College of Science, Engineering and Food Science - Doctoral Theses
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- ItemInvestigating the host-microbe dialogue in aging(University College Cork, 2022-12-16) Killian, Christina; Joyce, Susan; Clarke, David J.; Eli Lilly and CompanyThis thesis aimed to investigate the role of the gut microbiota and their associated functionality in the context of aging and life stage related diseases. The focus was maintained on key inter-kingdom signalling molecules, host produced but bacterially modified Bile acids (BAs) and on dietary microbially derived Fatty acids (FAs) as key elements to indicate gut microbial potential impactful roles. In the context of Chapter 3, this thesis examined the levels of BAs and FAs, to identify convergence on specific microbially produced, or modified, metabolites in the two neurological disease states representing (1) early life (murine model of Autism Spectrum Disorder (ASD)) and (2) later life (human Parkinson’s Disease (PD)). It further established the potential to redress the balance through microbial intervention, as a cause or consequence in the case of ASD. BAs converged during the healthy aging process, in Chapter 4, to determine the nature of these specific interactions, based on nuclear receptor conservation. The Caenorhabditis elegans nematode model of aging was employed to determine and genetically decipher potential individual BA influences. Chapter 5 built on recognising that gut microbes and the pathobiont Escherichia coli HM605 induced inflammation. This chapter also focused on the mechanisms by which Escherichia coli HM605 could influence the aging process.
- ItemRegulation of intracellular trafficking of the insulin-like growth factor I receptor(University College Cork, 2023) Godsmark, Grant; O'Connor, Rosemary; Science Foundation Ireland; Swiss Forum for International Agricultural ResearchInsulin-like Growth Factor 1 and its receptor (IGF-1R) are required for normal cellular growth, but aberrant expression is linked to the progression and development of many malignancies. Despite IGF-1R being a promising cancer therapeutic target, clinical targeting has not been generally successful. This has also highlighted gaps in our knowledge on IGF-1 signalling and how the IGF-1R and its regulatory regions function. Two tyrosine residues Tyr1250/1251 located within the 1248SFYYS1252 signalling motif of the IGF-1R are required for receptor internalisation, transformation and Golgi localisation. This thesis aimed to further investigate how this region and the C-terminal tail contribute to IGF-1R trafficking, sub-cellular localisation and regulation by using a range of cell lines and IGF-1R receptor mutants. Phosphorylation of Tyr1250/1251 was shown to be required for IGF-1R localisation to the Golgi and that a phosphomimetic EE (Y1250E/Y1251E) IGF-1R mutant is less stable, is more ubiquitinated and undergoes more rapid proteasomal degradation than wild type IGF-1R. Three lysine residues (Lys1256, Lys1294, Lys1324) were identified within the IGF-1R C-terminal tail as putative ubiquitin binding sites, but mutation of these to arginine in mutational studies established that all of these sites have a minor function in receptor ubiquitination. Peptides encompassing the hydrophobic Tyr1250/1251 site in the IGF-1R were recently proposed as a cargo-sorting motif that binds to the protein trafficking ESCPE-1 (SNX5/SNX6) complex, which rescues the IGF-1R from lysosomal degradation. This was tested this using full-length receptors expressed in different cell models and using siRNA-mediated suppression of SNX5/SNX6. However, our data did not replicate the published observations on SNX5/SNX6 knockout causing reduced IGF-1R protein expression. Furthermore, no effect of SNX5/SNX6 suppression on IGF-1R protein levels or location at different sub-cellular compartments including the Golgi was observed. Interestingly, SNX5/SNX6 suppression induced lysosomal accumulation at the leading edge of cells as well as decreased cellular migration. SNX5/SNX6 was observed to interact with the IGF-1R, but the hydrophilic Y1250E/Y1251E mutant exhibited a stronger interaction, than the hydrophobic Y1250F/Y1251F mutant, which suggest that this interaction may be modulated by phosphorylation in the full-length receptor. In summary, the findings confirm the importance of the IGF-1R C-terminal tail, in particular, Tyr1250/1251, in IGF-1R signalling and regulation. These tyrosines facilitate ubiquitin binding, SNX5/SNX6 interaction and Golgi localisation of the IGF-1R which all contribute to the transformed phenotype. Further research on the associated mechanisms should assist in tailoring future cancer therapy treatments to improve clinical efficacy.
