Evidence suggests haematopoietic stem cells (HSCs) can migrate to injured liver and influence tissue repair. However, the molecular adhesive mechanisms governing HSC recruitment to injured hepatic microcirculation are poorly understood. These mechanisms were investigated in vivo following murine hepatic ischemia-reperfusion (IR) injury. HSC adhesion was significantly enhanced in injured livers and could be reduced by blocking CD49d on HSCs or VCAM-1 in vivo. Blockade of HSC CD18, CD31 or CD44 did not alter adhesion. HSC adhesion in sham treated CD31-/- animals was raised compared to wild-type animals and IR injury did not further raise this adhesion. Studies in vitro demonstrated that HSC treatment with inflammatory cytokines or conditioned media/plasma did not upregulate adhesion molecule expression but CXCL12 and CXCL1 did significantly enhance HSC adhesion to endothelium. However, blockade of CXCR4 (CXCL12 receptor) failed to reduce HSC adhesion in vivo following IR injury. Furthermore, we demonstrated exogenous HSCs were identified primarily in the pulmonary circulation and intraportal injection raised recruitment within the liver irrespective of the presence of injury. This study provides novel evidence for the importance of the VLA-4/VCAM-1 pathway in HSC recruitment to IR injured liver, a pathway that may be manipulated in order to enhance hepatic engraftment of these cells clinically.

Fabry disease (FD) is an X-linked lysosomal storage disorder caused by a deficiency of the α-galactosidase-A (α-gal-A) enzyme. The lack of enzymatic activity results in the accumulation of glycosphingolipids (GSLs) in the lysosomes of various tissues and organs. Globotriaosylceramide (Gb3) and Globotriaosylsphingosine (Lyso-Gb3) and their isoforms/analogues have been identified and quantified as potential biomarkers. This study aimed to develop an HPLC-MS based method for the quantitation of plasma and urinary Lyso-Gb3 and its analogues in Fabry patients to evaluate its utility in diagnosis and monitoring FD. The results showed that plasma Lyso-Gb3 as a reliable diagnostic biomarker for FD, as plasma Lyso-Gb3 levels could easily discern classical Fabry patients from controls. Moreover, plasma Lyso-Gb3 could also distinguish male cardiac variant Fabry patients from control males. Nevertheless, cardiac variant Fabry females showed an overlap of Lyso-Gb3 levels with controls, hence a positive value in this group would be considered diagnostic but a negative value could not exclude FD. In a small cohort of our patients on ERT there was a trend towards falling Lyso-Gb3 levels with time suggesting that Lyso-Gb3 has a potential value in monitoring these patients. Urinary Lyso-Gb3 levels were substantially different between classical and cardiac variant Fabry patients, and the lack of detectable urinary Lyso-Gb3 and analogues in controls allowed us to differentiate between these patients and healthy controls. The total levels of urinary Lyso-Gb3 and its analogues proved particularly useful in differentiating between classical and atypical Fabry patients of both genders. In the course of the study, a novel rapid MALDI-TOF-MS based Method for measuring urinary Gb3 in Fabry patients has been established. Collectively, the final findings demonstrate that urinary Lyso-Gb3 is superior to urinary Gb3 as a diagnostic biomarker for FD, where the later has been shown to be found in healthy subjects. Our study subsequently led to development of regional laboratory service for testing Lyso-Gb3 in Queen Elizabeth Hospital, Birmingham, UK. This service is now open to Fabry patients across England. In conclusion both plasma and urinary Lyso-Gb3 levels are useful diagnostic and monitoring biomarker in classical and cardiac variant males patients, but have questionable utility in cardiac variant females due to overlap with healthy controls. Although we studied the role of Lyso-Gb3 in diagnosing FD further studies are needed to establish its role in disease severity assessment and a larger study required to test our initial finding related to monitoring disease in patients on treatment.

A large proportion of human pregnancies have the wrong number of chromosomes, known as aneuploidy, with the chances of having an affected pregnancy increasing with maternal age. The majority of these errors can be traced back to the egg (oocyte), which undergoes two meiotic cell divisions to generate a cell with half the number of chromosomes of a somatic cell. The first meiotic division is particularly error-prone and accounts for a significant proportion of aneuploidies in early embryos. This first division is a unique form of cell division because it entails separation of homologous chromosome pairs and co-segregation of identical sister chromatids at anaphase (in mitosis and meiosis II, by contrast, sister chromatids separate at anaphase). The kinetochore is a multiprotein structure that assembles on the centromeres of chromosomes and facilitates chromosome segregation by forming attachments to spindle fibres emanating from one of the spindle poles. For a successful meiosis I division, kinetochores on sister chromatids must act as a single functional unit. In mouse and yeast this is achieved through close physical association of meiotic sister kinetochores; in humans, however, little is known about the arrangement of sisters in oocytes, largely due to the limited availability of human oocytes for research. In this project, I show that in human meiosis I stage oocytes donated to research by women undergoing assisted reproduction, sister kinetochores are not physically fused and are each capable of forming individual attachments to spindle microtubule fibres. I also found a significant increase in the distance between sister kinetochores in patients over 35 years of age, which may indicate a decline in inter-kinetochore cohesion over time. These unique features of sister kinetochore geometry in human oocytes may shed light on why meiosis in humans is susceptible to error with increasing maternal age.

