College of Science, Engineering and Food Science - Doctoral Theses

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    Multiferroic investigations of Aurivillius phase thin films
    (University College Cork, 2023) Colfer, Louise; Keeney, Lynette; Long, Brenda; Royal Society; Science Foundation Ireland
    In recent years, the amount of data being created and processed is growing at a much faster rate than the rate of computational storage technology development. With CMOS technologies reaching their miniaturisation limits, new disruptive materials are needed to increase data storage capabilities. Technological road-maps have identified room temperature, non-volatile magnetoelectric multiferroic materials as promising candidates for memory scaling within future memory storage devices. Although multiferroic memory devices have the potential to revolutionise memory storage technologies, commercial devices successfully utilising multiferroics have not yet come to fruition. The focus of this thesis is to understand and optimise a rare example of a room temperature magnetoelectric multiferroic, Bi6TixFeyMnzO18 (B6TFMO; x = 2.80 to 3.04; Y = 1.32 to 1.52; Z = 0.54 to 0.64). Aurivillius phase materials, (Bi2O2)(An−1BnO3n+1), where ferroelectric perovskite units are interleaved between dielectric [Bi2O2]2+ layers, are flexible scaffolds for technological applications. While earlier studies indicated that B6TFMO is a promising material for future memory devices, my thesis presents significant advances in the characterisation, understanding and optimisation required towards implementing the material in fully realised devices. In this work, correlation between the octahedral tilting and atomic-level structural distortions with functional electronic and magnetic properties of B6TFMO were determined, revealing that crystal field splitting of the Ti4+ octahedra is influenced by its position within the Aurivillius unit cell. Theoretical calculations determined that this is predominantly driven by changes in the extent of tetragonal distortion along the c-direction. Atomic scale mapping of polar displacements reveals this has a direct impact on the ferroelectric properties. Polarisation is largest towards the outer perovskite cells, correlating with an increased extent of local tetragonal distortion of octahedral geometries. Experiments demonstrate that tilting of the BO6 octahedra competes with the extent of tetragonal distortion of the TiO6 octahedra, where the degree of octahedral tilting increases towards the central layers of this Aurivillius system, where the magnetic cations preferentially partition. This work presents the first indication that octahedral tilting might be an important enabler of long-range magnetic interactions and subsequent multiferroic behaviour in B6TFMO. Delving deeper into fundamental understandings of B6TFMO’s antipolar and magnetic behaviour, the purposeful inclusion of structural defects within the layered structure of B6TFMO and how they can impart elastic strain and electrostatic energy changes which in turn influence polar behaviour is explored. The findings show that the vicinal sapphire substrates (mis-cut angle 0.2 o to 10 o) are successful for promoting the propagation of sub-unit-cell defects and disruptions to the periodicity of the Aurivillius phases. This has a marked effect on the film morphology and ferroelectric properties. Macroscopic and local measurements show that defect, crystal grain and ferroelectric domain density increases with increasing substrate mis-cut angle. Atomic resolution polarisation mapping showed that charged domain walls alongside exotic polar vortices are facilitated by OPBs when two OPB defects are spaced 5 nm apart. This work provides insight into methods for successfully controlling defect levels and how polar vortex domain walls and charged domain walls are promoted within layered multiferroics by tailoring the underlying substrate that the film is grown on. Moving on from vicinal sapphire surfaces, patterned sapphire with 3D domes were used to encourage the growth of the Aurivillius grains towards an upright geometry. An increased number of non-(00l) reflections were present in the B6TFMO films on patterned sapphire along with evidence from STEM imaging showing that B6TFMO grains grow along the incline of the patterned sapphire domes. With the growth of the crystal grains towards an upright geometry it would be expected that access to the major a-axis polarisation via out-of-plane measurement would be improved, however with a maximum inclination angle of 60 ° achieved with the 3D dome architectures, the out-of-plane piezoresponse of the samples remained weaker than the in-plane piezoresponse. Studies of the magnetic properties of the films demonstrated that the B6TFMO samples were ferromagnetic at room temperature. These findings provide further evidence of room temperature multiferroic behaviour in B6TFMO. Lastly, the role of bismuth excess and substrate strain were investigated to optimise the epitaxial growth of B6TFMO via DLI-CVD. A single-step deposition method on epitaxial substrates was developed to allow the successful synthesis of continuous 45 nm thick B6TFMO films at thicknesses relevant to applications as piezoelectric actuators, sensors and energy harvesters. These films nucleated via a layer-by-layer growth mode and were found to have a strong in-plane ferroelectric response with isotropic domains. Film purity was enhanced with utilisation of epitaxial substrate with appropriate lattice match to B6TFMO and by optimising the amount of bismuth precursor used. In this work, progress was made towards the optimisation of epitaxially grown B6TFMO films, allowing greater control of film orientation and augmenting strain-induced enhancement of multiferroic properties in future data storage devices. Overall, this research has increased understanding of the fundamental mechanisms governing the ferroelectric and ferromagnetic properties of B6TFMO. The work has elucidated some of the key requirements fundamental to the manifestation of polar topologies and has created strategies for the tailoring of novel polar topologies. This combination of new material understanding and new growth optimisation of room temperature multiferroics contributes to solving the ‘big data’ problem. Application of B6TFMO in future technologies based on ultra-high density, energy efficient memory devices, spintronic devices, multilevel resistance control (memristive and synaptic devices) and energy-efficient neuromorphic “brain inspired” devices are envisioned.
