No description available.

No description available.

Cystic fibrosis (CF) is an autosomal recessive disorder. One of the characteristic hallmarks of the disease is infection of the lung. Over the life time of a CF patient, a number of pulmonary exacerbations occur which result in irreversible lung damage. Despite continuous treatment with antimicrobials, microorganisms continue to persist in the CF lung due to the formation of biofilms. The biofilm mode of growth can display up to a 1000x greater tolerance to antimicrobial treatments than their free living planktonic counterparts. Hence, anti-biofilm therapy strategies are required to break up these tolerant microbial communities. Nitric oxide (NO) is one of the proposed anti-biofilm therapies, which has been shown to successfully initiate biofilm dispersal of one of the most widely recognized CF pathogens, P. aeruginosa. The work in this thesis was undertaken to investigate the use of NO as a potential biofilm dispersing agent for the monospecies biofilms formed by other commonly identified CF microorganisms. The second chapter outlines the isolation of these microorganisms from CF sputum samples and describes the culture profiling results for the CF patient cohort sampled. The third results chapter examines the effect of NO on the monospecies biofilms formed the CF microorganisms isolated in chapter 2. A biofilm dispersal effect was not observed across a range of NO concentrations; however intriguingly there was an effect on biofilm cell viability for NO concentrations ≥ 100 pM. Chapter 4 outlines a mixed species biofilm model composed of CF isolates for P. aeruginosa and S. aureus and a method to recover and analyse genomic DNA from biofilms. Biofilm dispersal for this mixed species biofilm was not observed however a reduction in the S. aureus population fraction was noted. The work in Chapter 5 was undertaken to adapt a new fluorescence in situ hybridisation method CLASI-FISH for the identification of microorganisms within CF sputum.

Inflammatory Bowel Disease (IBD) can be classified into two main subtypes: Crohn’s Disease (CD) and Ulcerative Colitis (UC). The aim of this study is to identify the genetic contribution to the susceptibility to IBD. In the first part of the study, I focused on Crohn’s Disease, the subtype that shows the greatest heritability. Using both pooled and sub-phenotype data, followed by replication, the results reveal substantial genetic heterogeneity and the total number of confirmed CD susceptibility loci was increased from 71 published by others to 200. This was achieved by analyzing the data using a multimarker approach and high-resolution genetic maps in Linkage Disequilibrium Units. In the second part of the study, I focused on Ulcerative Colitis. The results show that although UC has a lower reported heritability, many loci were also found for Ulcerative Colitis. Some of these overlap with those found for Crohn’s Disease.

The incidence of oesophageal adenocarcinoma is rising rapidly in the UK with most, if not all, adenocarcinomas arising within Barrett's oesophagus (BO). Defining those patients at high risk is difficult because the histological diagnosis of high grade dysplasia (HGD) is imprecise. Surveillance is unlikely to prove to be of benefit to patients in its current form. Detection of aneuploidy may offer more accurate detection of patients at high risk but its measurement is not currently possible in routine clinical service. Until recently if a patient had HGD the only widely available option was oesophagectomy with all of its adherent risks. This thesis demonstrated that image cytometry can reliably diagnose aneuploidy, that this is correlated with histology and is accurate compared to flow cytometry performed in Seattle. Additionally, aneuploidy measured by image cytometry was validated in a case-control study of future risk of relapse following ablative therapy in BO. Elastic scattering spectroscopy (ESS) is a real-time in vivo technique sensitive to changes in the physical properties of tissue. This data demonstrated that ESS can reliably detect both HGD and aneuploidy in vivo in BO in algorithm generation and prospective testing. A proof of concept study also suggested that ESS could potentially be used for surveillance. Photofrin-PDT has led the way in minimally invasive therapy for HGD in BO. The best regimen of ALA-PDT identified in this thesis is 60mg/kg of ALA activated by 1000J/cm of red light. The early data from the RCT of ALA versus Photofrin-PDT appears to show that ALA may be a lower risk alternative with possibly a higher efficacy for the eradication of HGD. In summary these studies potentially offer the more accurate definition of high risk patients in BO and a reduction in the frequency of endoscopies for those at low risk. Additionally, it has advanced the understanding of ALA-PDT.