Background: Chronic low back pain (CLBP) is a highly prevalent condition that has substantial impact on patients, the healthcare system and society. Its aetiology is complex and the condition can be exacerbated by many psychological, physical and social factors. Pain management services (PMS), which aim to address the complex nature of back pain, are recommended in clinical practice guidelines to manage CLBP. Although the effectiveness of such services has been widely investigated in relation to CLBP, the quality of evidence underpinning the use of these services remains moderate. Given that these services are resource intensive, evidence is needed to determine their cost-effectiveness. Aim: This study aims to describe the patient characteristics, clinical outcomes and healthcare resource use of people with CLBP in a community-based pain management service (PMS) in Nottingham to derive an indicative estimate of the cost effectiveness of PMS compared with standard care (SC). Methods: The study followed the Medical Research Council (MRC) guidance for evaluating complex interventions. The MRC suggest conducting developmental and observational work before evaluating complex interventions on a larger scale. Therefore, this PhD research includes a service evaluation study, which was conducted in two community-based PMS in Nottingham. This was followed by a systematic review of the cost effectiveness of a PMS in CLBP. Finally, a decision analysis model was developed to assess the cost effectiveness of PMS compared with SC. In the service evaluation study, newly referred people with CLBP who provided written consent were included. Participants provided information on health status and healthcare resource use using postal questionnaires and diaries at baseline and then three and six months after recruitment. The outcome measures were the Brief Pain Inventory (BPI), the Roland Morris Disability Questionnaire (RMDQ) and the EuroQoL (EQ-5D-3L). In the systematic review, electronic searches were conducted in clinical and economic databases from their inception to August 2017. Full economic evaluations, undertaken from any perspective, conducted alongside randomised clinical trials (RCTs) or based on decision analysis models were included. The Cochrane Back Review Group (CBRG) risk assessment and the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) checklist were used to assess the methodological quality of eligible studies. These studies informed the development of an economic evaluation based on a decision analysis model to compare PMS with SC. Costs per extra quality-adjusted-life-year (QALY) were calculated from the UK National Health Service (NHS) England perspective using a lifetime horizon. Transition probability, utilities, healthcare resource use and costs were obtained from the service evaluation study and the published literature. Results: In the service evaluation study, 32 people were recruited over 10 months. The mean age was 58.8 years with an equal distribution of both genders. The dropout rates were 21% and 25% over three and six months respectively. Disability was measured using RMDQ (with a score range between 0-24), where higher scores mean more disability. The mean score at baseline was 13.5, after three and six months the means were 11.9 and 11.1 respectively. The BPI was used to measure pain intensity and interference. The scores ranged from 0 to 10, where higher scores mean more pain. The mean score for pain intensity was 5.9 at baseline, followed by a mean of 5.5 and 5.6 at three and six months respectively, whereas pain interference was 5.9 at baseline, followed by a mean of 5.2 at three and six months. As for the EQ5D-3L, higher scores in EQ5D means better health states. The mean score was 0.38 at baseline and, after three and six months, the scores were 0.46 and 0.40 respectively. The mean number of health visits per patient between baseline and three months was 4, whereas between four and six months the mean number of visits per patient was 2.9. In the systematic review, five studies fulfilled the eligibility criteria. The PMS varied significantly between studies in terms of the number of treatment modalities, intensity and the duration. The PMS was compared with either standard care, which varied according to the country and the setting or with two different surgical interventions. In this review, three out of five studies had a high risk of bias based on the design of the randomised controlled trials (RCTs). In addition, there were limitations in the statistical and sensitivity analyses in the economic evaluations. Therefore, the results from this systematic review need to be interpreted with caution. Finally, the indicative economic evaluation showed thatthe PMS was both more effective and more costly compared with SC. The mean incremental cost effectiveness ratio (ICER) was £761.43 and £706.98 per quality adjusted life years (QALY) gained in the deterministic and probabilistic analyses, respectively. At a ceiling willingness to pay (WTP) of £20,000, the PMS reaches a 51% probability of being cost-effective. This suggest that there is 51% probability that the PMS is both more effective and less costly. The incremental net monetary benefit generated a mean of £7884.07, indicating that the PMS is cost-effective compared with the SC at a WTP of £20,000. In the sensitivity analysis, the results were affected by changing the utility score for severe pain and increasing the initial cost of the intervention. In the scenario analysis, the incremental cost and QALYs were in favour of the PMS for people with severe pain at baseline. The probability of the PMS being cost effective for people with severe pain was 54%. In addition, the sustained reduction in treatment effect reduces the PMS probability of being cost effective to 48%. Finally, using the two months’ data to generate the transition probabilities demonstrated that PMS dominates the SC and the probability of the PMS being cost effective was 58% Conclusion: Community-based PMS have the potential to improve functional disability and pain interference for people with CLBP in primary care by providing well-timed access to a specialised pain management team, ensuring effective use of pain medicines and streamlining the treatment pathways based on the individual patient’s needs. A systematic literature review highlighted inconsistent evidence supporting PMS. The cost effectiveness studies included in this systematic review were alongside RCTs with a maximum follow up period of two years. As a result of the limitations of this type of economic evaluation, a decision analysis model was developed in order to assess the life time effectiveness of PMS compared with SC. The decision analysis model showed that PMS is more effective and costly compared with SC in the base case analysis; however, changing the source of transition probabilities from 12 months to two months demonstrated that PMS dominated SC, providing the potential for PMS to be cost-effective if high quality research is conducted to reduce the uncertainty around transition probabilities. The results were also sensitive to change in the utility score for severe pain, the initial cost of the PMS and using the PMS for people with severe pain at baseline. Therefore, further information is needed to assess the uncertainties in these parameters to provide a more robust estimate of the cost-effectiveness of PMS compared with SC.

