Previous studies have identified stem cell populations in articular cartilage using colony forming assays and mesenchymal stem cell (MSC) marker expression. The specificity of classical MSC markers for isolation of stem cells within articular cartilage is insufficient, with large and highly variable quantities being reported in the literature. This study has demonstrated, for the first time, a panel of stem cell markers specific for articular cartilage-derived stem cells (ACSC). ACSCs were isolated, quantified and cultured from healthy and OA joints. Stem cells were clonally-derived cell lines that proliferated beyond 50 population doublings whilst maintaining a phenotype, and demonstrated tri-lineage potential. We discovered that OA cartilage had a two-fold increase in stem cell number, consisting of two divergent stem cell sub-populations. These divergent populations varied in proliferative capacity with only 50% of stem cells from the OA joint capable of extended proliferation in vitro. Using transcriptomic next generation sequencing of culture-expanded chondrocytes and ACSCs we successfully identified differentially expressed genes and a panel of novel markers of cartilage-specific stem cells. Novel markers were validated using qPCR and protein labelling and, were specifically expressed in ACSCs, with no expression in the culture-expanded full-depth chondrocytes. Using immunofluorescence for novel stem cell markers we found articular cartilage-derived stem cells are localised within the transitional zone in normal cartilage and the superficial zone in OA cartilage. OA cartilage was found to contain a 2-fold increase in stem cells using immunofluorescence. Subsequently, we used the panel of novel markers and fluorescent active cell sorting to isolate a sub-population from full-depth cartilage with stem cell characteristics. These cells were plastic adherent, clonogenic, with proliferative capacity greater than 50PD and displayed tri-lineage potential, therefore meeting all criteria for classification as a MSC population. The use of specific markers to isolate ACSCs will allow for further characterisation of stem cells, including a more in-depth understanding of the mechanisms of proliferation, differentiation and degeneration within articular cartilage.

Duplication of the proteolipid protein (PLP1) gene, which encodes the most abundant protein of central nervous system (CNS) myelin, is the most common cause of Pelizaeus Merzbacher disease (PMD). Various animal models have been generated to study the effect of Plp1 gene overexpression on oligodendrocyte and myelin sheath integrity. The #72 line harbours 3 additional copies of the murine Plp1 gene per haploidic chromosomal set. Homozygous #72 mice appear phenotypically normal until three months of age, after which they develop seizures leading to premature death at around 4 months of age. An earlier study examining the optic nerve showed a progressive demyelination accompanied by marked microglial and astrocytic responses. Using electron microscopy and immunohistochemistry, I demonstrated that initial myelination of the #72 corpus callosum was followed by a progressive demyelination, probably mediated by a distal “dying back” phenomenon of the myelin sheath. No evidence of effective remyelination was observed despite the presence and proliferation of oligodendrocyte progenitor cells (OPCs). A marked increase in density and reactivity of microglia/macrophages and astrocytes, and the occurrence of axonal swellings, accompanied the demyelination. In situ and in vitro evaluation of adult #72 OPCs provided evidence of impaired OPC differentiation. Transplantation of neurospheres (NS) into adult #72 mouse corpus callosum confirmed that axons were capable of undergoing remyelination. Furthermore, NS transplanted into neonatal CNS integrated into the parenchyma and survived up to 120 days, demonstrating the potential of early cell replacement therapy. Taking advantage of the spatially distinct pathologies between the retinal and chiasmal region of the #72 optic nerve, I evaluated the capability of diffusion weighted MRI to identify lesion type. I found significant differences between #72 and wild type optic nerves, as well as between the two distinct pathological regions within the #72 optic nerve. These results confirm the potential of the #72 mouse to serve as a model to study chronic demyelination. The study also demonstrates the utility of the #72 mouse to evaluate cell transplant strategies for the treatment of chronic CNS white matter lesions and PMD. Additionally, DW MRI has potential as a modality capable of diagnosing myelin-related white matter changes, and may be applicable to the clinical setting.

EHMT1 is a gene that encodes an epigenetic regulator important for normal brain development. Disruption in EHMT1 is associated with a number of neurodevelopment and psychiatric conditions, like schizophrenia, Autism Spectrum Disorders, developmental delays and intellectual disabilities. In order to help elucidate the role of Ehmt1 in cortical development two models are examined: the differentiation of mouse pyramidal neurons lacking one copy of the gene and a forebrain-specific Ehmt1-haploinsufficient mouse model. Ehmt1+/- cells demonstrated changes in cell cycle, with significant differences in proliferation rates at embryonic stem cell and neural progenitor stages. Ehmt1+/- cells demonstrated significantly different transcriptional profiles in early and late stages of progenitor development, which suggested these cells, underwent precocious differentiation. In addition, the dysregulation of mRNA expression in a number of the Nrsf/Rest repressor complex members and Rest target genes was found; and Ehmt1+/- cells did not survive as post-mitotic neurons. The forebrain-specific Ehmt1-haploinsufficient mouse model, Ehmt1D6Cre/+, importantly showed normal Mendelian birth ratios, survival, motor coordination and function and no gross morphological changes in brain structure. However, these mice demonstrated differences in activity levels and anxiety-related measurements; deficits in sensorimotor gating and object recognition; and significant differences in a number of electrophysiological measurements, including abnormal event-related neural responses in the cortex and high frequency oscillatory patterns. Taken together, these data suggest that Ehmt1 expression is important for normal pyramidal development and Ehmt1 haploinsufficiency throughout development manifests cortical dysfunction, which leads to marked behavioural and electrophysiological abnormalities.

