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Theses - Clinical Medicine


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  • ItemOpen Access
    The role of natural killer cells in control of varicella zoster virus infection
    Allen, Caroline Patricia
    Varicella zoster virus (VZV) is a globally important virus with significant health and economic impacts. Although primary VZV infection is often perceived as a mild childhood illness, VZV infection is lifelong and reactivation of the latent virus in the elderly and immunocompromised population carries significant associated morbidity. Understanding precisely how the body responds to this virus is an important first step in tackling these challenges. As with other components of the innate response, Natural Killer (NK) cells respond directly to VZV infection, and VZV is likely to use evasion strategies to modulate the immune response. This thesis explores the details of this interplay between NK cells and VZV. First, I address the hypothesis that NK cell receptor expression in VZV seropositive patients is different to NK receptor expression in seronegative subjects. By comparing the expression of NK cell receptors in vivo in three cohorts of patients (seronegative, acutely infected, and recovered seropositive) I examine how these findings provide evidence to characterise the NK cell response to VZV, and to provide insight into viral mechanisms to evade detection by NK cells. Secondly, to address the hypothesis that specific ligand-receptor interactions between NK cells and infected cells responsible for control of VZV replication I examine changes in surface expression of NK cell ligands on infected cells in an vitro model of VZV infection using monocyte-derived dendritic cells (MDDC). Finally, using proteomic analysis, I explore changes in expression of proteins in in vitro infection in both monocyte-derived dendritic cells and an immortalised human keratinocytes cell line.
  • ItemOpen Access
    Preventing Retained Central Venous Catheter Guidewires
    Mariyaselvam, Maryanne
    Whole central venous catheter (CVC) guidewire retention is an iatrogenic error, with an incidence of 1:3,167, a morbidity of 4-53%, mortality of 4-6% and is a never event. Since 2011, health policy has mandated that all hospitals must implement national safety guidance to prevent this error, emphasising that the never event will be prevented if the guidance has been introduced. Despite this, the error continues to occur, and the reported frequency is rising. The aim of this thesis was to identify a novel approach to prevent this error occurring. A literature search, an analysis of ten years of CVC guidewire retention events nationally, and interviews with ten clinicians who had made the error were conducted. Guidewire retention was found to be an omission error, occurring due to distraction or interruption at a ‘critical point’ in the CVC procedure. Current safety mechanisms were mostly ineffective, and none were 100% effective. Strategies were operator focused interventions and performed after the error had occurred when correction may be difficult. An intraprocedural systems solution that prevents over-insertion or forces early recognition was required. Several solutions were developed and trialled. Finally, the locked procedure pack was deemed the best solution, due to ease of use and implementation, and low cost. A randomised controlled forced error simulation study was used to assess efficacy of the locked procedure pack and prevented CVC guidewire retention in 10/10 versus 2/10 with standard practice (p < 0.001). The locked procedure pack was developed into a clinical product and renamed ‘WireSafe’. When introduced into clinical practice, there were no demonstrable negatives compared to standard practice on procedure duration (10% noninferior margin, p = 0.44), with a significant improvement in sharps safety (100% WireSafe versus 47% standard practice, p = 0.0008) and wide staff acceptability of the device in terms of preventing guidewire retention and improving sharps safety (20/20). Finally, a novel ‘suck out’ suction technique was developed to aid guidewire retrieval. No difference in retrieval techniques were found when the guidewire was retained above skin level, however the ‘suck out’ technique was significantly better when the guidewire was retained below skin level in 9/10 versus 0/10 in withdrawal and 1/10 in clamp and withdrawal (p < 0.001). The WireSafe prevents CVC guidewire retention, protects clinicians from making this error and protects patients from the morbidity and mortality associated with this error. Further work is still required to evaluate the effectiveness of the solution through large scale clinical studies.
  • ItemOpen Access
    Cancer incidence by Ethnic Group in England, 2001-2007
    Ali, Raghib
    Background There are large unexplained variations in the incidence of many cancers globally and the incidence of cancer in migrant populations can contribute to our understanding of aetiology for cancers for which there are few established risk factors. Studying different ethnic groups in the same country overcomes the limitations of some international comparisons as similar diagnostic methods, reporting and registration procedures are used. The primary objective of this study is to compare the incidence of all major cancers in the six main ‘non-White’ ethnic groups in England to each other and to Whites using self-assigned ethnicity. A secondary objective is to compare these incidences with their countries of origin. Methods All cancer registrations from 2001–2007 in England were analysed. Ethnicity was obtained by linkage to the Hospital Episodes Statistics database and mid-year population estimates from 2001-2007 from the Office for National Statistics. Age-standardised incidence rates were calculated for all ethnic groups and incidence rate ratios (adjusted for age, sex and income) were calculated comparing the six non-White ethnic groups (and combined ‘South Asian’ and ‘Black’ groups) to Whites and to each other. Results There were significant differences in the incidence of nearly all cancers between the ethnic groups. In general, incidence was lower in non-White ethnic groups, but there was considerable variation with South Asians having higher rates of head & neck, liver, gallbladder, Hodgkin lymphoma and thyroid cancer and Blacks having higher rates of stomach, liver, gallbladder, prostate, endometrium, non-Hodgkin lymphoma, myeloma, thyroid and childhood cancers. There also was strong evidence of differences in risk between Indians, Pakistanis and Bangladeshis for most cancers and between Black Africans and Black Caribbeans for many.   Conclusions The risk of most cancers varies greatly by individual ethnic group, including within those groups that have traditionally been grouped together (South Asians and Blacks). Many of these differences are not readily explained by known risk factors and suggest that important, potentially modifiable causes of these cancers are still to be discovered. In order to understand why these differences exist and the relative contribution of genetic and environmental factors, a large, prospective cohort study of non-White ethnic groups in the UK with individual level risk-factor information is needed.
