College of Science, Engineering and Food Science - Masters by Research Theses
Permanent URI for this collection
Browse
Recent Submissions
Item Studies on protein standardisation of skim milk powder-based systems using milk ultrafiltration permeate and lactose(University College Cork, 2024) Cusack, Maeve A.; O'Mahony, Seamus Anthony; Kelly, Alan; Kerry GroupThe protein content of skim milk powder (SMP) is often standardised to achieve more consistent composition and functionality, whilst also ensuring the protein content of the powder product is as close as possible to the target. This requirement for protein standardisation arises largely from the changes in chemical composition of raw milk, as influenced by stage of lactation, breed of cow, genetics and also factors including diet and weather. To achieve standardisation, two standardising media are permitted to reduce the protein content of milk, i.e., lactose and milk permeate. When lactose or skim milk permeate are added, either in liquid (reconstituted) or powder form, they reduce the protein content by effectively diluting the protein present in the milk. The objectives of the work reported in this thesis were to develop an optimised standardisation process, focused in particular on the approaches used for reconstitution of the standardisation media, lactose and skim milk permeate powder, including the influences of temperature, mixing time and holding time (Chapter 2). Both powders were reconstituted at 50% total solids and the resultant dispersions were analysed for a range of parameters, including pH, viscosity, colour, sediment, conductivity and lactose crystallisation. Reconstitution of the skim milk permeate and lactose powders at 80°C, followed by cooling to 60°C, was determined to be the optimal process for preparing the standardisation material, resulting in dispersions with low viscosity and low levels of sediment, when compared to those reconstituted at lower temperatures. This optimised standardisation process was then used in Chapter 3 to examine the influence of protein content modification and standardisation media on the functionality of low-protein skim-based systems, standardised with lactose or permeate to 3.3 and 3.5% protein, with unstandardised skim milk, with an average protein content of 3.92 ± 0.13% used as the control. These are considered low-protein skim-based systems as, when spray-dried, they produce powders with 16-22% protein, and do not meet the the Codex Alimentarius standard for SMP. The functionality of the low protein skim-based systems was investigated by measuring heat stability, particle size, viscosity and ethanol stability. The permeate-standardised skim milk systems had shorter heat coagulation times (<2 min) and increased particle size on heating from 45 to 85°C when compared to the lactose-standardised skim milk systems and unstandardised control. Mineral contents of the permeate-standardised systems were higher than those of the lactose-standardised skim milk systems. The results of this work clearly demonstrate that the additional minerals in the skim milk permeate systems strongly affected the functionality of the protein-standardised skim milk systems, particularly in applications involving high heat treatments. Rheological analysis showed minimal changes in viscosity on heating of all systems, indicating that the systems were stable at 90°C for 2 min and could be used in lower heat-treated end applications. The original research described in this thesis contributes new scientific knowledge to the process of protein standardisation, as applied to SMP, and to our understanding of the effects of protein standardisation on functionality and applications of skim milk-based systems.Item The influence of powder composition, physical properties, and design configurations on the suitability of process analytical technologies for the in-line analysis of moisture in dairy powders(University College Cork, 2024) Murphy, Áine L.; O'Mahony, Seamus Anthony; O'Callaghan, Tom F.; Behan, Ger; Kerry GroupMoisture content is one of the most important parameters to monitor and control in dairy powders. Increased moisture contents in the finished powder can allow a higher yield to be achieved, but could also lead to increased risks of quality issues such as microbial growth, stickiness, caking and impaired flowability. For these reasons, within the dairy industry, there is growing interest in the development, and in-line integration of, moisture analysers within drying and powder handling equipment. Near infrared (NIR) spectroscopy is a quick, non destructive, non-product contact option in such scenarios. The studies presented in this thesis involve investigation and identification of the most suitable