http://theses.ncl.ac.uk/jspui/handle/10443/5256 Fri, 08 May 2026 23:06:05 GMT 2026-05-08T23:06:05Z http://theses.ncl.ac.uk/jspui/handle/10443/6766 Title: Understanding the role of transcription in organisation of the bacterial chromosome Authors: Norris, Jonathan Abstract: Bacterial chromosomes are organised by various proteins, types of supercoiling and other cellular processes. One such process, transcription, massively impacts the chromosomal structure from the local level up to overall organisation of the nucleoid. Uniquely to bacterial transcription, the process can be physically coupled with translation since they occur in the same cellular compartment. The processes and associated proteins can, thus, happen simultaneously and physically interact. This coupling can have further impact of the overall structure of the nucleoid. While transcription-translation coupling is well documented in E. coli, some work suggests that it does not happen in other bacteria, including the Gram-positive model bacterium Bacillus subtilis. To study transcription-dependent chromosome organisation at a single cell level in B. subtilis, I fluorescently labelled DNA in the vicinity of the promoter of an inducible gene coding for a transmembrane protein and followed the localisation of the gene locus using fluorescence microscopy. We found that, upon induction, the gene migrates from a central position in the cell towards the membrane, and back towards the nucleoid when induction is removed. This movement was further confirmed by monitoring the fluorescently labelled locus in vertically immobilised cells (Vertical Cell Imaging by Nanostructured Immobilisation), which provides a better optical viewing angle for the observed process. Inhibiting either transcription and translation, via antibiotics and mutations in respective initiation regions, abolished the movement of fluorescently labelled locus towards the cell periphery. This loss of gene movement indicates the involvement of both transcription and translation in the process. Our results are fully consistent with transertion; a postulated process in which transmembrane proteins are inserted in the membrane co-translationally and cotranscriptionally thereby pulling the gene locus from the nucleoid core to the periphery of the cell and provide the first direct experimental evidence for transertion in Gram-positive bacteria. Furthermore, these findings demonstrate that translation and transcription can indeed be coupled in B. subtilis, alongside translocation, at least for genes encoding for membrane proteins. Description: PhD Thesis Wed, 01 Jan 2025 00:00:00 GMT http://theses.ncl.ac.uk/jspui/handle/10443/6766 2025-01-01T00:00:00Z http://theses.ncl.ac.uk/jspui/handle/10443/6752 Title: Cyclin B1 regulation and APC/C processivity in mouse oocyte meiosis I Authors: Kerridge, Scott Thomas Abstract: Chromosome alignment is orchestrated by the activity of CDK1 bound to its coactivator cyclin B1. Equally important, chromosome segregation is orchestrated by termination of CDK1 activity, driven by cyclin B1 destruction. Both events must be precisely timed by APC/C (Anaphase Promoting Complex/Cyclosome) activity, an E3 ligase that targets cell cycle proteins for destruction via the ubiquitin-proteasome pathway. In mitosis, this system is rapid, robust, and well-studied. Critically, through oocyte chromosome alignment, CDK1 and APC/C activities must be regulated differently, over substantially longer time frames. Specifically, the APC/C must first be dampened to prevent cyclin B1 destruction and CDK1 activity loss as chromosomes begin aligning. This is followed by a period of non-CDK1-bound cyclin B1 destruction during final alignment stages, followed by CDK1-bound-cyclin B1 destruction once fully aligned. This ordering is vital to minimise errors that otherwise cause the cell to arrest (infertility) or produce an embryo that is incompatible with life (miscarriage). However, little is published regarding the molecular mechanism of cyclin B1 targeting in oocytes, or indeed the wider landscape of any APC/C substrate ordering. To address this, I have expressed fluorescent versions of cyclin B1 and APC/C substrates in live mouse oocytes, utilising time-lapse fluorescence microscopy to map their levels and movements through meiosis I. Additionally, I mutated short linear motifs within these substrates, as well as APC/C subunit knockdowns to obtain mechanistic insight into substrate targeting. I reveal that cyclin B1 harbours a motif which boosts its affinity for a specific form of the APC/C during chromosome alignment. This form of the APC/C does not exist globally across the cell, but likely in high priority areas across this spindle zone. This and other insights, describe novel aspects of cell cycle regulation that are critical to produce healthy eggs Description: Ph. D. Thesis. Wed, 01 Jan 2025 