The innate immune system is a generic response to infection or injury. Evidence shows the innate response has immunological memory capable of altering subsequent responses to stimuli. Fibroblasts are ubiquitous stromal cells capable of responding to inflammatory triggers, and of orchestrating endothelial cell and leukocyte behaviour during inflammation. Repeated challenge with cytokines (such as tumour necrosis factor (TNF) a) induced an augmented second response to stimulation. Fibroblasts from multiple anatomical locales significantly increased cytokine secretion upon second challenge with TNFa. The precise mediators augmented depended on fibroblast site of origin. Depending on site, memory was inherent, or only present in fibroblasts from chronically-inflamed tissue. This suggests a phenomenon intrinsic to some sites but pathological in others. The secreted mediators from the fibroblast initial or memory responses exerted differing effects on leukocytes, dependent upon fibroblast site of origin. Finally, examination of intracellular signalling showed the augmented response was at least partly due to prolonged activity of nuclear factor (NF) KB during the memory response. Innate immune memory exists in fibroblasts from multiple tissues, but may be pathologically acquired in some. The altered response to second challenge may represent a fibroblast mechanism for altering the recruitment and behaviour of the inflammatory infiltrate.

The liver contains a number of tissue-associated lymphocyte populations, of which many have been implicated in the pathogenesis of chronic liver diseases. γδ T cells, particularly the Vδ2^neg subset, are known to comprise a substantial proportion of tissue-associated lymphocytes, although their immunobiology remains poorly understood. Here, the localisation, TCR diversity, immunophenotype and function of human intrahepatic γδ T cells was explored with an emphasis on highlighting any potential role in chronic liver disease and also to further understanding of tissue-associated γδ T cells, using the liver as a model tissue. Intrahepatic γδ T cells were predominantly localised in the sinusoids and did not increase in frequency with chronic inflammation. Vδ2^neg cells exhibited private TCR clonal focussing, with complex CDR3 regions suggestive of antigen-driven expansions, concordant with a loss of naive-like CD27^hi cells present in the periphery. Expanded clonotypes were phenotypically T_EM- or T_EMRA-like, with T_EMRA-like clonotypes shared between liver and blood and resembling vasculature-associated virus-specific CD8⁺ T cells while T_EM clonotypes were identified only in the liver and resembled tissue-resident CD8⁺ T cells. These findings suggest that disease has minimal impact on intrahepatic γδ T cells, while supporting an adaptive paradigm for these cells in the formation of tissue-associated subsets.

