Biochemistry and Cell Biology - Doctoral Theses

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    The gut microbiome of the wild great tit (Parus major): drivers and fitness consequences
    (University College Cork, 2023) Somers, Shane Edmond; Quinn, John; Ross, R. Paul; Stanton, Catherine; Irish Research Council for Science, Engineering and Technology; European Research Council; Science Foundation Ireland
    The gut microbiome plays a vital role in its host’s ecology. Clinical studies have shown gut microbes increase host health and fitness by providing digestive and immune functions, as well as aiding development. Natural variation in the microbiome is widely believed to affect host fitness in the wild but we are lacking experimental studies to test this. The microbiome varies with both host and environmental factors but most studies to date have focussed on individual factors and not adequately addressed the multiple overlapping and hierarchical drivers of microbiome variation working at environmental, host and microbial scales. This thesis investigates the role of the gut microbiota in host fitness, and how this is affected by and varies across contexts. Additionally, we address sources of variation in the gut microbiota at a host and environmental level, accounting for host ecology and drivers at different scales. We find that the host’s weight is correlated with microbiome diversity during development but that the direction of this relationship is context dependent. This shows that the microbiome interacts with the environment to determine host fitness and is important because it helps explain the contradictory findings linking diversity to weight. We also show that the interaction between the host, its microbiome and environment change with developmental stage. Specifically, we found that the microbiome of developed individuals is remarkably resilient to environmental perturbation, while developing individuals are much more sensitive, with important implications for future experiments. We developed a novel method for experimentally perturbing the microbiome that will allow microbiome researchers to begin testing hypotheses linking the microbiome to host ecology and evolution in natural settings. Finally, we show that welfare measures, such as environmental enrichment may interact with the gut microbiota to impact on host health and behaviour. In summary, I show that variation in the microbiome is linked to host ecology and that this variation is linked to host fitness.
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    Functional characterisation of LNX1 and LNX2 proteins
    (University College Cork, 2022) Lenihan, Joan A.; Young, Paul; Irish Research Council
    Ligand of Numb protein X1 (LNX1) and LNX2 are E3 ubiquitin ligases that contain a catalytic RING (Really Interesting New Gene) domain and four PDZ (PSD-95, DlgA, ZO-1) domains. LNX1 and LNX2 can interact with Numb – a key regulator of neurogenesis and neuronal differentiation. LNX1 can target Numb for proteasomal degradation, and Lnx mRNAs are prominently expressed in the nervous system, suggesting that LNX proteins play a role in neural development. This hypothesis remains unproven, however, and our understanding of LNX protein function is very limited – largely because LNX proteins are present at very low levels in vivo. Chapter 2 of this thesis addresses this – investigating possible reasons for the low levels of LNX proteins observed in vivo, at both transcriptional and translational levels, and also in terms of protein stability. Luciferase reporter assays show that the 5’ untranslated region of the Lnx1_variant 2 mRNA, that generates the LNX1p70 isoform, strongly suppresses protein production. This effect is mediated in part by the presence of upstream open reading frames (uORFs), but also by a sequence element that decreases both mRNA levels and translational efficiency. By contrast, uORFs do not negatively regulate LNX1p80 or LNX2 expression. Instead, some evidence is presented that protein turnover via proteasomal degradation may influence LNX1p80 levels in cells. To gain functional insights into the LNX family, Chapter 3 details the first physiologically relevant affinity purification/mass spectrometry-based analysis of the LNX interactome. In the context of mammalian cells, this approach identified a large number of novel LNX1-interacting proteins, as well as confirming known interactions with NUMB and ERC2. Many of the novel interactions mapped to the LNX PDZ domains, particularly PDZ2, and many showed specificity for LNX1 over the closely related LNX2. It was shown that LIPRIN-α1, KLHL11, KIF7 and ERC2 are substrates for ubiquitination by LNX1. LNX1 ubiquitination of LIPRIN-α1 is dependent on a PDZ binding motif containing a carboxyl terminal cysteine that binds LNX1 PDZ2. Surprisingly, the neuronally-expressed LNX1p70 isoform, that lacks the RING domain, was found to promote ubiquitination of Liprin-α1 and KLHL11, albeit to a lesser extent than the longer RING-containing LNX1p80 isoform. Of several E3-ligases identified in the LNX1 interactome, interactions of LNX1 with MID2/TRIM1 and TRIM27 were confirmed. On this basis, a model is proposed, whereby LNX1p70 - despite lacking a catalytic RING domain, may function as a scaffold to promote ubiquitination of its ligands through recruitment of other E3-ligases. Proteomic analysis of LNX-interacting proteins in the context of brain tissue identified and/or confirmed interactions of LNX1 and LNX2 with proteins known to have presynaptic and neuronal signalling functions, including the presynaptic active zone constituents ERC1, ERC2, and LIPRIN-s (PPFIA1, PPFIA3), as well as the F-BAR domain proteins FCHSD2 (nervous wreck homolog) and SRGAP2. To examine the role of LNX proteins in vivo, mice lacking both LNX1 and LNX2 expression in the brain were generated. Surprisingly, these mice are viable, fertile and physically healthy (Chapter 4). Behavioural analysis of LNX1/LNX2 double knockout mice revealed decreased anxiety-related behaviour, as assessed in the open field and elevated plus maze paradigms. By contrast, no major defects in learning, motor or sensory function were observed. The proteomic analysis (Chapter 3) revealed several novel neuronal LNX-interacting protein candidates that might contribute to the anxiolytic phenotype observed. Overall, these findings provide novel functional insights into the LNX protein family and identify promising candidates to mediate LNX functions in the central nervous system.
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    An investigation of the development and evaluation of online approaches for improved kinaesthetic learning in science
    (University College Cork, 2023) Scanlan, Anna M.; McCarthy, Tommie V.; Kennedy, Declan
    Kinaesthetic learning approaches (KL) offer great potential to enhance learning in the advanced molecular sciences. However, online KL remains under-researched and poorly implemented on affordable, scalable platforms. Furthermore, there appears to be a disconnect between the fields of education psychology and neuroscience when discussing kinaesthetic techniques. This research connects what is known from both disciplines to provide a coherent overview of what constitutes kinaesthetic learning. Here, an online KL assembly model is presented which proved effective for learning advanced molecular science topics as exemplified by three different lessons: the Lac Operon gene regulation system in E. coli, DNA transcription and translation, and Salmonella virulence factors. A mixed-methods study was conducted including three pilot studies, three randomised control trials and two sub-studies. Study participants included over 100 students from a variety of secondary schools (typically aged 16-19 years), over 250 first-year undergraduate science and medicine students, and 18 postgraduate students from both science and non-science disciplines. Topics were chosen for which each cohort would have little to no prior learning. Results show that KL assembly was at least as effective and, in some instances better than, some top learning strategies identified in education psychology namely, computer notetaking (Trafton & Trickett, 2001; Bui et al., 2012; Chi & Wylie, 2014) and retrieval-practice (O’Day & Karpicke, 2021). KL assembly involving both movement and recall was most effective overall for long-term learning retention, and for learning science material that is represented in a complex graphical and text format.
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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.