location to install a non-product contact NIR in-line analyser in an industrial sized spray dryer used for drying a range of dairy powders, with different compositions and physical attributes. Two NIR analysers were studied (PAT 1 and PAT 2), across two spray dryers (Dryer A and Dryer B). The success criteria included (1) the analyser must be accurate to within 0.2% of the standard oven moisture test, (2) the analyser must last 6 months without re-validation, and (3) the analyser must enable operators to control spray dryer settings to achieve real-time optimisation of powder moisture content. This original research demonstrated that a vertical downpipe, located after the fluidised bed, before packaging, with an intermittent internal air purge system to clear any dust accumulation on the sapphire window was the optimal configuration. The main challenges with this configuration, as identified in Chapter 2, were powder (skim milk powder (SMP) and whey powder) accumulation on the sapphire window, and low powder flow rate, both contributing to insufficient powder passing in front of the NIR beam, leading to light reflection from other materials (e.g., stainless steel). To better understand and help mitigate these challenges, in Chapter 3, the physical and bulk handling properties (i.e., particle size and shape, bulk and tapped densities, surface free fat (SFF) and flowability) of SMP and full cream milk powder (FCMP) were investigated, using an in-house designed, laboratory-scale replication of the industrial analyser configuration. This work demonstrated that the analyser was accurate for measuring moisture content of both SMP and FCMP, provided that the flow of powder over the sapphire window was optimal (i.e., no gaps/patches in the flow of powder). This was confirmed by p-values greater than 0.05 for both powders, indicating that the differences between the oven and PAT tool moisture results were not significantly different. This work also clearly showed that particle size distribution (PSD) and particle shape were the main factors influencing the flowability of SMP (with larger, less spherical powder particles having the greatest flowability), which was supported by Pearsons correlation coefficient results, while PSD and SFF were the main factors influencing the flowability of FCMP (with larger particles having low SFF, demonstrating greatest flowability). From the new findings in this thesis, dairy researchers can have a greater scientific insight into the integration and optimisation of non-product contact, in-line moisture analysers such as PAT 2, into spray dryers, to control the moisture content in commodity dairy powders (SMP and FCMP). The conclusions drawn from Chapter 2 will simplify the process of locating a suitable installation point for such an analyser, and the knowledge created from Chapter 3 will enable dairy researchers to tailor processes (e.g., insert powder guiding systems to control the flow, install air purges) based on the properties of the respective powders (SMP or FCMP). The ultimate goal for the dairy research community when applying PAT tools in an industrial sized spray dryer in the future, would be that the spray dryer is automatically controlled (e.g., using an advanced process control system) based on the feedback of moisture results produced by the PAT tool, which will remove the risk of human error. This system will allow for increased powder yield (increased profit margin), the conservation of energy (sustainable processing), and a more consistent product with an increase in right-first-time material (satisfied consumers and a reduction in down-grade material).Item Assessment of the American crocodile (Crocodylus acutus) population in Ambergris caye, Belize(University College Cork, 2024) Ó Mórdha, Ciarán; Collins, Courtney; Ramsay, Ruth; Summers, Chris; Manzi, Christina; Crocodile Specialist Group; European Crocodile NetworkAmerican crocodiles (Crocodylus acutus) have the second largest global distribution of all extant crocodilians, ranging from South Florida through Hispaniola, Cuba, and Jamaica, along the Caribbean coast from southern Mexico to Venezuela, and along the Pacific coast from Mexico to Peru. The species is considered Vulnerable by the IUCN throughout its range, with habitat loss currently a major threat facing the species in parts of its range, including Belize. In Belize, C. acutus is found throughout the coastal zone. Ambergris caye, the country’s largest and most densely populated caye, is inhabited by C. acutus, but its present status there is poorly understood. The aim of this study was to address these data gaps by assessing the status and life history of C. acutus on Ambergris caye and comparing this information with that