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Ultra-high precision diamond turning of advanced contact lens polymers

  • Authors: Liman, Muhammad Mukhtar
  • Date: 2020
  • Subjects: Contact lenses , Electrostatic lenses Lenses -- Design and construction
  • Language: English
  • Type: Thesis , Doctoral , PhD
  • Identifier: http://hdl.handle.net/10948/46108 , vital:39496
  • Description: Contact lens polymer-based materials are extensively used in the optical industry owing to their excellent corrosion resistance, the possibility of mass production and their ability to be processed without external lubrication. Owing to the fast growth in optical industries, contact lens (CL) requires high accuracy and a high surface quality. The demand for high-accuracy and minimal surface roughness drives the development of ultra-high precision machining technology with regard to single point diamond turning (SPDT). Ultra-high precision diamond turning is an advanced manufacturing technique employed in the machining of CLs owing to its capability of producing high optical surfaces with complex shapes and nanometric accuracy. Yet, even with the advances in ultra-high precision machining (UHPM), it is not continuously easy to achieve a highquality surface finish during polymers machining as the adhesion of the tool chip around the tool dictates the presence of electrostatic charges. The electrostatic charges encountered by a cutting tool when turning advanced CLs are important as they reflect the quality and condition of the tool, machine, fixture, and sometimes even the finished surface, which is responsible for tool wear and poor surface quality. This study investigates the role of cutting parameters, namely cutting speed, feed rate and depth of cut on surface roughness (Ra), electrostatic charge (ESC) and material removal rate (MRR), which determines machine economics and the quality of machining contact lens polymers. The experiments were mainly conducted on two different advanced polymeric materials: polymethyl methacrylate (PMMA) and Optimum Extreme (Roflufocon E) CLs. Experimentation was carried out on the Nanoform 250 ultra-grind turning machine with a monocrystalline diamond-cutting tool for machining the PMMA and Roflufocon E CL polymers, covering a wide range of machining parameters. Before conducting the experiments, a design of experiment was conducted according to the response surface methodology (RSM) that is based on the Box-Behnken Design (BBD). In addition, the research study focused on the determination of the optimum cutting conditions leading to minimum Ra and ESC as well as maximum productivity in the SPDT of the PMMA and Roflufocon E CL polymers, using a monocrystalline diamondcutting tool. The optimization was based on RSM together with the desirability function approach. In addition, a mathematical model was developed for Ra, ESC and MRR using a RSM regression analysis for PMMA and Roflufocon E CL polymers by means of Design Expert software. RSM allowed for the optimization of the cutting conditions for minimal Ra and ESC as well as maximal MRR, which provides an effective knowledge base for process parameters to enhance process performance in the SPDT of CL polymers. Furthermore, this study also deals with the development of Ra, ESC and MRR prediction models for the diamond turning of PMMA and Roflufocon E CL polymers, using the fuzzy logic based artificial intelligence (AI) method. The fuzzy logic model has been developed in terms of machining parameters for the prediction of Ra, ESC and MRR. To judge the accuracy and ability of the fuzzy logic model, an average percentage error was used. The comparative evaluation of experiments and the fuzzy logic approach suggested that the obtained average errors of Ra, ESC and MRR using the fuzzy logic system were in agreement with the experimental results. Hence, the developed fuzzy logic rules can be effectively utilized to predict the ESC, Ra and MRR of PMMA and Roflufocon E CL polymers in automated optical manufacturing environments for high accuracy and a reduction of computational cost. Moreover, owing to the brittle nature of optical polymers, the Roflufocon E CL polymer requires ductile-mode machining for improved surface quality. Molecular Dynamics (MD) simulation methods are thus applied to investigate the atomistic reaction at the tool/workpiece surface to clearly study and observe conditions occurring at nanometric scale in polymer machining. This research study is particularly concerned with the comparative analysis of experiments and a MD study of the Roflufocon E optical polymer nano cutting approach to the atomistic visualization of the plastic material flow at the tool/workpiece interface during cutting. The simulated MD acting force, machine stresses, and the temperature at the cutting region were evaluated to access the accuracy of the model. Hence, the nanomachining simulations were found to have a correlation to the experimental machining results.
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  • Date Issued: 2020

Ultrasound promoted synthesis, characterization and computational studies of some thiourea derivatives

  • Authors: Odame, Felix , Hosten, Eric C , Lobb, Kevin A , Tshentu, Zenixole R
  • Date: 2020
  • Subjects: To be catalogued
  • Language: English
  • Type: text , article
  • Identifier: http://hdl.handle.net/10962/451199 , vital:75028 , xlink:href="https://doi.org/10.1016/j.molstruc.2020.128302"
  • Description: Synthesis of some thiourea derivatives have been achieved by using ultrasound, the compounds have been characterised using IR, NMR, GC-MS and elemental analysis. The single crystal X-ray structure of N-[(benzyloxy)methanethioyl]benzamide (IV), 1-benzoyl-3-(2-hydroxyethyl)thiourea (V) and 3-benzoyl-1-(1-benzylpiperidin-4-yl)thiourea (VI) has been presented and the bond lengths and bond angles contrasted with computed results. The HOMO and LUMO energy levels as well as the global chemical reactivity descriptors of the compounds have also been computed and discussed. Two comformers were obtained for compounds IV to VI in the molecular Electrostatic potential and the vibrational frequency computations and these have been discussed.
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  • Date Issued: 2020

UN Decade on Ecosystem Restoration 2021–2030: what chance for success in restoring coastal ecosystems?

  • Authors: Waltham, Nathan J , Elliott, Michael , Lee, Shing Yip , Lovelock, Catherine , Duarte, Carlos M , Buelow, Christina , Simenstad, Charles , Nagelkerken, Ivan , Claassens, Louw , Wen, Colin K-C , Barletta, Mario , Connolly, Rod M , Gillies, Chris , Mitsch, William J , Ogburn, Matthew B , Purandare, Jemma , Possingham, Hugh , Sheaves, Marcus
  • Date: 2020
  • Language: English
  • Type: text , article
  • Identifier: http://hdl.handle.net/10962/149836 , vital:38887 , https://doi.org/10.3389/fmars.2020.00071
  • Description: On 1 March 2019, the United Nations (UN) General Assembly (New York) declared 2021–2030 the “UN Decade on Ecosystem Restoration.” This call to action has the purpose of recognizing the need to massively accelerate global restoration of degraded ecosystems, to fight the climate heating crisis, enhance food security, provide clean water and protect biodiversity on the planet. The scale of restoration will be key; for example, the Bonn Challenge has the goal to restore 350 million km2 (almost the size of India) of degraded terrestrial ecosystems by 2030. However, international support for restoration of “blue” coastal ecosystems, which provide an impressive array of benefits to people, has lagged. Only the Global Mangrove Alliance (https://mangrovealliance.org/) comes close to the Bonn Challenge, with the aim of increasing the global area of mangroves by 20% by 2030. However, mangrove scientists have reservations about this target, voicing concerns that it is unrealistic and may prompt inappropriate practices in attempting to reach this target (Lee et al., 2019). The decade of ecosystem restoration declaration also coincides with the UN Decade of Ocean Science for Sustainable Development, which aims to reverse deterioration in ocean health. If executed in a holistic and coordinated manner, signatory nations could stand to deliver on both these UN calls to action.
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  • Date Issued: 2020

