College of Science, Engineering and Food Science - Masters by Research Theses
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Item Differential regulation of the phosphorylated and dephosphorylated forms of the ubiquitin-conjugating enzyme Ubc6e(University College Cork, 2024) Burns, Stephanie; Fleming, John V (Eoin)The ubiquitin proteasome system (UPS) is a key stress response employed by cells to remove damaged and misfolded proteins that accumulate in the endoplasmic reticulum (ER) during energy deprivation, hypoxia, or viral infection. Ubc6e is an ER-localised ubiquitin-conjugating enzyme that plays an important role in the proteasomal degradation of misfolded proteins and can be phosphorylated at serine residue 184. Recent studies have demonstrated that Ubc6e is co-localised with p62/sequestrosome, which is a cargo receptor that delivers ubiquitinated cargo to autophagosomes for degradation. To further investigate the possible role that Ubc6e may play in autophagy, we aimed to investigate any protein-protein interactions between p62 and Ubc6e and we also aimed to investigate the effect of autophagic-related proteins, p62 and ULK1 on Ubc6e. From our research, we saw that Ubc6e levels are reduced when co-expressed with proteins that promote autophagy. Specifically for the autophagy inducer ULK1, we saw differential regulation between the phosphorylated and dephosphorylated forms of Ubc6e, suggesting that the S184 phosphorylation may protect the protein from autophagic degradation. Degradation of the dephosphorylated form, on the other hand, did not depend on S184 phosphorylation, or the catalytic activity of Ubc6e. Neither was it dependent absolutely on ER localisation. Finally, although ULK1 co-expression did not appear to specifically alter the ER/ perinuclear localisation of Ubc6e, it was noted that amino-terminal tagging with GFP led to a ULK1-related localisation of Ubc6e to intracellular vesicles. Together our results suggest that Ubc6e does play a role in autophagy however it is not mediated by the direct interaction with p62.Item Impact of endosomal recycling inhibitors on drug resistant and immune tolerant breast cancers(University College Cork, 2024) Chembukavu, Suraj Narayanan; Lindsay, AndrewBreast cancer (BC) is the most common and frequent of all the cancers, accounting for about 25% of all the cases of cancer detected in women annually. There are a number of approved drugs on the market that have been developed or repurposed for the treatment of BC. However, a recurrent problem faced in the clinic is the emergence of drug resistance, which can be intrinsic or acquired. Prolonged periods of chemotherapeutics and targeted therapy have resulted in the development of acquired resistance through a number of different adaptations. This project focuses on resistance developed by various sub-types of breast cancer cells through drug-therapy-induced senescence. Previous studies have reported the cytoprotective nature of this cell cycle arrest phenomenon upon prolonged exposure to targeted- and chemotherapeutics. This study investigates specific molecular mechanisms governing therapy-induced senescence, the impact it has on the membrane trafficking of iron transporters, and the potential repurposing of endosomal recycling inhibitors (ERIs) to target these drug-tolerant cells. Additionally, we examined the effects of ERIs on the total protein and surface levels of immune checkpoint proteins. This was done by targeting the recycling of the glycoprotein PDL1, which is expressed on the surface of some tumour cells and binds to its cognate receptors on the surface of T cells. PDL1-PD1 binding leads to T cell inactivation. A number of ERIs were used to determine their impact on the levels of PDL1.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 An adaptive and reliable forward error correction mechanism for real-time video delivery from UAVs(University College Cork, 2023) Sarvi, Batoul; Sreenan, Cormac J.; Zahran, Ahmed; Science Foundation IrelandThis thesis introduces the Adaptive and Reliable Forward Error Correction (AR-FEC) mechanism, an advanced protocol designed to enhance real-time video transmission in Unmanned Aerial Vehicles (UAVs) communications. Addressing the challenge of unreliable wireless channels, AR-FEC leverages edge computing principles at the application layer to incorporate adaptive FEC, dynamic video quality, and Unequal Error Protection (UEP). By dynamically adjusting redundancy frames within each video Group of Pictures (GoP) based on packet loss, round-trip times, and cross-layer network information, AR-FEC optimizes the balance between video quality and transmission reliability. A significant portion of this work is dedicated to rigorous simulation-based validation using the NS3 network simulator. The simulation environment is carefully crafted to replicate a range of UAV operational conditions, including varying distances and network loads, to assess the AR-FEC protocol's performance under realistic scenarios. Key performance indicators such as deliverable frame count, latency, throughput, and real-time streaming constraints are meticulously evaluated, demonstrating AR-FEC's capacity to outperform existing error correction methods significantly. The simulations also explore the protocol's behavior with multiple UAVs as senders, highlighting its robustness in complex network settings. The results indicate a marked improvement in video delivery quality, showcasing AR-FEC's potential for broad application across different UAV uses, from surveillance to disaster management. In summary, the thesis articulates the development and validation of AR-FEC, illustrating its superior performance and adaptability. The proposed mechanism not only advances the field of UAV communication but also establishes a comprehensive simulation framework for future research, potentially guiding the development of more reliable and efficient UAV multimedia communication systems.