Faculty of Science (ETDs)
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Item Investigating 2-hydroxypropyl-β-cyclodextrin (HPβCD) as a novel therapeutic agent for breast cancer(University of the Witwatersrand, Johannesburg, 2019) Saha, Sourav Taru; Kaur, MandeepCancer cells have an increased need for cholesterol, which is required for cell membrane integrity. Cholesterol accumulation has been described in various malignancies including breast cancer. Cholesterol has also been known to be the precursor of estrogen and vitamin D, both of which play a key role in the histology of breast cancer. Elevated cholesterol levels have been linked to breast cancer therefore depleting cholesterol levels in cancer cells can be a viable strategy for treatment. 2-hydroxypropyl-β-cyclodextrin (HPβCD) is a cholesterol depleting compound which is a cyclic amylose oligomer composed of glucose units. It solubilizes cholesterol and is proven to be toxicologically benign in humans. This led us to hypothesise that it might deplete cholesterol from cancer cells and may prove to be a clinically useful compound. Our work provides experimental evidences to support this hypothesis. We identified the potency of HPβCD in vitro against two breast cancer cell lines: MCF7 (Estrogen positive, ER+), MDA-MB-231 [Triple negative breast cancer (TNBC)], and compared the results against two normal cell lines: MRC-5 (Normal Human Lung Fibroblasts) and HEK-293 (Human embryonic kidney) using cytotoxic, apoptosis and cholesterol based assays. HPβCD treatment reduced intracellular cholesterol resulting in significant breast cancer cell growth inhibition through apoptosis. The results hold true for both ER+ and TNBC. We have also tested HPβCD in vivo in MF-1 mice xenograft model and obtained 73.9%, 94% and 100% reduction in tumour size for late, intermediate and early stage TNBC. These data suggest that HPβCD can prevent cholesterol accumulation in breast cancer cells and is a promising anti- cancer agentItem Characterising the Role of Cholesterol in Hypoxia-induced Epithelial- Mesenchymal Transition in Breast Cancer(University of the Witwatersrand, Johannesburg, 2022) Abdulla, Naaziyah; Kaur, MandeepThe cellular epithelial-mesenchymal transition (EMT) process is a complex labyrinth dependent on subversion of critical cellular signalling pathways, which crosstalk extensively to confer cancer cells with characteristics that mediate metastasis. Based on the pleotropic role of cholesterol in the cell, it is not surprising that cancer cells have evolved several mechanisms to facilitate cholesterol dyshomeostasis. In addition to meeting the increased metabolic demands of cancer cells, deregulated cholesterol metabolism also facilitates increased cellular cholesterol availability which is crucial to regulating the activity of protein intermediates in EMT-related signalling pathways. Despite evidence indicating that cholesterol directly regulates signalling pathways related to EMT, no publication to date has attempted to address the effect of EMT induction on cellular cholesterol levels in cancer. To shed light on the dynamics of cholesterol in the relationship between hypoxia and EMT, cholesterol content in MCF-7 cells pre- and post-hypoxia induced EMT was assessed. This dissertation presents findings indicating increased levels of free cholesterol, cholesteryl esters as well as lipid raft cholesterol in MCF-7 cells following hypoxia-induced EMT. Interestingly, MCF-7 cells post- EMT induction displayed increased sensitivity to treatment with cholesterol targeting agents and presented with reversion to an epithelial state as evidenced by the increased expression of epithelial markers, decreased expression of mesenchymal markers and also reduced invasive potential. Importantly, treatment with cholesterol targeting agents is also seen to abrogate the drug resistant potential following hypoxia-induced EMT. Based on these observations, it is proposed that targeting cellular cholesterol could be a promising area to invest in the search for novel therapeutics effective in combatting cancer metastasisItem Influence of polyploidy on morphology, genetic differentiation and reproductive strategy amongst varieties of Rhodohypoxis baurii (Hypoxidaceae)(University of the Witwatersrand, Johannesburg, 2023) Ferreira, Bianca Tasha; Goodman-Cron, Glynis V.; Glennon, Kelsey L.Evolutionary mechanisms, such polyploidy (increase in chromosome sets), alters plant morphology, gene flow and reproductive strategies, which can facilitate the generation or loss of species. Rhodohypoxis L. (Hypoxidaceae) is a small near-endemic