Faculty of Science (ETDs)

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Exploring the Structure, Function and Stability of Glutathione Transferases Engineered from Intra- and Inter-class Consensus Sequences: How Forgiving is Nature?
(University of the Witwatersrand, Johannesburg, 2024-10) Mulenga, Thabelo; Achilonu, Ikechukwu; Sayed, Yasien
Protein folding is an enigmatic biochemical process that is foundational to the structural and functional requirements of a cell. The problem of protein folding, in a nutshell, concerns itself with the rate of protein folding as well as the conversion of amino acids from a linear sequence to a fully folded structure. This problem is partly answered by the existence of folding pathways. The folding funnel was conceptualised as a depiction of folding pathways, and it is a framework that illustrates that native proteins naturally favour the lowest energy state, encountering kinetic and thermodynamic barriers as they fold. Consensus protein design, based on this understanding, aims to: (1) enhance stability and (2) navigate the pitfalls of folding by modifying the folding funnel of a protein. This approach can also shed light on the significance of evolutionarily conserved residues. In this study, consensus protein mutants were generated for the Alpha and Mu glutathione transferases (GSTs) classes. The consensus proteins were then benchmarked against the parental proteins that were chosen (hGSTA1-1 and hGSTM1-1). The Alpha consensus mutant had 11 consensus mutations, including a notable M50L mutation, which affects the dynamic behaviour of helices α2 and α9, while the Mu consensus mutant had 13 unique mutations. Protein production and purification showed that the Mu consensus mutant had larger and purer yields. Data from far-UV circular dichroism studies and root-mean-squared-fluctuation (RMSF) from molecular dynamics (MD) simulations showed that the secondary structural components of the Alpha and Mu proteins remained largely the same, although the Alpha consensus mutant displayed a far lower molar residue ellipticity reading than its wildtype counterpart, indicating the disruption of secondary structural elements, likely caused by the M50L mutation. The ANS binding results showed that the M50L mutation in the Alpha consensus protein caused an increase in exposure of the surface area of the H-site, while the Mu consensus protein had a decrease in the solvent accessibility of its H-site. Thermal shift assay results indicated the consensus proteins had increased thermal stability. Enzyme kinetics results showed that the functionality of the proteins was severely diminished in the consensus mutants, particularly the Alpha consensus mutant. MD simulation results showed that there was an overall increase in the rigidity and compactness of the consensus mutant proteins, further affirming the improvement of thermal stability, while signalling the loss in functionality. The results produced herein have the potential to facilitate the proliferation of engineered GSTs for biotechnological applications that require proteins with an increased half-life and greater stability.
Natural resource use and environmental perceptions in different types of rural villages in Bushbuckridge, South Africa
(University of the Witwatersrand, Johannesburg, 2024-09) Mbedzi, Mary Emma; Twine, Wayne
Households in rural communities in developing countries rely on natural resources that contribute to income generation, food, medicine, cooking energy, and act as livelihood safety nets. Levels of dependency vary between households within communities due to factors such as wealth status and household size. However, less is known about differences in household resource use between communities within an area. Environmental awareness, values, and preferences are shaped by various factors including, local context. Thus, perceptions of the environment and natural resources, and changes in these are likely to vary between communities. This study aimed to compare the levels of natural resource use and environmental perceptions across village types and determine if there is any relationship between the level of natural resource use and environmental perceptions across village types in the Bushbuckridge Local Municipality in the Mpumalanga province, South Africa. Village types were defined and categorised based on their size, socio-economic factors, access to basic services, connectedness, and remoteness. The level of natural resource use was analysed using existing data from a survey of 491 households across 12 villages located in the Agincourt Health and Demographic Surveillance System (AHDSS) site. This included data on the use, collection, buying, and selling of a range of natural resources used locally (e.g., wild foods, firewood, and medicinal plants), annual consumptions, ownership of resources made from natural resources, and