  • ItemOpen Access
    Trend and Impact of Nephrectomy Centralisation on Renal Cell Carcinoma Survival
    Hsu, Ray
    Introduction The incidence of renal cell carcinoma is increasing worldwide. The widespread use of radiological imaging has contributed to disease stage migration with greater proportion of small, asymptomatic renal tumours often detected incidentally. Despite this, mortality from RCC has not changed substantially and over 4,000 patients still die from RCC in the UK annually, accounting for 3% of all cancer deaths. In response, the Improving Outcomes for Urological Cancers guidance from the National Institute for Health and Care Excellence was published in 2002, recommending the centralisation of oncological resections for urological cancers to high volume centres. During the same period, advancements in surgical technology such as laparoscopic and robotic surgery were emerging together with the adoption of nephron-sparing surgery in the management of small renal tumours. It is not known how implementation of new guidelines and surgical techniques have impacted on RCC patient outcomes, particularly on a national level. Many population-based studies examining RCC survival have focused on patients irrespective of treatment modality and may include those who have not received active treatment, diluting the true effect of surgical and public health interventions. While evidence has emerged that complex operations performed in high volume centres were more likely to yield better outcomes, it is not yet clear whether this benefit is seen in RCC nephrectomy. There is therefore a need to better understand how RCC nephrectomy has changed in recent years and whether patient outcomes, particularly survival, have improved. Characterisation of the benefit of nephrectomy centralisation is also required particularly as it can significant disrupt patients, clinicians and the wider healthcare system. This study hypothesizes that RCC nephrectomy has undergone substantial changes in recent years with improvements in patient survival. The study also hypothesizes that hospital nephrectomy volume is an important variable in predicting mortality risks both in the post-operative period and in the longer-term. Methods Information from English patients who underwent RCC nephrectomy between 2000 and 2010 were captured using linked datasets consisted of Hospital Episode Statistics and National Cancer Data Repository. Empirical analyses were performed to examine the temporal trends in RCC nephrectomy practice and outcomes and the relationship between hospital nephrectomy volume and intermediate and long-term patient survival. Meta-analysis was performed to determine the association between hospital nephrectomy volume and perioperative outcomes using studies published between 1990 and 2016. Results The annual number of RCC nephrectomy performed in England increased by 65.7% during the study period from 2,211 to 3,664. There was rapid adoption of nephron-sparing and minimally-invasive surgery, increasing annually by 13.2% and 54.6% respectively (p<0.01). The number of hospitals performing RCC nephrectomy declined by one quarter but the number of surgeons and nephrectomies performed in each unit steadily increased, suggesting surgical centralisation. In parallel, 30-day mortality decreased from 2.4% to 1.1% (p<0.01). 1 and 5-year age-standardised relative survival increased from 86.9% and 68.2% to 93.4% and 81.2% respectively (p<0.01). Greatest survival improvement was seen in patients with locally advanced disease (T3 or T4). The improvements could be explained, at least in part, by the centralisation of RCC nephrectomy. Meta-analysis of 12 studies containing 201,506 patients found that patients undergoing radical nephrectomy in high-volume hospitals had a 26% (OR 0.74, 95% CI 0.61- 0.90, p<0.01) reduction in postoperative mortality and 18% (OR 0.82, 95% CI 0.73-0.92, p<0.01) decrease in complications. Similar benefit was observed in nephrectomy with venous thrombectomy where patients treated in high-volume hospitals had a 52% reduction (OR 0.48, 95% CI 0.29-0.81, p<0.01) in postoperative mortality. Survival benefit was also seen beyond the immediate post-operative period. Patients with localised disease treated in high-volume hospitals were 34% less likely to die within the first year (HR 0.66, 95% CI 0.53-0.83, p<0.01). Survival benefit in high-volume hospitals did not appear to extend beyond the first year. Conclusions During the study period between 2000 and 2010, RCC care in England appears to be improving with factors such as newer surgical techniques and technology contributing to the improvement in patient survival. Nephrectomy centralisation is truly underway with growing evidence that adverse outcomes can be reduced through reconfiguration of surgical delivery.
