http://theses.ncl.ac.uk/jspui/handle/10443/1146 2026-02-04T10:56:10Z 2026-02-04T10:56:10Z Sowter, Peter http://theses.ncl.ac.uk/jspui/handle/10443/6431 2025-11-21T13:20:54Z 2024-01-01T00:00:00Z

Title: Identifying Candidates for Chemopreventative Aspirin Prophylaxis : Improving the Detection of MMRd Authors: Sowter, Peter Abstract: Lynch syndrome (LS) is an inherited cancer syndrome arising from germline mutations in genes of the DNA mismatch repair (MMR) system. To optimise clinical management, the efficient diagnosis of LS individuals is essential, with known carriers of LS-associated gene defects benefiting from various practices including daily aspirin prophylaxis. Current guidelines in the UK advocate the use of two techniques in the screening for LS: MMR protein immunohistochemistry and DNA microsatellite instability (MSI) testing. However, several limitations compromise the efficacy of this clinical guidance, including the restricted tumour spectrum recommended for analysis, and the ambiguous genetic diagnoses returned by existing assays. This project aimed to enhance the existing techniques for detecting mismatch repair deficiency (MMRd) and LS individuals by addressing both these issues. In considering the potential cancer spectrum for LS screening, I initially analysed 122 extracolonic cancer samples from LS gene carriers using a new MSI assay (the Newcastle Assay) to review MSI as a biomarker of MMR deficiency in these tumour types. An MSI-H classification was returned for the majority of tumours, including 80% (41/51) of those considered of the LS spectrum, but a comparatively low proportion of endometrial cancer (EC) samples (26/35 - 72%) were found with this phenotype. Further investigation of MSI in EC specifically involved the analysis of 363 well-characterised samples, from two external clinical trial cohorts, using the Newcastle Assay. In this study, the frequency of instability between the cohorts varied for samples with confirmed MMR deficiency, highlighting caveats when using MSI as a biomarker for MMRd in EC. With this approach also often failing to detect MMRd for which MSH6-deficiency is responsible, these findings support the prioritisation of IHC for LS screening of this tumour type as recently recommended by NICE, but also suggest that additional MSI testing could have clinical benefit. For the diagnosis of LS, confirmation of a germline pathogenic MMR variant is required, but this is complicated for the MMR gene PMS2 by the presence of multiple pseudogenes. To improve the analysis of this gene, I attempted to develop a sequencing-based assay using a combination of long-range PCR sequencing and Molecular Inversion Probe (MIP) technology. I established a MIP pool consisting of 42 ii exonic and 100 intronic probes to assess for sequence variants and CNVs/loss of heterozygosity respectively. In the review of samples for which PMS2 mutation was confirmed, detection of variants by the exon-tiling component of this assay was demonstrated. Validated point mutations were accurately identified, with all but one of the 22 pathogenic variant calls returned from the 66/138 samples with verified PMS2 changes. However, the proficiency of this assay for detecting copy-number variation and loss of heterozygosity remains to be analysed, curtailed by time constraints. Information derived from intronic SNPs has thus far been inconclusive, and in no samples has homozygosity or substantial deletions been indicated. Results from the assay did however suggest that LS patients may have mutations in both PMS2 alleles, and these will require further investigation. Both these studies endeavoured to further the guidance and techniques for LS screening, and subsequently the application of treatment practices such as aspirin prophylaxis. The results of MSI analysis in extracolonic tumour samples demonstrated the facility of this biomarker in various Lynch-spectrum cancers, with EC analysis highlighting conditions for its use. In contrast, development of a PMS2 assay was less successful, and alternative long-read sequencing approaches are likely to supplant it. This suggests that continued development of the assay is likely not viable, although the complexity of the results highlight the need for accurate mutation screening of this MMR gene. Description: Ph. D. Thesis.