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    Impact of protein genotypes on milk composition and processability
    (University College Cork, 2024) Gai, Nan; Kelly, Alan; O’Regan, Jonathan; Goulding, David A.; Uniacke-Lowe, Therese; Nestlé
    Milk protein genotypes are associated with differences in milk yield, composition, and processability due to direct effect of their structural differences, or their indirect effects on casein micelles, as well as differences in milk composition. This study investigated the effects of β-casein (β-CN) genotypes on milk physicochemical properties, functionalities, processability (Cheddar cheese processing), and proteolytic mechanisms. Influences of β-lactoglobulin (β-lg) genotypes on heat-induced whey protein denaturation were also investigated. The investigation of milk physicochemical properties, including gross composition, mineral content, casein micelle size, zeta potential, polydispersity index (PDI) and fat globule size distribution were initially focused on three main β-CN genotypes, A1A1, A1A2 and A2A2. No significant differences were detected on milk composition between three genotypes, and casein micelle sizes between three genotypes were similar. Better rennet coagulation properties and acid coagulation properties were determined in A1A1 milk, in comparison to A1A2 and A2A2 milk, but differences were not significant. A2A2 milk had smaller fat globule size and better stability than A1A2 and A1A1 milk against creaming. Cheddar cheese was produced using milk with A1A1, A1A2 or A2A2 β-CN genotypes. A2A2 cheese milk had significantly poorer rennet coagulation properties compared to the other two genotypes, which caused a delay in the cutting step. A1A1 cheese had a lower protein content, while A2A2 cheese had a lower fat content compared to the other two cheeses. Protein contents in both A1A1 and A2A2 cheese whey were higher than that in A1A2 cheese whey. Ripened A1A1 cheese was the softest, and the least fracturable. Dissociation and interfacial properties of purified A1 and A2 β-CN, obtained from milk with A1A1 and A2A2 β-CN genotypes using microfiltration (MF), were studied. A2 β-CN, on micellization, had smaller particle size than A1 β-CN, and A1 β-CN was more stable over heating and cooling than A2 β-CN. Foam stability and emulsifying ability of A2 β-CN was higher than that of A1 β-CN, while stability of emulsions produced using A1 β-CN was higher than that of A2 β-CN. The differences between the two β-CN genetic variants A1 and A2 in terms of proteolysis in milk were determined. A2A2 milk had higher plasmin activity than A1A1 milk, and A2 β-CN was more susceptible to plasmin than A1 β-CN. Referring to the different amino acid on sequence position 67, where proline (Pro) is in A2 β-CN and histidine (His) is in A1 β-CN, His67 was determined to be more susceptible than Pro67 in hydrolysis of β-CN. The influence of β-CN genotypes on milk characteristics was also studied for milk containing minor β-CN genotypes, A1B, A2B, A1I and A2I. Larger casein micelle size, better rennet coagulation and acid coagulation properties were associated with β-CN variant B compared to the I variant. The structural differences between the two genotypes, B and I, has also influenced their proteolysis, and the Arg122 in β-CN B variant was determined to be more sensitive to plasmin-derived proteolysis compared to the Ser122 in the I variant. In addition, the His67-Asn68 bond in the A1 and B variants was more susceptible than Pro67-Asn68 in the A2 and I variants to β-CN proteolysis driven by milk indigenous enzymes, while the activities of Ile66-His67 and Ile66-Pro67 were similar. The effect of κ-CN genotypes was determined to have a stronger influence on milk casein micelle size and fat globule size than that of β-CN genotypes, where milk with κ-CN A had larger casein micelle and smaller fat globule size than milk with κ-CN B. The acid coagulation and rennet coagulation properties of milk containing the B variant of κ-CN were better than milk containing the A variant, which was possibly due to the effect on casein micelle size and fat globule size. In the study of whey protein denaturation induced by heating, milk with β-lg A variant was more heat resistant than with the B variant. More denatured whey proteins with B β-lg were likely to interact with casein micelles to form insoluble aggregates compared to the A variant. In conclusion, the findings in this study suggest that milk protein genotypes are clearly associated with milk characteristics and technological properties, and opened new, related areas such as the potential linkage between specific peptides obtained from proteolysis to milk functional properties. These findings can be used as a reference for further studies, and as guidance for milk selection when dairy products with specific characteristics are required.
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    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.