Drakensberg genus comprising six species, one of which is Rhodohypoxis baurii. Rhodohypoxis baurii contains three morphologically distinct varieties, with varying ploidy-levels: R. baurii var. baurii (2×, 4×), R. baurii var. platypetala (2× 3× , 4×), and R. baurii var. confecta (2×). Therefore, R. baurii is an ideal system to evaluate whether polyploidy leads to lineage divergence or homogenization and contributes to biodiversity in this lineage. The aim of the study was to assess the influence of ploidy on morphology, genetic differentiation and reproductive strategy among varieties of Rhodohypoxis baurii (Hypoxidaceae), as well as to better recognise the three varieties. Thirty vegetative, floral, and reproductive traits were measured across 124 herbarium specimens and 43 individuals housed in the greenhouse. A matrix containing 20 quantitative and 12 qualitative characters was constructed and a Principal Coordinates Analysis (PCoA) and Non-Metric Multidimensional Scaling analysis (NMDS) were conducted. Important distinguishing morphological features that had a high eigenvalue (shown via a PCA) were selected for direct comparison using box and whisker box plots to compare means ± standard errors (SE). Certain morphological traits such as anther length, peduncle length and tepal sizes differed significantly among the varieties and ploidy levels, with polyploid individuals exhibiting the gigas effect. This was especially evident in R. baurii var. platypetala, which contained many polyploid individuals and exhibited larger flowers (longer and wider tepals) and larger anthers compared to other varieties. Rhodohypoxis baurii var. confecta and R. baurii var. platypetala are genetically, geographically and morphologically similar, differing only in flower colour, flower size and peduncle length. However, most of these differences can be attributed to differences in ploidy and /or altitude with R. baurii var. confecta occurring at higher altitudes and R. baurii var. platypetala containing multiple ploidy-levels. It is therefore evident that ecological differences and polyploidy have shaped the morphological differences in these two taxa. In addition, R. baurii var. baurii populations in the 7 Eastern Cape Drakensberg were morphologically, geographically and genetically distinct from all other populations and varieties, and may be a new/ undescribed taxon; however, this warrants further investigation. Out of 231 experimental crosses, 113 intra-varietal and inter-varietal crosses produced seeds. Rhodohypoxis baurii polyploid individuals show a shift away from sexual reproduction to asexual reproduction as they all showed higher rates of clonal reproduction than the diploid individuals. Moreover, crosses between polyploids yielded lower seed sets and lower germination rates than diploid-diploid crosses. Genetic differentiation and gene flow were quantified for 280 individuals among the varieties and ploidy-levels across 11 populations using 12 microsatellite markers labelled with the FAM NED dyes. Leaf material was collected from 237 individuals of Rhodohypoxis baurii (R. baurii var. confecta n = 88, R. baurii var. baurii n = 87, R. baurii var. platypetala n = 62) and flow cytometry conducted to estimate ploidy. A latitudinal ploidy gradient was evident across sampled populations that corresponds with shifts in reproductive strategy, and changes in the extent of gene flow. Population genetic structure coincided primarily with geographic localities, with diploid Northern Drakensberg populations having similar allelic diversity to one another. The Central and Southern Drakensberg mixed ploidy populations also showed similar allelic diversity but differed from the tetraploid Eastern Cape Drakensberg populations. Furthermore, gene flow was higher between geographically close populations irrespective of ploidy-level, with geographically isolated regions (such as the Eastern Cape Drakensberg) and outlying populations (i.e. Karkloof) showing unique genotypes, indicating little gene flow and allele sharing. Consequently, shifts in reproductive strategy and geographic isolation are likely changing gene flow patterns among varieties and ploidy levels which appears to be facilitating both lineage diversification and homogenization in this speciesItem Symmetry reductions and approximate solutions for heat transfer in slabs and extended surfaces(University of the Witwatersrand, Johannesburg, 2023-06) Nkwanazana, Daniel Mpho; Moitsheki, Raseelo JoelIn this study we analyse heat transfer models prescribed by reaction-diffusion equations. The focus and interest throughout the