Understanding a West African recreational fishery as a complex social-ecological system – a case study of the fishery for giant African threadfin Polydactylus quadrifilis (Cuvier, 1829) in the Kwanza Estuary, Angola

  • Authors: Butler, Edward C
  • Date: 2020
  • Subjects: Cuanza River (Angola) , Fishing -- Angola , Polynemidae , Fishes -- Angola -- Ecology
  • Language: English
  • Type: text , Thesis , Doctoral , PhD
  • Identifier: http://hdl.handle.net/10962/146688 , vital:38548
  • Description: Despite increasing global recognition of the importance of recreational fisheries, their management largely remains poor. This is because they exhibit unique human-nature relationships and are nested within complex social-ecological systems (SESs). Recreational fisheries in the developing world have large potential for socio-economic development, but are generally underappreciated, in terms of their value and their impact, and are poorly governed. This is particularly concerning as they are highly complex and often compete for resources with dependent artisanal and subsistence fisheries. Developing world recreational fishery SESs are not well understood and present an important research gap for improved governance. The general aim of this thesis was to explore the recreational fishery targeting Polydactylus quadrifilis on the Kwanza Estuary, Angola, and provide context for how managers should approach recreational fisheries in the developing world and in Africa. To do this, the recreational fishery SES was explored using a combination of methodologies including those characteristic of traditional fisheries science, and new methods involving biology, sociology, and economics. The thesis contains an introductory chapter, a chapter describing the background, study area and study species, five data chapters and a discussion chapter. Chapter 3 aimed to investigate the reproductive style of P. quadrifilis. Results identified the species as a protandrous hermaphrodite. Evidence to suggest this included degenerating testicular tissue and the presence of early developing ovarian tissues in transitional individuals. Early-stage oocytes were commonly found in the outer area of male regions and residual late-stage spermatids and spermatozoa were found in the luminal space of ovarian regions, suggesting a process of sex change from the outside inwards. Owing to the species’ reliance on large highly fecund females for reproduction, it is likely that P. quadrifilis will be sensitive to fisheries that target larger individuals, such as trophy recreational fisheries and line fisheries within other sectors. Chapter 4 aimed to investigate alternative methods for adequately describing the growth of P. quadrifilis individuals belonging to either one of two distinct hypothetical life-history pathways: pathway I (‘changers’) – initial maturation as a primary male followed by a sex change to female; pathway II (‘non-changers’) – initial maturation as a male fish with no subsequent sex change, using von Bertalanffy Growth Functions (VBGFs). Other specific objectives included determining the size- and age-at-maturity and size- and age-at-sex-change for P. quadrifilis. Otolith aging revealed rapid growth and early maturation (L50 = 399.2 mm FL, A50 = 1.50 years) and sex change occurred over a wide size (790–1125 mm FL) and age (3–8 years) range. There was strong evidence for partial protandry in P. quadrifilis with several extremely old male fish (up to 22 years) observed in the population. When compared to the conventional model produced for the entire population, there were significant differences in the models for the ‘non-changers’ (LRT, p < 0.01) and their parameters L∞ (full model = 130.8, ‘non-changers’ = 113.3, p < 0.01), k (0.32, 0.44, p < 0.01) and t0 (0.23, 0.43, p = 0.03) in the first approach and the models (LRT, p < 0.01) and their L∞ (‘changers’ = 113.7, p < 0.01) values in the second approach. This suggests that utilising conventional modelling techniques may be inappropriate for the stock assessment and management of P. quadrifilis and, potentially, other sequentially hermaphroditic fishery species. Chapter 5 aimed to assess the sensitivity of P. quadrifilis to recreational C&R within the foreign recreational fishery using a rapid assessment approach. To do this, a number of C&R variables including fight time, air exposure, hook placement, hooking injury, total time of the stress event, river depth and angling method were measured and related to two indicators of fish health and survival – the physiological stress indicators blood glucose and blood lactate concentration and reflex action mortality predictors (RAMPs). Air exposure was identified as a major contributor to motor impairment (Cumulative Link Model: p < 0.01) and fight time was an important contributor to motor impairment via its interaction with air exposure (Cumulative Link Model: p = 0.02). Handling practices appear to be particularly important for larger individuals as fish size was positively correlated with air exposure (Pearson’s r coefficient = 0.41, p < 0.01) and fight times (0.88, p < 0.01). The findings suggest that recreational C&R may result in mortalities directly, via C&R, and indirectly, via predation, and several recommendations were made for best practice. Chapter 6 aimed to assess the direct economic contribution of the recreational fishery for Polydactylus quadrifilis on the Kwanza Estuary. Results indicated that the recreational fishery for contributed significantly to the economy of an area that would otherwise likely receive little external input ($282 054 per four-month fishing season). However, high rates of economic leakage from the study area were identified (58.7%–92.9% of locally spent revenue) and were attributed to the sourcing of lodge supplies, services and staff outside of the local area and the repatriation of profit by foreign business owners. Capacity building within the local community is likely required to develop ‘linkages’ between the local community and the recreational fishery. Greater community involvement in the fishery is suggested to incentivise the protection of recreationally important fishery species and their associated ecosystems. Chapter 7 aimed to investigate the resource user groups involved within the SES. Results illustrated that artisanal and domestic recreational anglers are well-established and are characterised by long histories of participation. The artisanal fishery was highly valued as a source of livelihoods for the local community. Artisanal fishers were eager for involvement in the recreational sector, through the chartering of their vessels, due to the attractiveness of extra earnings. Both recreational and artisanal fishers reported recent decreases in P. quadrifilis catch and anticipated further declines. Domestic recreational anglers appeared to be highly consumptive in their use of the fishery and C&R angling was uncommon. User conflict may be problematic for future management as recreational anglers perceived the artisanal gill-net fishery to be a threat towards P. quadrifilis stocks. In conclusion, the open-access nature of the fishery was identified as the most pertinent threat to its sustainability and likely needs to be addressed. Potential solutions involve offering users the opportunity to purchase access rights (e.g. day permits), thus initiating the concept that users must pay for their use of public resources. Management should aim to protect large female fish due to their increased reproductive value and worth as trophy fish. Thus, C&R angling is likely to be an important interaction between users and the resource. However, angler behaviour will need to be manipulated to promote C&R and minimise C&R-related mortalities. Solutions include angler educational drives and interventions and the implementation of competitive C&R-only angling. Foreign recreational fisheries, although touted as potential ecotourism ventures, will only succeed in improving the lives of local people if they fully integrate the community into the operation of the fishery.
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  • Date Issued: 2020

