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
Understanding social-ecological changes in Fairbairn village, Eastern Cape
- Authors: Falayi, Menelisi
- Date: 2017
- Subjects: Fairbairn Village (Eastern Cape, South Africa) , Rural development -- South Africa -- Eastern Cape -- Fairbairn Village , Land tenure -- South Africa -- Eastern Cape -- Fairbairn Village
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/7408 , vital:21257
- Description: The world is becoming increasingly complex and uncertain due to increasing levels of social- ecological change. Rural communities in sub-Saharan Africa are faced with multiple interconnected challenges such as population growth, environmental change, economic recession and climatic changes, amongst others. Such challenges can play a key role in determining vulnerability and food security, particularly for natural resource product- dependent societies that have limited livelihood sources. Studies that consider understanding how society and ecosystems simultaneously interact and respond to new and exacerbated drivers are increasingly needed. Therefore, this study was conducted in Fairbairn village, Eastern Cape, South Africa with the purpose of exploring multi-scale historical processes and current related trends in livelihood and environmental change, and the implications for future trajectories under a changing climate. This study applied social-ecological thinking and several conceptual approaches were combined to provide a lens for exploring the changes taking place. These included, inter alia, complexity theory, social-ecological systems, the sustainable livelihoods approach and the Intergovernmental Platform on Biodiversity and Ecosystem Services framework and principles. The study employed a mixed method approach to gather data, which included a household survey, aerial photography, historical records as well as Participatory Learning and Action, focus group discussions, and Participatory Scenario techniques. Quantitative data were analysed using Excel and Statistica version 13, whilst coding was used for thematic analysis of qualitative data. The main objective of this study was to explore multi-scale historical processes and current related trends of livelihood and environmental change, and the implications of these for future trajectories under a changing climate. Livelihood and landscape changes in Fairbairn village are embedded within a history of direct state intervention and more recently, improvements in basic service delivery. The findings show that natural resource products still form an important part of people’s livelihoods despite many other changes over the last fifteen years. A high proportion of households continue to utilise different products to meet household needs. The most widely used products are fuelwood, wild herbs and wild fruits. More so, my results revealed a significant increase in the number of people commercialising natural resource products over the past fifteen years. Furthermore, I found that rural livelihoods in Fairbairn are heavily dependent on external income and consumption and have become increasingly divorced from local production patterns. My results depict a steady decline of cultivated fields with a corresponding increase of home gardens since the 1960s. Therefore, the results demonstrate that current livelihood strategies are an expression of historical processes interacting with current contextual complexities. Given the complex and multidimensional issues at play in Fairbairn, the study highlights that there is no straightforward answer regarding future livelihood strategies. However, participatory scenario deliberations revealed that the youth were much more open to diversified and even very different forms of livelihood strategies in the future, whilst community leaders and elders remained firmly attached to farming activities. Understanding the diversity of past livelihood changes, together with current trends, can help to better contextualise future livelihood trajectories and this can therefore help rural communities identify and avoid undesirable futures under a changing climate.
- Full Text:
- Date Issued: 2017
Relative contribution of wild foods to individual and household food security in the context of increasing vulnerability due to HIV/AIDS and climate variability
- Authors: Abu-Basutu, Keitometsi Ngulube
- Date: 2014
- Subjects: Wild foods -- South Africa -- Eastern Cape , Food security -- South Africa , Food supply -- South Africa , AIDS (Disease) -- Social aspects -- South Africa , HIV infections -- Social aspects -- South Africa , Chronically ill -- South Africa -- Economic conditions , Chronically ill -- South Africa -- Social conditions , Food -- Social aspects -- South Africa , Food -- Economic aspects -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4774 , http://hdl.handle.net/10962/d1010864 , Wild foods -- South Africa -- Eastern Cape , Food security -- South Africa , Food supply -- South Africa , AIDS (Disease) -- Social aspects -- South Africa , HIV infections -- Social aspects -- South Africa , Chronically ill -- South Africa -- Economic conditions , Chronically ill -- South Africa -- Social conditions , Food -- Social aspects -- South Africa , Food -- Economic aspects -- South Africa
- Description: Wild foods are an integral component of the household food basket, yet their quantified contribution to food security relative to other sources in the context of HIV/AIDS, climate change and variability remains underexplored. This study was carried out in Willowvale and Lesseyton which are rural communities in the Eastern Cape province of South Africa. Willowvale is a relatively remote, medium-rainfall coastal community, while Lesseyton is a peri-urban low rainfall inland community. Qualitative and quantitative methods were used to collect data from 78 HIV/AIDS afflicted households with 329 individuals and 87 non-afflicted households with 365 individuals in the two study sites. Households were visited quarterly over 12 months to assess food acquisition methods, dietary intake and quality, and levels of food security, and to determine strategies employed by households to cope with droughts. The wild foods investigated were wild meat, wild birds, wild fish, wild mushrooms, wild leafy vegetables and wild fruits. Diets were moderately well-balanced and limited in variety, with cereal items contributing 52 % to total calorie intake. Mid-upper arm circumference measurements showed that all respondents were adequately nourished. The bulk of the food consumed by households was purchased, with supplementation from own production, wild vegetables and wild fruits. In Willowvale, wild vegetables comprised 46 % of overall vegetable consumption for afflicted households and 32 % for non-afflicted households, while own fruit production comprised 100 % of fruit consumption. In Lesseyton, wild vegetables comprised only 6 % and 4 % of vegetable consumption for afflicted and non-afflicted households, while wild fruit comprised 63 % and 41 % for afflicted and non-afflicted households. More than 80 % of respondents from both afflicted and non-afflicted households had sufficient daily kilocalories, although the majority of afflicted households felt they were food insecure and sometimes collected wild foods as one of their multiple coping strategies. Hunting and gathering of wild foods was associated with site, household affliction status, gender, age and season. More than 80 % of respondents ate wild vegetables and said they were more drought tolerant than conventional vegetables, making them the most consumed wild food and approximately 16 % of respondents ate wild birds, making them the least consumed wild food. Approximately 14 % of respondents from afflicted households in Willowvale sold wild fish, whilst 34 % of respondents from afflicted households and 7 % from non-afflicted households sold wild fruits in Lesseyton. Strategies adopted by households to cope with droughts were different between the two study sites, and households in Willowvale used a wider range of strategies. Given the devastating effects of HIV/AIDS coupled with the drawbacks of climate change and variability on food security, wild foods represent a free and easy way for vulnerable households to obtain food.