work is on models for heat transfer in solid slabs (hot bodies) and extended surface. Different phenomena of interest are heat transfer in slabs and through fins of different shapes and profiles. Furthermore, thermal conductivity and heat transfer coefficients are temperature dependent. As a result, the energy balance equations that are produced are nonlinear. Using the theory of Lie symmetry analysis of differential equations, we endeavor to construct exact solutions for these nonlinear models. We will employ a number of symmetry techniques such as the classical Lie point symmetry methods, the nonclassical symmetry, nonlocal and nonclassical potential symmetry approach to construct the group-invariant solutions. In order to identify the forms of the heat source term that appear in the considered equation for which the principal Lie algebra (PLA) is extended by one element, we first perform preliminary group classification of the transient state problem. Also, we consider the direct group classification method. Invariant solutions are constructed after some reductions have been performed. One-dimensional Differential Transform Method (1D DTM) will be used when it is impossible to determine an exact solution. The 1D DTM has been benchmarked using some exact solutions. To solve the transient/unsteady problem, we use the two-dimensional Differential Transform Method (2D DTM). Effects of parameters appearing in the equations on the temperature distribution will be studied.Item Insights into silver(I) phosphine complexes in targeting cell death and metastatic mechanisms in malignant cell lines(University of the Witwatersrand, Johannesburg, 2023-09) Roberts, Kim Elli; Engelbrecht, Zelinda; Cronjé, Marianne J.Cancer is the leading cause of death worldwide, with 18.1 million new cases and 9.6 million deaths reported annually. Cisplatin, a popular chemotherapeutic drug, exhibits certain limitations in terms of selectivity and efficacy. This emphasizes the necessity for novel therapeutic approaches in addressing a variety of cancer types. Multiple studies have shown that silver-based compounds suppress cancer cell proliferation and induce apoptosis. Thirteen novel silver(I) mono-dentate phosphine complexes were investigated for their anticancer effects on seven different human malignant cell lines; A375 non-pigmented melanoma, A549 lung adenocarcinoma, HEP-G2 hepatocellular carcinoma, HT-29 colorectal adenocarcinoma, MCF-7 and MDA-MB-231 breast adenocarcinoma, and SNO oesophageal squamous cell carcinoma. Two non-malignant human cell lines, HEK-293 embryonic kidney cells and MRHF foreskin fibroblast cells, were used to assess the selectivity of the complexes. Cisplatin and the efficient silver(I) phosphine complexes were selected for dose-response experiments to determine IC50 concentrations for the respective cell lines. On the basis of these screening results (chapter two), five difficult-to-treat cancer cell lines, and their most efficient complexes were selected for further investigation. Various cellular characteristics were investigated in chapter three (A549, HEP-G2, HT-29); these included morphological changes, ATP levels, GAPDH levels, Ptd-L-Ser externalization, mitochondrial membrane potential, oxidative stress levels, and the activity of a metabolic enzyme, cytochrome P450 isoform CYP1B1. The antimetastatic activity of the selected complexes was assessed by evaluating their ability to impede the migration of A549 cells. The fourth chapter examines the anticancer effect of selected complexes on hormone-dependent (MCF-7) versus triple-negative (MDA-MB-231) breast cells. Changes in morphology, Ptd-L-Ser externalization, alterations in mitochondrial membrane potential, oxidative stress levels, cytochrome c release, and DNA damage were studied. Furthermore, in chapter five, molecular docking simulations were used to determine whether the most potent silver(I) phosphine complex across all cell lines bonds to estrogen receptor alpha (ER-α) and estrogen receptor beta (ER-β). Seven of the thirteen silver(I) phosphine complexes significantly reduced cell viability in malignant cell lines while being less toxic to non-malignant cells. Complex 4 best targeted all cancer types, with IC50 values ranging from 5.75 to 10.80 µM across malignant cell lines. In the malignant treated cells, morphological changes, reactive oxygen species production, mitochondrial membrane depolarization, and Ptd-L-Ser externalization were observed. Complexes 1 and 4 repressed cell migration in the A549 cells. The presence of damaged nuclei, metabolically inactive mitochondria and cytochrome