The superficial digital flexor tendon (SDFT) is one of the most frequently injured tendons in Thoroughbred racehorses. Exercise associated factors including hyperthermia are thought to lead to cellular dysfunction and cell death of the resident tendon fibroblasts, the cells responsible for the repair of the tendon lesions. The main aim of this thesis was to investigate the sensitivity of SDFT fibroblasts to hyperthermia and to compare this with the deep digital flexor tendon (DDFT), a non-injury prone tendon. Understanding the physiological mechanisms of the heat shock response in these cells e.g. the use of protein markers will allow preventative strategies to be devised to protect these cells from damage. I determined whether thermotolerance associated with the induction of heat shock proteins (a survival mechanism that allows cells to withstand a subsequent lethal shock) could be induced with both heat and cold shock in these cells. Firstly, the basal DNA damage levels were quantified in both SDFT and DDFT fibroblasts as the cell culture environment is known to damage cells. My research showed both SDFT and DDFT fibroblasts were susceptible to replication induced DNA damage in vitro. The SDFT in particular had high levels of DNA damage when cultured on a fibronectin matrix in ambient oxygen. SDFT and DDFT fibroblasts were shown to be susceptible to a lethal heat shock (52oC). When a preconditioned sub-lethal heat shock was given to both tendons, induction of thermotolerance occurred and these cells survived a lethal heat shock. Thermotolerance was induced in preconditioned cold shocked SDFT fibroblasts but not in DDFT fibroblasts. Finally, a useful protein marker, DAXX (that is involved in cellular stress pathways as a transcriptional repressor and in apoptosis) was shown to disperse into the nucleoplasm during a mild heat shock in SDFT fibroblasts. One of the limitations of this thesis is that sample size was small and as a result, larger numbers of animals will be required for future experiments to determine whether my results are of biological significance.

RA is a destructive and chronic autoimmune inflammatory disease. The inflammation of the synovium is associated with the local invasion of inflammatory cells across blood vessel endothelial cells (ECs), increases in synovial fluid volume and local pannus invasion of the connective tissues and bone. Synovial ECs in RA are involved in a wide range of processes, and chemokines are known mediators of inflammatory cell invasion into the tissue. Chemokines at the ECs of lymph vessels play a further role in attracting the infiltrates out of the tissue. This study used immunofluorescence to investigate the presentation of a number chemokines in RA tissue ECs, and also the presentation of CCL7, CCL14, CCL16 and CCL22 in lymphatic ECs. A number of chemokines were newly identified in synovial ECs, and continued investigation showed a marked dysregulation in blood vessel and lymphatic vessel chemokine presentation, including CCL7. In vitro studies showed that the chemokines also preferentially generated microvilli which may facilitate transendothelial migration in vivo. Mononuclear cells expressed the receptors for the chemokines and transmigration analysis showed CCL7 (among others) to significantly chemoattract monocytes. This suggests that dysregulation of chemokines may have a functional role in RA pathology. Furthermore, the analysis of these chemokines in matched synovial fluid (DF) and serum indicates that EC chemokines may be inflammatory markers in arthritic diseases. Overall, this study has shown that the EC interface between the influx and efflux of inflammatory cells in the RA synovium may offer currently unexplored therapeutic opportunities.