Chronic lymphocytic leukaemia (CLL) is a lymphoid malignancy which despite advances in the treatment options available is still incurable. Characterised by the gradual accumulation of CD5+ B cells, the paradigm that this is due to failed apoptosis has been challenged and a significant proliferative component has been identified. However, despite the crosstalk between pathways which regulate metabolism and proliferation the metabolic characteristics of these cells are not fully understood. Furthermore, there is a renewed interest in the field of cancer cell metabolism because of the Warburg effect, a hallmark of malignancy whereby cells preferentially switch to aerobic glycolysis and rapidly consume glucose. This has led to the development of new drugs such as AZD3965 an inhibitor of monocarboxylate transporter 1 (MCT1), which along with MCT4 mediates the export of lactate, a toxic bi-product of glycolysis, out of the cell. The aim of this project was to assess whether therapeutically targeting MCT -1 and -4 would be a viable approach for CLL. Chapter 2 of this thesis examines expression of MCT -1 and -4 as well as a specific chaperone protein needed for the surface expression of these proteins, CD147. This chapter confirms the presence of both MCT -1 and -4 and CD147 in normal B cells as well as demonstrating for the first time that these transporters are expressed in CLL cells using Western blotting and qRT-PCR to assess the MCTs and flow cytometry to measure CD147. The levels of both MCTs and CD147 are demonstrated to be significantly reduced in CLL cells in comparison normal B cells likely due to the adoption of a quiescent phenotype to aid cell survival. The following chapter investigates this further by assessing whether there are any changes in expression under the influence of microenvironmental stimuli, specifically CD40 ligand (CD40L). In this chapter it is demonstrated for the first time that MCT4 is upregulated in CLL cells in response to CD40L. Analysis of gene expression using a Fluidigm Biomark™ array suggests this is due to the induction of glycolysis and that CLL cells may promote fatty acid synthesis as well as instigating changes in the metabolism of the tumour stroma possibly to provide substrates. Finally, chapter 4 evaluates the sensitivity of CLL cell lines to AZD3965 using cell death and cell viability assays. Both MEC-1 and HG3 CLL cell lines are shown to be resistant to MCT1 inhibition using AZD3965 and silencing of MCT4 using siRNA cells also has no effect on the viability of MEC-1 cells. That MCT4 can compensate for MCT1 inhibition is shown by the transient expression of MCT4 in a Raji cell line where only MCT1 is expressed. Taken together, the data presented in this study indicates that while the inhibition of MCT1 is likely to be ineffective dual inhibition of both MCT -1 and -4 may be a viable strategy for the localised inhibition of CLL in the secondary tissues. Furthermore, MCT inhibition in this disease may have the potential to negate mechanisms of resistance and protection from oxidative stress mediated by CD40L.

Mutations of the WFS1 gene are responsible for most cases of Wolfram syndrome (WS), a rare, recessively inherited neurodegenerative disorder characterised by juvenile-onset non-autoimmune diabetes mellitus and optic atrophy. Variants of WFS1 are also associated with non-syndromic hearing loss and type-2 diabetes. Understanding the function of the WFS1 protein-product wolframin, would enable developments in targeted therapy for WS patients and important insights to its possible contribution to type-2 diabetes pathogenesis. This study was aimed at expanding the spectrum of WS-associated genetic mutations and clinical data, and investigating the molecular mechanisms responsible for phenotypic variation associated with WFS1-mutation. The mutational and phenotypic spectrum of WS is broadened by our report of novel WFS1 mutations and a case of WFS2-associated WS. New perspectives on the molecular mechanisms linking mutation to disease manifestation are also taken by characterisation of representative WFS1 mutations specific to phenotype, identification of potentially novel WFS1 interacting partners, and the first evidence linking WFS1 with the exocrine pancreas. Our data suggests that some WFS1-mutations may allow residual protein function and these findings lay the groundwork for future functional investigation of mutated wolframin to explore this hypothesis further.

Metabolite concentrations are fundamental biomarkers of disease. With increasing interest in personalised medicine, this work assessed the accuracy of non-invasive metabolite quantification with magnetic resonance spectroscopy (MRS) using a combination of simulations, phantom and in vivo data. No optimal echo time (TE) was found for measuring a range of key metabolites with quantification accuracy generally influenced more by data quality than TE choice. The T2 relaxation times of water and metabolites with MRS dominated by a singlet could be estimated using 2 TEs and were found to be significantly different in paediatric brain tumours compared with normal brain, varying between tumour types. The T2 relaxation times of paediatric brain tumours were significantly shorter at 3T compared with 1.5T. Metabolite concentrations for individual patients were most affected by changes in the T2 relaxation time of water which is quick to measure. A clinical JPRESS protocol was developed which aids assignment of overlapping metabolites using changes of MRS with TE. Overall, measurement of MRS with a short TE reduces inaccuracies associated with variability in metabolite T2 and does not tend to lead to worse quantification of overlapping resonances. Further improvements in concentration accuracy can be obtained by measuring case-specific water T2.