c translocation from the mitochondria’ intermembrane to the cytosol in MCF-7 cells were observed. These findings suggest that complexes 2, 4 and 7 induced apoptotic cell death. Furthermore, in silico computational predictions suggested a promising interaction between complex 4, and ER-α and ER-β. Overall, this study demonstrates the potential of silver(I) phosphine complexes as anticancer agents, with promising effects on various cancer cell lines.Item The application of machine learning methods to satellite data for the management of invasive water hyacinth(University of the Witwatersrand, Johannesburg, 2023-06) Singh, Geethe; Reynolds, Chevonne; Byrne, Marcus; Rosman, BenjaminBiological invasions are responsible for some of the most devastating impacts on the world’s ecosystems, with freshwater ecosystems among the worst affected. Invasions threaten not only freshwater biodiversity, but also the provision of ecosystem services. Tackling the impact of invasive aquatic alien plant (IAAP) species in freshwater systems is an ongoing challenge. In the case of water hyacinth (Pontederia crassipes, previously Eichhorniae crassipes), the worst IAAP presents a long-standing management challenge that requires detailed and frequently updated information on its distribution, the context that influences its occurrence, and a systematic way to identify effective biocontrol release events. This is particularly urgent in South Africa, where freshwater resources are scarce and under increasing pressure. This research employs recent advances in machine learning (ML), remote sensing, and cloud computing to improve the chances of successful water hyacinth management. This is achieved by (i) mapping the occurrence of water hyacinth across a large extent, (ii) identifying the factors that are likely driving the occurrence of the weed at multiple scales, from a waterbody level to a national extent, and (iii) finally identifying periods for effective biocontrol release. Consequently, the capacity of these tools demonstrates their potential to facilitate wide-scale, consistent, automated, pre-emptive, data-driven, and evidence-based decision making for managing water hyacinth. The first chapter is a general introduction to the research problem and research questions. In the second chapter, the research combines a novel image thresholding method for water detection with an unsupervised method for aquatic vegetation detection and a supervised random forest model in a hierarchical way to localise and discriminate water hyacinth from other IAAP’s at a national extent. The value of this work is marked by the comparison of the user (87%) and producer accuracy (93%) of the introduced method with previous small-scale studies. As part of this chapter, the results also show the sensor-agnostic and temporally consistent capability of the introduced hierarchical approach to monitor water and aquatic vegetation using Sentinel-2 and Landsat-8 for long periods (from 2013 - present). Lastly, this work demonstrates encouraging results when using a Deep Neural Network (DNN) to directly detect aquatic vegetation and circumvents the need for accurate water extent data. The two chapters that follow (Chapter 3 and 4 described below) introduce an application each that build off the South African water hyacinth distribution and aquatic vegetation time series (derived in Chapter 2). The third chapter uses a species distribution model (SDM) that links climatic, socio-economic, ecological, and hydrological conditions to the presence/absence of water hyacinth throughout South Africa at a waterbody level. Thereafter, explainable AI (xAI) methods (specifically SHapley Additive exPlanations or SHAP) are applied to better understand the factors that are likely driving the occurrence of water hyacinth. The analyses of 82 variables (of 140 considered) show that the most common group of drivers primarily associated with the occurrence of water hyacinth in South Africa are climatically related (41.4%). This is followed by natural land cover categories (32.9%) and socio-economic variables (10.7%), which include artificial land-cover. The two least influential groups are hydrological variables (10.4%) including water seasonality, runoff, and flood risk, and ecological variables (4.7%) including riparian soil conditions and interspecies competition. These results suggest the importance of considering landscape context when prioritising the type (mechanical, biological, chemical, or integrated) of weed management to use. To enable the prioritisation of suitable biocontrol release dates, the fourth chapter forecasts 