livestock (cattle) ownership. The survey included questions on respondents’ perception of the change in the supply of natural resources over the past 5 years (2016-2021) before the survey. Focus group discussions were conducted in 10 of the study villages asking participants their perception of the changes in the environment and the causes thereof, the importance of the natural environment, natural resources used for their daily needs, income generation, their culture, as well as the changes in availability of natural resources and impacts on their livelihoods. The study villages were classified according to a village typology that had been developed for the AHDSS study site, based on factors such as connectedness, access to amenities and services, and the proportion of Mozambican refugee households. ANOVA and Kruskal Wallis tests were used for continual data, while the Chi-squared test was used for categorical data to test for significant differences across village types in the quantitative part of the study. For qualitative, responses of participants from each village were recorded and grouped into village types, and an overall picture of the perception of natural resources that participants used was developed using the focus group schedule. Similarities and differences in natural resources between villages were assessed by grouping widely and less used resources by villages and village types. There was no significant difference (p>0.05) in the mean prevalence of use, collection, buying, and selling of natural resources, or annual consumption of natural resources across villages and village types. Households in better-serviced villages owned more products from natural resources and were to greater extent, likely to own livestock than poorly-serviced villages. As to environmental perceptions, more respondents in the poorly serviced village types (refugee settlements (RS) and underdeveloped communities (UC)) perceived a decrease in the supply of natural resources than better-serviced village types (established communities (EC) and central communities (CC)). Participants in focus group discussions also noted a decrease in the supply of natural resources across all villages and village types. There was a relationship between village types, natural resource use, and perceptions of change in natural resource availability. Established communities correlated with a higher number of resources perceived as decreasing and central communities were associated with higher cattle ownership. Underdeveloped communities and refugee settlements were associated with a higher number of resources used/owned, a higher number of resources used often (more than 4 times a month), and a higher number of resources sold, and buying firewood. Natural resources were more highly used in, better-serviced village types (central communities, and established communities), than in poorly-serviced village types (underdeveloped communities, and refugee settlements), which was unexpected. However, there were quite a few proxies such as the collection and selling of resources, which were used that did not help back up these findings which could explain the unexpected results. Most respondents in remote and poorly-serviced village types perceived a change in the supply of natural resources, the greatest decrease being in the supply of natural resources. This is associated with awareness of the value of the environment and high reliance on natural resources for these poorly-serviced village types. This study suggests that differences exist in resource use patterns between villages, even in the same area. However, village characteristics were not a good predictor of all aspects of resource use. Changes in proxies of resource utilization and re-evaluating the existing village typology would be useful for further studies.
Evaluating the impact of land use activities in and around Lake Kariba on the presence and levels of anions and cations in the water body
(University of the Witwatersrand, Johannesburg, 2024-09) Monyai, Mokgaetji Andelina; Chimuka, Luke; Tutu, Hlanganani; Cukrowska, Ewa; Richards, Heidi L.
Huge seas, lakes, and rivers come to mind when we think of surface water. Surface water is vulnerable to water pollution, with consequential repercussions for the well-being of both human and aquatic environments. Furthermore, the diminishing levels of oxygen have a profound effect on the natural ecological equilibrium within river and lake ecosystems. Lake Kariba, situated in the Southern African region, is a vital freshwater ecosystem supporting local communities, wildlife, and regional economies. However, it faces threats from human activities and erratic weather. This study investigated the influence of land use activities in and around Lake Kariba on water composition and the concentration of anions and cations. The research employed a combination of field surveys