http://theses.ncl.ac.uk/jspui/handle/10443/96 2026-02-07T03:16:01Z 2026-02-07T03:16:01Z Cottam, Curtis http://theses.ncl.ac.uk/jspui/handle/10443/6675 2026-02-06T15:59:16Z 2024-01-01T00:00:00Z

Title: Investigating the role of sugar uptake and metabolism in the regulation of Enterohaemorrhagic Escherichia coli virulence Authors: Cottam, Curtis Abstract: Enterohaemorrhagic Escherichia coli (EHEC) is a major foodborne pathogen of the human colon, and cause for zoonotic disease. Transmitted via the faecal-oral route, EHEC exhibits a remarkably low infectious dose, resulting in outbreaks that manifest in bloody diarrhoea, and in extreme cases, acute renal failure. Treatments against EHEC are limited due to conventional antibiotics exacerbating infection. Understanding EHEC pathogenesis is therefore crucial to the development of novel treatment strategies. EHEC have evolved to sense environmental nutrients as “signals” to fine-tune the expression of their primary virulence factor, the Type 3 Secretion System (T3SS), which is essential for host-cell colonisation. These signals include sugars and fats ingested as part of the diet, or by-products of metabolism by the gut microbiota. However, the mechanisms underlying how EHEC exploit these different nutrients are poorly understood. Here, a novel ATP-binding cassette (ABC) transporter in the murine pathogen Citrobacter rodentium, commonly used as a surrogate model for EHEC, was characterised. This system, known to be upregulated during murine infection, is specific for D-ribulose and likely aids colonisation of the mouse gut. Searches in EHEC revealed a similar ABC transporter encoded on a horizontally-acquired genetic element to be significantly enriched amongst EHEC strains. Transcriptionally, the locus was regulated exclusively by L-arabinose, in an AraC- dependent manner. Furthermore, growth on L-arabinose significantly enhanced T3SS expression and the ability to attach to host cells. Deletion of the genes required for L-arabinose uptake, metabolism and associated regulation revealed this phenotype to rely on L-arabinose breakdown and not merely “sensing” its presence in the environment. Collectively, this work suggests L-arabinose metabolism to be important in EHEC pathogenesis through providing a source of nutrition and enhancing virulence gene regulation. It is proposed that these systems and their substrates allow EHEC to outcompete the native gut microbiota, with their downstream metabolism contributing to virulence regulation. Description: Ph. D. Thesis.

2024-01-01T00:00:00Z Andres, Tim-Joshua Yannick http://theses.ncl.ac.uk/jspui/handle/10443/6674 2026-01-30T14:38:25Z 2025-01-01T00:00:00Z

Title: Finding what is normal - Consistency in individual behaviour and movement patterns in laboratory-housed Macaques and how this relates to individual personality traits Authors: Andres, Tim-Joshua Yannick Abstract: This thesis investigates individual differences in behaviour of laboratory-housed Rhesus macaques (Macaca mulatta) through the utilization of automated methods designed for in-cage observation. Animals consistently exhibit differences in movement and behaviour over time, this is often described as personality or temperament. Previous research found associations between temperament and physiological indicators, such as well-being and welfare of non- human primates. Rhesus macaques serve as a pivotal model species in biomedical research, and considerable efforts are made in optimizing their welfare for both ethical and scientific purposes. This emphasizes the significance of comprehending these individual differences and to specifically identifying behavioural needs. However, evaluating welfare and temperament typically entails labour-intensive processes reliant on human observers. Recent advancements in computational techniques to automate video analysis have transformed animal behaviour studies, reducing costs and labour intensity. Furthermore, such methods are non-invasive and less susceptible to observer bias. This thesis presents a self-built in-cage camera system (based on a Raspberry Pi), as well as a pipeline (based on YOLOv8, a state-of-the-art image-segmentation model) to automate video analysis and predict the location of pair-housed Rhesus macaques in their home cage. Utilising the camera module and pipeline, over 1500 hours of video material was collected and analysed. The findings reveal distinct individuality in movement trajectories as well as variations in space utilization across different contexts related to welfare and personality traits. In conclusion, the findings suggest the potential of automated methods to assess behavioural needs, temperament, and welfare on an individual level, thereby contributing to more precise and comprehensive care strategies for laboratory-housed macaques. Furthermore, I present an effective solution of monitoring home cage space use on a long-term basis during light periods (5:30AM-7PM). Description: Ph. D. Thesis.