Tolerance of the semi-allogeneic fetus presents a significant challenge to the maternal immune system. The effect of pregnancy on maternal cellular immunity was established by assessing maternal effector and regulatory T-cell subsets during human pregnancy. This demonstrated that an increase in maternal peripheral regulatory T-cells or a shift from a Th1 to Th2 phenotype was not a requirement for normal pregnancy. We also determined the profound impact of maternal Cytomegalovirus seropositivity on maternal T- cell dynamics. T-cells with specificity for fetal epitopes have been detected in women with a history of pregnancy but it has been thought that such fetal specific cells were deleted during pregnancy. We identified, using MHC-peptide multimers, fetal-specific CD8 T-cells in half of all pregnancies. The fetal-specific response increased during pregnancy and persisted in the post natal period. Fetal-specific cells demonstrated an effector memory phenotype and retained functional potential. These data show that the development of a fetal-specific adaptive cellular immune response is a normal consequence of human pregnancy. Women with recurrent miscarriage were found to have abnormal T-cell function, with increased IFN\(\gamma\) and Il-17 production. Fetal specific T-cells were also detected in this cohort and progesterone attenuated their function, which may have therapeutic implications.

Birt Hogg Dubé (BHD) syndrome is an inherited cancer susceptibility syndrome characterised by the development of fibrofolliculomas on the face and upper torso, and increased risk of lung cysts, spontaneous pneumothorax and renal cancer. The findings presented in this thesis advance knowledge into how the mutations in the FLCN gene cause the phenotypes associated with BHD syndrome, and provides novel insights into the functions of folliculin within the cell. The results presented provide further evidence of the association between BHD syndrome and increased risk of colorectal cancer in a subset of BHD syndrome families, and suggest that this association appears restricted to those patients with an exon 11 mononucleotide tract mutation. Evolutionary conservation analysis across the FLCN sequence suggests that pathogenic mutations could be expected throughout the gene, and identifies a region between codons 100-230 of increased evolutionary significance. The experiments undertaken demonstrate a practical strategy for determining the pathogenicity of non-truncating folliculin variants in vitro, and indicate that loss of protein stability is the main mechanism of pathogenicity for the previously reported non-truncating mutations within FLCN. Finally, this thesis reports the first identification of p0071 as a folliculin interacting protein. Folliculin deficiency exerts a functional impact on previously reported p0071 functions inducing RhoA signalling upregulation, mitotic defects and disruption of cell junctions. These results demonstrate the potential efficacy of using inhibitors downstream of RhoA as therapeutic targets in BHD tumours with dyregulated RhoA signaling, and provide novel directions for research into BHD syndrome.

In this thesis I report three related studies that utilise state-of-the-art technologies to investigate germline and somatic chromosomal rearrangements in humans. Firstly, 16 patients with cytogenetically detectable deletions of 3p25-p26 were analysed with high density single nucleotide polymorphism (SNP) microarrays; Affymetrix 250K SNP microarrays (n=14) and Affymetrix SNP6.0 (n=2). Assuming complete penetrance, a critical region for congenitalheart disease (CHD) susceptibility gene was refined to approximately 200 kb and a candidate critical region for mental retardation was mapped to ~1 Mb interval containing SRGAP3. Secondly, I used SNP microarray and molecular cytogenetic studies to characterize chromosome 11p15 in 8 patients with the imprinting disorder Beckwith-Wiedemann syndrome (BWS). In addition to characterising 11p duplications in three patients, the breakpoints in two patients with balanced rearrangements were mapped to two distinct regions. Thirdly, I used high resolution SNP arrays (Affymetrix 250K Sty1 and 6.0 arrays) to identify copy number changes in renal cell carcinoma (RCC) primary tumours (n=81) and cell lines (n=23). Copy number changes most frequently involved large segments (>10Mb) and loss of 3p and gain of 5q were the most common copy number changes. A comparison of copy number changes in RCC cell lines and inherited and sporadic primary tumours was made.

Myeloid and lymphoid malignancies are potentially curable through a graft versus leukaemia (GvL) effect following allogeneic haematopoietic stem cell transplantation. Whilst donor T cell are thought to be the main mediators of GvL, the effect of donor NK cells within HLA matched T cell depleted transplant setting is more unclear. Patient blood samples were analysed during the first month post-transplant, with higher reconstitution of NK cells at two weeks conferring a relapse protection association. Donor stem cell graft samples, from which NK cells within the patient at two weeks are thought to be derived, similarly displayed a strong association between high NK cell dose and protection from disease relapse. CD56dimDNAM+ NK cells were found to be the population with the most significant association. The ability of NK cells to kill AML blasts in a DNAM dependent manner was shown indicating that direct killing of residual tumour cells may be a valid mechanism of GvL. These findings suggest that optimising the number of NK cells within stem cell grafts should be considered as a means to prevent disease relapse.