and laboratory experiments to identify potential sources of ions. Sixty-nine (69) water samples (53 downstream and 16 upstream) were collected during different seasons in October 2021, July 2022 and April 2023. The Ion Chromatography, Inductively Coupled Plasma equipped with Optical Emission and Mass Spectroscopy detectors were used to concentrations of various anions (Fˉ, Clˉ, NO3ˉ, SO4 2ˉ, and PO4 3ˉ) and cations (Ca, K, Mg, Na, Si, Al, Cr, Fe, Mn, As, Cu, Ni, Ti, and Zn) respectively. Acidic water was notably observed upstream in two sampling areas, namely the Malasha and Kanzinze rivers. The Malasha River exhibited pH levels ranging from 3.71 to 4.81, while the Kanzinze River showed a pH of 6.01. The electrical conductivity (EC) for Malasha ranged from 1035 to 1484 µS/cm, whereas for Kanzinze, it measured 878.0 µS/cm. These areas exhibited significantly elevated levels of both anions and cations. In the Kanzinze River, the detected concentrations showed the following descending order: SO4 2ˉ> Clˉ > NO3ˉ> Fˉ> PO4 3ˉ (anions); Ca > Mg > Na > K > Si > Fe > Al > Zn > Cu > Mn > Ni > Cr > Ti > As (cations). Conversely, the Malasha River, exhibited the following order for anions: SO4 2ˉ > Clˉ > NO3 ˉ > Fˉ > PO4 3ˉ, and for cations: Ca > Fe > Mg > Na > Si > K > Al > Mn > Zn > Cr > Cu> Ni > Ti > As. The significant presence of SO4 2- and NO3 - indicates that human activities and agricultural practices in certain areas of Lake Kariba's catchment can have a considerable impact on the lake's water quality. Despite this, the corresponding Water Quality Index (WQI) indicated that the water quality from Kanzinze and Malasha rivers was unsuitable for drinking purposes. The findings revealed variations in ions concentration at different sampling points, with discernible patterns corresponding to specific land use types, such as mining in the upstream that elevated the levels of SO4 2- and some heavy metals and also NO3 - levels in the downstream due to commercial cage fish farming. Statistical analysis showed significant downstream variations (p < 0.05) in water chemistry parameters related to land use, while upstream areas exhibited no significant differences (p > 0.05). Water quality index ranged from 13.1 to 230.0, categorizing water quality from "excellent" to "very poor." The study underscores the complex interplay between land use activities and water chemistry in Lake Kariba, emphasizing downstream impacts. These findings contribute valuable insights for sustainable management and conservation efforts in the region, considering the dynamic nature of the ecosystem and potential threats posed by anthropogenic activities. Continuous monitoring and mitigation strategies are crucial to reserving the ecological balance of Lake Kariba and safeguarding the well-being of its surrounding communities and wildlife.
Surface water dependencies and activity patterns of mammalian herbivores in South Africa
(University of the Witwatersrand, Johannesburg, 2024-10) Padayachy, Janiel; Hetem, Robyn; Strauss, Willem Maartin; Venter, Jan
An increase in aridity in Africa may make water availability more variable, thus understanding how animals respond to these conditions is important for future wildlife management and conservation. However, mammalian herbivores with varying water requirements may respond differently to changes in water availability and predation. Using camera trap data, I analysed the spatial distribution relative to surface water sources and the 24-hour activity of 16 mammalian herbivores across 10 sites in South Africa. As expected, water dependent herbivores were generally closer to water, but only at sites where lions were absent. Herbivores with low water requirements were more nocturnal, potentially reducing water required to cool themselves evaporatively when active during the heat of the day. But that nocturnal activity was reduced when lions were present, likely reducing predation risk, increasing water requirements to dissipate heat and forcing herbivores to remain close to water. Nocturnal activity increased with body size in mixed-feeders and grazers, but decreased with body size in browsers, potentially reflecting more time spent foraging by large browsers. Using a novel approach of multistate diel occupancy models I showed that herbivores were generally active during both the day and night, and that the presence of lions impacted occupancy of preferred prey species (blue wildebeest, gemsbok and zebra). Diel occupancy of water-dependent prey (blue wildebeest) was influenced by an interaction between lion presence and distance to water sources. Thus environmental factors as well as physiological and morphological features affected the timing of activity and spatial distribution of several mammalian herbivore species in South African wildlife areas, which should be considered for future research and management of these species.