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- Date Issued: 2014
The contribution of trees to local livelihoods in urban areas
- Authors: Kaoma, Humphrey
- Date: 2013
- Subjects: Trees in cities - -South Africa , Urban ecology (Sociology) -- South Africa , Poverty -- South Africa , Urban forestry -- South Africa , Urban poor -- South Africa , Natural resources -- South Africa , Human-plant relationships -- South Africa
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4734 , http://hdl.handle.net/10962/d1003911 , Trees in cities - -South Africa , Urban ecology (Sociology) -- South Africa , Poverty -- South Africa , Urban forestry -- South Africa , Urban poor -- South Africa , Natural resources -- South Africa , Human-plant relationships -- South Africa
- Description: Trees in natural forests are widely known for their essential contribution to rural livelihoods in developing countries, providing both consumptive and non-consumptive products to rural inhabitants. These benefits are also obtained from trees in urban forests and used by urban households. In the past decades, the role of urban trees to urban livelihoods, municipalities, local and global environment has often been overlooked by researchers and development agencies, and hence are poorly documented, especially in Africa. In South Africa, the increase in urbanisation and urban poverty means many urban residents are expected to be dependent on trees and tree products from homesteads, neighbourhoods and edges of towns. There is however a paucity of literature on the potential of trees in sustaining livelihoods and poverty alleviation in urban areas. This study determined the magnitude and nature of the direct contribution of trees and tree products to local livelihoods in different urban residential areas (informal, Reconstruction and Development Programme (RDP), and township) in three South African towns (Tzaneen, Bela Bela and Zeerust) which cover a rainfall gradient from relatively higher (775 mm p.a.) to low (575 mm p.a.). The results showed that most (90%) households had an interest in planting and managing trees on homesteads, given the high proportion (71%) of residents who had planted trees on their homesteads. The abundance of trees followed the moisture gradient, with Tzaneen having a larger share (46.4%) of trees, followed by Bela Bela (27.5%) and Zeerust (26.1%). Larger plots in the informal residential area accounted for the larger proportion (42.8%) of trees on homesteads, followed by the township (32.9%) and RDP households being the least because they were recently established. Tree density was also higher in the high rainfall town but the pattern did not follow the moisture gradient between Bela Bela and Zeerust due to little rainfall difference. However, tree density across residential areas was similar to the proportion of trees on homesteads in residential areas. The most common tree species were alien, and mainly exotic fruit trees made up two-thirds of the trees encountered. Most households collected various products from urban trees, particularly fuelwood, from edges of towns. This was especially by households with lower cash income. However, fruits were collected from homesteads regardless of the wealth status. Tree products had a significant contribution to the total annual household income, with fuelwood alone contributing up to R5 663 per household per annum, equivalent to two-three month household cash income. Tree products added 20% to total household income, which represents the amount of money households save by collecting tree products for free. Therefore, trees within and around urban areas contribute significantly to livelihoods and reduce poverty in urban areas. There is need therefore to encourage urban residents to plant trees on homesteads and policy makers to come up with policies that promote sustainable harvesting of tree products from areas surrounding urban areas.
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- Date Issued: 2013
Towards modelling the formation of ore bodies initial results dealing with the fluid mechanical aspects of magma chamber convection
- Authors: Botha, André Erasmus
- Date: 1999
- Subjects: Ore deposits , Fluid mechanics , Magmatism
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5492 , http://hdl.handle.net/10962/d1005278 , Ore deposits , Fluid mechanics , Magmatism
- Description: This thesis forms part of a larger effort which aims to establish the means of assessing the fluid mechanical behaviour of magma 1 as it cools inside a magma chamber surrounded by porous country rock. The reason for doing so is to advance the understanding of some types of mineral deposits; for example,the Platinum Group Elements (PGEs). The magma is modelled with the governing equations for a single-phase incompressible Newtonian fluid with variable viscosity and density. In this thesis, thermal conductivity and specific heat are approximated as constants and the country rock is treated as a conducting solid so as to save on computational time in the initial phases of the project. A basic review of the relevant literature is presented as background material and three basic models of magma chambers are discussed: crystal settling, compositional convection and double diffusive convection.The results presented in this thesis are from finite element calculations by a commercial computer code: ANSYS 5.4. This code has been employed in industry for over 26 years and has a long and successful benchmark history. In this context, finite element methods that are applicable to the code are discussed in chapter 5. In chapter 6, results that were obtained in the course of this research are presented. The thesis concludes with an indication of the possible geological significance of the results and various refinements that should be made to future models.
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- Date Issued: 1999