Biochemistry and Cell Biology - Doctoral Theses

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    Regulation 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 Research
    Insulin-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.
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    IGF-1 signalling controls mitochondrial morphology and basal mitophagy in cancer
    (University College Cork, 2023-01-06) Murray, Joss; O'Connor, Rosemary; Science Foundation Ireland
    Insulin-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.
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    Development of a sensor-based rapid microbial testing platform for the Irish meat industry
    (University College Cork, 2023-01-05) Elisseeva, Sophia; Papkovsky, Dmitri B.; Kerry, Joseph; Department of Agriculture, Food and the Marine, Ireland; Dawn Meats
    Microbial spoilage and foodborne diseases cause significant productivity and economic losses for the food industry. There is a need for novel approaches to extend shelf life of products, improve quality and microbial safety, reduce spoilage and waste, and new assessment methods. Traditional methods are time consuming, labour intensive, centralised, have lengthy time to result, and some cannot analyse crude food homogenates. In this project, funded by the Irish Department of Agriculture, Food and the Marine and performed in partnership with a large Irish food company (Dawn Meats), several new optical oxygen sensor based systems were devised to increase efficiency and accuracy of testing. Chapter 1 (Literature review) describes the state of the art in the area, the range of existing approaches and analytical systems, and their capabilities. Experimental methods used in this study are summarised in Chapter 2 (Materials and Methods). The first experimental section (Chapter 3) describes the development of new multi-parametric toxicity testing platform based on the soluble oxygen probe, MitoXpress-Xtra, and 96 well plate format, which was used to investigate the antimicrobial effects of the compound Lauroyl Arginate Ethyl Ester (LAE) on pure cultures and whole meat microbiota. Through the measurement and analysis of the oxygen probe time profiles (phosphorescent lifetime) under different assay settings, we were able to assess and quantify the toxicity of LAE on different bacterial species, generating dose-response curves, and calculating EC50. The assay allowed for the simultaneous assessment of multiple variables and conditions such as bacterial species, temperature, growth media, sample type, and antimicrobial concentrations. The second experimental part (Chapter 4) describes the new portable and autonomous system(s) based on disposable vials integrated with solid state sensors, tailored for the analysis of meat samples, carcass swabs, and environmental swabs. Along with the disposable sensor vials, the system is composed of two additional parts; a handheld, autonomous sensor reader and a portable incubator/heater. Up to 20 samples were prepared using the standard methods (ISO 4833-1:2013; ISO 18593:2018) in sensor vials, incubated at 30C and measured hourly in a non-invasive, contactless manner. Such a simple system with manual measurements also revealed dissolved oxygen time profiles which were used to determine the threshold time of the sensor signal, which in turn, was used to calculate TVC values (CFU/ cm2 or CFU/g) using developed calibration equations. The method was validated using: i) meat samples and carcass swabs obtained from Dawn Meats, ii) brush swabs of artificially contaminated surfaces with E. coli, iii) swabs of surfaces contaminated with meat microbiota and iv) environmental swabs. No statistical difference was found between the sensor based method and reference method, providing the opportunity for the former to potentially replace the latter. Finally, in Chapter 5 the new respirometric sensor based system was applied to a shelf-life study with four different types of MAP mincemeat samples: beef, turkey, lamb and pork, together with the analysis of these samples by 16S rRNA sequencing. Respirometric profiles revealed unusual linear profiles for pork and lamb mince, the origins of which remain to be investigated. In addition to respirometric microbiological assessment, the whole microbiome of each mincemeat type was analysed using 16S rRNA sequencing, which revealed an overall decrease in alpha diversity with some taxa exhibiting statistically significant changes over shelf-life and after exposure to respirometry. Beta diversity was seen to be dictated by mincemeat type. Overall, the new optical oxygen respirometry systems are highly efficient and attractive for the food industry, with both developed systems improving existing methods in time to result, accuracy, user-friendliness, and on-site use. Furthermore, the portable sensor based system can be combined with cutting edge techniques such as next generation sequencing to provide more detailed information on the microbiota of food samples.