- ItemIGF-1 signalling controls mitochondrial morphology and basal mitophagy in cancer(University College Cork, 2023-01-06) Murray, Joss; O'Connor, Rosemary; Science Foundation IrelandInsulin-Like Growth Factor 1 (IGF-1) signalling is known to support oncogenic transformation and the promotion of cancer development. A growing body of evidence has outlined the protective effect IGF-1 signalling has on the mitochondria, however this has been relatively underexplored in cancer. Therefore, this thesis aims to elucidate the mechanisms by which IGF-1 promotes mitochondrial protection in cancer. Previously, we determined that the mitophagy receptor BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3) is induced by IGF-1 to support mitochondrial turnover and protection. Here, we analysed a publicly available gene expression dataset of breast cancer cells stimulated by IGF-1. Gene ontology classification revealed a signature of genes induced and repressed by IGF-1 involved with mitochondrial functions. Further analysis to classify genes by biological process suggested that genes involved with apoptosis and suppression of mitochondrial metabolism were most enriched in the gene groups regulated by IGF-1. To further interrogate mitochondrial dynamics downstream of IGF-1 signalling, we assessed mitochondrial morphology. Lack of IGF-1R promoted mitochondrial fusion, while IGF-1 stimulation promoted mitochondrial fragmentation. Mitochondrial fragmentation was associated with increased mitochondrial transport to the leading edge of invasive breast cancer cells. Pharmacological inhibition of mitochondrial fission inhibited the migration of cells expressing the IGF-1R but was ineffective at moderating migration of cells lacking the IGF-1R. Finally, we interrogated the function of BNIP3 downstream of IGF-1 stimulation. While BNIP3 is induced, IGF-1 stimulation suppressed mitophagy. However, BNIP3 turnover was higher in basal cell culture conditions than in nutrient deprived conditions, suggesting that BNIP3-mediates basal mitophagy in cancer cells. Indeed, IGF-1R knockout reduced the basal turnover of BNIP3 implying that IGF-1 regulates basal mitophagy via BNIP3. In totality, this thesis presents evidence that IGF-1 signalling promotes mitochondrial protection by regulating genes involved with redox homeostasis while tempering mitochondrial metabolism. Mitochondrial fragmentation is induced by IGF-1 and can also regulate cancer cell migration, while also supporting basal mitophagy mediated by BNIP3. These findings demonstrate that targeting IGF-1 signalling in cancer could impair mitochondrial protection mechanisms, which offers an avenue for novel therapeutic opportunities.
- ItemDietary quality of school-aged children and teenagers in Ireland by demographic characteristics and eating location(University College Cork, 2022-10) Rusu, Ioana; Kehoe, Laura; Flynn, Albert; Cashman, Kevin; Walton, Janette; O'Mahony, Jim; Department of Agriculture, Food and the Marine, IrelandBackground: Childhood and the teenage years are distinctive life stages characterised by unique dietary needs. Data from nationally representative dietary surveys of children and teenagers across Europe have shown that intakes of key nutrients are not in line with recommendations. Furthermore, demographic characteristics such as sex, age, socio-economic status and weight status may have an influence on dietary quality in children and teenagers. It has also been shown that eating location can influence dietary quality, with some locations such as ‘home’ and ‘school’ being associated with better dietary quality than other locations, such as ‘restaurants’, ‘takeaways’ and ‘shops’. Objectives: The overall aim of this thesis was to examine the dietary quality of school-aged children and teenagers in Ireland by demographic characteristics and eating location using data from the National Children’s Food Survey II (NCFS II) (2017-18) and the National Teens’ Food Survey II (NTFS II) (2019-20). Methods: The analyses for this thesis were based on data from the NCFS II and the NTFS II which are two nationally representative dietary surveys of children (5-12 years, n 600) and teenagers (13-18 years, n 428) living in the Republic of Ireland. Dietary intake data were collected at brand level using a 4-day weighed food diary for both surveys. Dietary quality was determined using energy-adjusted (%E or /10MJ) intakes of nutrients and food groups. ‘Eating location’ was defined as the location where food was prepared or obtained, irrespective of where it was consumed. For eating location analysis, consumers were defined as those who consumed food at a given location at least once during the four-day recording period. Results: The overall dietary quality of children and teenagers in Ireland was found to be unfavourable and there were very few differences observed across sex, age groups, categories of socio-economic status and weight status. Intakes of key food groups were not in line with food-based dietary guidelines (FBDG) for either children or teenagers. In both children and teenagers, dietary intakes were driven by the ‘home’ location. While the majority of children (73%) and teenagers (78%) consumed food from ‘outside of the home’, most eating occasions took place at ‘home’ for both children (89%) and teenagers (85%), accounting for a large proportion of the energy consumed (88% in children and 81% in teenagers). The contribution of food consumed from ‘outside of the home’ was higher in teenagers than in children (19% vs. 12%). Younger children had higher intakes of energy from food consumed from ‘other homes’ than older children and both children and teenagers of parents with primary/intermediate education only had higher intakes of energy from food consumed from ‘fast food/takeaways’ than children and teenagers of parents with tertiary education. There were no other differences observed in these eating patterns across any other demographic characteristics examined. Food consumed from ‘home’ and the ‘participant’s own home’ was better in terms of dietary quality than food consumed from ‘outside of the home’ and ‘other homes’. Of all ‘out of home’ locations, ‘school’ provided the best dietary quality for children, but this was not seen in teenagers. Conclusion: This thesis has shown that the overall dietary quality of children and teenagers in Ireland was unfavourable and there were few differences found across demographic characteristics. Dietary intake was driven by the home environment, with ‘home’ and the ‘participant’s own home’ being associated with better dietary quality than ‘other homes’ and ‘outside of the home’. The findings of this thesis can be of use to policy makers when introducing healthy eating policies aimed at school-aged children and teenagers such as taxation on unhealthy foods and regulation around marketing of unhealthy foods.
