Biochemistry and Cell Biology - Doctoral Theses
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Item Defining gut mediated metabolism for health and disease(University College Cork, 2024) Quilter, Karina; Joyce, Susan; Melgar Villeda, Silvia; Science Foundation IrelandWe investigated the overall concept that human produced and microbially modified bile acids could act as an indicator of health. In chapter 3 we verified disruptions to bile acid metabolism in a porcine model of metabolic syndrome towards cardiovascular disease, induced by diet. In chapter 4 we applied dietary means to address mild hypercholesterolemia and followed BA readouts to indicate health parameters, we determined that BAs could be used as indicators of return to health but only when acute fed conditions were initiated and tracked over a 6 hour time period. Furthermore we noted that baseline fold change in BAs could be applied to distinguish responder and non-responder directions in a clinical setting. In chapter 5 we examined the metabolic outputs relating to elite athletes through 3 intervention studies. Our indications were that a key subset of BAs -significant in westernized MetS and CVD induction- were reduced in elite athletes. We further noted that different sports elicited different correlations with both microbes and metabolites in these small cohorts. In chapter 6, the key BAs associated with MetS-CVD and their interchangeable intermediates were examined at the cellular level and shown to have different outcomes in cell organelle functionality and related gene expression systems depending on dietary lipid interventions. In all, certain BAs are convergent indicators of disease, they diversify in health, just like microbiome composition. A number of key BAs were identified that can indicate health and the push towards disease in this study.Item Identifying dietary additive regulated mechanisms impacting intestinal epithelial cell responses and inflammatory processes(University College Cork, 2024) Saiz-Gonzalo, Gonzalo; Melgar Villeda, Silvia; Joyce, Susan; Irish Research Council for Science, Engineering and TechnologyIn recent years, research has increasingly identified diet as a crucial factor in the development and management of gastrointestinal diseases like Inflammatory Bowel Disease (IBD). Dietary influences are now considered significant in determining an individual's susceptibility to IBD, yet much remains unknown about the long-term effects of various dietary components. The main objective of my thesis is to explore the mechanisms through which dietary emulsifiers and sweeteners influence gut epithelial cell health, with a focus on their potential role in exacerbating gastrointestinal diseases. The first part of the study focuses on the effects of food emulsifiers, specifically lecithin, carboxyethyl cellulose (CMC), and polysorbate 80 (p80), on human intestinal epithelial cells and murine organoids. While lecithin and CMC showed no significant harmful effects, p80 was found to induce cell death at concentrations below the FDA-approved level. Through RNA sequencing and a series of assays including cell viability, cell death, lipid peroxidation autophagy, Seahorse, transmission electron microscopy, western blotting to name a few, the study reveals that p80 triggers ferroptosis—a type of cell death associated with iron-dependent lipid peroxidation—causing mitochondrial damage. Interestingly, p80 did not seem to activate the inflammatory response in the cells. The second part of the thesis explores the role of the lipid peroxidation and lipid accumulation prompted by p80 and ferroptosis. Using lipidomic analysis and inhibitors targeting lipid formation enzymes, the research identifies specific lipid changes and profiles, especially in the polyunsaturated fatty acids, associated with ferroptosis. The study highlights how lipid metabolism is intricately linked to cell death mechanisms, suggesting potential therapeutic approaches to mitigate emulsifier-induced lipotoxicity by using lipid and ferroptosis inhibitors. In the final part, the study examines the effects of rare sugars and sweeteners on human intestinal epithelial cells under both steady-state and inflammatory conditions such as IBD. Among the sweeteners tested—Psicose, Lactitol, Maltitol, Saccharin, and Tagatose—saccharin shows a significant impact on gene expression, particularly under inflammatory conditions. Saccharin activates JAK/STAT pathway related to inflammation by increasing cytokine secretion and cell death, indicating a potential role of saccharin in potentiating intestinal inflammation. Collectively, this thesis provides comprehensive insights into how specific dietary additives influence intestinal cell health. By unravelling the complex interactions between diet and cellular processes, the