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    Fractionation of β-casein, the formation of complex coacervates between β-casein preparations and lactoferrin, and an assessment of their digestion, technofunctional and biofunctional properties
    (University College Cork, 2024) van der Schaaf, Jasper Melle; Kelly, Alan; O'Mahony, James; O'Regan, Jonathan; Goulding, David A.; Fondation Nestlé
    Lactoferrin (LF) and β-casein are major human milk proteins that have individually been recognized for their significant roles in infant health and nutrition. However, the impact of their complex coacervation, involving liquid-liquid phase separation, and the behavior of these structures during gastrointestinal digestion remains largely unexplored. This thesis aims to advance the fractionation and use of β-casein as a dairy ingredient and knowledge on the formation of LF:β-casein complex coacervates, their physicochemical properties, and biological functionalities within the context of infant in vitro gastrointestinal development. The commercial production of β-casein is limited, due to challenges with yield, associated costs, and industrial application of the final ingredients. Combining fractionation treatments may improve the overall composition and purity of these ingredients. A novel approach for β-casein isolation was developed in Chapter 1, that included membrane filtration combined with chymosin treatment, which removed the κ-casein fraction from the final enriched β-casein ingredient. The second chapter focused on the factors influencing the formation of complex coacervates, including pH, ionic strength, stoichiometry, temperature, protein concentration and protein profile, and demonstrated a strong interdependence of these factors. The coacervate yield varied based on the starting material, and optimal conditions at which the highest phase separation yield were identified. Understanding these factors, and their interactions, is essential for optimizing complex coacervate-based formulations for infant nutrition. The effects of spray-drying or freeze-drying on the stability and reformation of complex coacervates were also investigated. The complex coacervation of LF with β-casein increased the denaturation point of LF by 5 °C; however, this increase does not permit commercial thermal treatment of LF without anticipated denaturation and functional loss. Importantly, drying the complex coacervates and rehydrating them showed no significant impact on the rheological behavior and all complex coacervates exhibited a frequency-dependent viscoelastic behavior. Potentially, freeze-drying complexes formed under infant nutrition grade conditions and dry-blending coacervated ingredients into infant formula may avoid further thermal processing and ensure that bioactivity is retained. The final chapters investigated the stability of complex coacervates during infant in vitro digestion and their potential impact on gastrointestinal health. Complex coacervates showed altered gastric proteolysis, which resulted in different peptide profiles, and could potentially influence bioactivity. Additionally, the digested complex coacervates were studied for their anti-inflammatory properties using a cell T84 epithelial cell model. The bifidogenic properties were studied by stimulating Bifidobacterium longum ssp. infantis with digested complex coacervates; all digested samples exhibited bifidogenic effects, with undigested β-casein stimulating bifidogenesis the most. The findings of this research will further support the development of LF and β-casein complex coacervation as a potential way of producing novel ingredients, as their unique physicochemical properties, increased heat-stability, and altered peptide profiles upon digestion, potentially leading to different biological activities, make them interesting to consider for inclusion in infant nutrition.
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    Impact of food, environmental and pharmaceutical antimicrobials on the gut microbiome
    (University College Cork, 2024) Walsh, Lauren; Ross, R. Paul; Hill, Colin; Science Foundation Ireland
    This thesis is concerned with antimicrobials (both protein, peptide and chemical based) and their role in the gut microbiome from a functional and compositional perspective, as well as the isolation and development of new bacteriocins against pathogenic bacteria of interest. Pharmaceutical antimicrobials such as antibiotics, bacteriocins, phages and their endolysins are discussed in chapters 1, 3, 4 and 5. Chapter 1 describes antibiotic alternatives that could potentially be used to treat nosocomial methicillin resistant Staphylococcus aureus (MRSA) infection. Some alternative options include bacteriocins, phages and phage lysins. In Chapter 5, the antibiotics fidaxomicin and vancomycin were compared with the two bacteriocins, thuricin CD and nisin, as potential therapeutics to combat CDI and to assess their overall impact on the gut microbiome. Chapters 3 and 4 specifically discuss the isolation of novel bacteriocin producing bacteria. Chapter 3 describes the isolation of two bacteriocin producing strains termed AS1 and AS2. Chapter 4 outlines the isolation of Paenibacillus ottowii FAA_942_34, which demonstrated activity against IBD-associated bacteria. Environmental antimicrobials and food antimicrobials were examined in chapters 2, 6 and 7. Chapter 2 is a review focusing on the herbicide glyphosateTM, specifically focusing on the compositional and functional changes that glyphosate elicits in the gut microbiome. In chapter 6, glyphosate and four food preservatives were analysed for their effect on the gut microbiome. In Chapter 7, the heavy metal cadmium was used as a selective agent to identify transformants acquiring pJOS01. Following electroporation of pJOS01 into Staphylococcus aureus RN4220, a smaller 21 Kb plasmid termed pJOS02 was recovered in transformants. Formation of pJOS02 from pJOS01 is thought to result from the presence of inverted repeat regions at either end of pJOS02. Overall, the results of this thesis outline a variety of antimicrobials and their effect on the gut microbiome and their potential as therapeutics. This research builds on the growing amount of knowledge around the antimicrobials, the gut microbiome and the effect antimicrobials have on the composition and functionality of the gut microbiome.