Understanding biotic interactions in invaded pond communities in the Sundays River irrigation network, South Africa

  • Authors: Mofu, Lubabalo
  • Date: 2020
  • Subjects: Mozambique tilapia -- South Africa -- Sundays River (Eastern Cape) , Western mosquitofish -- South Africa -- Sundays River (Eastern Cape) , Gobiidae -- South Africa -- Sundays River (Eastern Cape) , Clupeidae -- South Africa -- Sundays River (Eastern Cape) , Reservoirs -- South Africa -- Sundays River (Eastern Cape) , Zooplankton -- South Africa -- Sundays River (Eastern Cape) , Freshwater ecology -- South Africa -- Sundays River (Eastern Cape) , Biotic communities -- South Africa -- Sundays River (Eastern Cape) , Fishes -- Effect of temperature on -- South Africa -- Sundays River (Eastern Cape) , Stable isotopes , Relative Impact Potential
  • Language: English
  • Type: Thesis , Doctoral , PhD
  • Identifier: http://hdl.handle.net/10962/167089 , vital:41436
  • Description: The Sundays River valley irrigation ponds provide a unique opportunity to investigate biotic interactions within a biological invasions context, as they contain both native and non-native fish species. This study focusses on two native species (Glossogobius callidus and Gilchristella aestuaria) and two non-native species (Oreochromis mossambicus and Gambusia affinis). The ecology of the ponds was driven by physico-chemical variables, mainly temperature, but the interactions between fishes were a complex interplay between temperature, pond community ecology and food web structure. Seasonal changes in temperature and subsequent fluctuations in water levels resulted in changes in zooplankton community. Chlorophyll-a, temperature, G. callidus and G. affinis were the drivers of the seasonal changes in macroinvertebrate composition. Stable isotope analysis identified substantial ontogenetic dietary shifts in all species, corresponding to changes in body size. Stable isotope analysis revealed that the niche space occupied by G. affinis was broad and overlapped with that of the other three focal species. Stable isotope metrics showed that G. affinis and O. mossambicus utilised a wide range of resources compared to G. callidus and G. aestuaria. Stomach content analysis showed that G. callidus, O. mossambicus and G. affinis fed predominantly on benthic resources, while G. aestuaria fed mainly plankton resources. Functional response experiments revealed that G. callidus and G. affinis both displayed Type II functional responses. In single fish trials, G. affinis had significantly higher functional responses than G. callidus. In heterospecific G. callidus-G. affinis combinations the functional response of G. callidus was reduced by the presence of G. affinis, whereas, this combination greatly enhanced G. affinis functional response magnitudes. The functional response of G. callidus, O. mossambicus and G. affinis under two temperature treatments along with fish abundance data was used to determine temporal differences in the ecological impacts of each fish species between seasons. The relative impact potential of O. mossambicus was consistently higher than that of G. callidus and G. affinis. This study demonstrates how seasonal temperature fluctuations affect the relative impact capacities of introduced species. Overall, this thesis showed that high temperature along with life-history traits contributes to the biotic interactions between native and non-native species in novel environments.
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  • Date Issued: 2020

Understanding climate change and rural livelihoods in Zimbabwe: adaptation by communal farmers in Ngundu, Chivi District

  • Authors: Nciizah, Elinah
  • Date: 2020
  • Subjects: Agriculture -- Zimbabwe , Agriculture -- Climatic factors -- Chivi District (Zimbabwe) , Chivi District (Zimbabwe) -- Rural conditions
  • Language: English
  • Type: text , Thesis , Doctoral , PhD
  • Identifier: http://hdl.handle.net/10962/118765 , vital:34666
  • Description: Climate change and variability is a global phenomenon which has deeply localised patterns, dynamics and effects. Amongst those people who are particularly vulnerable to climate change effects are small-scale farmers who are dependent in large part on rain-fed agriculture in pursuing their livelihoods. This is true of small-scale farmers in contemporary Zimbabwe and, more specifically, farmers in communal areas. At the same time, at international and national levels, there are attempts currently to minimise the effects of, and to adapt to, climate change. However, adaptation measures also exist at local levels amongst small-scale farmers, such as communal farmers in Zimbabwe. In this context, as its main objective, this thesis examines climate change and small-scale farmer livelihood adaptation to climate change with specific reference to communal farmers in Chivi District in Zimbabwe and, in particular, in Ward 25 which is popularly known as Ngundu. In pursuing this main objective, a number of subsidiary objectives are addressed, including a focus on the established livelihoods of Ngundu farmers, the perceptions and concerns of Ngundu farmers about climate change, the coping and adaptation measures of Ngundu farmers, and the enablements and constraints which affect attempts by Ngundu farmers to adopt such measures. The fieldwork for the thesis involved a diverse array of research methods, such as a questionnaire survey, life-history interviews, key informant interviews, focus group discussions and transect walks. In terms of theoretical framing, the thesis makes use of both middle-level theory (the Sustainable Livelihoods Framework) and macro-theory in the form of the sociological work of Margaret Archer. Combined, these two theories allow for a focus on both structure and agency when seeking to understand livelihood adaptations to climate change by communal farmers in Ngundu. The thesis concludes that there are massive constraints inhibiting adaptation measures by Ngundu farmers, but that this should not distract from the deep, often historically-embedded, concerns of Ngundu farmers about climate change and the multiple ways in which they express agency in and through adaptation and coping activities. It also highlights the need for more specifically sociological investigations of climate change and small-scale farmer adaptation, as well as the need for localised studies which are able to identify and analyse the specificities of adaptation.
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  • Date Issued: 2020

Understanding individual, family and community perspectives on delaying early birth among adolescent girls

  • Authors: Samandari, Ghazaleh , Sarker, Bidhan Krishna , Grant, Carolyn , Talukder, Aloka , Mahfuz, Sadia Nishat , Brent, Lily , Nitu, Syeda N.A. , Aziz, Humaira , Gullo, Sara
  • Date: 2020
  • Subjects: To be catalogued
  • Language: English
  • Type: text , article
  • Identifier: http://hdl.handle.net/10962/281153 , vital:55697 , xlink:href="https://doi.org/10.1186/s12905-020-01044-z"
  • Description: Background: Pregnancy among adolescent girls in Bangladesh is high, with 66% of women under the age of 18 reporting a first birth; this issue is particularly acute in the northern region of Bangladesh, an area that is especially impoverished and where girls are at heightened risk. Using formative research, CARE USA examined the underlying social, individual and structural factors influencing married girls’ early first birth and participation in alternative opportunities (such as education or economic pursuits) in Bangladesh. Methods: In July of 2017, researchers conducted in-depth interviews of community members in two sub-districts of northern Bangladesh (Kurigram Sadar and Rajarhat). Participants (n = 127) included adolescent girls (both married and unmarredi), husbands of adolescent girls, influential adults in the girls’ lives, community leaders, and health providers. All interviews were transcribed, coded and organized using Dedoose software. Results: Participants recognize the health benefits of delaying first birth, but stigma around infertility and contraceptive use, pressure from mothers-in-law and health provider bias interfere with a girl’s ability to delay childbearing. Girls’ social isolation, lack of mobility or autonomy, and inability to envision alternatives to early motherhood compound the issue; provider bias may also prevent access to methods. While participants agree that pursuit of education and economic opportunities are important, better futures for girls do not necessarily supersede their marital obligations of childrearing and domestic chores. Conclusions: Findings indicate the need for a multi-level approach to delaying early birth and stimulating girls’ participation in economic and educational pursuits. Interventions must mitigate barriers to reproductive health care; train adolescent girls on viable economic activities; and provide educational opportunities for girls. Effective programs should also address contextual issues by including immediate members of the girls’ families, particularly the husband and mother-in-law.
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  • Date Issued: 2020