  • ItemOpen Access
    Senescence and the Innate Immune System
    Robins, Anne
    Cellular senescence prevents deoxyribonucleic acid (DNA) damaged cells undergoing proliferation, protecting against malignancy but at the expense of senescent cell accumulation. Since the proportion of senescent hepatocytes in man correlates closely with liver injury, fibrosis stage and mortality, prevention of malignancy occurs at the expense of impaired organ function. Senescent cells are removed by the innate immune system, including natural killer (NK) cells, during normal embryonic development and healthy wound healing. Since eradication of senescent cells in mice leads to resistance against age-related disorders and functional restoration, one approach to liver injury might be enhanced removal of senescent cells. The non-classical human leukocyte antigen class I molecules E, F and G (HLA-E, -F and -G), are ligands for NK cells and considered tolerogenic. For example, HLA-E binds with the NK inhibitory receptors, killer cell lectin like receptor D1 (CD94) and c-type lectin family receptors (NKG2), preventing NK mediated killing. Less is known of HLA-F, which is present in placental trophoblasts, possibly promoting foetal tolerance. HLA-G is expressed on trophoblast cells and is considered a key component mediating foetal tolerance. Little is known of HLA-E or -F in liver injury, while a few immunohistochemical studies have suggested increased hepatocyte expression of HLA-G in cirrhosis of all aetiologies. My hypothesis was that senescent hepatocytes are removed from healthy liver by the innate immune system, as in healthy wound healing, but that in chronic liver disease there is a failure of this response, leading to an accumulation of senescent cells, fibrosis and cirrhosis. I examined hepatic expression of HLA class I, HLA-E, -F and -G in health and disease. Secondly, I assessed the major histocompatibility complex (MHC) class I ligands, the NK cell receptors. Finally, the various components of the antigen presentation pathway in health and disease were studied. My study shows that HLA class I is present in liver tissue and is upregulated in cirrhosis. HLA-E and -F are expressed in liver tissue and are also upregulated in cirrhosis. The expression of HLA-E and -F was largely intracellular and consistent with retention within the golgi, however, some less marked surface expression was also seen. The data on expression of HLA-G was clear, but inconsistent with the literature. While immunohistochemistry work was indeed congruent with published data showing widespread expression in cirrhosis, all other biochemical techniques, undertaken herein for the first time, failed to detect HLA-G protein in healthy liver tissue or cirrhosis; HLA-G RNA was only identified in hepatitis B infected liver tissue. These data suggest that previous reports linking HLA-G and liver injury may be artefactual. The NK cell receptors, killer cell immunoglobulin like receptors 2DL2 and 2DS2, were significantly upregulated in cirrhosis although the numbers studied were low suggesting that NK cell activation is altered in cirrhosis. Tapasin and tapasin related protein (TAPBPR), both parts of the antigen presentation pathway are expressed in liver tissue and show a slight upregulation in liver disease. This suggests that aberrant expression via this pathway may be part of the liver pathology. In summary there is altered expression of HLA molecules in cirrhosis, specifically HLA-E and -F. Secondly, altered expression of inhibitory molecules is seen on circulating NK cells. These findings are consistent with reduced clearance of damaged hepatocytes in cirrhosis. It proved impossible, despite numerous approaches, to link increased hepatocyte HLA expression with senescence. My study concludes that HLA class I is present in liver tissue and is upregulated in cirrhosis. The expression of HLA-E and -F is identified in liver tissue for the first time and is shown to be upregulated in cirrhosis. This altered expression of HLA-E and -F may have a role in the pathogenesis of cirrhosis and could be a target for future therapies.
  • ItemOpen Access
    Individualising prognostic stratification in non-metastatic prostate cancer: the development, validation and clinical impact assessment of the Predict Prostate tool.
    Thurtle, David; Thurtle, David [0000-0001-5636-7088]
    Decision-making around treatment for non-metastatic prostate cancer is complex, with radical treatment associated with significant potential morbidity despite some tumours being relatively indolent. Prognostic stratification should therefore help inform management. However, models currently used in clinical practice have significant flaws. This thesis sets out the rationale for a new individualised prognostic model, describes the development and validation of a novel model called Predict Prostate, and then evaluates the impact of this model in clinical practice. Chapter 1 introduces the topic and includes a systematic review of existing tools, including a collaborative screening process of 6,597 records. Very few individualised prognostic tools were identified, and those found were considered inadequate by failing to include treatment effect, and disregarding non-cancer mortality. Chapter 2 describes the development of an algorithm to estimate individualised 15-year prostate cancer-specific, non-prostate cancer, and overall mortality. Data on 10,089 men from the UK National Cancer Registration and Analysis Service were split into model-development and validation cohorts. An additional validation was performed in a small international cohort. Chapter 3 describes the updating and large external validation of the model in a geographically independent cohort of 69,206 men from the Swedish Prostate Cancer database. Overall the model was well calibrated, and discriminatory performance of the model generally exceeded existing models. Chapters 4 and 5 evaluate the application of the model. First, model estimates were presented to health care professionals in a randomised online format using hypothetical clinical vignettes. Clinicians were found to overestimate cancer lethality, and were less likely to recommend radical treatment when shown Predict Prostate estimates. Chapter 5 describes the multi-centre randomised controlled trial assessing the impact of the model among newly diagnosed prostate cancer patients. Here, the model was shown to shift perceptions around prognosis, reduce decisional conflict and uncertainty, and was popular with patients. Chapter 6 summates and concludes the thesis.
  • ItemOpen Access
    Anaemia, Iron-Deficiency and Reducing Transfusion in Cardiac Surgery and A Novel Method for the Measurement of Total Haemoglobin Mass
    (2021-11-11) Yeates, James
    Anaemia and iron-deficiency are common in cardiac surgical patients and are associated with poor surgical and patient-centred outcomes. Pre-operative anaemia is increasingly being treated with intravenous iron despite a lack of high-quality evidence of its effectiveness. This thesis explores these topics with a literature review exploring anaemia, iron-deficiency, and transfusion in cardiac surgery, and further review on transfusion risk-prediction models, and treatment of pre-operative anaemia with intravenous iron. The first methodological chapter describes the development of a new risk-scoring system to predict those who are likely to require peri-operative red blood cell transfusion in cardiac surgical patients in the UK. This risk scoring system was then modified and recalibrated using an Australian database to create an alternative score applicable to that population, described in the subsequent chapter. The use of intravenous iron has increased significantly since the introduction of new iron-preparations and is now recommended in many treatment pathways and guidelines. This has occurred in advance of high-quality evidence that it can effectively treat anaemia in the pre-operative period and have any meaningful effect on patient outcomes. Chapter 5 describes a UK-wide multicentre trial that demonstrated that IV iron can significantly increase haemoglobin concentration in cardiac surgical patients in the pre-operative period. Challenges in the recruitment for larger studies of anaemic cardiac surgical patients led to an exploration of the recent trends in anaemia-rates over the last 5-7 years, which is described in Chapter 6. This demonstrates that anaemia rates in certain areas have decreased significantly over that period and there has been concomitant exponential increase in the use of intravenous iron preparations in various medical settings. The final methodological chapter describes a novel method for measuring total haemoglobin mass, which may be a more appropriate method of assessing anaemia in various disease states. Previous methods have been difficult to establish and complicated to undertake. This chapter describes a new method using a modification of an existing respiratory function test which is demonstrated to be a simple method of estimating total haemoglobin mass and plasma volume.