XRCC4-like factor (XLF) is the most recently discovered core member of the nonhomologous end joining (NHEJ) machinery. XLF enhances ligation of DNA ends by DNA ligase IV (LIG4) and functionally interacts with KU70. Previous results showed that some polymorphic changes in LIG4 impact on the efficiency of double strand breaks (DSBs) repair. A random Caucasian population sample was screened for XLF polymorphic mutations with similar functional impact. This analysis identified two novel noncoding single nucleotide polymorphisms (SNPs). To address the regulation of XLF and KU70, the acetylation status of both proteins were analysed. It has been found that XLF undergoes acetylation both in vitro and in vivo and the acetylation sites were mapped in vitro by mass spectrometry. Preliminary analysis has indicated that XLF deacetylation might be histone-deacetylase (HDAC3) dependent. For KU70, it has been found that lysine residues K317, K331 and K338 are critical for NHEJ. Cells overexpressing aceto-mimicking or aceto-blocking mutants of these residues are radiosensitive and defective in DSBs repair (DSBR). This indicates that the dynamic regulation of the acetylation/deacetylation status of these residues is critical for DSBR in response to ionizing radiation. These findings establish the importance of non-histone repair protein acetylation in the regulation of NHEJ and define new possible therapeutic targets for cancer treatment.

Fibroblasts are the most abundant cell type of the stroma, producing extracellular matrix components which provide mechanical strength to tissues. Recent work has shown that fibroblasts are not simply passive structural cells but active participants in the immune response. In this study fibroblast heterogeneity within the murine lymphoid system was investigated by analysing gene and protein expression. Fibroblasts were isolated from a range of lymphoid and peripheral sites. Our findings show that fibroblasts grown from different sites show heterogeneous gene and protein expression and are functionally different in their response to lymphotoxin α treatment in vitro and in their capacity to recruit host leucocytes in vivo. A novel in vivo functional assay has been developed using a collagen sponge as a three-dimensional structure in the kidney capsule transfer model, to assess the ability of fibroblasts to form and maintain lymphoid structures. We conclude that murine fibroblasts isolated from lymphoid tissues, display site specific features. They are phenotypically distinct with site specific gene and protein expression profiles. This suggests that murine lymphoid fibroblasts have positional memory and help impart anatomical identity. An important implication of these findings is the effect this diversity may have in conveying site specificity to immune responses.

Histone modifications are thought to act as a layer of epigenetic information, because of their strong association with gene expression, and their potential role in transcriptional memory. However, although specific histone modifications correlate with transcriptional status, whether they play a causative role or act in the long term inheritance of gene expression patterns is unclear. In order to explore this, the histone deacetylase inhibitor valproic acid (VPA) was used to induce hyperacetylation of histones in embryonic stem cells. Surprisingly, although global levels of acetyl marks were highly increased by VPA treatment (up to 16-fold), only 10% of genes showed transcriptional changes. Interestingly, these global changes in histone modification were not reflected in the changes at individual genes where increases in acetylation were rarely greater than 2-fold. Furthermore, changes in acetylation levels did not correlate with transcriptional effects. Wash-out experiments showed that transient VPA treatment could not induce long term effects on transcription, even during ES cell differentiation when histone modifications play a crucial role. Finally, the role of polycomb silencing in the response to VPA treatment was assessed using an ES cell line in which the polycomb components Eed and Ring1b had been knocked out. Target genes showed small up-regulation in knockout cells but VPA did not further induce transcription. It was concluded that histone acetylation plays an important role in transcription but additional signals are required for transcriptional induction and cellular memory. My results suggest the existence of protective mechanisms against hyperacetylation and highlight the complexity of epigenetic regulation, potentially involving many layers of control.

All classes of leukocytes must be able to move from the circulation into tissue to carry out their protective functions. To achieve this transfer, the flowing cells must adhere to the endothelium and migrate through the vessel wall. Though this process follows common stages during recruitment in different organs, there is also specialisation in underlying molecular mechanisms. The main aim was to further elucidate these organ specific phenotypic differences and relate them to the functional ability of EC to recruit subsets and total leukocytes from flow. Murine models of human disease are an incredibly useful experimental tool allowing investigation of whole diseases to the effect of one gene on a disease outcome. Isolation of primary human endothelial cell populations is well-defined (hLSEC/HUVEC) however obtaining and culturing the murine counterparts is more challenging. Primary mLSEC were isolated from murine livers using ɑ-CD146 magnetic beads and the phenotype compared to immortalised cell lines from heart (mUCEC-1), skin (s.END) and brain (b.End.5). The expression of a number of adhesion molecules and endothelial markers varied within cell type in response to pro-inflammatory insult (Endoglin, CD34, CD31). Some artefacts of immortalisation were also apparent (VCAM-1 expression in mUCEC-1 and LYVE-1 in s.END). Functional assays indicated small differences in cell types and further microarray analysis elucidated further candidates, including chemokines, which could be involved in regulating leukocyte recruitment processes in the different organs examined.