Understanding of biological teleology from a naturalistic perspective

  • Authors: Abrahams, Sanaa
  • Date: 2020
  • Subjects: Teleology , Biology -- Philosophy , Evolution (Biology) -- Philosophy
  • Language: English
  • Type: text , Thesis , Masters , MA
  • Identifier: http://hdl.handle.net/10962/140534 , vital:37896
  • Description: To the extent that teleological thinking is metaphysically suspect, many theorists attempt to shift the stigma of functional explanations by reducing function ascriptions, and aim thus to de-legitimise an appeal to teleological causal relations in an analysis of function. The point is to dispel the mystery which envelops the application of function concepts by reformulating biological functional explanations so as to dispense with teleology. My project is to interrogate the success with which teleological explanations have thus been eliminated in the biological sciences, and, over the course of this thesis, I conclude that a kind of teleological causation nevertheless remains the most adequate explanatory ground of natural products. My proposal is that functional explanations are causal explanations for the presence and maintenance of self-reproducing systems. I contend that, insofar as the attribution of function presupposes the valuation of a function-bearing system as a causal necessity for its constituent parts, functional explanation references distinct and irreducible holistic properties. Using Kantian metaphysics to frame the discussion, this thesis aims first to explore critically the subject of functional characterisations of biological phenomena, and second, the metaphysical basis of modern science. Its chief contributions to the philosophical function debate reside in proposing novel arguments in justification of what I consider is an improved formulation of an attempted definition of biological function, in which teleological causal powers are explicitly recognised and accommodated in functional explanation. Moreover, this thesis attempts a naturalistic reconstruction of the metaphysical entailments of the real causality of a whole
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  • Date Issued: 2020

Understanding of the underlying resistance mechanism of the Kat-G protein against isoniazid in Mycobacterium tuberculosis using bioinformatics approaches

  • Authors: Barozi, Victor
  • Date: 2020
  • Subjects: Mycobacterium tuberculosis , Isoniazid , Drug resistance in microorganisms , Proteins -- Microbiology
  • Language: English
  • Type: text , Thesis , Masters , MSc
  • Identifier: http://hdl.handle.net/10962/146592 , vital:38540
  • Description: Tuberculosis (TB) is a multi-organ infection caused by rod-shaped acid-fast Mycobacterium tuberculosis. The World Health Organization (WHO) ranks TB among the top 10 fatal infections and the leading the cause of death from a single infection. In 2017, TB was responsible for an estimated 1.3 million deaths among both the HIV negative and positive populations worldwide (WHO, 2018). Approximately 23% (roughly 1.7 billion) of the world’s population is estimated to have latent TB with a high risk of reverting to active TB infection. In 2017, an estimated 558,000 people developed drug resistant TB worldwide with 82% of the cases being multi-drug resistant TB (WHO, 2018). South Africa is ranked among the 30 high TB burdened countries with a TB incidence of 322,000 cases in 2017 accounting for 3% of the world’s TB cases. TB is curable and is clinically managed through a combination of intensive and continuation phases of first-line drugs (isoniazid, rifampicin, ethambutol, and pyrazinamide). Second-line drugs which include fluoroquinolones, injectable aminoglycoside and injectable polypeptides are used in cases of first line drug resistance. The third-line drugs include amoxicillin, clofazimine, linezolid and imipenem. These have variable but unproven efficacy to TB and are the last resort in cases of total drug resistance (Jilani et al., 2019). TB drug resistance to first-line drugs especially isoniazid in M. tuberculosis has been attributed to single nucleotide polymorphisms (SNPs) in the catalase peroxidase enzyme (katG), a protein important in the activation of the pro-drug isoniazid. The SNPs especially at position 315 of the katG enzyme are believed to reduce the sensitivity of the M. tuberculosis to isoniazid while still maintaining the enzyme’s catalytic activity - a mechanism not completely understood. KatG protein is important for protecting the bacteria from hydro peroxides and hydroxyl radicals present in an aerobic environment. This study focused on understanding the mechanism of isoniazid drug resistance in M. tuberculosis as a result of high confidence mutations in the katG through modelling the enzyme with its respective variants, performing MD simulations to explore the protein behaviour, calculating the dynamic residue network analysis (DRN) of the variants in respect to the wild type katG and finally performing alanine scanning. From the MD simulations, it was observed that the high confidence mutations i.e. S140R, S140N, G279D, G285D, S315T, S315I, S315R, S315N, G316D, S457I and G593D were not only reducing the backbone flexibility of the protein but also reducing the protein’s conformational variation and space. All the variant protein structures were observed to be more compact compared to the wild type. Residue fluctuation results indicated reduced residue flexibility across all variants in the loop region (position 26-110) responsible for katG dimerization. In addition, mutation S315T is believed to reduce the size of the active site access channel in the protein. From the DRN data, residues in the interface region between the N and C-terminal domains were observed to gain importance in the variants irrespective of the mutation location indicating an allosteric effect of the mutations on the interface region. Alanine scanning results established that residue Leucine at position 48 was not only important in the protein communication but also a destabilizing residue across all the variants. The study not only demonstrated change in the protein behaviour but also showed allosteric effect of the mutations in the katG protein.
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  • Date Issued: 2020

Understanding popular resistance to xenophobia in South Africa: ‘people think’ and the possibility of alternative politics