An ageing population increases the number of frail, elderly individuals. Physiotherapists are increasingly treating frail individuals due to the associated rate of rapid muscle atrophy. A rapid loss of muscle strength can result in difficulty performing activities of daily living, making individuals more susceptible to other age‐related pathologies. Some frail individuals are unable to contract their muscle sufficiently to complete a rehabilitation programme. This can be exacerbated in a neurological population such as stroke. The aim of this thesis was to prevent muscle atrophy associated with age to allow rehabilitation to commence with a quicker onset. Neuromuscular electrical stimulation (NMES) is a treatment modality capable of producing muscle contraction. Its use is poorly understood, with little guidance surrounding optimal parameters or muscular response to treatment. This thesis has identified optimal stimulation parameters for strength training with NMES, and tested them on a healthy population of varying ages, and in a stroke population. A muscle measurement device was designed and tested to allow accurate measurements of moments about joints. Results indicate that the protocol is effective in inducing hypertrophy, as indicated by advances in pennation angle and maximal isometric force production. The protocol was effective at producing a small decline in force associated with a hypertrophic stimulus. Results indicate that treatment should be administered with the highest available stimulation amplitude to achieve optimal results. NMES appears to be able to advance internal muscle architecture, despite lack of volitional muscle control post stroke. Variability of response was investigated through blood biomarkers (Creatine Kinase) which was demonstrated to increase in line with volitional strength training literature. The exercise status of the individual appears to be correlated with muscle response. It is recommended that NMES could be administered in the acute period of a physiotherapy protocol to prevent muscle atrophy associated with ageing. Further work should focus on developing the strength measurement device used throughout this thesis, and investigating a protocol suitable for other applications to allow a smooth transition into clinical settings.

Iron is vital for healthy brain function. However when present in a redox-active form or in excess concentrations it can be toxic. Interestingly, increased levels of redox-active iron biominerals have been shown to exist in Alzheimer’s disease (AD) tissues, including lesions comprised of the AD peptide β-amyloid (Aβ). These iron phases are capable of producing reactive oxygen species, resulting in the generation of oxidative stress manifesting as neuronal injury. As oxidative stress and the accumulation of iron are recognised as early stage events in AD, the presence of redox-active iron may prove fundamental in the development of AD pathology. The origin of these redox-active iron biominerals is unclear but recent studies suggest their formation may involve the interaction of Aβ with unbound brain iron and/or the malfunction of the iron storage protein ferritin. Despite these observations, the relationship between Aβ and iron is poorly understood, and the products of Aβ/iron interaction remain unknown. In this thesis, synchrotron-based x-ray techniques are combined with traditional biological approaches to examine the interactions between Aβ and various synthetic and naturally occurring iron forms. Through this methodology Aβ is shown to incorporate ferric iron phases into its fibrillar structure in vitro, with this interaction resulting in the chemical reduction of iron into a redox-active state. Further to this, Aβ is demonstrated to disrupt ferritin structure resulting in the chemical reduction of its redox-inactive iron core in vitro. Additionally the interaction of Aβ with crystalline iron phases is shown destroy iron crystal structure. Finally, redox-active iron is shown to be associated with regions of AD pathology, including fibrillar Aβ-like structures, within a transgenic mouse model of AD in situ. These findings suggest an origin for the redox-active iron forms and oxidative stress previously witnessed in AD tissue, thereby shedding light on the process of AD pathogenesis.