The Gene Catalogue and Functional Analysis of the Gut Microbiome of Lions in Etosha National Park
(University of the Witwatersrand, Johannesburg, 2024-09) Belger, Carl Warner; Hetem, Robyn; Hazelhurst, Scott
Characterising the microbiomes of free-living mammals may aid conservation efforts, yet the gut microbiome of carnivores is underrepresented. This study represents the first description of the gut microbiome of free-living African lions (Panthera leo melanochaita). Faecal samples from 20 lions were collected in Etosha National Park, Namibia and microbial DNA was extracted. Samples were then whole genome sequenced, and classified using MetaPhlAn and Genome Taxonomy Database toolkit. The two most abundant bacterial genera in the lions’ gut microbiomes were Bacteroides (16.9%) and Phocaeicola (16.6%). Microbiome diversity was similar between the sexes and across seasons as assessed through Bray-Curtis dissimilarity and Shannon diversity index. The genus Clostridium_AH was more abundant in male lions (P = 0.007; d.f. = 22), while Aphodousia (P = 0.003; d.f. = 22) was more abundant in females. Lions captured in winter had a high abundance of Plesiomonas relative to those captured in summer (P = 0.008), whereas lions captured in summer a high abundance of Dysosmobacter (P = 0.038; d.f. = 22), Pelethomonas (P = 0.021; d.f. = 22), Metalachnospira (P = 0.033; d.f. = 22) and Clostridium Q (P = 0.012; d.f. = 22) compared to those captured in winter. Following various taxonomic classification approaches, a third of the reads (33.6%) present in the lion gut microbiome remained unclassified. We constructed 272 metagenome assembled genomes, from seven bacterial phyla, representing mostly new species which will contribute to understanding of the carnivore gut microbiome.
The Taxonomy and Phylogeny of Varanopidae from the Middle Permian of outh Africa
(University of the Witwatersrand, Johannesburg, 2024-08) Sibiya, Zoleka; Rubidge, Bruce; Benoit, Julien
Varanopidae are the only pelycosaur-grade tetrapods from the Abrahamskraal Formation of the Beaufort Group. Although four varanopid species are described from the middle Permian of South Africa (SA), their validity has been questionable. Moreover, Ford and Benson recently proposed that varanopids belong to the Diapsida rather than Synapsida. Given this taxonomic and phylogenetic turmoil, this study i) describes two newly discovered and well-preserved varanopid specimens from the middle Permian of SA using CT scanning and ii) re-assesses the validity of the four species from the Karoo Basin in the light of new data from these specimens. The phylogeny of the group is addressed by updating two pre-existing cladistics matrices. The two new specimens exhibit a mosaic of features that overlap the diagnoses of several species of SA varanopids. In addition, some diagnostic traits of the four SA varanopid genera and species are found to be variable across ontogeny. For instance, dorsal osteoderms and the extent of ornamentation on the angular bone co-vary with size. It is therefore proposed that all varanopid specimens currently described from the middle Permian of South Africa belong to a single valid species, Heleosaurus scholtzi, with differences between specimens being accounted for by ontogenetic changes. The phylogenetic analysis supports Ford and Benson’s hypothesis but contra the prediction of this hypothesis, no differences in cranial fenestration during ontogeny are observed. Morphological characters preserved in the manus of the newly discovered specimen BP/1/8499 suggest that Heleosaurus was arboreal.
Investigating the impact of the land reform policy on land use and land cover changes, in Ngaka Modiri Molema district of the North West province
(University of the Witwatersrand, Johannesburg, 2024) Mmangoedi, Molebogeng Precious; Adam, Elhadi
The purpose of this study was to assess how land reform policies affected changes in land use and land cover in the province of North West's Ngaka Modiri Molema district municipality. The study employed remote sensing technologies to analyse changes in land use and land cover (LULC) resulting from the implementation of land reform programs between 1985 and 2015. The primary objective of the research was to systematically map Land Use and Land Cover types across five-year intervals spanning from 1985 to 2024, leveraging Landsat earth observation data in conjunction with a random forest classifier. These methodologies were employed to facilitate the identification of spatial patterns and trends associated with the implementation of land reform policies within the study area. Furthermore, the study utilized Landsat data and advanced change detection algorithms to quantitatively assess LULC changes over the specified timeframes. Through the application of spatial analysis techniques, the research aimed to elucidate the relationship between the implementation of land reform measures and corresponding shifts in LULC patterns across the research study area. The findings of the investigation indicated a noticeable expansion in built-up areas between the years 1985 and 2024 which was approximately 10.86%. This expansion was primarily attributed to the growth experienced by the municipality during this period. Additionally, more opportunities might have risen from the agricultural farming activities and also from the land reform policy being implemented. However, as the ownership changed due to land redistribution and more land was being acquired by black people through the land reform policy, agricultural farming decreased slightly throughout the years. The reduction was due to the factors that arose from inefficient policy implementation. The study also recommends that remote sensing techniques should be utilised to carry out studies to determine LULC changes that derive from land policies aiming at dealing with socio-economic factors and urbanisation. An incorporated agrarian reform sustainable programme has vast potential in cultivating the production of the projects, particularly if it involves packages in rural infrastructure, support services, and co-operatives. The major role of such an approach should be in the trainings conducted for the farmers, obtaining, and distributing agricultural resources and equipment to agrarian reform or beneficiaries of the land reform projects. Additionally, there should be an allowance for special grants which will be useful in supporting the government’s efforts.