  • Authors: Parker, Jemima
  • Date: 2020
  • Subjects: Xenophobia -- South Africa , Nationalism -- South Africa , Lazarus, Sylvain -- Political and social views , Political culture -- South Africa , Social change -- South Africa -- Political aspects , Discourse analysis -- South Africa -- Political aspects , South Africa -- Politics and government -- 1994- , Political science -- Philosophy , Political sociology -- South Africa
  • Language: English
  • Type: text , Thesis , Masters , MA
  • Identifier: http://hdl.handle.net/10962/115006 , vital:34069
  • Description: This thesis is concerned with the crisis of xenophobia in South Africa. It argues, firstly, that xenophobia itself is not primarily a reaction to poverty, inequality, or any other set of social conditions. Rather, xenophobia must be considered to be a collective political discourse which has arisen in post-apartheid South Africa from an exclusionary conception of state nationalism. Where this work may be distinguished from the majority of research on xenophobia in South Africa is in the fact that its particular focus is on instances where ‘ordinary’ South Africans have challenged and resisted xenophobic violence in their communities through collective political mobilisation. I suggest that these sites of resistance deserve careful consideration in their own right. I argue that they may demonstrate a subjective break with the oppressive politics of state nationalism through the affirmation of alternative political conceptions. Drawing on the political theory of Sylvain Lazarus, and his principal thesis that people are capable of thinking politics in ways which can subjectively think beyond the social and the extant (underscored by his political and methodological axiom, people think), this thesis argues that these sites of resistance show that people – and especially those who are considered to be marginalised from the domain of legitimate politics – can and do think politically, and it is in the thought of people that new and potentially emancipatory visions of politics may emerge.
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  • Date Issued: 2020

Understanding the context of multifaceted collaborations for social-ecological sustainability: A methodology for cross-case analysis

  • Authors: Cockburn, Jessica J , Schoon, Michael , Cundill, Georgina , Robinson, Cathy , Aburto, Jamie A , Alexander, Steve M , Baggio, Jacopo A , Barnaud, Cecile , Chapman, Mollie , Llorente, Marina G , Garcia-Lopez, Gustavo A , Hill, Rosemary , Speranza, Chinwe I , Lee, Jean , Meek, Chanda L , Rosenberg, Eureta , Schultz, Lisen , Thondhlana, Gladman
  • Date: 2020
  • Subjects: To be catalogued
  • Language: English
  • Type: text , article
  • Identifier: http://hdl.handle.net/10962/370725 , vital:66371 , xlink:href="https://doi.org/10.5751/ES-11527-250307"
  • Description: There are limited approaches available that enable researchers and practitioners to conduct multiple case study comparisons of complex cases of collaboration in natural resource management and conservation. The absence of such tools is felt despite the fact that over the past several years a great deal of literature has reviewed the state of the science regarding collaboration. Much of this work is based on case studies of collaboration and highlights the importance of contextual variables, further complicating efforts to compare outcomes across case-study areas and the likely failure of approaches based on one size fits all generalizations. We expand on the standard overview of the field by identifying some of the challenges associated with managing complex systems with multiple resources, multiple stakeholder groups with diverse knowledges/understandings, and multiple objectives across multiple scales, i.e., multifaceted collaborative initiatives. We then elucidate how a realist methodology, within a critical realist framing, can support efforts to compare multiple case studies of such multifaceted initiatives. The methodology we propose considers the importance and impact of context for the origins, purpose, and success of multifaceted collaborative natural resource management and conservation initiatives in social-ecological systems.
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  • Date Issued: 2020

Understanding the links between smallholder farmers and retailers of meat and vegetables in Mthatha, Mqanduli, Port St Johns and Tsolo, Eastern Cape, South Africa

  • Authors: Qaula, Nyaniso
  • Date: 2020
  • Subjects: Farms, Small
  • Language: English
  • Type: Thesis , Masters , M Agric
  • Identifier: http://hdl.handle.net/10353/18823 , vital:42811
  • Description: It is commonly accepted that one of the challenges faced by smallholder farmers is lack of ‘access to markets’. This is especially so against the backdrop of the supermarket revolution, which is widely understood to give advantage to large-scale farmers at the expense of smallholder farmers. Given that supermarkets are now well established even within South Africa’s former homelands, it would seem that even smallholders based in these areas are at a disadvantage. The purpose of this study is to delve deeply into the market access of smallholder farmers, and to do so specifically across a large area of the former Transkei where supermarkets are well established and where there are also a range of other types of retailers. The study seeks to understand the links between smallholder farmers and diverse kinds of food retailers of meat and vegetables in and around the towns of Mthatha, Mqanduli, Port St Johns and Tsolo in the Eastern Cape Province, South Africa. For each of these sites, the study estimates the share of purchased fresh vegetables and meat that ultimately originates from smallholder farmers within the former Transkei, as opposed to large-scale farmers further afield. The findings suggest that smallholder farmers account for about 24% to 40% of fresh vegetables, and 3% to 13% of meat. The difference between vegetables and meat seems to relate to the fact that smallholder farmers account for a reasonably large share of the fresh vegetables sold by at least some supermarkets, whereas smallholder livestock farmers are largely unable to sell to abattoirs, whether because of the standards imposed by these abattoirs (including the one in Mthatha), or because of the cost of transport. The differences between sites appear to be a function of several factors, including differential market size and resource endowments. The study further sought to identify the advantages and disadvantages that the different types of retailers experience in procuring from smallholder farmers, and also identify the advantages and disadvantages that smallholder farmers experience in supplying different types of retailers. The advantage of working with smallholder farmers is that smallholder farmers can even deliver on Sundays, unlike other suppliers that refuse to work during weekends. The disadvantage is that smallholder farmers are not producing consistently therefore it is hard to rely on smallholder farmers for supply of produce. The advantage that smallholder farmers experience in supplying different types of retailers is that they get their cash right away after delivering the produce. The challenge that smallholder farmers experience when supplying food retailers is that some of the supermarkets will see the sample and order large quantity, however on the day of delivery they will complain and say it is not the good quality so the farmers must reduce the selling price. The study suggests the strategy that could be used by farmers to access the market, which is to enhance knowledge of market skills and negotiation skills.
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  • Date Issued: 2020

Understanding the livelihoods of Zimbabwean informal traders in South Africa: the case of Makhanda

  • Authors: Musiyandaka, Tariro Henrietta
  • Date: 2020
  • Subjects: Informal sector (Economics) South Africa Makhanda , Foreign workers, Zimbabwean South Africa Makhanda Economic conditions , Foreign workers, Zimbabwean South Africa Makhanda Social conditions , Street vendors South Africa Makhanda
  • Language: English
  • Type: Master's theses , text
  • Identifier: http://hdl.handle.net/10962/164535 , vital:41127
  • Description: Increasingly, Zimbabweans are migrating from their country for both economic and political reasons, with South Africa being the primary destination. In seeking employment in South Africa, Zimbabweans face numerous initial problems, including the high unemployment rate in the country alongside restrictions on their employment in the formal economy. In this context, Zimbabweans often turn to work in the informal economy, including as informal traders. This thesis seeks to understand the lives and livelihoods of Zimbabwean informal traders in Makhanda in the Eastern Cape Province of South Africa. Drawing upon the Sustainable Livelihoods Approach, and in the light of existing literature on Zimbabweans more broadly in South Africa, the thesis examines the livelihoods of a purposeful sampled grouping of six informal traders from Zimbabwe in Makhanda. It discusses their reasons for leaving Zimbabwe, their journey from Zimbabwe to Makhanda, relationships amongst themselves and their ongoing relationships with family back home, as well as their hopes and plans for the future. It also examines more specifically their livelihood activities, the daily challenges they face in pursuing their livelihoods and concerns about their livelihood status in South Africa. Despite the many deep-rooted systemic obstacles confronting these Zimbabwean informal traders, the thesis concludes that they demonstrate significant micro-level ingenuity in pursuing their livelihoods in South Africa. , Thesis (MA) -- Faculty of Faculty of Humanities, Sociology, 2020
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  • Date Issued: 2020