  • ItemOpen Access
    Multi-omic characterisation of Barrett’s oesophagus reveals a molecular continuum in the progression to oesophageal adenocarcinoma
    (2020-07-01) Katz-Summercorn, Annalise
    Barrett’s oesophagus (BE) is the main risk factor for the development of oesophageal adenocarcinoma (OAC) yet only 0.4%/year of non-dysplastic (ND) BE cases progress to cancer (Bhat et al., 2011). In this thesis I present the first comprehensive, multi-omics study comparing indolent, non-progressors with those who progressed to a range of dysplasia grades. BE is highly heterogeneous with regards to mutational load, copy-number aberrations (CNAs) and structural variants (SVs). Mutational signatures are laid down early and persist regardless of progression status. Hence, Cosmic signature 17 (T:A>G:C in a CTT context), the hallmark of OAC, is visible in indolent, ND samples. TP53 mutation, GATA6 amplification, ERBB2 amplification, APC mutation and whole genome doubling are confined to cases that have progressed with TP53 being by far the most prevalent. In contrast CDKN2A alteration occurs early in around 50% of indolent cases. SV analysis reveals a dominance of translocations from the early ND grade. Spatial analysis of multiple samples from across BE segments shows that the total mutation burden, total CNAs and total numbers of SVs to be surprisingly constant. However, clonality analysis highlights the complexity of BE, with some cases arising from a clear ancestral clone while others having minimal sharing of variants and showing heterogeneity in terms of driver events. Transcriptomic analysis reveals a clear but gradual differential gene expression between ND and dysplastic biopsies. We demonstrate a loss of the intestinal metaplasia phenotype with progression and downregulation of the HNF4A pathway, a transcription factor with roles in intestinal development. In progression there is an upregulation of genes in the ERK/MAPK pathway. In summary, BE is a heterogeneous disease that shows a continuum of abnormalities in the progression towards cancer. We hypothesise that it is the accumulation of events that tip the balance to progression, rather than a stepwise model through the phenotypic dysplasia grades. Selected features could help to differentiate indolent from high risk disease and further work is being performed to identify scoring systems and biomarkers to apply in the clinical setting.
  • ItemOpen Access
    Computer modelling of metabolic adaptions during mitochondrial dysfunction and machine learning to predict novel mitochondrial disease genes
    Smith, Alexander Gary
    Mitochondria are organelles found in almost every eukaryote and are primarily responsible for generating chemical energy in the form of adenosine triphosphate. This thesis investigates two main causes of mitochondrial dysfunction: mitochondrial toxicity arising from side-effects of drugs; and mitochondrial diseases arising from defects in nuclear-encoded genes. Novel chemical entities being developed as drug leads are screened for cellular toxicity in which mitochondrial dysfunction is a major cause. However, our lack of understanding of the metabolic adaptations to mitochondrial dysfunction limits the accurate screening of mitochondrial dysfunction for pharmaceutical companies, thus preventing potentially useful drugs from being developed. To further our understanding of these adaptations, I analysed a large-scale metabolomics data set of rats administered a known mitochondrial complex III inhibitor. The analyses revealed many perturbed pathways which can be exploited as biomarkers of mild mitochondrial dysfunction, a condition which is currently clinically undetectable during the drug development process. To direct future studies on mitochondrial dysfunction, a multi-organ model of mitochondrial metabolism was generated and used to simulate inhibition of the mitochondrial respiratory complexes. The simulations of complex III inhibition accurately predicted many of the metabolite behaviours identified in the metabolomics analyses and provided theories for their significance. Simulations of the other complexes’ inhibitions identified many unique behaviours which can be used to direct future studies, studies which would greatly improve our understanding of the metabolic adaptations and provide higher confidence biomarkers. Mitochondrial dysfunction is linked to many late onset diseases such as Parkinson’s, and inborn errors of mitochondrial metabolism cause severe neurological and physiological diseases. Patients with suspected mitochondrial disease have their DNA sequenced and analysed. Diagnosis of mitochondrial disease by sequencing requires knowledge of the mitochondrial proteome, which is currently incomplete. A predicted mitochondrial proteome was generated using a support vector machine trained using the abundance of protein localisation data available in the MitoMiner database. The support vector machine identified 442 novel mitochondrional proteins. The current success rate of diagnosing mitochondrial disease using sequencing is currently limited by our inability to filter and prioritise a patient’s DNA variants. Patients which do not have a variant in one of the already known mitochondrial disease genes are usually left with over hundreds of potential disease-causing variants. A probability of being disease-causing for each gene in the mitochondrial proteome was generated using two trained neural networks. The networks were trained on a large amount of different data sources for differentiating mitochondrial disease genes including protein-protein interaction network metrics, gene tissue expression and protein evolution. The predicted probabilities allow for better filtering and prioritisation of a patient’s variants for candidate disease-causing genes to be experimentally verified. The predicted mitochondrial proteome and their predicted disease-causing probabilities are currently used in an NGS analysis pipeline at the MRC Mitochondrial Biology Unit for diagnosing mitochondrial disease patient samples.