2025-01-01T00:00:00Z Lambton, James http://theses.ncl.ac.uk/jspui/handle/10443/6673 2026-01-29T16:17:40Z 2025-01-01T00:00:00Z

Title: Investigating the mechanisms underlying ATG7 related neurological disease Authors: Lambton, James Abstract: The key catabolic process of autophagy relies on a set of core autophagy related (ATG) genes, of which very few have been directly associated with human disease. Genetic variants in ATG4D, ATG5, ATG7 and ATG9B have been associated with developmental delay, intellectual disability, ataxia, cerebellar and corpus callosum abnormalities. Here the first de novo heterozygous missense ATG7 subject is described, presenting similarly to the previously described ATG7 cohort, with impairment of the LC3-conjugation system and accumulation of the autophagy adapter p62 (PMID: 34161705). Also described is the first foetal ATG7 subject, harbouring segregating loss-of-function variants. Immunohistochemical and immunofluorescence analyses of subject brain tissue shows clusters of unmigrated neurons and p62 accumulation in neurons and cerebellar proliferative zone. To investigate these findings further, I have developed and characterised an induced pluripotent stem cell derived neural model of ATG7 pathology. CRISPR/Cas9 genome editing was used to engineer ATG7+/+, ATG7+/- and ATG7-/- iPSCs. Differentiation of these iPSCs into neuron-astrocyte co-cultures showed that ATG7-/- co-cultures recapitulated the biochemical phenotype of ATG7 primary fibroblasts. ATG7+/- and ATG7-/- co-cultures also demonstrated altered proliferation and morphology. Astrocyte specific changes were observed, with differing astrocyte differentiation and cell death among the ATG7+/- and ATG7-/- cultures. Functional Ca2+ live cell imaging showed increased intracellular Ca2+ in ATG7+/- neurons, with both ATG7+/- and ATG7-/- neurons having impaired Ca2+ recovery. Finally, a cohort of 3 subjects from 2 unrelated families harbouring rare, damaging ATG12 variants is also described, with affected individuals presenting with developmental delay, ataxia and cerebellar abnormalities. Biochemically they had mild impairment of either the ATG5-ATG12 or LC3-II conjugation pathway. The equivalent atg12 variants were also unable to fully recover autophagy in atg12-null Saccharomyces cerevisiae models. Together these data expand the existing cohorts of ATG related congenital disorders, with the ATG7 iPSC derived neural model elucidating disease mechanisms in addition to ATG7’s core role in autophagy. Description: PhD Thesis

2025-01-01T00:00:00Z Gopee, Nusayhah Hudaa http://theses.ncl.ac.uk/jspui/handle/10443/6671 2026-01-29T15:41:16Z 2025-01-01T00:00:00Z

Title: Decoding the development of human prenatal skin Authors: Gopee, Nusayhah Hudaa Abstract: Skin, the largest organ in the human body, is composed of diverse cell types strategically organised in defined strata and specialised appendages. Its morphogenesis is underpinned by precise spatiotemporally distributed cellular and molecular changes which remain incompletely deciphered. It is seeded by immune cells, including macrophages, early during gestation but their contribution, if any, to prenatal skin and hair follicle development is uncertain. To investigate de novo hair follicle formation and the role of immune cells in skin development, a multi-omics atlas of human prenatal skin (7 to 17 post-conception weeks) was assembled, integrating single-cell and spatial transcriptomic data. Comparative assessments with adult skin and hair follicle datasets and cross-species analyses were performed to determine features specific to human prenatal skin. A hair-bearing skin organoid model, derived from human embryonic stem cells and induced pluripotent stem (iPS) cells, was benchmarked against prenatal skin to evaluate the faithfulness of skin organoids to in vivo skin and their utility to functionally interrogate skin morphogenesis. Findings were validated using immunofluorescence, in situ hybridization and tissue culture experiments. The systematic charting of prenatal skin (433,961 single cells) identified the precursor cell states, differentiation trajectories and cross-compartmental cellular interactions underpinning human hair follicle neogenesis. Re-mapping of these single cells in situ, using spatial transcriptomic data, uncovered microanatomical tissue niches where immune cells co-located and cross-talked with non-immune cells, contributing to early hair follicle development, angiogenesis, neurogenesis, and scarless healing. In particular, macrophages promoted prenatal skin vascular endothelial development. Skin organoids, which lacked immune cells, concomitantly had markedly reduced quantity and heterogeneity of endothelial cells. Addition of iPS cell-derived macrophages significantly improved vascular network formation in skin organoids. Overall, this study revealed that immune cells function as critical players in skin morphogenesis and provided a potential blueprint to enhance future skin and organoid tissue engineering. Description: PhD Thesis

2025-01-01T00:00:00Z