70-day open water proportion post-release as a reward for effective biocontrol. This enabled the simulation of the effect of synthetic biocontrol release events under a multiarmed bandit framework for the identification of two effective biocontrol release periods (late spring/early summer (mid-November) and late summer (late February to mid-March)). The latter release period was estimated to result in an 8-27% higher average open-water cover post-release compared to actual biocontrol release events during the study period (May 2018 - July 2020). Hartbeespoort Dam, South Africa, is considered as a case study for improving the pre-existing management strategy used during the biocontrol of water hyacinth. The novel frameworks introduced in this work go a long way in advancing IAAP species management in the age of both ongoing drives towards the adoption of artificial intelligence and sustainability for a better future. It goes beyond (i) traditional small-scale and infrequent mapping, (ii) standard SDMs, to now include the benefits of spatially explicit model explainability, and (iii) introduces a semi-automated and widely applicable method to explore potential biocontrol release events. The direct benefit of this work, or indirect benefits from derivative work outweighs both the low production costs or equivalent field and lab work. To improve the adoption of modern ML and Earth Observation (EO) tools for invasive species management, some of the developed tools are publicly accessible. In addition, a human-AI symbiosis that combines strengths and compensates for weaknesses is strongly recommended. For each application, directions are provided for future research based on the drawbacks and limitations of the introduced systems. These future efforts will likely increase the adoption of EO-derived products by water managers and improve the reliability of these products.Item Assessing aquifer vulnerability to landfill pollution using drastic method in Gauteng, South Africa(University of the Witwatersrand, Johannesburg, 2023) Mphaphuli, Idah; Abiye, TamiruThis study integrated the DRASTIC method and field investigations into mapping the degree of vulnerability of aquifers to landfill pollution in the Gauteng Province, which is one of the most populated provinces in South Africa. In order to investigate the aquifer vulnerability of Gauteng's heterogeneous and complex geology, the DRASTIC method was used to generate intrinsic and specific vulnerability maps. Three vulnerability classes were generated from the DRASTIC index, namely, low vulnerability, moderate vulnerability and high vulnerability, which covered 46%, 37% and 17% of the study area, respectively. The highly-vulnerable areas were associated with the karst aquifer of Malmani dolomite, permeable vadose zone, high hydraulic conductivity and loamy sand/sandy loam soil type, whilst moderately-vulnerable areas were associated with fractured/weathered aquifers, high recharge and low topography. The intrinsic vulnerability was validated using average NO3+NO2-N (nitrate + nitrite as nitrogen) and the results of water samples from field investigations conducted in Marie Louise and Robinson landfill sites. Elevated NO3+NO2-N concentration (9.85-16.03 mg/l) was observed in the highly-vulnerable areas. Water samples were collected, in order to analyse the water chemistry, stable isotopes and radioactive isotopes (tritium). Gibbs and Piper diagrams were used to evaluate the main mechanism controlling the groundwater chemistry and the dominant major ions that influence it. Pollution by leachate was detected in the Marie Louise landfill site, where the groundwater showed high tritium and ammonia concentration. The main hydrochemical facies detected in Marie Louise were Mg SO4, Ca-SO4, Na-SO4 and Na-Cl. The hydrochemical facies detected in Robinson were Na-SO4, Ca-HCO3, Na-Cl and Ca-Cl. The DRASTIC method was shown to be effective in assessing groundwater vulnerability on a regional scale, provided that there is adequate input data.Item Antibacterial activity and susceptibility testing of bacterial isolates from nematodes (Cruznema spp.)