Haliotis midae is an economically important marine invertebrate that is farmed in South Africa, contributing more than half of the revenue generated by the aquaculture industry. However, the future sustainability of abalone farming in South Africa is threatened by the ongoing climate crisis. The effect of climate change is unrelenting for organisms such as abalone, which rely on a succinct balance of physico-chemical environmental properties. Indeed, the ocean environment is susceptible to these imbalances and has already witnessed changes in seawater temperatures and pH. Over the last century, global ocean surface temperatures have increased by 0.74°C and seawater pH has declined by 0.1 units, while global predictions for 2100 suggest oceans will experience a decline in pH by 0.3-0.5 units. Thus, ocean acidification (OA) is a growing cause for concern since it adversely affects marine organisms such as corals and calcareous marine invertebrates. Research focusing on the effect of climate change on marine life has grown tremendously over the last two decades, with an emphasis on molluscs such as mussels and oysters which are considered ideal proxies for measuring environmental change and the underlying molecular effects thereof. However, research on abalone in this arena has primarily focused on larvae and the physiological effects of OA on development and shell growth. The underlying molecular mechanisms involved in the Haliotis midae stress response to ocean acidification have largely remained unexplored. Thus, this study sought to elucidate the effect of OA on the haemocyte proteome of H. midae, as well as to gain insight into the underlying molecular mechanisms that characterize the stress response of this abalone species. This study employed a comparative shotgun proteomics approach using isobaric tagging for relative and absolute quantification (iTRAQ) coupled with LC-MS/MS to investigate the proteomic response of H. midae haemocytes to reduced pH conditions representative of future predictions of ocean acidification. Four independent iTRAQ experiments were conducted where haemocytes were sampled after 12, 72 and 168 hours of exposure of H. midae to OA conditions (pH 7.5, represents OA predicted by 2100). Following quantitative analysis, 227 non-redundant and differentially expressed proteins were detected across the four independent experiments. Proteins of statistical significance (p ≤ 0.05) and biological relevance (foldchange ≥ 1.2) were identified. The 227 proteins were grouped according to their expression profile using Weighted Gene Cluster Network Analysis to gain an insight into the biological processes underlying the stress response of H. midae to OA conditions. Sequence similarity, Gene Ontology, data mining and network modelling based on other molluscs and well-characterised genomes were utilized for assigning putative functions to the grouped proteins. This revealed a multifaceted interplay of various biological processes and signaling pathways in H. midae, such as the induction of anaerobic metabolism, cytoskeletal stabilization and the induction of the ERK/MAPK signaling cascade. Notably, this analysis demonstrated a possible link between the stress and immune responses which has only been observed in other molluscs. Their complex association suggests an overlap of pathways, with putative dual functionality of proteins such as MAPK, CAMK, Serpin B2 and haemocyanin, all of which have been implicated in the stress and innate immune responses of other organisms. The combined data from the quantitative (Chapter 2) and functional analyses (Chapter 3) resulted in the compilation of a group of 33 candidate protein biomarkers of OA stress. A holistic and complete picture of the potential regulatory mechanisms involved in the stress response of H. midae was generated through protein-protein interaction network modelling and data mining. Aquarium-based experiments were conducted to validate the candidate OA biomarker proteins, as well as a set of previously identified biomarker candidates of acute temperature stress in H. midae (Calder and Coyne, unpublished), using label-free protein quantification (LFQ) coupled to LC-MS/MS analysis. Furthermore, the effect of a combination of reduced pH and elevated temperature on the candidate biomarker proteins was investigated. Five candidate biomarker proteins of OA stress were validated, while 10 candidates of acute temperature stress were detected and validated. The combined stress condition identified 7 potential biomarkers. Candidate biomarkers of OA stress were predominantly associated with the innate immune system, while those responding to temperature stress were largely associated with energetics and oxidative stress. Potential biomarkers of the combined stressors were found to be associated with signal transduction and intracellular trafficking. This component of the research project not only highlighted the importance of validation in biomarker discovery, but demonstrated the usefulness of an LFQ-proteomics approach for biomarker validation. This is the first time high-throughput shotgun proteomics has been employed to investigate the H. midae haemocyte stress response. This study provides a solid foundation for elucidating the putative functional stress response of a non-model organism, and highlights the complex dynamics and interplay between the stress and immune responses of H. midae. On-farm experimentation will be conducted to test whether the candidate biomarkers identified in this project can reliably detect abiotically stressed H. midae and whether they can be integrated into a “suite” of biomarkers for a health monitoring program for farmed H. midae. Successful implementation of such a health monitoring program will ensure the future sustainability of the South African abalone aquaculture industry despite the severity of climate change.

