The development of high-throughput assays to screen for potential anticancer and antimalarial compounds that target ADP-ribosylation factor 6 and its signalling machineries
- Authors: Khan, Farrah Dilshaad
- Date: 2019
- Subjects: ADP-ribosylation , Proteins -- Metabolism , Nucleoproteins , Malaria -- Chemotherapy , Cancer -- Chemotherapy
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/92952 , vital:30810
- Description: ADP-ribosylation factors (Arfs) are small GTP-binding proteins that cycle between active GTP-bound forms and inactive GDP-bound forms. GDP/GTP cycling is regulated by large families of guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). ArfGEFs activate Arfs by mediating the exchange of GDP for GTP, while ArfGAPs terminate Arf function by stimulating the hydrolysis of the terminal phosphate group of GTP. Arf6 is a major regulator of endocytic trafficking and reorganization of the actin cytoskeleton in eukaryotic organisms. Owing to its participation in wide range of fundamentally distinct cellular processes, Arf6 may be a drug target for cancer and malaria amongst other diseases. As with cancer cells, rapid growth and viability of eukaryotic pathogens likely places a heavy burden on their endocytic pathways and a critical reliance on Arf6 activity. A putative malarial homolog of Arf6 (PfArf6) localises to numerous puncta along the periphery of the parasite in the mature trophozoite life stage of the parasite (T. Swart, MSc dissertation). Owing to highly inefficient parasite transfection procedures and a relative shortage of well described and validated parasite organelle markers, the possible functions of PfArf6 were explored using HeLa cells as a surrogate model for parasites by fluorescence microscopy of cells transfected with GFP-tagged PfArf6. Partial co-localisation was observed with the mammalian markers HsArf6 and LC3, which suggested possible roles in Arf6-dependent endocytosis and autophagy, respectively. While these possible roles are currently under investigation in parasites, an overall long-term goal which was initiated in this study was to determine whether PfArf6 is a valid drug target. To chemically validate PfArf6 as a drug target, a potent inhibitor needs to be identified. This requires the development of assays that may be employed for high-throughput screening of compound libraries. To support this goal, a novel plate-based assay was developed using human Arf6. The assay relies on the selective binding of an Arf effector protein domain (GGA3) fused to glutathione-S-transferase (GST), to His-tagged Arf6 immobilised on a nickel-coated plate. The assay format was developed and could robustly distinguish HsArf6-GDP (inactive) from HsArf6-GTP (active). Furthermore, it could be employed to detect the deactivation of Arf6 by ArfGAP1-stimualted GTP hydrolysis, but not Arf6 activation by ARNO-stimulated GDP/GTP exchange (ARNO is an ArfGEF). The ArfGAP1 deactivation assay was chemically validated using a known ArfGAP inhibitor, QS11. An improved assay was developed that employs JIP4 as an Arf6-specific binding partner instead of GGA3. In addition to superior performance, the alternative assay format could potentially be exploited for cancer drug discovery, since Arf6-JIP4 interaction has been implicated in cancer cell invasion and metastasis. Both assays may be employed to explore alternative ArfGEFs and ArfGAPs that act on Arf6 and contribute to the advancement of cancer. In parallel experiments, where development of PfArf6 assays was the focus, several issues arose. Firstly, we could not prepare GDP- and GTP-bound forms of PfArf6 since EDTA-mediated nucleotide exchange appeared to irreversibly destabilise the protein. However, PfArf6 activation (i.e. the preparation of PfArf6-GTP) was possible when mediated by ARNO and assessed by tryptophan fluorescence kinetic assays, suggesting that PfArf6 may be expressed in GDP-bound form in E. coli. As with human Arf6, ARNO-mediated GDP/GTP exchange on PfArf6 was not detectable in the immobilised PfArf6-GGA interaction GST assay format. However, a more sensitive assay was developed which relies on the use of nickel-horseradish peroxidase to detect the binding of His-tagged PfArf6 to JIP4-GST immobilised on glutathione plates and could detect ARNO-mediated PfArf6 activation. Since we could not prepare PfArf6-GTP (that did not rely on the presence of the ArfGEF, ARNO), malarial ArfGAP deactivation studies were conducted using PfArf1 instead of PfArf6 in the GGA-GST interaction assay. Both PfArfGAP1and PfArfGAP2 stimulated GTP hydrolysis by PfArf1, but only the former was inhibited by the standard human ArfGAP inhibitor, QS11. The development of these simple, cost-effective assays can be used in the high-throughput screening of novel anticancer and antimalarial compounds that target Arf signalling machineries. In theory, the assay could be extended as a tool to identify novel inhibitors of the multitude of Arfs, ArfGEFs and ArfGAPs originating from any organism and hence has broad clinical significance.
