College of Science, Engineering and Food Science - Masters by Research Theses
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Item Novel RNA-based biomarkers for ovarian cancer – uncovering how LINC01132 is regulated by the tumour suppressor protein, p53(University College Cork, 2023) Hartigan, Shaun; Dean, Kellie; McKenna, Sharon L.; Irish Research CouncilOvarian cancer is one of the deadliest female cancers worldwide. Most cases are detected in advanced stages, as it is difficult to diagnose ovarian cancer early due to nonspecific symptoms. Presently, there are no sensitive biomarkers to identify early-stage disease. Over 60% of ovarian cancer cases have a mutation in p53, a tumour suppressor transcription factor which counteracts cell stress and oncogenic signals. Most mutations occur within p53’s DNA-binding domain, a vital region which facilitates its anchorage to target gene promoters. Previous data found numerous long-non-coding RNAs (lncRNAs) are differentially expressed in ovarian cancer cells with mutant TP53, compared to those with the wildtype gene. Here, we show that three p53 mutants (R175H, I195T and R248Q) commonly found in ovarian cancer patients, were unable to activate firefly luciferase expression from a synthetic p53-responsive promoter, reflecting the impact of p53 core domain mutations on its ability to bind target promoters and transactivate gene expression. In silico genome-wide ChIP-seq analysis of differentially expressed lncRNAs identified three (MEG3, LINC01132, and LINC2960) containing regions within -1 kb of their transcription start sites, showing interaction with p53. Another four (EMX20S, PRICKLE2-DT, LINC00887 and LINC02610 contained p53-interacting regions within -5 kb. Division of the region up to -6,995 bp of the LINC01132 transcription start site into a distal, middle, and proximal segment, and subsequent cloning upstream of firefly luciferase, allowed us to assess p53 activity at the LINC01132 promoter. Western blot analysis could not detect luciferase expression from either segment under wildtype p53 overexpression, despite the proximal segment containing six p53-interacting sites. To determine the true response of wildtype and mutant p53 binding to the LINC01132 promoter, future quantitative reverse-transcriptase PCR and luciferase assays should be conducted, given their higher sensitivity compared to Western blot analysis. To improve patient prognosis in ovarian cancer, there is vital necessity to discover specific biomarkers, which can diagnose and monitor disease progression. LncRNAs can be detected in blood, so linking expression of differentially expressed lncRNAs to the mutational status of p53 in ovarian cancer, and studying how these change with the therapies, is novel, previously unexplored, and may aid in biomarker discovery.Item Evaluation of dynamic light scattering as a technique for the routine monitoring of therapeutic proteins on stability(University College Cork, 2023) O'Kennedy, Laura; Moore, Eric; Crowley; Lowney, Declan; Crowley, StephenThroughout the course of biotherapeutic development and manufacturing, the detection and quantification of protein aggregates and particles is critical to ensure patient safety. Size Exclusion Chromatography (SEC) is one of the most widely used techniques for the separation of proteins and allows for the detection and quantification of aggregates. In terms of aggregate detection at drug substance (DS) and drug product (DP) release, as well as for stability testing, the ICH guidelines reference SEC as an example. However, it is acknowledged that new analytical techniques are continuously being developed and can be used where appropriate. Dynamic Light Scattering (DLS) is one such technique, that has been described on several occasions as an early indicator of protein aggregation. Some benefits of DLS over other aggregation-monitoring techniques, is its ability to analyse proteins in their native environment, its fast analysis time, and its ability to detect a wide particle size range. Additionally, for high molecular weight species (HMWS) detection, DLS is an exceptionally sensitive technique. This sensitivity and level of detection are particularly important in the biopharmaceutical industry as the presence of even a small number of aggregates can significantly increase upon long-term storage. However, this sensitivity can also be a weakness, as scattering from traces of other large particulates can interfere with results, and overall, the quantification of results can be difficult. Consequently, the technique is typically used in a complementary fashion with more widely used techniques such as SEC. The capabilities and applications of DLS have been improved over time with the invention and incorporation of e.g. multi angle DLS (MADLS), extended size range analysis and diffusion interaction parameter (kD) analysis, into the one instrument and software. The aim of this research was to explore