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    Investigation of the TNF superfamily of ligands as potential therapeutic targets in inflammatory bowel diseases
    (University College Cork, 2022-05-10) Rajaram, Subhasree; Nally, Ken; Melgar, Silvia; Science Foundation Ireland; AbbVie
    Inflammatory bowel diseases (IBD) are chronic diseases of the gut affecting a significant proportion of the population in the western world with increasing incidence globally. They are multifactorial diseases with susceptibility mediated by a combination of host genetics and environmental factors leading to altered gut barrier function with ongoing non-resolving innate and adaptive inflammatory responses to components of the gut microbiota. There are two major forms - Crohn's disease (CD) and Ulcerative colitis (UC) differing in their location along the gastrointestinal tract, their pathophysiology, and characteristics of the ongoing innate and adaptive immune responses. While CD is largely characterised by a mixed Th1/Th17 immune response, UC is characterised by a so-called Th2 immune response. As reviewed in Chapter1 of the thesis, various cytokines, particularly IFN-γ and TNF-α, produced by activated immune cells, play a significant role in the pathogenesis of IBD. TNF-α blocking antibodies, known collectively as anti-TNFs, revolutionised the treatment of IBD in the mid to late 1990s; however, about 60% of patients are classified as inadequate responders, experiencing either no response or losing the response over time to both these anti-TNF drugs and newer targeted therapies. Hence there is a large therapeutic gap in IBD, leading to a clinically unmet need for new potential therapeutic targets that can be targeted in addition to TNF-α to obtain maximum efficacy. In the first study (Chapter 2 of this thesis), we investigated potential mechanisms underpinning synergy between IFN-γ and TNF-α and their ability to induce cell death of intestinal epithelial cells. Through weighted gene co-expression network analysis (WGCNA) of microarray data from HT-29 cells treated with IFN-γ and TNF-α, we identified gene modules that suggested both cytokines can cross regulate components of each other’s receptor-mediated signalling pathways. We confirmed these results through wet-lab experiments and propose the activation of a positive feedback loop when cells are treated with both IFN-γ and TNF-α, leading to enhanced activation of the signalling pathway and cell death. Though genes associated with the TNFR-related apoptotic and necroptotic pathways are increased when cells are treated with IFN-γ+TNF-α, perturbation of these canonical cell death pathways using commercially available inhibitors could not block the cytokine-induced cell death. STAT1 was identified as the main component required for IFN-γ+TNF-α-induced cell death, and we propose that cytokine-mediated pathway cross-regulation drives a positive feedback signalling loop culminating in enhanced activation of STAT1 and STAT1-induced cell death. In the second study (Chapter 3 of this thesis), we identified TNF-α-like cytokines that can exhibit synergy with IFN-γ as an approach to finding new targets to overcome resistance to anti-TNFs. For this, we screened ligands of the TNF superfamily of which TNF-α is a member. We utilised IFN-γ+TNF-α induced cell death and chemokine production as phenotypic readouts to screen a library of recombinant TNFSF ligands. We identified three major hits - TRAIL, TWEAK and LIGHT that could synergise with IFN-γ to induce pro-inflammatory chemokines and cell death in colon cancer cell lines and primary human colonic organoids. In the last chapter (Chapter 4 of this thesis), we selected one of these hits- TRAIL and investigated its synergistic effects with IFN-γ. IFN-γ and TRAIL synergised to induce cell death and production of chemokines, particularly the Th1 chemokine CXCL10. Similar to IFN-γ+TNF-α synergism, the IFN-γ+TRAIL-induced synergistic cell death was independent of canonical TRAIL-R associated apoptosis and necroptosis and was regulated via a caspase-8-JAK1/2-STAT1 pathway with STAT1 activation playing a significant role. Our results show that TRAIL is a TNF-α like molecule from the TNF superfamily and is a potential target for the treatment of IBD. The Casp-8/JAK1/2-STAT1 pathway plays a major role in modulating synergistic responses by the cytokine combinations. These results also explain, to an extent, the success of JAK inhibitors in the treatment of IBD. With further pre-clinical and clinical studies with antibodies targeting the hits from the screen, namely, TWEAK, LIGHT and TRAIL, either alone or in combination with anti-TNFs, new therapies can be developed for clinical trials in IBD.
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    Structural basis of ribosomal frameshifting
    (University College Cork, 2021-10-10) Bhatt, Pramod R.; Atkins, John F.; Irish Research Council
    Versatility in genetic decoding enriches gene expression. Programmed shifting of the reading frame during translation is a prominent feature productively utilized in probably all life forms. While the occurrences, function, and mechanistic components have been identified for many instances of frameshifting, structural understanding of their basis at the atomic level has been limited to the individual components in isolation. Here, I present atomic level information of the decoding apparatus relevant to both -1 and +1 frameshifting. The +1 frameshifting event investigated is that of a sensor and effector of an autoregulatory circuit present in the common ancestor between yeast and humans. In regulating intracellular polyamine levels, the mammalian antizyme 2 frameshifting studied interacts with ornithine decarboxylase and c-myc. The polyamine spermidine is shown here to be present proximal to but not interfering with the peptidyl transferase center, making stabilizing interactions with the terminal phosphate of the P-site tRNA. Spermidine occupancy at this location is inferred to preclude binding of the translation factor eIF5A by occlusion of its hypusine moiety. Visualization of the nascent chain reveals a network of interactions with key residues of the peptide exit tunnel that gate the transit of the growing polypeptide. The -1 frameshifting event investigated is that required by SARS-CoV-2, the causative virus of the COVID-19 pandemic. The downstream RNA element that stimulates frameshifting is seen to adopt a corkscrew-shaped 3-stemmed pseudoknot structure that lodges itself at the entrance of the ribosomal mRNA channel. Distinct functional interactions of the nascent chain with the ribosome exit tunnel are observed while the distal end of the nascent peptide begins to undergo co-translational folding into a zinc finger motif. The study highlights the frameshift-inhibitory properties of Merafloxacin, a small molecule that inhibits propagation of SARS-CoV-2.