Diversity and Abundance of Arthropods on Conventional Sugarcane under Field Conditions in South Africa
(University of the Witwatersrand, Johannesburg, 2024-09) Smith, Roshay; Malinga, Lawrence; Bouwer, Gustav
Insect diversity and abundance are often the base for formulating strategies that involve the appropriate application of pest control methods, considering the ecosystem services provided by insects. Therefore, the aim of this study was to provide recent baseline data on the diversity and abundance of insects in conventional sugarcane based on two sugarcane fields in KwaZulu-Natal. Three sampling methods, namely pitfall, sticky and water pan traps, were used to sample insects in rain-fed and irrigated sugarcane in Gingindlovu and Pongola from March to October 2022. This study collected 12 493 insects belonging to 14 insect orders and 88 families in rain-fed sugarcane and 22 309 insects belonging to 14 orders and 94 families in irrigated sugarcane. Significant differences in the diversity indices were found between the sampling methods and the sampling periods. This study provides recent baseline data on the diversity and abundance of insects in sugarcane.
Machine Learning Algorithms-Based Classification of Lithology using Geophysical Logs: ICDP DSeis Project Boreholes, South Africa
(University of the Witwatersrand, Johannesburg, 2024-09) Atita, Obehi Chapet; Durrheim, Raymond; Saffou, Eric
One of the most significant geosciences tasks is the accurate classification of lithologies for metal and mineral resources exploration, characterization of oil/gas reservoir(s), and the planning and management of mining operations. With the availability of abundant, huge and multidimensional datasets, machine learning-based data-driven methods have been widely adopted to assist in solving geoscientific problems such as the efficient evaluation and interpretation of large datasets. The adoption of machine learning-based methods aims to improve lithological identification accuracy and extract information required for accurate and objective decision-making with respect to activities such as exploration, drilling, mine planning and production. Practically, this helps to reduce working time and operating costs. We aim to evaluate the feasibility of machine learning-based algorithms application to geophysical log data for the automated classification of lithologies based on the stratigraphic unit at the formation level for the purpose of distinguishing and correlating the quartzites between boreholes, and mapping key radioactive zones within the mining horizon. This study implemented four different machine learning algorithms: gradient boosting decision trees, random forest, support vector machine, and K-means clustering models. Analyzed features and labelled datasets are multivariate downhole geophysical and lithology logs from the two ICDP DSeis project boreholes drilled in the Klerksdorp gold field, respectively. To mitigate misclassification error and avoid model overfitting/underfitting, the optimal combination sets and optimal values for each implemented supervised model’s hyperparameters were obtained using the Grid search and 10-fold cross-validation optimization methods. The input dataset was randomly split automatedly into training and testing subsets that made up 80% and 20% of the original dataset, respectively. The models were trained and cross-validated using the training subset, and their performances were assessed using the testing subset. The classification performance of each model was evaluated using F1 scores and visualized using confusion matrices. The best supervised classification model for our study area was selected based on the testing subset F1 scores and computational cost of training models. The testing subset results shows that Random Forest and Support Vector Machine classifier models performed much better relative to the Gradient Boosting Decision Trees classifier model, with F1 scores over 0.80 in borehole A and B. In borehole A and B, Random Forest classifier has the least computational training time of about 14- and 6- hours, respectively. The feature importance results demonstrate that the logging feature P-wave velocity (Vp) is the highest predicting feature to the lithology classification in both boreholes. We find that the quartzite classes at different stratigraphic positions in each borehole are similar and they are correlated between the DSeis boreholes. The K-means clustering revealed three clusters in this study area and effectively map the radioactive zones. This study illustrates that geophysical log data and machine learning-based algorithms can improve the task of data analysis in the geosciences with accurate, reproducible and automated prediction of lithologies, correlation and mapping of radioactive zones in gold mine. This study outputs can serve as quality control measures for future similar studies both in the academic and industry. We identified that availability of large data is the major factor to high accuracy performance of machine learning-based algorithms for classification problems.