study contributes to a better understanding of potential dietary risks and offers guidance for future research aimed at improving dietary management in IBD and GI-related disorders.Item Evaluation of biomarkers for the diagnosis, management and follow-up of women with gestational trophoblastic disease(University College Cork, 2024) Joyce, Caroline Martha; O'Donoghue, Keelin; McCarthy, Tommie V.; Coulter, John; Irish Research CouncilGestational Trophoblastic Disease (GTD) describes a spectrum of disorders arising from the abnormal proliferation of trophoblastic tissue. Human chorionic gonadotrophin (hCG) is an excellent biomarker for most forms of this disease as its concentration accurately reflects trophoblastic activity. Hydatidiform mole (HM) or molar pregnancy is the most common form of GTD which may be suspected on ultrasound but requires pathological confirmation for diagnosis. Prognosis is generally excellent however close surveillance with hCG monitoring is imperative to detect cases progressing to malignant disease, referred to as gestational trophoblastic neoplasia (GTN). Clinical management and treatment of women with GTN in specialist centres achieves cure rates approaching 100%, emphasising the importance of accurate hCG monitoring for early detection of disease persistence to ensure optimal outcome. This thesis aims to evaluate pathological, genetic, and biochemical biomarkers used in the diagnosis, monitoring, and follow-up of women with GTD. In this thesis, a scoping review was performed to generate evidence and identify gaps in the GTD knowledge base to inform areas for future research. Key findings were the need to standardise hCG immunoassays for effective GTD surveillance, to improve diagnostic reporting pathways and to establish more accurate incidence rates. In order to identify patient priorities and inform initiatives to enhance the quality of care, a service evaluation was performed with women enrolled in the National GTD Registry invited to participate. Feedback revealed a knowledge gap regarding GTD amongst healthcare professionals outside GTD centres. This study also revealed shortcomings in psychological support and bereavement counselling offered to women after a molar pregnancy. To enhance the diagnostic accuracy of HM reporting, an in-situ hybridisation ploidy assay was developed with a customised scoring system to aid partial hydatidiform mole (PHM) diagnosis. This innovative technique provides a reliable adjunct to morphological assessment for PHM classification. The accuracy of this ploidy technique was confirmed by evaluation with molecular short tandem repeat genotyping. A national pathology questionnaire was performed to gather information on GTD diagnostic rates. Data collected was cross-referenced with cases documented in the National GTD Registry. This revealed a concerning under-enrolment of women with GTD (42%) onto the National GTD Registry by their clinicians. An audit of HM diagnostic rates over a 3-year period was performed following the Implementation of the new ploidy technique to aid diagnosis . This revealed a higher local PHM incidence rate (1 in 296 births) than reported nationally in the pathology audit (1 in 484 births). An electronic questionnaire was distributed to all European laboratories offering hCG for GTD management to gather insights on 5 key areas: hCG methodology, quality parameters, reporting and operational procedures, and use of hCG for non-GTD testing. This revealed considerable inter- and intra-laboratory variability in practice, emphasising the need to promote standardisation and harmonisation of immunoassays for GTD-derived hCG. A review of five challenging cases managed at the Irish GTD centre was conducted to evaluate the role of serum hCG and molecular genotyping in guiding management decisions, treatment strategies, and risk stratification for women with molar pregnancy, GTN and GTD mimics. These complex cases confirmed the pivotal role of serum hCG and molecular genotyping in monitoring disease persistence, prognostic stratification, and clinical management Novel strategies to measure hCG were pursued to address the lack of centralised hCG testing for GTD in Ireland. A proof-of-concept study for remote capillary blood collection was performed to evaluate the clinical efficacy and user acceptability of this method of collection for hCG monitoring in early pregnancy. The study confirmed the equivalence of capillary and venous blood hCG testing, demonstrated complete clinical concordance, thereby offering an alternative to venepuncture for hCG measurement. To explore the accessibility of hCG testing in the community, a point-of-care testing (POCT) device was evaluated for its suitability to triage women with pregnancy of unknown location (PUL) in the early pregnancy unit. The