the possible benefits and limitations associated with DLS, in order to determine if the technique has the potential to be used more widely for testing protein therapeutics on stability. A range of biotherapeutics including monoclonal, bispecific and trispecific antibodies were tested, that varied in concentration from 2 mg/mL to 160 mg/mL. Multiple stressed samples were included in the test panel to explore the techniques’ ability to detect large particles and aggregates. The particle size and size distribution results were compared to those generated from SEC, to explore whether DLS could give additional insight into the molecules stability. It was found that the technique showed great sensitivity and, in some cases, detected an increase in large particles where SEC failed to do so. These results were then substantiated by looking at data from sub-visible particle analysis via light obscuration. The zetasizer instrument, used to measure DLS, can also perform various other measurements that are indicators of colloidal and/or thermal stability. These include electrophoretic light scattering (ELS), extended size range analysis, particle concentration, kD determination, osmotic second virial coefficient (B22) determination and molecular weight (Mw) measurements. These measurements were performed to determine their respective abilities to provide additional insight into therapeutic protein stability. For most of the measurements however, the results and trends weren’t as consistent and as reliable as the particle size and particle distribution measurements. Further evaluation of these measurements, protein concentration optimisation and additional knowledge of the sample properties would be needed to provide more accurate results. However, overall, DLS is seen to be a highly sensitive and fast analysis tool for the determination of particle size and can provide additional insight into molecule stability when used in conjunction with SEC.Item Design and use of novel metal catalysts in organic synthesis(University College Cork, 2023) Fitzgerald, Deirbhile; Maguire, Anita; Collins, Stuart; Irish Research CouncilThis project focuses on the design and synthesis of novel dirhodium carboxylate catalysts, and their precursor ligands, for use as enantioselective catalysts in transformations of α-diazocarbonyl compounds. Chapter One focuses on the literature background over the past thirty years in transition metal-catalysed asymmetric transformations of α-diazocarbonyl compounds. This overview aims to give an insight into the use of both copper and rhodium-based catalysts in intramolecular and intermolecular C-H insertion reactions, cyclopropanation and desymmetrisation reactions. The evolution of dirhodium carboxylate design since the 1980s is explored. This summary provides insight into their use as enantioselective catalysts in asymmetric transformations of α-diazocarbonyl compounds, thus providing context for this work. Chapter Two describes the design and synthesis of novel, enantiopure dirhodium carboxylate complexes, drawing inspiration from the rhodium (S)-mandelate skeleton. Initially, two enantiopure rhodium carboxylates were synthesised, incorporating carboxylic acid ligands bearing a fenchyl chiral auxiliary. Synthesis of carboxylic acid ligands bearing a bulky adamantyloxy group or a tert-butoxy group in place of the fenchyl substituent was investigated to simplify the stereochemical features, with just one stereogenic centre in each ligand. Two target carboxylic acids were obtained in racemic form, one bearing an adamantyloxy group and the other bearing a tert-butoxy group adjacent to the carboxylic acid moiety. Resolution of these carboxylic acids was explored through formation of diastereomeric salts or enzymatic kinetic resolution; while enantioenrichment was observed, further work is required to obtain the ligands in an enantiopure form for catalyst preparation. Chapter Three describes the synthesis of both racemic mandelamide and enantiopure (S)-mandelamide. In addition, a pair of diastereomerically pure mandelamides with α-methylbenzylamine were synthesised and characterised. The mandelamides were prepared to enable exploration of their crystal landscape in collaboration with another research group in UCC. Chapter Four contains the full experimental details and spectroscopic and analytical characterisation of all compounds synthesised in this project, while details of chiral stationary phase HPLC analysis are included in Appendix 1.Item Development of a synergistic synbiotic containing arabinoxylan and Bifidobacterium longum using in vivo selection(University College Cork, 2024) Jones, Evan; Walter, Jens; van Sinderen, Douwe; University College Cork; Synbiotic HealthColonisation and metabolic activity of orally ingested bacteria in the colon rely on competitive ecological and niche-based factors that often limit the functionality of commonly used probiotics. Synergistic synbiotics, which