Naulikha
- Date: 2017
- Subjects: Heat shock proteins , Nuclear structure , Nuclear membranes , Cancer Treatment , Molecular chaperones , Cytoskeleton , Cytoplasm
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/59230 , vital:27485 , DOI 10.21504/10962/59230
- Description: A vital component of the integral nuclear membrane is emerin, a Lamin Emerin and Man1 (LEM) domain protein whose concentration determines the levels of partner proteins that together constitute the structure of the nuclear envelope. Deficiencies in any of these proteins causes the failure of the structure and assembly and disassembly of the nuclear envelope, which disrupts chromosome segregation and nuclear compartmentalization that are both associated with disease. Emerin also localizes in the cytoplasm where it is implicated in the structure of the cytoskeleton via interaction with tubulin and actin and thus its deficiency may equally contribute to the collapse of the cytoskeleton. The Hsp70-Hsp90 organising protein (Hop) functions as a cochaperone for entry of client proteins into the Hsp90 folding cycle. Hop is upregulated in cancer and regulates a number of cell biology processes via interactions with proteins independently of Hsp90. In a previous study using global whole cell mass spectrometry, emerin was shown to be the most significantly down regulated protein in Hop depleted cell lysates. In this current study, it was postulated that emerin interacts with Hop, and this interaction regulates the stability, and level of emerin in the nucleus which impacts on the structure of the nuclear envelope. We used HEK293T cell lines stably expressing shRNA against Hop, emerin and a non-targeting control alongside the over expression of Hop in HEK293 cells to determine the effect of Hop levels on emerin expression and vice versa via Western blotting. The effect of Hop on the localization of emerin was assessed via subcellullar fractionation and confocal microscopy, while the impact on the structure of the nucleus was determined by transmission electron microscopy (TEM). We established that the depletion of Hop using shRNA and the over expression of Hop both result in the proteasomal and lysosomal degradation of emerin. Co-immunoprecipitation assays confirmed that Hop and emerin are in a common complex, which was not dependent on the presence of Hsp90. Loss of Hop or emerin led to a deformation of nuclear structure and a statistically significant decrease in nuclear size compared to control cells and was associated with an increase in the levels of nuclear protein, lamin A-C. Loss of emerin and Hop resulted in increased long term cell survival, but only after restriction of the nucleus when the cells had migrated across a transwell membrane. Taken together, the results obtained suggest that Hop acts as a scaffold for the stabilization of emerin and that the effects of Hop depletion on the structure of the nucleus and long term survival are mediated via the depletion of emerin. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2017
- Full Text:
- Date Issued: 2017
- Authors: Kituyi, Sarah Naulikha
- Date: 2017
- Subjects: Heat shock proteins , Nuclear structure , Nuclear membranes , Cancer Treatment , Molecular chaperones , Cytoskeleton , Cytoplasm
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/59230 , vital:27485 , DOI 10.21504/10962/59230
- Description: A vital component of the integral nuclear membrane is emerin, a Lamin Emerin and Man1 (LEM) domain protein whose concentration determines the levels of partner proteins that together constitute the structure of the nuclear envelope. Deficiencies in any of these proteins causes the failure of the structure and assembly and disassembly of the nuclear envelope, which disrupts chromosome segregation and nuclear compartmentalization that are both associated with disease. Emerin also localizes in the cytoplasm where it is implicated in the structure of the cytoskeleton via interaction with tubulin and actin and thus its deficiency may equally contribute to the collapse of the cytoskeleton. The Hsp70-Hsp90 organising protein (Hop) functions as a cochaperone for entry of client proteins into the Hsp90 folding cycle. Hop is upregulated in cancer and regulates a number of cell biology processes via interactions with proteins independently of Hsp90. In a previous study using global whole cell mass spectrometry, emerin was shown to be the most significantly down regulated protein in Hop depleted cell lysates. In this current study, it was postulated that emerin interacts with Hop, and this interaction regulates the stability, and level of emerin in the nucleus which impacts on the structure of the nuclear envelope. We used HEK293T cell lines stably expressing shRNA against Hop, emerin and a non-targeting control alongside the over expression of Hop in HEK293 cells to determine the effect of Hop levels on emerin expression and vice versa via Western blotting. The effect of Hop on the localization of emerin was assessed via subcellullar fractionation and confocal microscopy, while the impact on the structure of the nucleus was determined by transmission electron microscopy (TEM). We established that the depletion of Hop using shRNA and the over expression of Hop both result in the proteasomal and lysosomal degradation of emerin. Co-immunoprecipitation assays confirmed that Hop and emerin are in a common complex, which was not dependent on the presence of Hsp90. Loss of Hop or emerin led to a deformation of nuclear structure and a statistically significant decrease in nuclear size compared to control cells and was associated with an increase in the levels of nuclear protein, lamin A-C. Loss of emerin and Hop resulted in increased long term cell survival, but only after restriction of the nucleus when the cells had migrated across a transwell membrane. Taken together, the results obtained suggest that Hop acts as a scaffold for the stabilization of emerin and that the effects of Hop depletion on the structure of the nucleus and long term survival are mediated via the depletion of emerin. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2017
- Full Text:
- Date Issued: 2017
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