No description available.

No description available.

Mucosa-associated adherent, invasive Escherichia coli (AIEC), found in increased number in Crohn’s disease (CD) ileal and colonic mucosae, can survive and replicate within underlying host immune competent cells (e.g. macrophages and dendritic cells) without triggering host cell death. The intra-macrophage environment plays an essential role in bacterial killing where engulfed bacteria are exposed to a hostile environment of low pH, high levels of proteolytic/lysosomal enzymes, high nitrosative and high oxidative stress, and the activation of a respiratory burst with generation of superoxide ions. Although a few stress response genes have been identified that likely support the paradigm ileal AIEC isolate LF82 to survive and replicate within the macrophage, the key molecular mechanisms involved in supporting Crohn’s disease (CD) mucosa-associated AIEC to resist killing by host mucosal macrophages within harsh environment of the phagolysosome still remains largely unclear. Here we aimed to compare the ability of a number of E. coli strains to survive and replicate inside macrophages, including a number of clinical isolates (from CD, colorectal cancer (CRC) and ulcerative colitis (UC) patients and other infective or non-inflamed sources), and this to toleration of growth in chemical-induced stress conditions mimicking the intra-phagolysosome environment. In addition, a focus was to further understand the molecular mechanisms responsible for acid tolerance of the paradigm CD isolates and examine their replication within macrophages defective in NF-κB pathway signalling. Finally, to also assess whether CD AIEC possess ability to alter host oxidative stress response gene expression in macrophages to support their survival/replication. Both ileal and colonic CD isolates (AIEC) were found to possess ability to either survive and/or replicate within murine macrophages (i.e. J774-A1 cell-line and wild-type (WT) C57BL/6 bone marrow derived macrophages [BMDM]) and to tolerate all stress conditions mimicking those within the phagolysosome, e.g. low nutrient, high acid, high nitrosative, high oxidative stress including exposure to superoxide ions. Interestingly pathogenic E. coli isolates from urinary tract infection (UTI) and some healthy-mucosa associated E. coli strains behaved similarly. Crohn’s AIEC were unable to survive and replicate inside Nfκb1-/- and Nfκb2-/- BMDM, whilst they survived/replicated within WT and c-Rel-/- BMDM. Thus Crohn’s AIEC survival and replication appears dependent on host NFκB signalling within the macrophage. Conversely, all CRC and UC isolates tested and the majority of laboratory E. coli strains studied were unable to survive inside murine J774-A1 macrophage phagolysosomes and they were also intolerant to most stress conditions, in particular superoxidative stress. Colonic CD AIEC isolate HM605 showed higher initial levels of expression of acid response genes gadA and gadB that may support adaptation to the intra-macrophage phagolysosome niche. Adaptation to an intra-macrophage lifestyle appeared not to be through any ability to alter host macrophage oxidative stress response to infection as no differential changes were observed in the expression of 84 host genes related to oxidative stress to that seen with non-replicating laboratory E. coli strain. Overall this study provides new insight into how CD mucosa-associated E. coli isolates resist killing by mucosal macrophages through adaptation to the acidic, high oxidative environment within the macrophage phagolysosome. The data may support future development of new therapeutic strategies that target the fundamental pathology of CD, in particular support a reduction in bacterial persistence/increased killing of intra-macrophage E. coli in CD patient mucosae.

