DSpace Community:

Filling a critical knowledge gap in androgen receptor variant splicing to enable development of new prostate cancer therapies

2025-01-01T00:00:00Z

Title: Filling a critical knowledge gap in androgen receptor variant splicing to enable development of new prostate cancer therapies Authors: Nelson, Ryan Abstract: Androgen receptor (AR) signalling has long been recognised as critical in the initiation and progression of prostate cancer (PC). Therapeutic interventions therefore focus on interrupting AR signalling through suppression of circulating testosterone with androgen deprivation therapy (ADT) or direct inhibition of the AR with anti-androgens, such as enzalutamide. Whilst initially effective, a significant proportion of patients will see progression of their disease despite castrate levels of androgens. Castrate-resistant prostate cancer (CRPC) arises through several mechanisms including alternative splicing of the androgen receptor leading to the generation of androgen receptor spliced variants (AR-Vs). Cryptic exons (CEs) of AR-Vs replace the ligand-bind domain (LBD) of the AR consequently rendering them constitutively activated and able to drive disease progression. AR-Vs are currently resistant to all therapeutic interventions and themselves are complex targets due to their high level of structural disorder. Histone demethylase (HDMs) can influence splicing decisions and may offer an alternative therapeutic option to direct targeting of splicing factors (SFs) which often lack specificity to pathophysiological splicing events. KDM6A is one such HDM which is known to be highly mutated in CRPC. However, its role in disease progression is relatively unknown. Data obtained during this PhD reveals that KMD6A contributes to PC progression to CRPC through both catalytic-dependent and independent mechanisms. The influence of KDM6A on splicing appeared to be through the chromatin-adapter model, recruiting SFs to CE3 driving the generation of AR-V7. Critically, this work revealed that in models of enzalutamide resistance, KDM6A inhibition can lead to enzalutamide re-sensitisation by affecting both genes and pathways essential for the emergence of resistance. Results from this work show KDM6A to be an attractive target in CRPC for which there are currently no curative treatment options. Description: PhD Thesis

Understanding the role of transcription in organisation of the bacterial chromosome

2025-01-01T00:00:00Z

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

The impact of glucocorticoids in Duchenne muscular dystrophy throughout the lifespan:implications for clinical care and clinical trial design

2025-01-01T00:00:00Z

Title: The impact of glucocorticoids in Duchenne muscular dystrophy throughout the lifespan:implications for clinical care and clinical trial design Authors: Schiava, Marianela Abstract: This publication-based PhD analysed two large databases of genetically confirmed individuals with Duchenne muscular dystrophy (DMD) treated with glucocorticoids to inform clinical care and trial design. The paediatric database included longitudinal data on boys with DMD (4-<8 years old) from the Finding the Optimum Corticosteroid Regime for Duchenne Muscular Dystrophy study (FOR-DMD, 2013-2016), a randomized, parallel-group, doubleblind clinical trial comparing the benefits and side effects of the three most prescribed glucocorticoid regimens: daily prednisone, daily deflazacort, and prednisone 10 days off/10 days on. The adult database contained retrospective data from individuals with DMD (>16 years old) followed at the John Walton Muscular Dystrophy Research Centre (JWMDRC, 1986-2022) according to evolving Standards of Care. Using the FOR-DMD database, I investigated the effect of genotype on motor performance in young glucocorticoid-naïve boys, the clinical factors influencing early motor trajectories after glucocorticoid initiation, time to motor performance peak, and the short- and long-term correlation between anthropometric measures and motor performance. This data provides important considerations for clinical trials randomization, and analysis adjustments, while also supporting clinical care decisions for glucocorticoid initiation. Using the adult JWMDRC database, I explored the effect of glucocorticoid on long-term motor, respiratory, and cardiac outcomes when glucocorticoids are continued after loss of ambulation (LOA), investigate DMD-related comorbidities and predictive factors and the correlation between age at LOA and cardiac function in adults with DMD. I described the current practice regarding glucocorticoid use in adults with DMD in a highly specialised neuromuscular centre. These findings support clinical care discussion on glucocorticoid prescription after LOA, guide clinicians on minimal glucocorticoid dose in adults and inform adult clinical service requirements. Finally, the PhD included a lab-based project investigating for the first time the novel biomarker Chitinase-3-like-1 protein in serum samples from individuals with DMD to better understand its role in DMD pathophysiology Description: PhD Thesis

Understanding and ameliorating late-effects in childhood medulloblastoma survivors

2025-01-01T00:00:00Z

Title: Understanding and ameliorating late-effects in childhood medulloblastoma survivors Authors: Castle, Jemma Abstract: Medulloblastoma (MB) is the most common malignant paediatric brain tumour, with 5-year survival rates over 70%. Survivors frequently suffer a wide variety of late-effects due to their tumour and its multi-modal treatment: tumour resection, chemotherapy and craniospinal radiotherapy with posterior fossa boost (PFB). Treatment induces deleterious late-effects through damage to normal tissues and increases the risk of neurocognitive impairment, endocrine impairment, ototoxicity, secondary tumours, cardiotoxicity, poor physical function and premature ageing/frailty. Sadly, approaches to ameliorate treatment-induced late-effects are lacking; a paucity of appropriate model systems hinders their development. This thesis aims to develop clinically-relevant models of MB treatment to enable investigation of the biological mechanisms that underpin late-effect onset, and facilitate the appraisal of interventions. To recapitulate childhood medulloblastoma radiotherapy and late-effect profile in vivo, age-equivalent mice received CT image-guided, human-equivalent cranial radiotherapy (CRT) or CRT+PFB and were longitudinally assessed for over 1 year. Following CRT, mice were significantly more frail, had reduced physical functioning and exhibited neurocognitive deficits. Receipt of PFB did not induce a more severe late-effect profile. The biological underpinnings of radiation-induced late-effects were explored, ex vivo. Assessment of transcriptional modifications 1 year post-irradiation, via RNA-sequencing, showed CRT did not induce consistent global changes, instead pathways including interferon-α/γ and epithelial-mesenchymal-transition were downregulated. Quantification of pro-inflammatory proteins, using immunohistochemistry, showed higher abundance following CRT, though response was not dose-dependent. Altered DNA methylation patterns are associated with premature ageing. Utilising a prebuilt epigenetic-clock, predicted age increased with chronological age, though this was more accurate in DNA from peripheral blood than brain tissue. CRT did not induce a significantly accelerated epigenetic age. To understand acute response to radiation-insult, human-equivalent radiation was delivered in vitro and the Luminex assay was utilised to develop a novel, multi-analyte assessment of molecular insult response to MB-equivalent radiation. Markers of inflammation increased 1 hour post-irradiation, and typically increased further at 48 hours. Increased inflammation was expected and is thought to play a major role in the development of radiation-induced late effects. To provide a baseline model for future investigation of chemotherapy-induced late-effects, a close-MB chemotherapy regimen was developed in vivo that replicated the popular Packer-style chemotherapy. The development of clinically-relevant, treatment-induced late-effect models enables the elucidation of novel/target mechanisms underpinning MB late-effects and the development of novel interventions for their amelioration. Description: PhD Thesis