Pancreatic cancer is common and aggressive: the main type is pancreatic ductal adenocarcinoma (PDAC). It is the fifth most common cause of cancer death in the UK with an overall five year survival of only 2-3%. Establishing the diagnosis of PDAC is important for optimal patient management but can be difficult and relies on imaging and cytology/pathology. Although imaging may be highly suggestive of PDAC, a pathological diagnosis is preferred prior to definitive treatment; therefore tissue samples are required. Cytology samples are obtained at endoscopy. Cytological analysis requires the identification of different cell types and in particular the distinction of malignant pancreatic epithelial cells from reactive pancreatic cells and other gastrointestinal contaminants. This requires experience and expertise and can be difficult. A tissue diagnosis is not achieved in a significant proportion of PDAC cases. Hence, an unmet clinical need exists for the diagnosis of PDAC from cytological samples. One potential way of improving the cytological diagnosis is to use immunohistochemistry (IHC) biomarkers as an adjunct to cytology in difficult to diagnose cases. Diagnostic IHC biomarkers have been investigated, but to date none has entered into routine clinical practice. The aim of this study was to improve the diagnosis of PDAC from cytology samples. It is hoped that the identification and validation of IHC biomarkers in PDAC will help their clinical translation. For biomarker identification a meta-analysis of potential IHC diagnostic biomarkers investigated in PDAC was performed. Sixteen biomarkers were quantified in meta-analysis and the highest ranked biomarkers were KOC, maspin, S100P, mesothelin and MUC1. These biomarkers have not entered into clinical practice partly because they were investigated in separate studies with relatively small sample sizes and without a uniform and clinically appropriate cut-off. Biomarkers identified in the meta-analysis were validated in a resection cohort from patients with pancreatico-biliary adenocarcinoma. The aim was to identify better biomarkers and cut-offs that could potentially be investigated in cytology samples. KOC, S100P, mesothelin and MUC1 were investigated in one set of tissue microarrays, while maspin was investigated in another set. Five cut-offs were carefully chosen for sensitivity/specificity analysis using receiver operating characteristics curve analysis. Using 20% positive cells as a cut-off achieved higher sensitivity/specificity values: KOC 84%/100%; S100P 83%/100%; mesothelin 88%/92%; MUC1 89%/63%; and Maspin 96%/99%. Analysis of a panel of KOC, S100P and mesothelin achieved almost 100% sensitivity and specificity if at least two biomarkers were positive for both 10% and 20% cut-offs. Clinical translation of biomarker requires a reliable and reproducible cut-off for interpretation of IHC. We identified three cut-offs for investigation to establish a consensus based cut-off(s) that could potentially be used by pathologists for PDAC and other cancers. A series of IHC images of microarray cores were used to investigate observer agreements for 10%, 20% and +2/+3 cut-offs. Seven pathologists participated in this study. The inter- and intra-observer agreements using the three cut-offs were reasonably good. For all three cut-offs a positive correlation was observed with perceived ease of interpretations (p<0.01 for all cut-offs). Finally, cytoplasmic only staining achieved higher agreement than cytoplasmic/nuclear and cytoplasmic/membranous staining. All three cut-offs investigated achieve reasonable strength of agreement modestly decreasing inter and intra-observer variability in IHC interpretation but 10% is slightly better than 20% and +2/+3 cut-offs. Finally, KOC, maspin, mesothelin and S100P were investigated in archival cytology samples with the aim to generate a diagnostic panel which could potentially help as an adjunct to cytology. Using 10% cut-off achieved higher sensitivity/specificity values: KOC 92%/100%; maspin 54%/100% and mesothelin 72%/100%. But no staining was observed for S100P. In addition, analysis of a panel of KOC, maspin and mesothelin achieved 82% sensitivity and 100% specificity for 10% cut-off if at least two biomarkers in the panel were positive. The inter-observer agreement for 10% positive cells as IHC cut-off in cytology samples was very good for all three biomarkers. In conclusion, a panel of KOC, maspin and mesothelin is a suitable diagnostic panel and 10% cut-off is a reasonable cut-off achieving high observer agreement. Their diagnostic accuracies approach those of optimal conventional cytology. These markers may be appropriate for further clinical validation and potentially routine use in difficult cases.

