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Cognitive symptoms in Primary Biliary Cholangitis (PBC)

2025-01-01T00:00:00Z

Title: Cognitive symptoms in Primary Biliary Cholangitis (PBC) Authors: Phaw, Naw April Abstract: Patients with primary biliary cholangitis (PBC) may suffer from cognitive symptoms in addition to the primary disease burden. These symptoms impact on quality of life but, to date, we have no pharmacological therapies demonstrated to improve these symptoms. It is crucial to understand the nature of cognitive symptoms and underlying neuropathological processes in PBC for the development of effective future clinical trials. In this thesis, neuropsychometric assessments and magnetic resonance Diffusion Tensor Imaging (DTI) measurements were used to determine the nature of cognitive symptoms in PBC, and to explore white matter pathology, as a potential explanation, respectively. In this thesis, cognitive deficits in PBC were found in multiple domains with pronounced impairments in memory, attention, and emotional cognition. Neuropsychometric testing demonstrated cognitive deficits in both patients reporting significant cognitive difficulties and asymptomatic patients, compared to a normative population. Therefore, cognitive symptoms in PBC may be influenced by behavioural variation. Nevertheless, those reporting cognitive symptoms are typically the group with the highest unmet clinical need, and likely to be prioritized for interventional studies. We found that PBC-40 cognitive scores were not closely associated with fractional anisotropy (FA) values from deep white matter tracts, contrary to what we had hypothesised. This suggests that cognitive symptoms do not arise from white matter tract dysfunction, although we acknowledge that we only studied a small number of a priori regions. Only patients with cholestasis demonstrated significant cognitive deficits. Based on these findings, we propose that cognitive symptoms may only become apparent when cholestasis is established, and therefore aggressive treatment for cholestasis at an early stage may be needed. Overall, the characterisation of cognitive symptoms in PBC, together with exploratory brain imaging, are important steps for informing the design of future PBC therapeutic trials by establishing reference endpoints for future studies. Description: MD Thesis

Investigating the systemic immune landscape of thymectomised paediatric transplant patients at risk of EBV-associated post-transplant lymphoproliferative disease

2025-01-01T00:00:00Z

Title: Investigating the systemic immune landscape of thymectomised paediatric transplant patients at risk of EBV-associated post-transplant lymphoproliferative disease Authors: Offor, Ugonna T. Abstract: Epstein-Barr virus-associated post-transplant lymphoproliferative disease (EBV-PTLD) are rare but life-threatening lymphoid malignancies that disproportionately affect paediatric heart transplant recipients. Recent discourse has focussed on the role of early removal of the thymus gland, which is critical for normal antiviral immunity, in the aetiology of EBV-PTLD after heart transplantation. This Fellowship aimed to investigate the systemic immune landscape of thymectomised paediatric transplant patients at risk of EBV-PTLD. Fifty-four patients awaiting transplant were recruited to the Immunology of THymectomy And childhood CArdiac transplant (ITHACA) study for immune profiling using full spectrum flow cytometry before transplant and at 3-, 6-, 12-and 24-months post-transplant. Patients were stratified into early (<6 months old) and late (≥6 months old) thymectomy for comparison to age-matched non thymectomy controls. Induction immunosuppression with anti-thymocyte globulin was associated with diminished antiviral CD16+ monocytes compared to Basiliximab induction. Failed expansion of CD16+ monocyte subsets was notable among patients with EBV DNAemia alongside a negative correlation between EBV DNA load and their expression of activation markers. Mycophenolate Mofetil was associated with an overall reduction in NK cells and a phenotypic shift towards immunosenescence. EBV DNAemia was associated with a marked increase in CD16dim CD56dim NK cells but a failure to expand the CD56dim NKG2A+ KIR- subset required for effective EBV control. Early thymectomy was associated with the depletion of regulatory, naïve CD4+ and CD8+ T-lymphocytes and expansion of senescent CD57+ memory T-lymphocytes. Most of these changes persisted after transplant and included the emergence of terminal effector memory subsets with increased expression of immune checkpoint proteins within the memory compartment of early thymectomy patients. Increased immunosenescence and exhaustion of EBV-specific T-lymphocytes were associated with early thymectomy and persistent EBV DNAemia. These data demonstrate that both early thymectomy and choice of immunosuppression are associated with distinct features of immunosenescence, exhaustion and impaired EBV control, all likely contributing to the risk of developing to EBV-PTLD. Description: PhD Thesis