Liver sinusoidal endothelial cells (LSEC) constitute a unique population of endothelial cells with specialised liver-specific morphologic features and functions. LSEC are the only endothelial cells with fenestrations and which lack an organised basement membrane. They are involved in hepatic stellate cell (HSC) quiescence, endocytosis of small particles, selective transfer of substances from the blood, in the hepatic sinusoid, to the parenchymal cells and in liver regeneration. As the group of cells that form the inner lining of the capillaries of the liver sinusoids, and being the first to be in contact with blood-borne particles, pathogens, and xenobiotics, they are prone to the deleterious effects of these. The aims of this thesis were to investigate the unique features of human liver sinusoidal endothelial cells (HLSEC) in comparison with endothelial cells from other vascular beds, evaluate the sensitivities of HLSEC to a range of hepatotoxic drugs, including small-molecule receptor tyrosine kinase inhibitors (RTKIs), such as regorafenib, and to explore the role of HLSEC in a triculture human liver microtissue. Results obtained from this study showed that HLSEC expressed phenotypic features of vascular and lymphatic endothelial cells, particularly vascular endothelial growth factor receptor 2 (VEGFR-2) which could be activated by VEGF-A to stimulate cell proliferation, migration and tubular morphogenesis. HLSEC also expressed functional VEGFR-3. Transcriptomic analysis indicated that HLSEC expressed specialised genes, such as plasmalemma vesicle associated protein (PLVAP), that support its liver-specific structure and functions. HLSEC were more sensitive to a range of small-molecule receptor tyrosine kinase inhibitors than other hepatic cells. (primary human hepatocytes [PHH] and human hepatic fibroblasts [HHF]) and endothelial cells from other vascular beds (human dermal microvascular endothelial cells and human dermal lymphatic endothelial cells). Regorafenib inhibited the activation of VEGFR-2 thereby abrogating cell proliferation, migration, tubular morphogenesis as well as upregulation of angiocrine factors involved in liver regeneration following activation by vascular endothelial growth factor A (VEGF-A). Regorafenib also caused a disruption of cytoskeletal structure of HLSEC and induced apoptosis via activation of caspase 3. Triculture liver microtissues formed with PHH, HLSEC and HHF were vascularised with higher expression of liver-specific drug-metabolising enzymes in comparison with the same combination of cells cultured as a monolayer. However, metabolic competence of triculture liver microtissues was significantly lower than in their monoculture counterparts (consisting of PHH only). This study has further confirmed the uniqueness of HLSEC as a specialised endothelial cell adapted to its anatomical role, which could respond to a range growth factors to initiate endothelial cell-specific functions. It has also been demonstrated that HLSEC are a direct target of hepatotoxic drugs. Triculture liver microtissues generated with PHH, HLSEC and HHF showed less metabolic competence than their PHH-only counterparts. Future studies need to investigate the role of RTKIs in vascular toxicity using in vivo models of sinusoidal obstruction syndrome (SOS) and liver regeneration. Finally, it would be informative to investigate the possibility of identifying HLSEC-specific biomarkers of liver toxicity.

Obesity is an increasing health problem worldwide as a result of the changing environment, with calorie-dense food and a sedentary lifestyle. However, numerous twin studies indicate that genetics plays a significant role in determining which individuals become obese or stay lean while sharing the same ‘obesogenic’ environment. Genetic research in obesity has two major goals: the first being to elucidate the pathophysiological basis of obesity, and the second is to provide an evidential foundation for personalised medicine. An obesity cohort was established, consisting of over a thousand severely-obese individuals (mean BMI 48.1 kg/m2 [± 8.67 SD]) undergoing bariatric surgery, as the basis for genetic and phenotypic analysis of the severely obese. The relatively high proportion of metabolically-healthy, but severely-obese individuals in this cohort illustrated some of the many unknown pathophysiological mechanisms, while a consistent increase of public distress among the more severely obese confirmed the ongoing stigmatisation of the obese in UK society. Screening for the most common form of Mendelian obesity, MC4R deficiency identified a lower than anticipated prevalence (0.77%) in the bariatric cohort, but analysis of treatment outcomes indicated that bariatric surgery both (RYGB and VSG) is effective for the individuals affected. Lifestyle intervention for children with MC4R deficiency, on the other hand, appeared to have less beneficial outcomes long term. Using whole exome sequencing on 40 super-obese bariatric participants, a higher than anticipated prevalence of putative Mendelian obesity (20.5%) was found, including several novel disruptive variants in known obesity genes. Finally, a novel Mendelian obesity and diabetes syndrome was detected, in a consanguineous family with a complex obesity phenotype, caused by a homozygous truncating mutation of the CPE gene (c.76_98del; p.Glu26ArgfsX68).