Abbott iSTAT®1 POCT instrument was chosen for this purpose and was clinically concordant with central laboratory hCG results facilitating the use of clinical decision thresholds for PUL. The research presented in this thesis provides valuable clinical insights through a series of nine research studies and has broadened the knowledge base in GTD. The findings offer an opportunity to positively impact diagnostic practices. The knowledge gap regarding GTD amongst healthcare professionals revealed in the patient survey points to a need for targeted educational initiatives in this area. Widespread integration of the HER2 ploidy technique to support morphology and standardise HM reporting could improve diagnostic accuracy, addressing a suspected underdiagnosis of PHM nationally and possibly internationally. It also suggests the need to re-evaluate incidence rates following the introduction of advanced diagnostic techniques such as ploidy assessment and molecular genotyping. This thesis identified an under-registration of women with GTD in Ireland supporting the need for initiatives such as mandatory registration or advocacy by professional organisations to increase registration rates. The inter and intra-laboratory variation in hCG immunoassays revealed in the European laboratory survey merits renewed harmonisation efforts through pilot external quality assurance and sample exchange programmes. The use of novel methods to enhance the accessibility of hCG monitoring for GTD in the community, through capillary blood collection or use of point-of-care analysis, warrants further study. The positive patient feedback indicated a preference for this approach, suggesting better compliance with serial test monitoring, a priority for GTD management.Item Investigation of a novel cell penetrating protein for the delivery of biomolecules(University College Cork, 2024) Cantillon, Emer A.; Fleming, John V (Eoin); Sanghamitra, Nusrat; CyGenica Ltd.Cellular delivery of therapeutically valuable macromolecules such as proteins and nucleotides, or genome editing systems such as CRISPR-Cas9, are key to advancing the treatment of many diseases. To make this a therapeutic reality however, the safe delivery of cargo across the cellular membrane remains a key obstacle. Furthermore, the development of molecular tools that target intracellular compartments and tissue specific subtypes is necessary in order to improve the therapeutic efficiency of macromolecules and negate off-target effects. In this context, several viral and non-viral systems have been developed to varying degrees, but there remain problems with immunogenicity, carcinogenesis, toxicity and low in-vivo delivery efficiency (1–3). Here we describe a protein, termed GEENIE, and identify its novel cell penetration capabilities, demonstrating that it can translocate across the plasma membrane of mammalian cells. We show that GEENIE can penetrate cells in a time dependent manner and can proceed in the presence of serum. Additionally, using a combination of biochemical and pharmacological experiments, we demonstrate that the mechanism of GEENIE uptake is not limited by endocytosis and confirm GEENIE uptake in red blood cells that lack endocytic machinery. Having demonstrated that GEENIE can cross the plasma membrane, we then extended its application to the delivery of biomolecules. As for other non-viral delivery systems, we first assessed the ability of GEENIE to deliver protein cargo such as fluorescent proteins. Using recombinant DNA technology, we expressed GFP tagged GEENIE chimeras. This strategy allowed for the quick and affordable purification of chimeric proteins, whereafter their cellular uptake was visualized using confocal microscopy. We were able to successfully demonstrate the ability of GEENIE to deliver GFP protein cargo intracellularly. The delivery of biotherapeutics can often be limited by the off-target effects that cause excessive cytotoxic to healthy, non-diseased cells. The cell specific delivery of therapeutics is therefore an important stipulation for delivery vehicles in order to maximize therapeutic output and reduce off target effects. The development of delivery vehicles that contain a moiety to target and deliver to a specific cell surface receptor is a common strategy to improve cell specific targeting. To this end, we investigated a newly identified peptide that has shown specificity to the HER2 receptor. Incorporating the peptide within GEENIE, allowed for increased cell specificity to HER2 expressing cells (SKBR-3) compared to HER2 negative cells (MDA-MB-468). Having demonstrated that GEENIE can be targeted to cell specific subtypes, can traverse the cellular membrane and deliver protein cargo, we next evaluated a strategy to deliver biomolecules involved in gene therapies. Gene therapies are