from other areas within the species’ range to inform the development of a local American crocodile management plan. During 19 nocturnal eyeshine surveys covering 171.5 km of shoreline on Ambergris caye carried out between May and September 2023, 199 American crocodiles were observed, an encounter rate of 1.16 crocodiles/km consisting of 22% adults, 10% subadults, 16% juveniles, 22% yearlings/hatchlings and 30% whose size class could not be determined. These results were similar to those of previous surveys carried out in 2010-2011, but with a higher encounter rate in 2023, suggesting a stable, if not slowly increasing, population. Crocodile nests were located by visiting previously identified nest sites, conducting additional surveys by boat and drone, and utilising social media to investigate reports of nesting activity by residents. A total 9 nests were found during the study. More than half (56%) of these were concentrated along the sand banks adjacent to the caye’s three sewage treatment ponds. Utilizing the 13 years of crocodile capture data (n=865) collected by ACES Wildlife Rescue in Ambergris caye, this study investigated growth rates, morphometrics, and sexual size dimorphism of the local crocodile population. Analysis revealed the growth rate (total length [TL]) of 115 crocodiles averaged 0.045 ± 0.027 cm/day. The growth rates found in adults differed by sex, with females (n=20) exhibiting a slightly lower average growth rate (0.039 ± 0.028 cm/day) compared to males (n=47) (0.048 ± 0.028 cm/day); however, the difference was not statistically significant. Notably, as total length (TL) increased with age, growth rates slowed more significantly in females than in males, with no significant relationship observed between TL and growth rates in males. Morphometric analysis encompassed 332 juveniles (TL<120cm), 25 female sub-adults (120≤ TL<180 cm), 31 male sub-adults (120≤ TL<180 cm), 40 female adults (TL≥180 cm) and 62 male adults (TL≥180 cm). Allometric relationships between TL and dorscal cranial length (DCL), snout length (SL), snout vent length anterior (SVLa) and snout vent length posterior (SVLp) revealed both positive and negative associations, all of which followed linear regressions. The dorsal cranial length to cranial width ratio (DCL: CW) of 2.7 to 4.2 indicates a broad-snouted skull morphotype akin to that observed in other island populations of C. acutus in the Yucatan region. The sexual dimorphism index of 1.07 toward males suggests a relatively minor disparity between the TL of males and females on Ambergris caye, indicating less pronounced sexual dimorphism compared to other populations of C. acutus. The sex ratio 2.39(M):1.0(F) was biased towards males. While Ambergris caye, likely the most human-populated caye in Belize, faces increasing development, pollution, and human-crocodile interactions, it may harbor one of the largest and most stable American crocodile populations in the country. The encounter rate of 1.16 on Ambergris caye is notably higher than that reported for nearby Turneffe atoll (0.34 in 2009) and the Belizean cayes overall (0.43 in 2000), suggesting this population is among the most robust in Belize. However, the study’s findings indicate that insufficient nesting habitat could pose a risk to the long-term stability of the population if not addressed through targeted conservation efforts. To mitigate this risk, it is imperative to conduct annual surveys to ascertain long-term population trends. Additionally, implementing protective measures for nests and intensifying surveys within the Bacalar Chico National Park and Marine Reserve to investigate nesting activity are vital steps in formulating a robust species management plan. Addressing these research gaps will not only enhance our understanding of the ecology of C. acutus in Belize but also inform targeted conservation efforts essential for safeguarding this population's viability and ecological role in the region.Item Engineering therapeutic messenger RNA for triple-negative breast cancer(University College Cork, 2024) Leahy, Mark; Krajewska, Malgorzata; Kowalski, Piotr; O'Connor, RosemaryBackground: Targeted therapies are revolutionising the landscape of cancer treatment, providing better therapeutic responses and minimising the adverse effects of conventional treatments such as radiation and chemotherapy. Human epidermal growth factor receptor 2 (HER2) is overexpressed in multiple cancer types and remains one of the therapeutic flagship targets in oncology. The HER2-targeting monoclonal antibody trastuzumab has dramatically improved the survival of patients with HER2-positive breast cancer. More recently developed antibody-drug conjugates, such as trastuzumab deruxtecan (T-DXd), have