(University of the Witwatersrand, Johannesburg, 2023-09) Mothapo, Maletjema Magdeline; Lephoto, Tiisetso E.Nematodes are unsegmented worms found in different niches associated with a diverse range of bacteria. Various types of nematodes exist including those that are parasitic to insects, known as entomopathogenic nematodes (EPNs). EPNS of genera Steinernema, Heterorhabditis and Oscheuis are symbiotically associated with Xenorhabdus, Photorhabdus and Serratia, respectively. The symbiotic bacteria of EPNs have been reported to produce a broad spectrum of antimicrobial compounds active against human pathogens. The aim of this study was to isolate and identify nematodes and their associated bacteria from soil samples collected from a vegetative farm in Lesotho and study their antimicrobial activity against four species of pathogenic bacteria (E. coli, S. aureus, E. faecalis and P. aeruginosa). An uncharacterized species of Cruznema was isolated and named Cruznema NTM-2021 (GenBank 18S rDNA accession number: OQ408141). Based on the BLASTN search incorporating the phylogenetic analysis of the 16S rDNA region, three genera of bacteria were identified as Alcaligenes sp., Enterobacter sp. and Elizabethkingia sp. The study revealed that all three bacterial isolates were pathogenic to Tenebrio molitor. Symbiosis tests, using lipid agar method demonstrated the ability of the host nematodes to develop and reproduce in the presence of their associated bacteria. Bacterial supernatants of Alcaligenes sp. and Enterobacter sp. showed some inhibitory activity against Escherichia coli and Enterococcus faecalis, by disk diffusion method. Staphylococcus aureus and Pseudomonas aeruginosa were the most resistant bacteria to supernatants of the three isolates. This study also showed that the Alcaligenes, Enterobacter, and Elizabethkingia species isolated from Cruznema NTM-2021 were resistant to ampicillin, amoxicillin, cefuroxime/sodium, vancomycin and cephalothin but susceptible to gentamicin.Item Detecting and Understanding COVID-19 Misclassifications: A Deep Learning and Explainable AI Approach(University of the Witwatersrand, Johannesburg, 2023-08) Mandindi, Nkcubeko Umzubongile Siphamandla; Vadapalli, Hima BinduInterstitial Lung Disease (IDL) is a catch-all term for over 200 chronic lung diseases. These diseases are distinguished by lung tissue inflammation (Pulmonary fibrosis). They are histologically heterogeneous dis eases with inconsistent microscopic appearances, but they have clinical manifestations similar to other lung disorders. The similarities in symptoms of these diseases make differential diagnosis difficult and may lead to COVID-19 misdiagnosis with various types of IDLs. Be cause the turnaround time is shorter and more sensitive for diagnosis, imaging technology has been mentioned as a critical detection method in combating the prevalence of COVID-19. The aim of this research is to investigate existing deep learning architectures for the aforementioned task, as well as incorporate evaluation modules to determine where and why misclassification occurred. In this study, three widely used deep learning architectures, ResNet-50, VGG-19, and CoroNet, were evaluated for detecting COVID-19 from other IDLs (bacterial pneumonia, nor mal (healthy), viral pneumonia, and tuberculosis). The baseline results demonstrate the effectivities of Coronet having a classification performance of 84.02% for accuracy, specificity of 89.87%, a sensitivity of 70.97%. Recall 84.12%, and F1 score of 0.84. The results further emphasize the effectiveness of transfer learning using pre-trained domain-specific architectures, resulting in fewer learnable parameters. The proposed work used Integrated Gradients (IG), an Explainable AI technique that uses saliency maps to observe pixel feature importances, to understand mis classifications. This refers to visually prominent features in input im ages that were used by the model to make predictions. As a result, the proposed work envisions future research directions for improved classi fication through misclassification understanding.Item Late Triassic to Early Jurassic ecology: An insight into diet and trophic levels using non-traditional Ca isotopes(University of the Witwatersrand, Johannesburg, 2023-07) Davechand, Priyanka; Bybee, Grant; Choiniere, JonahThe diet and trophic structuring of organisms in deep time is poorly understood, making comparison of ancient and modern ecosystems challenging. Proxy data (e.g., dental morphology, jaw muscle reconstruction) remain the most common mode of palaeodietary inference, but the correlative strength of these proxies remains untested due to a lack of direct evidence and an incomplete sampling of palaeobiodiversity. These major challenges in palaeodietary reconstruction can be overcome using novel geochemical markers in fossilised tooth enamel, which provide direct evidence of palaeodiet and trophic relationships. Traditional stable isotopes of carbon, oxygen, and nitrogen have been used in the past to infer palaeodiets, but these elements are susceptible to diagenetic alteration