Understanding the Pyrimethamine drug resistance mechanism via combined molecular dynamics and dynamic residue network analysis:

  • Authors: Amusengeri, Arnold , Tata, Rolland B , Tastan Bishop, Özlem
  • Date: 2020
  • Language: English
  • Type: text , article
  • Identifier: http://hdl.handle.net/10962/163022 , vital:41005 , https://doi.org/10.3390/molecules25040904
  • Description: In this era of precision medicine, insights into the resistance mechanism of drugs are integral for the development of potent therapeutics. Here, we sought to understand the contribution of four point mutations (N51I, C59R, S108N, and I164L) within the active site of the malaria parasite enzyme dihydrofolate reductase (DHFR) towards the resistance of the antimalarial drug pyrimethamine. Homology modeling was used to obtain full-length models of wild type (WT) and mutant DHFR. Molecular docking was employed to dock pyrimethamine onto the generated structures. Subsequent all-atom molecular dynamics (MD) simulations and binding free-energy computations highlighted that pyrimethamine’s stability and affinity inversely relates to the number of mutations within its binding site and, hence, resistance severity.
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  • Date Issued: 2020

Understanding the relationship between mangrove composition, stand dynamics and resource use in the Ntafufu estuary, Eastern Cape, as basis for sustainable resource use

  • Authors: Mvakade, Monica , Rajkaran, Anusha
  • Date: 2020
  • Subjects: Estuarine ecology -- South Africa -- Eastern Cape , Aquatic ecology , Marine sciences , Fresh water
  • Language: English
  • Type: Thesis , Masters , MSc
  • Identifier: http://hdl.handle.net/10948/49215 , vital:41611
  • Description: Traditionally, coastal communities along the Wild Coast in the Eastern Cape, used mangrove forests for their livelihoods and socio-economic needs. Mangroves along the Eastern Cape coast are threatened by both natural and human factors. The wood obtained from the mangroves by rural households was used as poles for house and fence construction, firewood, and traditional medicine. This is a threat to the status of several mangrove species in this and surrounding areas. The harvesting of mangrove trees will have a direct and indirect effect on the other biotic and abiotic components of the mangrove ecosystems. The Department of Agriculture, Forestry and Fisheries (DAFF) (currently the Department of Environment, Forestry and Fisheries, or DEFF), through the National Forest Act no 84 of 1998, was directly responsible for the wellbeing of the national forests in South Africa. This also includes the mangroves as a specific national forest type. Several estuaries with mangroves occur within state forests. The harvesting and clear-felling of trees for timber and land conversion and associated waste of harvested mangrove poles and laths causes the many changes in mangrove forests. The aim of this study was to develop a framework for sustainable management of the mangroves for the Mtambalala forest management unit. This was done in relation to the requirements of the current legislation, policy guidelines and business plan of the management authority. The aim was pursued through two specific objectives. The first objective was to understand the variation in the composition in different parts of the Ntafufu Estuary, and the population dynamics of the main mangrove species, as basis for the development of sustainable resource use practices. Plots along transects were used to measure seedling to adult ratios, population structure and abiotic characteristics. The second objective was to understand the community use and requirements of mangrove wood from the Ntafufu Estuary. A participatory approach was followed to collect demographic data from each community, the requirements of mangrove poles and behaviour of harvesters. This was used to set up a framework to guide the sustainable management of mangroves based on the current status and stand dynamics of the mangrove communities. There are three mangrove species found in the Ntafufu Estuary: Bruguiera gymnorhiza (black mangrove) regenerates under the mangrove canopy and forms layers of even-aged stands. Avicennia marina (white mangrove) does not regenerate under a mangrove canopy i.e. the seedlings establish outside the canopy. A few Rhizophora mucronata (red mangrove) trees exist in the lower intertidal zone and form multiple stems on stilt roots. The mangrove associate, Hibiscus tiliaceus (wild cotton tree- mangrove associate), forms dense clusters of scrambling stems. Some of the Avicennia individuals show signs of mangrove bleeding and one individual of Bruguiera showed indications of disease. Mngazana and Ntafufu Estuaries had the highest tree densities of 2594 and 1402 trees ha-1 respectively, typical of riverine mangrove forest. The seedling to adult ratio was high in this study: 12.8 for B. gymnorrhiza and 24.3 for A. marina. Abandoned cut stems and laths were found during the study. Bark harvested for traditional medicine and cattle feeding in mangroves was also prevalent and expected to reduce the health of the forest. Mangroves in the Ntafufu Estuary are expanding, and the population structure of the three mangrove species showed a healthy balance between young and mature stems. The harvesting of Black mangrove (B. gymnorrhiza) poles for mainly roof construction is relatively limited. As part of the functions of the DAFF, the use of the mangrove resources has to be balanced with the resource availability from the mangroves, and this should result in a healthy functioning mangrove forest. Development of a participatory forest management (PFM) plan for a forest estate and its forest management unit, require an understanding of the mangrove communities and their dynamics, and the current level of resource use associated with the mangroves. The study recommended that a Mangrove Pole Harvesters Group is needed to practice silviculture through guidance of relevant governmental personnel.
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  • Date Issued: 2020

Understanding the underlying resistance mechanism of Mycobacterium tuberculosis against Rifampicin by analyzing mutant DNA - directed RNA polymerase proteins via bioinformatics approaches