  • ItemOpen Access
    The Genetics of Anti-Neutrophil Cytoplasmic Antibody Associated Vasculitis (AAV)
    (2019-09) Wong, Limy
    Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a multi-systemic autoimmune disorder with evidence of circulating pathogenic ANCA. There are two main antigenic targets: proteinase 3 (PR3) and myeloperoxidase (MPO). Previous genome-wide association studies (GWAS) have provided evidence that PR3-AAV and MPO-AAV are genetically distinct autoimmune syndromes, though only three loci specific to PR3-AAV and one to MPO-AAV have been identified to date. With the European Vasculitis Genetics Consortium, we conducted a larger GWAS, powered to discover additional risk loci in both PR3-AAV and MPO-AAV independently. A meta-analysis of two European cohorts was conducted, comprising 1,610 PR3-AAV cases, 870 MPO-AAV cases and 11,947 controls. For PTPN22 (rs6679677), a further replication cohort, previously genotyped using the Sequenom MassARRAY platform, and comprising 1,122 PR3-AAV and 347 MPO-AAV cases and 1,531 controls was included in the combined analysis. This is the largest genome-wide association study of AAV to date and we have identified a total of 12 AAV susceptibility loci. Previously genome-wide significant loci were confirmed, including HLA class II, SERPINA1, PRTN3 and PTPN22. Seven new genome-wide significant loci were identified: three associated with PR3-AAV (BCL2L11-MIR4435-2HG, EBF3-MGMT, IGHV1-69), two with MPO-AAV (BACH2, ANKRD11-SPG7) and two shared by both (CTLA-4 and DGUOK-TET3). Further analyses based on common variants suggested that a substantial component of the genetic architecture was shared between PR3-AAV and MPO-AAV, similar to that observed between ulcerative colitis and Crohn’s disease. In addition, Mendelian randomisation analysis confirmed that a higher eosinophil count increased the risk of PR3-AAV but not MPO-AAV, and this effect might, in part, be modulated by MIR4435-2HG through prolongation of eosinophil survival. MIR4435-2HG encodes a long non-coding RNA that plays a critical role in the regulation of BCL2L11 transcription (a Bcl2 family member essential for controlling apoptosis) in myeloid cells and hence their lifespan. We have also identified a missense variant in IGHV1-69 (rs11845244) that leads to a loss in neutralising function of antibodies generated against the NEAT2 domain of Staphylococcus aureus. This therefore provides a plausible host genetic factor in determining the susceptibility to infectious disease and as a potential driver of PR3-AAV. Overall, this study provides key novel insights into disease biology for AAV and potential therapeutic targets.
  • ItemOpen Access
    Improving Data Quality for High Resolution Functional MRI in Cognitive Neuroscience Applications
    Pei, Huang
    Since the first successful Magnetic Resonance Imaging (MRI) image was produced by Paul Lauterbur in 1973, the field of MRI has been improving by leaps and bounds. The number of MRI and functional MRI (fMRI) papers have sky rocketed over the last decade, alongside with advancements in MRI field strength and techniques. In this thesis, I explore various methods for improving data quality for high resolution fMRI in 3T and 7T MRI scanners. Firstly, I studied the effect of Prospective Motion Correction (PMC) on 3T data using a simple visual paradigm. In contrast to most conventional techniques that use retrospective motion correction (RMC), PMC collects real-time motion data and uses it to update the acquisition field of view prior to each radiofrequency (RF) pulse. This allows for the correction of spin-history effects and intra-volume distortions. In this study, I utilized a secondary optical camera in the bore of the scanner to track a Moiré phase marker attached to the participant via a custom-moulded dental mouthpiece. I demonstrated that the camera is capable of accurately tracking the participant’s head motion. While simple metrics such as temporal signal-to-noise ratio (tSNR) and functional contrast-to-noise ratio (fCNR) showed no difference between the two methods, more complex analysis such as the Linear Discriminant Contrast (LDC) showed that the PMC data was indeed cleaner than the RMC data for higher resolution data. Next, I compared the sensitivity of two multi-voxel pattern analysis (MVPA) methods, Support Vector Machines (SVM) and Linear Discriminant Contrast (LDC). MVPA attempts to capture the relationship between the spatial fMRI activity and the experimental manipulations by treating it as a supervised learning problem. This is a promising technique that can capture spatial activation patterns that are lost in univariate analysis. I demonstrated through both actual fMRI data and computer simulations that LDC is a better MVPA metric than SVM. This agrees with our theory that SVM has more inherent variability and less sensitivity due to its limitations, discretization of results, rigid decision boundaries and ceiling effects. Subsequently, I analysed the quality of fMRI data acquired in a 3T Prisma scanner vs a 7T Terra scanner using a visual attention paradigm. While 7T scanners are becoming increasingly commonplace with over 70 of them worldwide now, the higher field strength also comes with its own host of problems. Field inhomogeneities and artefacts are a larger problem at 7T, and the smaller voxel sizes also cause data to be more susceptible to motion. As such, it is important to establish if there is a real benefit to using a 7T scanner. I observed that both 3T and 7T data showed similar trends with comparable z-scores and concluded that both scanners yielded comparable results. However, the 7T data was acquired at a much higher resolution (64x smaller volume per voxel) and thus, these results indicate a benefit of 7T as comparable results were achieved in spite of the smaller voxel volume. I hypothesized that acquiring data in a 7T scanner would be informative if studies sought to probe further into laminar or columnal structures which require submillimetre resolution, while a 3T scanner should suffice for studies looking at coarse regional activations. I did not explore the benefits of using 7T MRI at coarser resolutions. I also assessed the utility of boundary-based registration (BBR) realignment to improve on conventional RMC techniques to realign fMRI time series. Some motion artefacts affect the image in non-rigid ways and thus, voxel-based registration (VBR), generally utilized in conventional RMC, might be insufficient to properly realign fMRI time series. I demonstrated that BBR realignment outperforms VBR realignment across multiple metrics at submillimetre resolution, but no difference was observed at lower resolutions. Lastly, I examined the process of cleaning up 7T fMRI data for laminar analysis. Gradient echo (GE) sequences have been widely used for fMRI studies due to the high signal-to-noise ratio (SNR) and low specific absorption rate (SAR) relative to other sequences. However, GE sequences have been shown to exhibit superficial bias due to the presence of draining veins. I employed two methods- excluding venous voxels and utilizing a regression analysis, to remove superficial bias in an attempt to unmask any laminar effects for a visual attention task. In summary, I have explored various methods of optimizing fMRI data, ranging from initial setup decisions, such as which field strength scanner to use, to final MVPA analysis methods. I also analysed methods to remove motion artefacts, through both PMC and RMC, as well as post-processing methods to remove superficial bias in laminar data.