an important class of therapeutics that have potential in the treatment of a wide variety of diseases, while also providing an opportunity to progress personalized treatment strategies. Two important groups include nucleic acids, such as siRNA, and gene editing toolkits, such as CRISPR. Employing a genetic engineering approach, we produced two separate GEENIE chimeras, R9-GEENIE and Cas9-GEENIE, to achieve delivery of siRNA or Cas9-sgRNA ribonucleotide protein respectively. Despite our optimization attempts we were unable to produce a functional R9-GEENIE to achieve siRNA delivery and protein knockdown. On the other hand, we demonstrated a potential strategy for the delivery of Cas9-RNPs via GEENIE. Future studies to optimize gene knockout may provide extra insight into whether GEENIE can be used for this strategy, and whether it has clinical potential for the delivery of Cas9.Item A family of multi-functional phosphorescent bioprobes based on the PtPFPP scaffolding moiety(University College Cork, 2023) Zanetti, Chiara; Papkovsky, Dmitri B.; Maguire, Anita; Science Foundation IrelandAccurate monitoring of O2 levels is crucial for understanding the growth, differentiation, and function of individual cells and tissues. While phosphorescence-based oxygen sensing offers great potential for such investigations, current probes still show limitations in terms of their bio-distribution and analytical performance. New improved O2 probes are highly needed. In this project we synthesized a family of new hetero-substituted phosphorescent derivatives of Pt(II)-tetrakis(pentafluorophenyl)porphyrin (PtPFPP) dye, through click-modification of its para-fluorine atoms with 1-β-D-thio-glucose, neutral (mPEG) and carboxy (cPEG) thiol-PEGs, named as PtmPEG31Glc and PtcPEG31Glc respectively, or cysteamine (CA), named PtCA31Glc. Derivatisation with 2-thioelthyl-β-D-glucopyranoside was also performed to evaluate the effects of alternative SH-Glucose structures on cellular uptake, obtaining the corresponding 2Glc derivatives. The new probes comprise one cell-targeting moiety and three polar moieties, forming a hydrophilic shell. We also synthesized the di-glucosilated-di-PEGylated derivative, named as PtmPEG21Glc2 (trans), in which 1-thio-β-D-glucose (1Glc) and thio-methyl-polyethylene-glycol (mPEG) moieties were covalently attached to PtPFPP in trans- position. Tetra-substituted derivatives Pt1Glc4, Pt2Glc4, PtmPEG4 and PtcPEG4 were also synthesised and characterized for benchmarking. The chemical synthesis and purification procedures were optimized for all the new derivatives to achieve high reaction yields and scalability, and new chemical structures were confirmed by HR-MS and NMR. Then, the most promising derivatives were assessed for their spectral properties and cell penetrating ability in 2D mammalian cell cultures using time-resolved fluorescence (TRF) on a standard plate reader, and confocal microscopy in PLIM mode (phosphorescence lifetime imaging). Probes' ability to function as either cell permeable or impermeable was found to depend on polarity, molecular charge, size, and substitution position of the bioconjugate. Notably, the hetero-substituted tri-PEGylated (PtcPEG31Glc, PtmPEG31Glc) and tetra-PEGylated (PtcPEG4, PtmPEG4) derivatives showed reduced cell internalization compared to the 3:1 cysteamine (PtCA31Glc) derivative and 4:1 glycosylated probe (Pt1Glc4). This finding opens possibilities for their extracellular applications. In contrast, PtmPEG21Glc2 (trans), demonstrated efficient cell staining and suitability for intracellular oxygen sensing (IcO2) in murine embryonic fibroblast (MEFs) cells. Moreover, biocompatibility studies indicated no significant cytotoxicity for all probes at any tested concentrations, except for PtCA31Glc which caused reduced cell viability at concentrations above 10 µM. The chemical modifications did not alter absorption and emission spectra of the PtPFPP moiety but reduced its brightness and lifetimes. Some of the PEGylated derivatives showed tendency to aggregate in biological media. PtmPEG4 and Pt1Glc4 were also evaluated in microbial respirometric assays with Escherichia coli (E. coli) cells, however due to the lack of shielding, their performance in complex media was not as good as the macromolecular MitoXpress probe and the nanoparticle based NanO2 probe. Overall, our new phosphorescent bioprobes, particularly the hetero-substituted structures, extend the library of extracellular and intracellular probes for monitoring and imaging O2 in mammalian cell cultures. They are compatible with standard TRF plate readers and PLIM microscopes, making them promising candidates for physiological studies with cells. The established structure-activity relationships (SARs) will also help to develop new probe structures for future studies.