An initial estimation of the concentrations of methane (CH4) emitted from the Wakkerstroom wetland and Majuba power station in Mpumalanga province, South Africa
(University of the Witwatersrand, Johannesburg, 2024-08) Bobo, Christine; Scholes, Mary C.; Furniss, David. G.
The continuous increase in methane (CH4) emissions to the atmosphere poses a formidable challenge to Earth's climate resilience and exacerbates the impacts of global warming. This study compared microbial CH4 concentrations from the Wakkerstroom wetland to fossil fuel concentrations from the coal stockpiles at the Majuba power station in Mpumalanga, South Africa, using three methodological approaches: flexfoil bags, isotopic analysis, and satellite data. Seasonal ground measurements were collected in 3L flexfoil bags and analysed for CH4 mole fraction using a Picarro 2301 calibrated to the WMO X2004A CH4 scale. Samples were prepared for isotopic analysis by continuous-flow isotope-ratio mass spectrometry using an Isoprime Trace Gas system. Findings from ground sampling measurements showed that for three out of the four seasons, Phragmites australis at the Wakkerstroom wetland had the highest CH4 concentration [spring (0.209 ± 0.076 ppm), autumn (0.537 ± 0.239 ppm), and winter (0.132 ± 0.065 ppm)], and the coal stockpiles at the Majuba power station had the lowest CH4 concentration [summer (0.022 ± 0.021 ppm), autumn (0.014 ± 0.053 ppm), and winter (0.002 ± 0.004)]. Isotopic measurements, which identify and differentiate gas sources by examining distinct isotopic signatures, showed that the Wakkerstroom wetland had consistently light, 13C depleted isotopic signatures seasonally ranging between -61.4‰ to -55.6‰. In contrast to this, the Majuba power station coal stockpiles had inconsistent isotopic signatures through the sampling seasons- heavier CH4 emission isotopic signatures with 13C enrichment during spring and summer at -35.3 ± 1.0 ‰ and -43.5 ± 3.5 ‰ respectively, and lighter isotopic signatures with 13C depletion during autumn and winter of - 62.0 ± 1.6 ‰ and - 87.0 ± 10.3 ‰ respectively. On a larger temporal and spatial scale, satellite-based measurements from the Tropospheric Monitoring Instrument (TROPOMI) showed no significant differences between Wakkerstroom (1855.4 ± 18.0 ppb) and Majuba power station (1857.1 ± 22.9 ppb) in terms of the total column averaged dry-air mole fraction of methane (XCH4). An increasing trend in CH4 mole fraction, in Southern Africa as measured at Cape Point Global Atmospheric Watch (GAW) station over a 39-year period was also observed. Meteorological variables, particularly wind direction, had a more significant impact on CH4 mole fractions at Cape Point than at the Wakkerstroom wetland. The multi-faceted approach of utilizing the three measurement techniques provided a comprehensive view of atmospheric CH4 showing that wetlands could be more significant CH4 hotspots in comparison to coal stockpiles at power stations. Measuring methane concentrations is challenging due to its inherent complexity, thus the utilisation of different measurement methodologies further highlighted the spatial and temporal variability of CH4 concentrations at both the wetland and the coal stockpiles.

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