demonstrated unprecedented efficacy in HER2-overexpressing cancers. However, only a small subset of patients can benefit from HER2-targeted treatments (15-30% of breast cancers). Triple-negative breast cancer (TNBC) is characterised by the absence of HER2 and hormone receptors. TNBC is an aggressive subset of breast cancers for which few targeted therapies are available. It has recently been shown that artificial overexpression of HER2 could sensitise TNBC xenograft models to trastuzumab, however the clinical relevance of this strategy was limited by the use of viral vectors. Messenger RNA (mRNA) technology has been cemented as a safe and effective therapeutic modality, as evidenced by its success in SARS CoV-2 vaccination. Its potential to revolutionise cancer therapy is now becoming increasingly evident. We hypothesised that mRNA technology could be used to transiently overexpress HER2 in TNBCs to sensitise them to anti-HER2 therapeutics. Methods: Molecular cloning techniques were used to engineer a plasmid vector for in vitro transcription (IVT) of a truncated variant of HER2 (TrHER2), lacking the intracellular domain (ICD) to eliminate the potential for proliferative signalling. Modified nucleosides were incorporated into IVT reactions and mRNA was enzymatically capped and poly(A) tailed. TrHER2 and control (Luciferase and WtHER2) mRNA were delivered to TNBC cells using conventional transfection reagents. TrHER2 protein expression and HER2 signalling were measured by means of western immunoblot. TrHER2 localisation was assessed by immunofluorescent microscopy and flow cytometry. The impact of TrHER2 expression on migration and clonogenicity was measured by scratch and colony formation assays. Trypan blue and CellTiter-Fluor assays were used to assess the impact of TrHER2 expression on the efficacy of T-DXd and trastuzumab. Induction of apoptosis was determined by flow cytometry with Annexin V and propidium iodide stain. The effect of TrHER2 mRNA on trastuzumab function was determined by co-culture of TNBC cells with human immune cells. Results: TrHER2 plasmids were engineered and validated by sanger sequencing. IVT reactions with these plasmids produced pure, high yield TrHER2 mRNA. TrHER2 protein was shown to be approximately 100 kDa, lacking expression of the HER2 ICD. TrHER2 mRNA expression demonstrated cell surface localisation with a >110-fold increase in expression, exceeding that of WtHER2 mRNA (50-fold) at an equal dose. TrHER2 mRNA delivery did not active the HER2 signalling pathway, instead reducing cell migration and clonogenicity compared to WtHER2 mRNA. TrHER2 mRNA expression significantly increased the efficacy of T-DXd, causing an 80% reduction in viability (p<0.0001) by inducing DNA damage and apoptosis. TrHER2 mRNA also sensitised TNBC cells to antibody-dependent cell-based cytotoxicity by trastuzumab, causing an 37% reduction in viability compared to cells transfected with reporter mRNA (p<0.001). Significance: These findings demonstrate proof-of-concept for utilising mRNA-based overexpression as a strategy to enhance the sensitivity of TNBC cells to anti-HER2 therapies. Additionally, the results validate the safety profile of the engineered TrHER2 mRNA. The observed high efficacy suggests promising in vivo effectiveness. When paired with a suitable delivery system, this approach could have the potential for clinical translation.Item Modelling the tumour microenvironment and reversible and irreversible chemotherapy resistance(University College Cork, 2024) Quinlan, Thomas; Wieczorek, Sebastian; Alkhayuon, Hassan; Mulchrone, Kieran F.; University College CorkThe tumour microenvironment (TME) comprises various cell types, including cancer cells, immune cells, and stromal cells, which all engage in complex, dynamical interactions. Understanding these interactions, particularly their role in promoting cancer growth and the emergence of drug resistance, is crucial for developing effective treatment strategies. This project constructs a comprehensive mathematical model of the TME that incorporates the effects of chemotherapy. We systematically derive a series of process-based equations to describe the tripartite interactions between cancer cells, immune cells and stromal cells within the TME, as well as their response to chemotherapy. Throughout the model development, we analyse sub-models to ensure we capture key qualitative behaviours. Finally, we conduct a qualitative study by simulating our model under various hypothetical TME scenarios and different chemotherapy dosing schedules. Our simulations demonstrate how variations in stromal activity and treatment regimens can lead to diverse patient outcomes, highlighting the need for personalised treatment strategies.