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- Date Issued: 2019
Where dreams become reality: professionalism in flight training in the Eastern Cape
- Authors: Allison, Martin
- Date: 2019
- Subjects: Professional socialization , Flight training -- South Africa , Flight schools -- South Africa -- Case studies , Air pilots -- Training of -- South Africa
- Language: English
- Type: text , Thesis , Masters , MA
- Identifier: http://hdl.handle.net/10962/95376 , vital:31150
- Description: This study explores the construction of the identity of professional pilots through a case study of a flying school in South Africa. Here, a 15-20-month period of intensive study and training of students, fresh from school or college, leads in most cases to the attainment of a Commercial or Airline Transport Pilot’s Licence. The construction of identity is a continuous process and a lifelong project and hence this study can only reflect upon the factors influencing the early stages of a pilot’s career, until the point where the licenced Pilot leaves the Air School and enters full time employment with a commercial undertaking, but it is argued that this is a crucial step in the formation of professional identity, habitus in Bourdieu’s terms. The culture of the air School reflects the military background of the founders of the school and the staff employed in senior positions. The school, which is residential, observes a strict regime of Ground School and Practical Flying Training and a high standard of performance and personal conduct is demanded, both during training and in off duty hours and excessive consumption of alcohol and smoking are discouraged, and drug use absolutely taboo. Progress with training at the school is closely monitored and a disciplined environment maintained by surveillance cameras, house monitors and security guards; in Foucauldian terms, a modern version of the Panopticon, but somewhat less than Goffman’s Total Institution. It was found that the construction of a flying identity for most of the students entering the air school commenced in childhood or early adulthood, through the influence of friends and relatives and they enter the school with the firm intention of becoming Professional pilots. Full participation of the author in the Ground School revealed how professionalization is implemented through the discipline and rigor of the training methods employed. Through mastery of a complex body of theoretical knowledge in the Ground School and the practical skill of learning to fly in a one-on-one relationship with an instructor, the students gain confidence and efficacy which contributes to their self-respect and maturity. The international reputation of the school, confers prestige upon its graduates and they benefit from membership of a profession which commands respect and a high level of income. In large measure, the thesis shows, the success of the School is a function of the founders’ ‘invention of tradition’ focusing on the wartime training school that existed on the site and the many echoes of those times in the (re)construction of its buildings and facilities, continuing in the approach of the multinational that now owns the School.
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- Date Issued: 2019
Transformation through engagement: developing Grade 9 leadership opportunities through activity system using change laboratory intervention in a secondary school in Omusati region of Namibia
- Authors: Vaino, Loide Mwasheka
- Date: 2018
- Subjects: School management and organization Namibia Omusati , Student participation in administration Namibia Omusati , Educational leadership Namibia Omusati , Student government Namibia Omusati
- Language: English
- Type: text , Thesis , Masters , MEd
- Identifier: http://hdl.handle.net/10962/61569 , vital:28038
- Description: The evolution of traditional educational leadership theory to contemporary leadership theory came as a response to an increased demand for better services in schools by creating platforms for learner leadership such as distributed leadership where learner leadership is located. This study of learner leadership is conducted in a secondary school in Omusati region of Namibia. Drawing on distributed leadership theory, the study sought to promote the distribution of leadership opportunities amongst all educational stakeholders, including learners, as provided for by policy and projected by educational leadership theory. The motivation of this study was the need to understand the problems associated with learner leadership as identified by past research. In addition, this study hoped to address the gap in the literature by exploring learner leadership development opportunities developing agency in learners through a Change Laboratory (CL) intervention. This study was as a transformative case study, how the grade 9s in the case study school were involved in leadership, the constraining factors that hindered the involvement of grade 9 learners in leadership, the enhancement of learner leadership at school, the positive contributions of CL workshops to participants and the leadership growth brought about by an intervention. This study generated data through observation, questionnaires, semi-structured interviews, focus group interviews, and Change Laboratory Workshops. The findings revealed that the grade 9 learners were marginally involved in leadership at the school. The most substantial challenge relates to traditional and outdated views of leadership on the part of teachers and educational managers. Additionally, the findings from the Change Laboratory workshops revealed that despite grade 9 learners being the youngest at school, they have the potential to be learner leaders. Hence in addition to several practical recommendations, the study recommends a change of mindset towards learner leadership so that opportunities are provided to contribute to the growth and development of learners. Finally, these research study findings will help my professional colleagues and policy makers in education to better understand the significance role of learner leadership involvements in schools.
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- Date Issued: 2018