2024-01-01T00:00:00Z Hao, Yao http://theses.ncl.ac.uk/jspui/handle/10443/5584 2022-10-14T11:30:24Z 2021-01-01T00:00:00Z

Title: The role of miR-140 AND miR-455 in murine skeletal development and osteoarthritis pathogenesis Authors: Hao, Yao Abstract: Introduction: Osteoarthritis (OA) is the most common musculoskeletal disorder, which is complex and characterised by degradation of articular cartilage of joint tissue. Epigenetic changes alter transcriptional regulation and disrupt signalling pathways involved in cartilage homeostasis leading to OA, and dysregulation of and by microRNA (miRNA) (e.g. miR-140 and miR-455) correlate with bone and OA development. Bioinformatic analysis of RNAsequencing (RNA-seq) could reveal the mechanism of the function of miR-140 and miR-455 in murine skeletal development and OA pathogenesis. Aim: Generation of Mir140-null and Mir455-null mouse model to understand their role in relation to murine skeletal and OA development. Material and Methods: By CRISPR/Cas9 technology we generated two mouse lines, for Mir140 (miR-140-/- ) and Mir455 (miR-455-/- ). These mice were interbred to derive mice with deletions of both Mir140 and Mir455 (DKO). On the mutant mice, we performed phenotyping of skeletal X-ray, weight growth curve, and tibial growth plate analysis, including histological staining and immunocytochemistry (BrdU labelling to monitor proliferation and TUNEL labelling to monitor apoptosis). Destabilisation of the medial meniscus (DMM) surgery was performed on mutant mice to assess knee articular cartilage integrity following joint trauma. Differential expression of genes within the growth plate of the mutant mice were identified by RNA-seq and potential upregulated miRNA predicted targets validated by 3’UTR luciferase analysis. The expression of two growth plate targets (Creb3l1 and Nrf2) were assessed by western blot analysis in isolated chondrocytes. Transcriptome analysis was also performed on micro-dissected knee articular cartilage (preand post-DMM surgery) and on laser micro-dissected growth plates. Results: Genotyping analysis confirmed the deletion of miRNAs in our mutant mice. Phenotyping of our Mir140-null mice matched that of previously published work, including mild short stature, domed skull, and a short tail. Our Mir455-null mice were dwarf when compared to control mice, however, Mir455-null mice housed at our collaborator were undisguisable from age-matched control mice. Our DKO mice shared a similar phenotype III with Mir140-null mice. Histological staining revealed abnormal tibial growth plate morphology and delayed secondary ossification in 1-week and 3-week-old mice. A reduction in tibial growth plate chondrocyte proliferation and increased apoptosis was detected in mutant mouse lines. Mutant mice exhibited different transcriptome profiles of costal chondrocyte by RNA-seq, and three miR-140 growth plate predicted target genes (Creb3l1, Nrf2 and Zeb1) were confirmed to be targets and to attenuate chondrocyte proliferation by WST-1 assay analysis. Following DMM-surgery all mutant mice showed higher OA score with either Glasson scoring system or OARSI scoring system, and all exhibited significant proteoglycan staining loss compared to control mice. Comparison of transcriptome profiles before and after DMM surgery had led to understand the function of miR-140 during OA pathogenesis. Discussion and conclusion: Our mutant mice showed clear growth phenotypes compared to control mice, however the phenotyping of our Mir455-null mouse line was different to recent literature and to similar animals at our collaborator facility. Histological and immunohistochemistry analysis confirmed that mutant mice exhibited abnormal growth plate morphology, with reduced chondrocyte proliferation and increased apoptosis. Transcriptome profiling from costal chondrocyte and knee cartilage chondrocyte (pre and post-DMM surgery) represents an important tool to identify miRNA target genes and, herein, to gain insights about miR-140 and miR-455 in skeletal developments and OA pathogenesis. Description: Ph. D. Thesis.