Kidney stones contain various combinations of chemicals, however, 80% consist of calcium oxalate. Oxalate in humans is either absorbed into the urinary tract and excreted in urine or degraded by gut bacteria. Oxalate-degrading gut bacteria play a critical role in human oxalate homeostasis, evidenced by increased research interest in them as potential probiotics in the management of kidney stone disease. In South Africa, kidney stones are more prevalent in the white than in the black population, despite the latter having a diet that puts them at greater risk of developing kidney stones. It was, therefore, postulated in this study that differences in oxalate-degrading bacteria contributes to the observed South African kidney stone statistics. The major aims of this study were, consequently, to investigate the faecal micro biota of black and white South Africans, with respect to oxalate-degrading bacteria; and to identify and characterise novel oxalate-degrading probiotic candidates. The study population comprised twenty stone-free black and white South African males on a normal diet. Results obtained using PCR detection and denaturing gradient gel electrophoresis (DOGE) analyses showed differences in the oxalate-degrading bacteria between the two populations, with the black population recording a higher incidence of known oxalate-degrading bacterial species (70%) vs. the 30% recorded in the white population. Furthermore, culturable faecal bacteria isolated from the black population had greater oxalate-degrading capacities than those isolated from their white counterparts. Oxalate-degrading gut bacteria could, therefore, contribute to the lower incidence of kidney stones in the black South African population, relative to the white one. Two novel oxalatedegrading Escherichia coli and Clostridium innocuum strains were isolated from the faecal microbiota of a black test subject and physiologically characterised. Both species grew in oxalate-enriched media and the E. coli isolate degraded oxalate under both aerobic and anaerobic conditions. In silico genome screening of Lactobacillus genomes identified an oxalate-degrading strain of Lactobacil/us gasseri. Its oxalate-degrading mechanism was physiologically and transcriptionally characterised using in vitro growth studies coupled with RNA hybridisation analyses and reverse transcriptase PCR. In addition, the bacterium had significant oxalate-degrading ability under simulated in situ conditions in a continuous culture simulator of the human colonic microbiota. This bacterium is a viable candidate for use in the therapeutic management of kidney stone disease.

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The T-box transcription factor TBX3, plays critical roles in development including the formation of the limbs, heart and mammary glands. While haploinsufficiency of TBX3 results in ulnar mammary syndrome, its overexpression is linked to several cancers. We and others have shown that TBX3 drives tumour formation, invasion and metastasis of several sarcoma subtypes as well as melanoma, cervical cancer and breast cancer. TBX3 has thus been proposed as a novel therapeutic target to treat these cancers. Direct targeting of transcription factors for therapies however continues to represent a serious challenge and therefore an understanding of the molecular mechanisms that regulate and mediate its oncogenic activity may reveal more amenable anti-cancer drug targets. This project therefore aimed to (1) identify signalling molecules that upregulate TBX3 expression in MCF-7 breast cancer cells as well as (2) identify and characterize protein partners that cooperate with TBX3 to drive its oncogenic functions in these cells. The overexpression of the basic helix-loop-helix oncogenic transcription factor c-Myc has been widely reported in breast cancer progression and c-Myc-driven pathways are elevated in aggressive drug resistant breast cancer cells and tumours. Our laboratory has previously shown that c-Myc directly binds and activates the TBX3 promoter in several sarcoma subtypes, and it was hypothesised that c-Myc may also activate TBX3 in breast cancer. To investigate this, the impact of transiently knocking down c-Myc on TBX3 mRNA and protein levels was firstly assessed by qRT-PCR and western blotting respectively. Results show that when c-Myc is depleted, TBX3 mRNA and protein levels decrease, suggesting that c-Myc may be transcriptionally upregulating TBX3. To confirm this, c-Myc was ectopically overexpressed in MCF-7 breast cancer cells in the presence or absence of Actinomycin D, an inhibitor of de novo transcription, and TBX3 mRNA and protein levels were measured by qRT-PCR and western blotting respectively. Indeed, results show that when de novo transcription is inhibited, the c-Myc mediated activation of TBX3 expression is abolished. To identify and characterize TBX3 protein partners, MCF-7 breast cancer cells that stably overexpress FLAG-TBX3 were firstly established to enable effective immunoprecipitation for mass spectrometry. The overexpression of FLAG-TBX3 was confirmed by western blotting and immunocytochemistry and the anti-proliferative and pro-migratory roles of TBX3 overexpression in breast cancer cells was confirmed using growth curves and scratch motility assays respectively. Through affinity purifications coupled with mass spectrometry a myriad of putative TBX3 protein co-factors were identified and from this list three partners viz nucleolin, Hsc70 and HnRNP K were validated by immunoprecipitation and colocalization experiments. Importantly, results show that the interaction of TBX3 with Hsc70 is required for TBX3 protein stability and that nucleolin and TBX3 cooperate to promote MCF-7 breast cancer cell migration. Furthermore, treatment of MCF-7 cells with the nucleolin targeting aptamer, AS1411, mis localizes TBX3 and nucleolin to the cytoplasm and causes a reduction in cell viability while having no effect on the viability of normal skin fibroblasts. Together the results from this study show that c-Myc/TBX3/nucleolin/Hsc70 may be an important oncogenic pathway in breast cancer and that AS1411 may be a potentially important aptamer for the treatment of TBX3-driven breast cancer.

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