during fossil preservation and require large sample amounts for assays. In contrast, non-traditional calcium (δ44/42Ca) isotopes are less susceptible to diagenesis and require significantly smaller sample amounts. This, together with the fractionation that Ca isotopes undergo as a bio-essential element, allows δ44/42Ca to be utilised on a broad range of palaeontological questions including assessing dietary range and trophic level. The diverse ecosystems of the Elliot Formation (Karoo Supergroup) in South Africa are represented by abundant fossils of a variety of reptilian and mammalian stem lineages that coexisted during the latest Triassic–earliest Jurassic interval (218–190 Ma). The broad range of body sizes, inferred dietary preferences, and phylogenetic positions make the Elliot palaeoecosystems an ideal natural laboratory in which to apply palaeodietary isotopic tools. This dissertation aims to assess the palaeotrophic divisions of the Elliot Formation vertebrates using non-traditional δ 44/42Ca isotopes. This research uses ion-exchange chromatography on vertebrate tooth enamel to assess the palaeodietary preferences of Elliot Formation reptilian and mammalian lineages. To obtain these data, existing techniques for sample preparation of non-traditional δ 44/42Ca isotopes were modified and optimised at the Wits Isotope Geoscience Laboratory (WIGL) at the University of the Witwatersrand. δ 44/42Ca analysis was conducted on a variety of specimens across a broad range of amniote lineages, ranging from: dinosaurs such as presumed herbivorous sauropodomorphs Massospondylus and Aardonyx, the presumed omnivorous ornithischian (Lesothosaurus and Heterodontosaurus), and the presumed carnivorous theropod Megapnosaurus; to cynodont therapsids (Tritylodon, Pachygenelus and Scalenodontoides); to pseudosuchians such as the crocodylomorphs Protosuchus and Orthosuchus and earlier branching taxa (‘rauisuchians’ and poposauroids). A leaching procedure was also tested to ensure that the results produced were not influenced by diagenetic biases. Once consistent and reproducible methods were finalised, column chemistry and Multicollector-Inductively Coupled Plasma Mass Spectrometery (MC-ICPMS) analysis was conducted on the different Karoo-aged specimens. There are various outcomes from this dissertation. One important outcome was the optimisation of time for Ca separation using ion-exchange chromatography. This allowed for a shorter chemical preparation time and increased the number of analyses completed per session. Another improvement of the method was that the leaching procedure can be used to control for any diagenetic biases by removing secondary calcite in samples as old as those from the Triassic–Jurassic period. Elliot taxa were then analysed, and significant differences were found between δ 44/42Ca values of large carnivorous pseudosuchians (‘rauisuchians’; -0.45 ‰ to -1.17 ‰) and co occurring herbivorous sauropodomorph genera (-0.26 ‰ to -0.69 ‰). These results indicated that non-traditional δ 44/42Ca isotopes can be used to understand trophic structures and palaeodiets in ecosystems at least 210 million years old. We also found that while some taxa had δ44/42Ca isotope values in-line with their presumed diets, other taxa had more diverse diets than initially presumed. δ 44/42Ca-enriched values in this study provide evidence for herbivory in crocodylomorph and the oldest theropod. There is also a possibility of an omnivorous diet for presumed herbivorous Lesothosaurus as the δ 44/42Ca values are relatively depleted to other herbivores. In addition to diet, calcium plays a major role in the formation of reptilian eggs and there are documented changes in δ 44/42Ca values during the reproductive cycle. To assess this in a living system, Crocodylus niloticus, was analysed to understand if δ 44/42Ca could be used to identify the difference in sex based on the δ 44/42Ca values. No isotopic differences were found between the juvenile male and female Crocodylus niloticus samples. Testing these important ecological principles in temporally constrained formations allows us to understand the historical nature of biodiversity changes, especially across periods when environments on Earth were experiencing extreme conditions. The ability to determine factors such as palaeodiet and palaeotrophic range will enable the development and improvement of palaeoecological analysis. This research presents the first ever δ 44/42Ca values on Karoo-aged vertebrate fossils and will have a large impact on how palaeoecological reconstruction is conducted in the future of palaeosciences.