2021-01-01T00:00:00Z De Las Heras Ruiz, Thais http://theses.ncl.ac.uk/jspui/handle/10443/5376 2022-04-08T13:57:54Z 2020-01-01T00:00:00Z

Title: Tissue engineering approaches to study biomechanical sensing in pseudoachondroplasia Authors: De Las Heras Ruiz, Thais Abstract: Articular cartilage is a dense and avascular tissue present at the ends of diarthrodial joints that protects the underlying bone from shear and compressive forces. Pseudoachondroplasia (PSACH) is a skeletal dysplasia resulting from mutations in cartilage oligomeric matrix protein (COMP), a large glycoprotein found in cartilage. The presence of mutant protein in the cartilage matrix affects tissue stability and mechanosensing, leading to early-onset OA, muscle weakness and tendon abnormalities, thus COMP mutations affecting the C-terminal domain represent a good model of musculoskeletal ageing. Cartilage disease and ageing studies are often hindered by the difficulty in obtaining human material and the use of costly animal models. The aim of this study was to create a model that could recapitulate this disease in a mechanosensing model for the very first time, that could be applied in further ageing and other disease studies. In order to generate a novel mechanosensitive tissue-engineered model of cartilage that would allow the study of the pathomolecular mechanism of COMP PSACH, chondroprogenitor ATDC5 cells were seeded in pellets and 2% agarose constructs, cultured in chondrogenic medium with and without growth factor supplementation, and compared to the established 2D chondrogenesis model. ATDC5 cells transfected with wild type (WT) and mutant COMP constructs were then grown in pellets and 2% agarose cyclically compressed for 2 weeks for 30min/day at 10kPa 0.33Hz to mimic physiological compression of cartilage. Supplementation with both BMP7 and TGF-β3 improved ATDC5 chondrogenesis. Moreover, chondrogenic markers increased in ATDC5 cells upon compression, whilst dedifferentiation markers decreased. The p.T585M COMP ATDC5 models recapitulated the phenotype of the disease, with abnormal ECM deposition, an increase in cell apoptosis and a decrease in cell proliferation also allowed us for the very first time to see its effects in a novel mechanosensitive compressed in vitro model. These data suggest that this novel mechanosensing 3-dimensional 2% agarose and pellet model could be employed to study and discover possible targets to treat rare skeletal diseases and reduce the use of animal sin research in the future. Description: PhD Thesis

2020-01-01T00:00:00Z Cocks, Erin Twynham http://theses.ncl.ac.uk/jspui/handle/10443/5375 2022-04-08T14:00:04Z 2021-01-01T00:00:00Z

Title: Use of Serial Block Face-Scanning Electron Microscopy to Study the Ultrastructure of Vertebrate and Invertebrate Biology Authors: Cocks, Erin Twynham Abstract: The development of Serial Block Face Scanning Electron Microscopy (SBF-SEM) allows for acquisition of serially sectioned, imaged data of ultrastructure at high resolution. In this project, optimisation of both SBF-SEM methodology and 3-D image segmentation analysis was applied to the ultrastructural examination of two types of biological tissues, each requiring a different experimental approach. The first project was a connectomic based study, to determine the relationship between the neurons that synapse upon the Lobula Giant Movement Detector 2 (LGMD 2) neuron, within the optic lobe of the locust. A substantial portion of the LGMD 2 neuron was reconstructed along with the afferent neurons, enabling the discovery of retinotopic mapping from the photoreceptors of the eye onto the LGMD 2 neuron. A sub-class of afferent neurons was also found, most likely vital in the process of signal integration across the large LGMD 2 neuron. For the second project, two types of skeletal muscle (psoas and soleus) obtained from fetal and adult guinea pigs were analysed to assess tissue-specific changes in mitochondrial morphology with muscle maturation. Distinct mitochondrial shapes were found across both muscles and age groups and a classification system was developed. It was found that, in both muscles, by late fetal gestation the mitochondrial network is well developed and akin to that found in the adult. Quantitative and qualitative differences in mitochondria morphology and complexity were found between the two muscles in the adult group. These differences are likely to be related to functional specialisation. All data collected during the experiments have also been made available online on Zenodo, roughly 240GB, which can be used for further studies. Overall SBF-SEM was proven to be a robust method of gaining new insights into the ultrastructure in both models and has wide ranging capabilities for a variety of experimental objectives. Description: PhD Thesis

2021-01-01T00:00:00Z