  • ItemOpen Access
    Mitochondrial Metabolism Elucidated by Rapid Fractionation from Tissue
    (2020-03-21) Allen, Fay Marie
    Mitochondria are metabolic hubs, with many diseases found to have altered metabolism and mitochondrial dysfunction, such as ischaemia-reperfusion injury. A detailed understanding of the metabolic changes in different cellular pools would aid diagnosis and treatment. However, the current methods of mitochondrial isolation are too slow to provide a snapshot of purely mitochondrial metabolism, meaning that current metabolic data is only from whole cell. This project has developed and used a novel technique to rapidly isolate mitochondria from tissue by density centrifugation through silicone oil, with a view to assess the mitochondrial metabolic changes during ischaemia-reperfusion injury. This method has minimal cytosolic contamination and is completed in under 5 minutes, and mass spectroscopy analysis has shown enrichment of mitochondrial metabolites. Seahorse and Oroboros analysis have shown that the mitochondria are functional and capable of coupled respiration. Data is presented on how the method optimisation was analysed and developed. This largely reduced time frame gives the advantage over other methods to enable the study of metabolism in mitochondria.
  • ItemOpen Access
    Phenotype – genotype associations in a large cohort of patients with pulmonary arterial hypertension
    (2019-10-01) Hadinnapola, Charaka Mayura Bandara; Hadinnapola, Charaka Mayura Bandara [0000-0002-7794-3432]
    Idiopathic and heritable pulmonary arterial hypertension (PAH) are rare diseases with a poor prognosis. There is significant heterogeneity in clinical features at diagnosis, and in a proportion of patients there is a genetic cause of the disease. This clinical and genetic heterogeneity has hindered accurate risk stratification and the development of personalised treatments. The National Institute of Health Research BioResource – Rare Diseases PAH Study and the Medical Research Council / British Heart Foundation National Cohort Study of Idiopathic and Heritable PAH were established to investigate disease pathogenesis through whole genome sequencing and deep phenotyping. One thousand and seventy patients were sequenced and 391 clinical variables were captured for each patient. In patients with idiopathic PAH the age at diagnosis, gender, right atrial pressure and the transfer coefficient for carbon monoxide were identified as independent prognostic variables. However, the presence of rare and predicted deleterious variants in BMPR2 was not of prognostic significance. Variants in genes previously associated with disease pathogenesis were identified in 204 patients (19 %). Patients with variants in BMPR2 were younger at diagnosis and had more severe pulmonary haemodynamic impairment compared to patients with idiopathic PAH. Novel associations between BMPR2 variant status and both haemoglobin concentration and white blood cell count were observed. Four patients carried variants in both SMAD9 and either BMPR2 or EIF2AK4. The clinical significance of this requires further study. Unexpectedly, biallelic variants in EIF2AK4 were identified in patients with a clinical diagnosis of idiopathic PAH. These patients had a significantly worse prognosis compared to patients with idiopathic PAH. The spectrum of phenotypic, radiological and histological changes associated with biallelic EIF2AK4 variants was broader than previously recognised. As these datasets mature, further analyses assessing the response to specific treatments and the outcomes of specific subgroups will be possible.
  • ItemOpen Access
    Sleep apnoea and cardiac surgery: Screening, prevalence and postoperative outcomes
    (2019-04-25) Mason, Martina
    Introduction: An excess of postoperative complications have been reported in patients with Obstructive Sleep Apnoea (OSA) following surgical procedures, however, studies reporting outcomes in patients with OSA following cardiac surgery are sparse and of limited quality. The cause of worse surgical outcomes in the OSA population is unknown but deleterious effects of opiates/opioids, common pain relieve medication following surgery have previously been proposed. There is a move towards pre-operative screening for OSA prior to surgery but the best screening methodology has not yet been established and more importantly the effect of treatment, in particular Continuous Positive Airway Pressure (CPAP), on surgical outcomes in patients with OSA is unknown. Aim: This thesis examined the prevalence of sleep apnoea and its association with postoperative outcomes in patients undergoing major cardiac surgery. It also explored the usefulness of the STOP-Bang questionnaire, as a screening tool for OSA prior to cardiac surgery. In addition, current evidence regarding the effects of opiates/opioids and sedatives on patients with OSA was investigated and summarised in the Systematic Cochrane Review. The effect of morphine on severity of sleep apnoea in patients with moderate OSA was examined in a separate study. Methods: The prevalence and association of sleep apnoea with postoperative outcomes in patients undergoing cardiac surgery and the usefulness of the STOP-Bang questionnaire in identifying patients at risk of OSA prior surgery was examined in a prospective, observational cohort study. The Systematic Cochrane review included randomised controlled trials examining the effects of opioids and sedatives, compared to placebo on severity of OSA in patients with established diagnosis of OSA. The effect of intravenous morphine sulphate on the severity of sleep apnoea was examined in a prospective, paired design trial which recruited patients with moderate OSA. Results: A high prevalence of sleep apnoea (47%) and a significant association between its severity and postoperative complications was found in 122 participants undergoing major cardiac surgery. The most significant risk factor for complications was found to be oxygen desaturations during the night reflecting the severity of sleep apnoea (OR=1.1 for each unit increase in oxygen desaturation index (ODI), 95% CI 1.02-1.17; p=0.014). It was found that the STOP-Bang scores between 0-2 would with high confidence exclude patients with at least moderate sleep apnoea prior surgery. The best diagnostic performance for diagnosis of at least moderate sleep apnoea was found at higher STOP-Bang scores of ≥6 which could identify those patients who might benefit from a sleep study before cardiac surgery. A systematic Cochrane review found that opiates/opioids, sedatives and hypnotics have no deleterious effect on the severity of OSA but most of the studies included in the review were of short duration, small size and with indiscernible methodological quality. The results of the Systematic Cochrane Review informed the development of my study, studying the effect of opiate, morphine sulphate, on patients with moderate OSA. This showed no change in Apnoea/Hypopnoea Index (AHI) where median difference (MD) was -12.95, IQR 9.45, p=0.173 but showed significant improvement in sleep apnoea indices including: obstructive apnoea index (MD -2.7, IQR 7.37, p=0.03), central apnoea index (MD – 0.35, IQR 0.83, p=0.04 ).However there was a fall in median nocturnal oxygen saturation. Conclusion: This thesis reports high prevalence of sleep apnoea which was also found to be a risk factor for postoperative complications in patients undergoing major cardiac surgery. In this population, STOP-Bang score ≥6 could identify patients in need of a sleep study to identify those who may be at increased risk of postoperative complications. To date there is no strong evidence supporting deleterious effects of opioids/opiates on patients with OSA but larger studies are needed to clarify its effect.
