Biochemistry and Cell Biology - Doctoral Theses

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    Interrogating annotated protein coding regions for hitherto undetected translation
    (University College Cork, 2023) Fedorova, Alla; Baranov, Pavel V.; Science Foundation Ireland
    Ribosome profiling (Ribo-seq) is a technique that allows to capture ribosome protected fragments and sequence them. This powerful method enables discovery of not yet annotated proteoforms and translated open reading frames (ORFs), even ones that are hidden in annotated protein coding regions. Here we employed the Ribo-seq data together with comparative genomics analysis in order to discover non-AUG initiated proteoforms derived via alternative translation start sites that are in-frame with annotated starts. Production of such non-AUG proteoforms can be split into two scenarios. First, some nonAUG proteoforms are generated as alternative proteoforms in addition to annotated AUG- initiated ones. This phenomenon is called PANTs - Proteoforms with Alternative N-termini. The second scenario is when a non-AUG codon is used exclusively as the translation start for the generation of the main protein product from mRNA. In addition to discovery of non-AUG proteoforms, we rebuilt and upgraded an instance of the Galaxy platform for processing Ribo-seq data called RiboGalaxy. This update enabled prediction of novel translated ORFs from raw Ribo-seq reads by using only an internet browser with no need of local software. This update made working with Ribo-seq data more accessible to the scientific community. Chapter 1 is an introductory chapter which describes Proteoforms with Alternative N termini - PANTs. In particular, it covers different sources of PANTs, their functions and methods for their discovery. Chapter 2 covers the development of a pipeline for detection of non-AUG N-terminally extended proteoforms in the human genome which constitutes a phylogenetic approach and Ribo-seq-based approach. It also narrates the discovery of novel non AUG N-terminal extensions using the aforementioned pipeline and an attempt to describe the functionality of those non-AUG N-termini. Chapter 3 describes the phenomenon of exclusive non-AUG initiation when only non AUG initiated proteoform is generated from mRNA unlike Proteoforms with Alternative N-termini (PANTs) when both non-AUG and AUG proteoforms are generated from the same mRNA. Reported proteoforms were analysed and novel candidates predicted using Ribo-seq data. Chapter 4 reports the development of an update of RiboGalaxy - an interactive user friendly online platform for the processing Ribo-seq data which covers all the steps from preprocessing raw reads and quality control to transcriptomic and genomic alignments which then can be visualised and analysed in Trips-viz and GWIPS-viz - transcriptomic and genomic browsers for ribosome profiling data which altogether comprise the resource. This platform enables preparing ribosome profiling data for subsequent detection of translated ORFs in Trips-viz. This update includes its backend moving to configuration manager (ansible), updating tools, their dependencies and reference indices and adding novel tools that allow to prepare files for easy upload to GWIPs-viz and Trips-viz.
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    Exploring the camouflaged and non-coding genome: analysing difficult genomic regions in human disease genetics
    (University College Cork, 2023) Becerra Rodríguez , Maria de los Ángeles; Moore, Thomas F.; Baranov, Pavel V.; Science Foundation Ireland
    The main objective of this thesis is to highlight the importance of investigating camouflaged regions, specifically segmental duplications, and non-coding regions, in the human genome. These regions, often overlooked due to their complexity, hold immense potential for uncovering novel insights into disease genetics. In pursuit of this objective, this thesis first focused on the study of camouflaged and non-coding regions in the context of schizophrenia. Schizophrenia is a complex psychiatric disorder specific to humans characterized by a combination of altered cognitive function, distorted perception, and disrupted social behaviour. Understanding the genetic underpinnings of schizophrenia is crucial for advancing our knowledge of its aetiology and developing more effective diagnostic and therapeutic approaches. Through comprehensive genomic analyses, novel insights were gained, which identified a novel duplication in a locus affecting a dopamine receptor implicated in neurotransmission. Additionally, small deletions in constrained non-coding regulatory regions were implicated in schizophrenia for the first time. Moreover, this thesis characterizes a long non-coding RNA (lncRNA) originating from the segmentally duplicated Pregnancy-Specific Glycoprotein locus. The lncRNA was expressed exclusively in oligodendrocytes, implicating it in the regulation of myelination processes in the brain. This lncRNA is human-specific, further emphasizing the biological relevance of camouflaged and non-coding regions in the context of human evolution. Throughout this thesis, a human-specific perspective was adopted, recognizing the unique genomic features that shape our species. By expanding our knowledge of difficult genomic regions, such as camouflaged and non-coding regions, this thesis aims to close the gap in the missing heritability problem and to gain a comprehensive understanding of the genetic architecture underlying complex disorders and uniquely human traits like schizophrenia.
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    Investigating the host-microbe dialogue in aging
    (University College Cork, 2022-12-16) Killian, Christina; Joyce, Susan; Clarke, David J.; Eli Lilly and Company
    This 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.
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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.