- ItemThe design, synthesis and characterisation of selected chitosan-based thin films and studies of their use as materials for antimicrobial, heavy metal adsorption, and wound dressing applications(University College Cork, 2022-09) Pemble, Oliver James; Bardosova, Maria; Povey, IanChitosan is a naturally-derived polymer, sourced from the chitin present in crustacean shells and insect exoskeletons. It is a highly diverse molecule, with heavy metal adsorption, antibacterial and pH-based swelling properties. However, these properties may be hindered by the lack of strength of a singular polymer structure. Chitosan has the ability to cross-link with other polymers or reagents to create interpenetrating polymer networks (IPNs) and blends to improve the mechanical strength or application mechanisms. These composites can be cast into films which have superior properties for the applications listed above. The first half of this PhD thesis sets out to describe the process of synthesising thin films made from combining chitosan with cross-linking reagents and other polymer networks. These materials comprise two siloxane networks Tetraethyl-orthosilicate (TEOS) and 3-aminopropyltriethoxysilane (APTES), glutaraldehyde (GA), and the polymer polyacrylamide (PAM). Aqueous acidic solutions of low molecular weight chitosan were made and combined with one of the four materials at different ratios to produce hydrogels that could then be cast into thin films. These chitosan-based films were characterised as to their molecular structures via FT-IR spectroscopy and their mechanical elasticity/plasticity via tensile strength tests. The method of casting the films was primarily drop-casting in a Petri dish and drying for 24 hours, but an alternative procedure that utilises a doctor blade slot-die head was also developed. This novel method was studied in-depth, and the findings were published. For this reason, the results of the slot-die casting studies are presented in their published form. Briefly, the slot-die casting technique produced high quality thin films of chitosan-based composites in under 2 hours. Further, there was no influence of the direction of travel of the films during deposition on their mechanical properties. This study served as a proof-of-concept that high volumes of chitosan-based films could be made quickly, cheaply, and reproducibly. The second half of this thesis explores the applications of the composite chitosan-based films, by use of their adsorption, antimicrobial, and swelling abilities. The adsorption of radioactive technetium pertechnetate 99mTcO4- ions via chelation from solution was evaluated and correlated based on the Langmuir and Freundlich isotherms. It was found that the chitosan-glutaraldehyde films showed the best ability to adsorb 99mTcO4- ions and that a Langmuir-type, monolayer-based adsorption process was most likely in operation. A preliminary study of the antimicrobial properties of the chitosan-based films was undertaken via Gram-negative and Gram-positive bacterial growth and attachment assays. The intent of the study was to test the antibacterial abilities of the films while also speculating as to the mechanism of said abilities. The chitosan-tetraethyl-orthosilicate films were found to be the most effective against both types of bacterial growth and attachment. However, the chitosan-glutaraldehyde films showed little-to-no antibacterial ability, suggesting the antibacterial mechanism may be affected by the cross-linking reagent. Finally, a prototype for a smart wound dressing device that incorporates a pH sensitive sensor for chronic wounds was designed and developed using the chitosan-based films. The device utilised chitosan’s natural swelling ability and colloidal photonic crystal technology to create an indicator that produced a visual colour change in response to fluctuating pH levels, which may occur in chronic wounds. The mechanism was based on the distortion of photonic crystal layers by the swelling of the chitosan-based films to give a distinct red to green colour change. This preliminary study shows the potential of chitosan /photonic crystal-based sensors for use in medical devices that do not require an external power source to operate.