Targeting non-canonical NF-κB signalling in CYLD defective tumours

2025-01-01T00:00:00Z

Title: Targeting non-canonical NF-κB signalling in CYLD defective tumours Authors: Hodgson, Kirsty Abstract: CYLD cutaneous syndrome (CCS) is a rare, autosomal dominant, hair follicle tumour predisposition syndrome where non-surgical therapeutics are needed. In this thesis I delineate non-canonical NF-κB signalling in CYLD defective tumours and evaluate the utility of targeting this pathway with novel small molecule IKKα inhibitors. Such information may underpin the potential development of non-surgical approaches for CCS. First, I sought to establish the genetic landscape of tumours in CCS. Using whole genome sequencing, I showed that biallelic mutation of the cylindromatosis gene (CYLD) was present in almost all tumours studied. In addition, I discovered that a subset of tumours had mutations in epigenetic modifiers that modulate Wnt signalling, a known oncogenic dependency in these tumour cells. CCS has not been successfully modelled in mice, limiting mechanistic insight into how truncating mutations in CYLD result in a skin tumour phenotype. To overcome this, I explored both the canonical and non-canonical NF-κB signalling pathways in CCS whole tumour tissue. I also developed a patient-derived tumour spheroid model that recapitulated in vivo deregulation of both NF-κB signalling pathways. In the non-canonical pathway, I showed processing of the p52 precursor p100 was altered in CCS tumour tissue and spheroids. Given non-canonical NF-κB signalling is targetable by inhibiting IKKα, I inhibited IKKα in CCS spheroids with a novel compound, designated Compound Z, and showed reduction in viability following treatment. To determine the impact of biallelic CYLD truncating mutations on the transcriptome, I used a CD45 depletion method to isolate CD45- CCS tumour keratinocytes by FACS. RNA-sequencing of tumour keratinocytes identified the ectodysplasin a receptor EDAR as highly differentially expressed. Measurement of gene expression after treatment with Compound Z confirmed that EDAR overexpression is IKKα-dependent in CCS spheroids. In summary, I have highlighted the oncogenic dependency of CCS tumour cells on non-canonical NF-κB signalling and demonstrate the effectiveness of targeting IKKα for the treatment of CYLD cutaneous syndrome. Description: Ph. D. Thesis.

New methods for the discovery and characterization of bacterial natural products

2025-01-01T00:00:00Z

Title: New methods for the discovery and characterization of bacterial natural products Authors: Sumang, Felaine Anne Abstract: Actinomycetes are renowned for their ability to produce a wide array of natural products (NPs), with a broad range of applications. NPs are typically encoded by biosynthetic gene clusters (BGCs). Bioinformatic analysis of sequenced Actinomycetes genomes have shown that they possess many BGCs whose potential products are not detected under standard laboratory culture conditions. Advances in genome mining and molecular biology have led to the development of diverse strategies to activate these “cryptic” BGCs, but further improvements in these methods are warranted. This thesis explores multiple approaches for discovering novel bioactive compounds from Actinomycetes and investigating their BGCs. Chapters 3 and 4 focus on the development of a new bacterial artificial chromosome (BAC) vector, designated pJE2, and optimization of a protocol for genomic library construction enabling the isolation of large BGCs. This approach was applied to Actinomadura madurae T576, a producer of unusual sulphated metabolites, resulting in a BAC library from which a clone harbouring the complete 80 kbp BGC responsible for these metabolites was identified. Heterologous expression of this clone in Streptomyces albus successfully led to production of the sulphated metabolites. Screening of additional clones also revealed the successful capture of several previously uncharacterized BGCs. Chapter 5 focuses on a chemical elicitation method, using plant extracts to stimulate NP production in soil-derived Actinomycetes. Notably, hibiscus flower extract induced the production of the antibiotic thiolutin by Streptomyces strain MBN 2-2. Further analysis identified hibiscus acid and hydroxycitric acid as the elicitor compounds. The final chapter centres on associating putative BGCs with the synthesis of three novel antibiotics demurilactone A, persiathiacin, and quinovosamycin. In all three cases, insertional mutagenesis led to the abolition of antibiotic synthesis, thereby validating assignment of the BGCs to their respective antibiotics. Description: Ph. D. Thesis.