  • ItemOpen Access
    Early life phenotypes of type 1 diabetes genetic risk
    (2019-07-19) Eleftheriou, Antigoni
    Observational studies have shown that children who develop type 1 diabetes (T1D) have larger size at birth — a marker of foetal growth — or rapid postnatal weight gains. Recent studies have explored the contribution of the T1D susceptibility human leukocyte antigen (HLA) alleles, which explain half of disease heritability, to perinatal growth but findings are inconsistent. In the Cambridge Baby Growth Study (CBGS), a birth cohort which follows children over the first 2 years of life, I conducted genotype-phenotype correlation analyses with the objective of identifying T1D susceptibility single nucleotide polymorphisms (SNPs) and candidate genes that influence the archetypal phenotypes of the disease prodrome, which starts early in life, as a means of shedding light on the heterogeneity of data and sign-posting mechanisms. Based on prior ‘biological’ knowledge I selected T1D SNPs conjectured to affect growth mediated by hormones, vitamin D, or the microbiome. Genotypes were determined in ~600 children and used to derive high-risk T1D HLA haplotypes and genetic risk scores. Examination of data in the CBGS defined the contributors to physiological growth and set the framework for analyses. My findings concurred with most prior studies on the lack of an effect of the SNP rs689 in INS on early growth. A genetic contribution to size at birth was detected by variants in the imprinted DLK1 gene, the vitamin D metabolism CYP2R1 gene and the gene encoding TYK2 known for its susceptibility to infections. Examination of longitudinal phenotypes identified that infancy growth is influenced by genes with a key role in innate immunity, e.g. IFIH1. The strongest associations were found between skinfold thickness, a proxy for adiposity, and variants in genes whose products play a role in immune and inflammatory pathways, exemplified by the rs653178 in SH2B3, previously known for its association with cardiovascular disease. Taking on the lead from my findings with anthropometry, I explored genetic-hormonal correlates and found evidence in support of IGF-1 mediating the association that I identified, more strongly in boys than in girls, between the SNP tagging the high-risk HLA-DR3 and rapid linear growth. I made the novel finding that the rs12785878 in DHCR7, known to strongly associate with vitamin D levels, inversely correlated with leptin levels in girls but not in boys. This study elucidated the effects of T1D susceptibility SNPs on infancy adiposity, which might serve as a better disease marker than weight. It identified a link between leptin and a common polymorphism in DHCR7, opening up the possibility that vitamin D suppresses leptin production in a sex-specific manner with protective effects against autoimmunity.
  • ItemOpen Access
    An investigation into the prevalence, impact and management of cancer-related fatigue in teenage and young adult patients
    (2019-04-27) Spathis, Anna Olga
    Teenage and young adult (TYA) cancer patients describe debilitating fatigue. The impact of fatigue is particularly detrimental at this age because it hinders key developmental needs, such as independence. Although exercise and psychological treatments are the most effective management approaches in older adults, TYAs have unique healthcare needs and research findings cannot be extrapolated to younger patients. The aim of this programme of research was to increase understanding of cancer-related fatigue (CRF) in TYAs, in order to develop a non-pharmacological fatigue intervention. Three multimethod studies have been conducted, underpinned by self-efficacy theory. 1) A systematic literature review synthesised existing research evidence. 2) A multicentre electronic survey evaluated the experience of TYA CRF and its current management across the UK. 3) A multiphase, longitudinal, qualitative study, involving patient-parent dyads, has led to the co-design of a fatigue intervention. This work has confirmed that fatigue is the most prevalent and distressing symptom experienced by TYAs with cancer, and is an independent predictor of poor quality of life. Fatigue persists long after the end of cancer treatment, perpetuated by vicious cycles of emotional and behavioural responses to the symptom. Cognitive fatigue has a particularly negative impact and influences the ability to work, be in education or socialise. Parents also experience adverse consequences, including fatigue-related interpersonal tensions. Despite the enormity of the problem, fatigue is currently inadequately managed. TYAs described extensive, age-related intervention needs, including a preference for videos over written information. The co-designed intervention is predominantly cognitive in approach with, for example, mindfulness techniques being more acceptable than energy conservation. TYAs require structured activity support, given the physical reserve of youth and fear that activity may worsen fatigue. Young patients have inspired, and helped drive, this programme of work. Engagement with the research process was high, and participants embraced the innovative research methods. Information has been generated that will optimise the design of the future definitive trial to test intervention effectiveness.