  • Authors: Monama, Mokgerwa Zacharia
  • Date: 2020
  • Subjects: Mycobacterium tuberculosis , Rifampin , Drug resistance , Homology (Biology) , Tuberculosis -- Chemotherapy
  • Language: English
  • Type: text , Thesis , Masters , MSc
  • Identifier: http://hdl.handle.net/10962/167508 , vital:41487
  • Description: Tuberculosis or TB is an airborne disease caused by the non-motile bacilli, Mycobacterium tuberculosis (MTB). There are two main forms of TB, namely, latent TB or LTB, asymptomatic and non-contagious version which according to the World Health Organization (WHO) is estimated to afflict over a third of the world’s population; and active TB or ATB, a symptomatic and contagious version which continues to spread, affecting millions worldwide. With the already high reported prevalence of TB, the emergence of drug-resistant strains has prompted the development of novel approaches to enhance the efficacy of known drugs and a desperate search for novel compounds to combat MTB infections. It was for this very purpose that this study was conducted. A look into the resistance mechanism of Rifampicin (Rifampin or RIF), one of the more potent first-line drugs, might prove beneficial in predicting the consequence of an introduced mutation (which usually occur as single nucleotide polymorphisms or SNPs) and perhaps even overcome it using appropriate therapeutic interventions that improve RIF’s efficacy. To accomplish this task, models of acceptable quality were generated for the WT and clinically relevant, RIF resistance conferring, SNPs occurring at codon positions D516, H526 and S531 (E .coli numbering system) using MODELLER. The models were accordingly ranked using GA341 and z-DOPE score, and subsequently validated with QMEAN, PROCHECK and VERIFY3D. MD simulations spanning 100 ns were run for RIF-bound (complex) and RIF-free (holo) DNA-directed RNA polymerase (DDRP) protein systems for the WT and SNP mutants using GROMACS. The MD frames were analyzed using RMSD, Rg and RMSF. For further analysis, MD-TASK was used to analyze the calculated dynamic residue networks (DRNs) from the generated MD frames, determining both change in average shortest path (ΔL) and betweenness centrality (ΔBC). The RMSD analysis revealed that all of the SNP complex models displayed a level instability higher than that of the WT complex. A majority of the SNP complex models were also observed to have similar compactness to the WT holo when looking at the calculated Rg. The RMSF results also hinted towards possible physiological consequences of the mutations (generally referred to as a fitness cost) highlighted by the increased fluctuations of the zinc-binding domain and the MTB SI α helical coiled coil. For the first time, to the knowledge of the authors, DRN analysis was employed for the DDRP protein for both holo and complex systems, revealing insightful information about the residues that play a key role in the change in distance between residue pairs along with residues that play an essential role in protein communication within the calculated RIN. Overall, the data supported the conclusions drawn by a recent study that only concentrated on RIF-resistance in rpoB models which suggested that the binding pocket for the SNP models may result in the changed coordination of RIF which may be the main contributor to its impaired efficacy.
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  • Date Issued: 2020

Underutilisation of information communication and technology in the building construction industry case study : department of public works in the Amathole district in the Eastern Cape

  • Authors: Camngca, Vuyokazi Precious
  • Date: 2020
  • Subjects: Construction industry -- Technological innovations -- South Africa -- Amathole District Municipality.
  • Language: English
  • Type: Thesis , Masters , MBE
  • Identifier: http://hdl.handle.net/10948/49407 , vital:41718
  • Description: This study investigated the causes and effects of the underutilisation of Information Communication Technology (ICT) in the building section of the Amathole Region. The construction industry’s daily processes demand both heavy usages of data and data communication between project participants to meet client requirements. This industry is characterised by inaccurate and untimely communications that often result in costly delays and extensions of the project timeframe. ICT is a potential solution to this problem. The objectives of the study are, therefore, to determine why the above, a technically skilled occupational government institution in the Eastern Cape, does not employ ICT efficiently and optimally during this time of the looming global fourth industrial revolution. The literature review detailed the causes of and impact level of ICT’s utilisation and its potential benefits. For data collection, the researcher held unstructured interviews with a Chief Quantity Surveyor, Chief Architect, Engineer, Building Inspectors, and a Quantity Surveyor who dealt directly with construction projects. The key findings revealed that a lack of understanding of existing and newly available ICT software and hardware technology existed amongst certain senior officials within the building technology. The previously alluded, combined with the fear of the inability to learn and apply such new technology, has led to a culture of resistance to change from these more senior officials to utilise applications such as AutoCAD. The above-mentioned resistance, has also triggered the prevention of access to and lack of ICT resources, training, and inadequate funding, thereby eventually resulting in the underutilisation of ICT within the whole building section. This change also adversely affects all officials, especially the junior officials who have graduated using the most recent ICT technology during their studies. The researcher deduced, from the above findings, that adequate change management and continuous development, combined with the allocation of proper resources, would be necessary for all staff members. It was also determined that investments had to be made in the ICT equipment through the provision of a sufficient budget in the building section at AR.
  • Full Text:
  • Date Issued: 2020

Underutilisation of information communication and technology in the building construction industry case study: Department of Public Works in the Amathole district in the Eastern Cape

  • Authors: Camngca, Vuyokazi Precious
  • Date: 2020
  • Subjects: Construction industry -- Information technology , Construction industry -- Data processing Information technology Communication and technology
  • Language: English
  • Type: Thesis , Masters , MSc
  • Identifier: http://hdl.handle.net/10948/50632 , vital:42280
  • Description: This study investigated the causes and effects of the underutilisation of Information Communication Technology (ICT) in the building section of the Amathole Region. The construction industry’s daily processes demand both heavy usages of data and data communication between project participants to meet client requirements. This industry is characterised by inaccurate and untimely communications that often result in costly delays and extensions of the project timeframe. ICT is a potential solution to this problem. The objectives of the study are, therefore, to determine why the above, a technically skilled occupational government institution in the Eastern Cape, does not employ ICT efficiently and optimally during this time of the looming global fourth industrial revolution. The literature review detailed the causes of and impact level of ICT’s utilisation and its potential benefits. For data collection, the researcher held unstructured interviews with a Chief Quantity Surveyor, Chief Architect, Engineer, Building Inspectors, and a Quantity Surveyor who dealt directly with construction projects. The key findings revealed that a lack of understanding of existing and newly available ICT software and hardware technology existed amongst certain senior officials within the building technology. The previously alluded, combined with the fear of the inability to learn and apply such new technology, has led to a culture of resistance to change from these more senior officials to utilise applications such as AutoCAD. The above-mentioned resistance, has also triggered the prevention of access to and lack of ICT resources, training, and inadequate funding, thereby eventually resulting in the underutilisation of ICT within the whole building section. This change also adversely affects all officials, especially the junior officials who have graduated using the most recent ICT technology during their studies. The researcher deduced, from the above findings, that adequate change management and continuous development, combined with the allocation of proper resources, would be necessary for all staff members. It was also determined that investments had to be made in the ICT equipment through the provision of a sufficient budget in the building section at AR.
  • Full Text:
  • Date Issued: 2020