involve the parallel administration of a microorganism with its cognate substrate, have the potential to improve persistence and ecological performance of putative probiotic microbes. However, real synergism has not yet been established for synbiotics in human trials, and most synbiotic combinations have not been designed using an approach that accounts for the ecological constraints of the GI tract. Here we use in vivo selection (IVS) to identify strains of Bifidobacterium longum that are adapted toward the utilization of arabinoxylan (AX) in the human gut. To achieve this, bifidobacteria were quantitatively cultured from fecal samples collected during a human trial which showed that a high dose of corn bran AX leads to a significant but highly individualised increase of B. longum. Isolates were randomly picked and genotyped by a rapid, high throughput gyrB sequencing method that was developed for this project. Bacterial counts and strain composition were compared between baseline and week 6, and B. longum strains enriched in vivo were then tested through in vitro fermentations to investigate their growth on AX and its constituents. These monoculture experiments confirmed the ability of representative isolates to use free arabinose, xylo-oligosaccharides (XOS) and the complete corn AX fibre, which suggests that these B. longum strains are primary AX degraders. Viable cell counts revealed a high level of consistency in growth patterns among the fecal isolates compared to reference strains on AX. Whole genome sequencing (WGS) of selected strains followed by comparative genomic analysis revealed an enrichment of relevant glycoside hydrolase family 43 (GH43) genes and the presence of three specific carbohydrate utilisation clusters associated with xylan and AX metabolism in a number of in vivo-selected isolates which was not observed in reference strains. Finally, gas production experiments helped to further characterise the fermentation profiles of the AX-degrading isolates and highlighted their capacity to facilitate cross-feeding with other members of the microbiota. This study demonstrates the value of an ecologically relevant process for selecting improved synbiotic combinations, with the B. longum strains identified here representing promising candidates based on their predicted ecological performance in vivo.Item The impact of bile and anaerobic stress on Listeria monocytogenes isolates(University College Cork, 2023) Lynch, Mary Jane; Gahan, Cormac G.; Hill, ColinListeria monocytogenes is regarded as an important food pathogen that can cause serious illness. All individuals are susceptible to L. monocytogenes infection with pregnant women, immunocompromised people and the elderly particularly at high risk. L. monocytogenes infection can cause febrile gastroenteritis and a more serious invasive form where the bacteria cross the blood/brain barrier or the blood/foetal barrier. In this study the effect of bile and anaerobic stress on L. monocytogenes was assessed. Bile has antimicrobial properties and it is one of the stressors that L. monocytogenes must overcome in the human gastrointestinal tract to establish infection. L. monocytogenes uses an enzyme, bile salt hydrolase (BSH), to detoxify bile. The bile resistance response may be triggered by anaerobic conditions. In total 154 food/environmental isolates and 74 clinical isolates were screened. A bile tolerance assay and a BSH enzymatic agar plate assay were developed. Experiments were carried out at a lowered pH and/or anaerobic conditions to simulate conditions in the human gastrointestinal tract where L. monocytogenes is exposed to bile. Results indicate that there are bile tolerance variations in L. monocytogenes strains from food, environmental and clinical sources. In this large-scale study, cultures grown anaerobically were less tolerant to bile than cultures grown aerobically and generated more varied resistance patterns. The effect of anaerobiosis is not as well understood as aerobic and results provide an insight into the influence of environmental factors. Interestingly, the bile resistance assay revealed that the clinical isolates were significantly more tolerant to bile than the food and environmental isolates (P < 0.001). This important finding demonstrates the evolutionary pressure on these strains to survive in the gut. Correlation of results from the BSH agar plate assay with the bile tolerance assay demonstrated that the food and environmental isolates had slightly higher levels of BSH activity than the clinical isolates. However, the difference between the groups was not as significant as the bile resistance results. Results were also analysed on the level of lineage and clonal complex to identify possible links to the bile response. The results generated from this study provide insight into important associations between bile tolerance of individual strains/lineages and efficacy of L. monocytogenes infection.