  • ItemOpen Access
    Examination of ex-vivo lung perfusion in porcine model
    (2016-10-14) Roman, Marius Andrei
    Lung transplantation is a lifesaving therapeutic option for a variety of endstage cardiopulmonary conditions. There is a critical mismatch between the availability of suitable lungs and the number of patients awaiting a lung transplant. Ex-vivo lung perfusion (EVLP) is an established technique that may potentially allow more donor lungs to be reconditioned and provide waiting list patients with a better opportunity for transplantation. During EVLP, lungs are connected to a circuit similar to a cardiopulmonary bypass circuit and are perfused and ventilated under normothaermic conditions. In this thesis I have validated a donor after circulatory death (DCD) large animal model, by using porcine lungs initially from local abattoirs followed by change to a more suitable lungs source. As part of the experimental validation, I have shown that there is an initial bacterial load within the perfusate prior starting EVLP, which improved or disappeared during the procedure. I have characterised the perfusate solution and have demonstrated equivalence of alternative perfusate solutions to the current best practice; I have described the inflammatory profiles of the lungs and studied the impact of novel drug and cell therapies on these lungs. In the first phase I have compared cellular and acellular solutions currently used in practice, aiding in the decision of which solution would be most appropriate for my subsequent experiments. By comparing physiological, immunological, ultrastructural parameters, I have found improved but comparable trends in the blood-based solutions. Due to the cost effectiveness and physiological effects on lungs, I have decided to use the Papworth blood-based perfusate out of the three compared solutions. Lung inflammation and rejection processes are interlinked during lung transplantation. In order to characterise the occurring inflammatory processes, I have quantified the cytokine profile in the airway and perfusate; determined the leukocytes trapped in the leukocyte filters and present in the airway. By excluding the leukocyte filters at an earlier time point than the normal use, I have measured reduced levels of lnterleukin-8 and demonstrated that a shorter period use is enough to achieve the intended benefits of these filters while avoiding the deleterious effects. EVLP offers the opportunity to recognise and recondition injured lungs. I have used this quality to develop a one lung and a lobar warm ischaemic injury model, based on the initial DCD model. Apelin is a vasoactive peptide, with previously described roles in and vasodilatation through the eNOS pathway. I have investigated the modulatory effects of Apelin in the model of one lung injured by warm ischaemia. While this model produced sufficient injury, the vasodilator effects were minimal. In continuation to the reconditioning novel therapies, I have performed a transit and engraftment study of blood human derived endothelial cells. These cells were isolated and cultured in vitro previously, with potential to engraft and promote angiogenesis. I have demonstrated that injured lung lobes treated with these cells showed lower extravascular lung water and decreased warm ischaemic lung injury when compared to the injured lung without cell therapy. In conclusion, I have demonstrated that my porcine model of EVLP is a robust and promising technique that enables the assessment of lungs ex-vivo and may provide a platform for innovative therapeutic strategies.
  • ItemOpen Access
    The Satellite Cell of Skeletal Muscle
    (1968-06-10) Church, J. C. T.
    This thesis is centred on a unique feature of the skeletal muscle fibre, the satellite cell. This small fusiform cell, lying in a subsarcolemmal position, is the most recently discovered cytological entity of skeletal muscle. It can be readily distinguished from other better-known mononuclear cells found lying between muscle fibres, such as endomycial fibroblasts, vascular pericytes, mast cells and histiocytes. It is unique in that it is the only mononucleated cell lying within the basement membrane of the normal adult muscle fibreo Mononuclear cells are, however, a prominent feature of developing and regenerating muscle. In both, myoblasts fuse to form myotubes, which in turn develop into mature fibres. The immediate inference from this is that the satellite cell could be related to the mononuclear cells seen in these two situations.
  • ItemOpen Access
    Effects of Advanced Radiotherapy techniques on Normal Tissue Toxicity in Breast Cancer
    (2018-11-24) Mukesh, Mukesh Bindlish
    Whole breast radiotherapy after breast conserving surgery is an effective adjuvant treatment, but is associated with some long-term side effects and breast tissue toxicities including breast shrinkage, breast fibrosis, skin changes and poor overall cosmesis. These breast-specific complications can have a detrimental psychological impact. New radiotherapy techniques including IMRT and IGRT have the potential to reduce late treatment related toxicities. The aim of this thesis was to investigate if the use of IGRT and IMRT can reduce the breast tissue related late complications after whole breast radiotherapy. The project evaluated if the use of clip-based IGRT technique allows the use of smaller safety margins around the tumour bed in breast radiotherapy. It also involved the development of normal tissue complication probability (NTCP) model for breast fibrosis to quantify the clinical benefits of IGRT. The benefits of IMRT were assessed using clinician-based assessment, serial photographs for overall cosmesis and patient-reported outcome measures (PROMs). The project also evaluated if the type of surgical technique used to close the tumour bed has an impact on late breast tissue toxicities. Using set-up errors data from IMPORT HIGH study, this work confirmed that the use of clip-based IGRT technique reduces tumour bed PTV margins as compared to standard portal imaging. The use of clip-based IGRT had a small but significant reduction on the heart dose, especially for left sided breast cancer patients. The NTCP model for breast fibrosis suggests that for moderate-severe fibrosis, the breast tissue behaves as a serial organ and the maximum radiotherapy dose is most predictive of the complication. These results were verified in the independent START trial dataset. This research project confirmed that patients receiving IMRT have superior overall cosmesis and reduced risk of skin telangiectasia as compared to patients receiving standard radiotherapy. However, the benefits of simple IMRT could not be demonstrated using PROMs. The project showed that breast seroma is associated with increased rates of post-operative infection and haematoma. It is also an independent risk factor for tumour bed induration and inferior breast cosmesis at 5 years. The future research in breast cancer radiotherapy would be directed at quantifying patient’s individual risks and benefits and offering risk adapted radiotherapy.