Unfair discrimination relating to mental illness

  • Authors: Skosana, Hlengiwe
  • Date: 2020
  • Subjects: Discrimination -- Mental health
  • Language: English
  • Type: Thesis , Masters , LLM
  • Identifier: http://hdl.handle.net/10948/47443 , vital:39991
  • Description: Section 9 of the Constitution of the Republic of South Africa, 1996 provides that everyone has the right to equality. The Employment Equity Act, the Promotion and Prevention of Unfair Discrimination Act and the Labour Relations Act were promulgated to fulfil the Constitution’s goals of promoting equality and prohibiting unfair discrimination. Section 6(1) of The Employment Equity Act promotes equality in the workplace and prohibits unfair discrimination; section 187 of the Labour Relations Act provides that dismissals on the grounds listed in the section are automatically unfair; and section 6 of the Promotion and Prevention of Unfair Discrimination Act prohibits unfair discrimination in general. International law prohibits unfair discrimination. Article 1 of the International Labour Organisation’s Convention concerning Discrimination in Respect of Employment and Occupation provides that discrimination in the workplace that has the effect of impairing or nullifying equal opportunity or treatment is prohibited; Article 7 of the United Nations Declaration of Human Rights provides that all are equal before the law and are entitled to protection against any discrimination; and the United Nations Charter and the African United Charter on Human and People’s Rights both promote the right to equality. South Africa has signed and/or ratified these international anti-discrimination laws, and is, accordingly, bound by them. The South African anti-discrimination legislation and international anti-discrimination laws prohibit unfair discrimination, whether it is direct or indirect, or on a listed (that is, a specified ground such as race or disability) or unlisted ground, on a policy, rule or practice. Discrimination based on a listed ground is presumed to be unfair. If discrimination is based on an unlisted ground, the complainant must prove that the discrimination is unfair by applying the Harksen v Lane NO 1997 (11) BCLR 1489 (CC) test. The Harksen v Lane NO test asks whether the discrimination has the attributes and characteristics which have the potential to impair the fundamental human dignity of persons as human beings or to affect them adversely in a comparably serious manner. If the answer is yes, the discrimination is unfair. Mental illness is not a listed ground, meaning it is unlisted. The Labour Appeal Court and Labour Court have, therefore, had to consider whether discriminating against an employee on the basis of their mental illness is unfair, or dismissing them because of their mental illness constituted an automatically unfair dismissal. In the leading case of New Way Motor & Diesel Engineering (Pty)Ltd v Marsland [2009] 12 BLLR 1181 (LAC), the Labour Appeal Court applied the Harksen v Lane NO test and held that the employee’s dismissal was automatically unfair in terms of section 187(1)(f) of the Labour Relations Act. In the recent case of Jansen v Legal Aid South Africa (JA121/2014) [2018] ZALCCT 17, the Labour Court agreed with the Labour Appeal Court decision in New Way Motor & Diesel Engineering (Pty) Ltd v Marsland that dismissing an employee because of their mental illness is automatically unfair in terms of section 187(1)(f) of the LRA, and unfair discrimination in terms of section 6(1) of the EEA. Despite the consistency in case law, the anti-discrimination legislation does not list mental illness as an unfair ground, which means unfairness cannot be presumed. The complainant must, accordingly, prove the dismissal is automatically unfair in terms of section 187(1)(f) of the LRA or that it is unfair discrimination in terms of section 6(1) of the EEA.
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  • Date Issued: 2020

Unravelling the replication biology of Providence virus in a cell culturebased model system

  • Authors: Jarvie, Rachel Anne
  • Date: 2020
  • Subjects: Virology -- Research , RNA viruses , Viruses -- Reproduction , Providence virus
  • Language: English
  • Type: text , Thesis , Masters , MSc
  • Identifier: http://hdl.handle.net/10962/142339 , vital:38071
  • Description: There has been an increase in the number of viral outbreaks in the last decade; the majority of these are attributed to insect-human or animal-human transfer. Despite this awareness, there is limited understanding of the replication biology of the viruses causing the outbreaks and there are few model systems that are available to study RNA virus replication and viral persistence. In this study, we describe a Providence (PrV)-based model system to study virus replication biology. PrV is a single-stranded RNA virus that can cross Kingdom boundaries; it is capable of establishing a productive infection in insect and mammalian cell culture and it is also capable of replicating in plants. Only one other virus has been reported to infect a similar host range - the Nodavirus, Flock House virus (FHV). First, we performed a bioinformatic analysis of the PrV genome and validated the tools that were currently available to work with this model system in mammalian cells. Our data indicate that PrV infection of human cervical cancer (HeLa) cells results in the production of p130, p104/p40 and VCAP, albeit at low levels. While PrV replication in insect cells is associated with the Golgi apparatus and secretory vesicles, in HeLa cells, PrV replication is associated with the mitochondria. It is interesting to note that FHV replication factories are located on the outer mitochondrial membrane. In an attempt to study PrV virus replication in vitro, we adapted the BioID system reported by Roux et al. (2012). Here a promiscuous biotin ligase enzyme (BirA) was fused to a protein of interest and the expression of the fusion protein in mammalian cells resulted in the proximitybased biotinylation of proteins associated with the protein of interest. Using p40 as the protein of interest, we studied the fusion protein (BirA-p40) in transiently transfected HeLa cells and in a stable cell line, using western blot analysis and confocal microscopy. We faced challenges comparing the data collected using the two antibody-based detection techniques and the lack of BirA-p40 detection when using western analysis was attributed to the associated of p40 with detergent resistant membranes. BirA-p40 was subsequently expressed using in vitro coupled transcription/translation reactions, in the presence of excess biotin. While BirA-p40 was robustly expressed under these conditions, biotinylation of BirA-p40 was not detected. We attributed this to the conditions used in the experiments and given additional time, we would extend the duration of biotinylation, in vitro. PrV replication in mammalian cells was detectable using confocal microscopy however the levels of fluorescence were relatively low. The knowledge that p40 was associated with detergent resistant membranes led us to question the impact of detergent treatment of live cells on the detection of PrV replication. PrV-infected HeLa cells were treated with detergents with varying biochemical characteristics and the impact of these treatments on the detection of PrV replication were evaluated. We observed that linear and non-ionic detergents, namely NP-40 and Triton X-100, were most effective at enhancing the detection of viral replication in PrV-infected HeLa cells. Our data confirm that detergent treatment results in enhanced detection, and not enhanced PrV replication, in HeLa cells. Using the stable BirA-p40 expressing HeLa cell line, we showed that the protein is associated with membranes in vitro, and that the enhanced expression of BirA-p40 results in the formation of greater volumes of detergent-resistant membranes. In addition, detergent treatment of unfixed PrV-infected HeLa cells revealed the presence of the PrV p40 protein in the nucleoli of the cells. This is the first report of PrV proteins, which are translated in the cytosol of the mammalian cells, occurring in the nucleus. Our study has resulted in a deeper understanding of PrV replication in mammalian cell lines. A ‘simple RNA virus’ with only three predicted open reading frames has exhibited high levels of complexity within its elegant simplicity. This study has also highlighted the challenges associated with studying RNA virus replication biology in vitro. Looking forward, the identification of detergent-based enhancement for the detection of PrV replication provides the opportunity to perform more targeted PrV replication studies. The PrV-based model system can also be applied to the identification and analysis of potential broad-spectrum antiviral drugs in vitro. The latter application is particularly relevant considering the increase in the number of viral outbreaks over the last decade.
  • Full Text:
  • Date Issued: 2020
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