00:00:00 GMT http://theses.ncl.ac.uk/jspui/handle/10443/6752 2025-01-01T00:00:00Z http://theses.ncl.ac.uk/jspui/handle/10443/6750 Title: Causal neural mechanisms for decision making : putting rules into context Authors: Slater, Benjamin James Andrew. Abstract: Episodic memory, an important cognitive process in humans, relies on both contextual and temporal information to recall sequences of events accurately. While nonhuman animals are capable of understanding rules based on context and have shown capability in learning temporal sequences, the ability to flexibly shift between these types of information has yet to be demonstrated. This thesis aimed to explore the cognitive mechanisms behind adapting memory sequences to changing contexts in three species. Following work in Lister hooded rats, the study was extended to New world (Common marmosets) and Old world monkeys (Rhesus macaques) to assess each species’ proficiency in learning context-guided sequences. In addition, a noninvasive technique called transcranial ultrasound stimulation (TUS) was employed with the macaques to investigate potential underlying neurobiological pathways. The task design, consistent across all three species, involved learning visual object sequences (e.g., A-B or C-D) where the correct choice was contingent on one of two background contexts (e.g., blue or yellow). Results showed that rats and marmosets were proficient in learning sequences that remained constrained to a singular context but faced challenges with sequences involving a mid-trial contextual shift. Conversely, the macaques quickly mastered both fixed and context-switching sequences, facilitating further investigation into potential neurobiological mechanisms using TUS. Prior research suggests a possible role for the prefrontal-hippocampal circuitry in context-dependent learning, prompting us to apply targeted modulation to the hippocampus and prefrontal cortex separately. Notably, TUS of the anterior hippocampus enhanced performance during the initial stages of learning. In contrast, TUS of the prefrontal cortex enhanced performance in the later phases, particularly in trials that required a context shift mid-trial. Overall, this research highlights the evolutionary foundation of flexible learning and offers insights into neural modulation in primate cognition, particularly in how the brain adapts dynamically to contextual shifts when guiding memory sequences Description: PhD Thesis Wed, 01 Jan 2025 00:00:00 GMT http://theses.ncl.ac.uk/jspui/handle/10443/6750 2025-01-01T00:00:00Z http://theses.ncl.ac.uk/jspui/handle/10443/6734 Title: Identifying the mechanism behind mitochondrial changes in the cholinergic neurons of the pedunculopontine nucleus in patients with Parkinson’s disease Authors: Lu, Huiwen Abstract: n Parkinson’s disease (PD), patients often exhibit higher levels of somatic mitochondrial DNA (mtDNA) damage, particularly large-scale deletions, in their brains. This damage is associated with neuronal loss and has been seen in multiple neuronal types including nigral dopaminergic of the substantia nigra (SN) and cholinergic neurons in the Pedunculopontine Nucleus (PPN). Unlike dopaminergic neurons, PPN cholinergic neurons tend to maintain wild-type mtDNA levels by increasing mtDNA copy number (mtCN) in response to mtDNA deletions. This study employed ultra-deep sequencing to analyse single cholinergic neurons isolated in an unbiased fashion from postmortem PPN tissue of PD and controls, examining both the mtDNA deletions, and point mutations across various parameters. Comparative analysis of mtDNA deletions was also conducted with nigral dopaminergic neurons to elucidate compensatory response to mtDNA damage. Additionally, singleneuron qRT-PCR was used to quantify the expression of three nuclear genes involved in mitochondria biogenesis, maintenance and mitophagy. Our findings confirm significantly higher levels of large mtDNA deletions in PD patients compared to aged controls, with deletions ranging from 5-140bp (small) to >1,400bp (large). The distribution of large deletions suggests that replication errors are the primary mechanism underlying their generation, aligning with patterns observed in POLG patients and aged controls. Conversely, the low prevalence of small deletions suggests that oxidative damage is not a major contributor of mtDNA damage in these neurons. Point mutation levels did not differ significantly between groups across various parameters assessed. At the transcriptional level, PINK1 expression was upregulated in PD neurons, whereas no significant changes were observed in TFAM or PGC1α expression. These findings suggest that the mitophagy pathway plays a prominent role in regulatory response to mitochondrial changes in PD. Description: PhD Thesis Wed, 01 Jan 2025 00:00:00 GMT http://theses.ncl.ac.uk/jspui/handle/10443/6734 2025-01-01T00:00:00Z