Direct electrospray ionisation-mass spectrometry (direct ESI-MS), by omitting the chromatographic step, has great potential for application as a high-throughput approach for untargeted urine metabolomics analysis compared to liquid chromatography-mass spectrometry (LC-MS). The rapid development and technical innovations revealed in the field of ambient ionisation MS such as nanoelectrospray ionisation (nanoESI) chip-based infusion and liquid extraction surface analysis mass spectrometry (LESA-MS) suggest that they might be suitable for high-throughput metabolomics analysis. In this thesis, LC-MS and high-throughput direct ESI-MS methods using high resolution orbital trap mass spectrometer were developed and validated for untargeted metabolomics of human urine. Three different direct ESI-MS techniques were explored and compared with LC-MS: flow injection electrospray ionisation-MS (FIE-MS), chip-based infusion and LESA-MS of dried urine spots on a cell culture slide. A high-throughput sample preparation protocol was optimised using in-house artificial urine. Urine samples after consumption of green tea and healthy controls were used as a model to explore the performance and classification ability of the direct ESI-MS. High-throughput data pre-processing and multivariate analysis protocols were established for each method. The developed methods were finally applied for the analysis of clinical urine samples for biomarker discovery and to investigate the metabolic changes in osteoarthritis and malaria. Also, the methods were applied to study the effect of oligofructose diet on the gut microbial community of healthy subjects. The analytical performance of the methods for urine metabolomics was validated using quality control (QC) and principal component analysis (PCA) approaches. Rigorous validation including cross-validation, permutation test, prediction models and area under receiver operating characteristic (ROC) curve (AUC) was performed across the generated datasets using the developed methods. Analysis of green tea urine samples generated 4128, 748, 1064 and 1035 ions from LC-MS, FIE-MS, chip-based infusion and LESA-MS analysis, respectively. A selected set of known green tea metabolites in urine were used to evaluate each method for detection sensitivity. 15 metabolites were found with LC-MS compared to 8, 5 and 6 with FIE-MS, chip-based infusion and LESA, respectively. The developed methods successfully differentiated between the metabolic profiles of osteoarthritis active patients and healthy controls (Q2 0.465 (LC-MS), 0.562 (FIE-MS), 0.472 (chip-based infusion) and 0.493 (LESA-MS)). The altered level of metabolites detected in osteoarthritis patients showed a perturbed activity in TCA cycle, pyruvate metabolism, -oxidation pathway, amino acids and glycerophospholipids metabolism, which may provide evidence of mitochondrial dysfunction, inflammation, oxidative stress, collagen destruction and use of lipolysis as an alternative energy source in the cartilage cells of osteoarthritis patients. FIE-MS, chip-based infusion and LESA-MS increased the analysis throughput and yet they were able to provide 33%, 44% and 44%, respectively, of the LC-MS information, indicating their great potential for diagnostic application in osteoarthritis. Malaria samples datasets generated 9,744 and 576 ions from LC-MS and FIE-MS, respectively. Supervised multivariate analysis using OPLS-DA showed clear separation and clustering of malaria patients from controls in both LC-MS and FIE-MS methods. Cross-validation R2Y and Q2 values obtained by FIE-MS were 0.810 and 0.538, respectively, which are comparable to the values of 0.993 and 0.583 achieved by LC-MS. The sensitivity and specificity were 80% and 77% for LC-MS and FIE-MS, respectively, indicating valid, reliable and comparable results of both methods. With regards to biomarker discovery, altered level of 30 and 17 metabolites were found by LC-MS and FIE-MS, respectively, in the urine of malaria patients compared to healthy controls. Among these metabolites, pipecolic acid, taurine, 1,3-diacetylpropane, N-acetylspermidine and N-acetylputrescine may have the potential of being used as biomarkers of malaria. LC-MS and FIE-MS were able to separate urine samples of healthy subjects on oligofructose diet from controls (specificity/sensitivity 80%/88% (LC-MS) and 71%/64% (FIE-MS)). An altered level of short chain fatty acids (SCFAs), fatty acids and amino acids were observed in urine as a result of oligofructose intake, suggesting an increased population of the health-promoting Bifidobacterium and a decreased Lactobacillus and Enterococcus genera in the colon. In conclusion, the developed direct ESI-MS methods demonstrated the ability to differentiate between inherent types of urine samples in disease and health state. Therefore they are recommended to be used as fast diagnostic tools for clinical urine samples. The developed LC-MS method is necessary when comprehensive biomarker screening is required.

The work presented in this thesis uses Selected Ion Flow Tube Mass Spectrometry to investigate if hydrogen cyanide (HCN) is a marker of Pseudomonas aeruginosa (PA). The initial in-vitro studies measure the HCN released into the gas phase by cultures of PA after various durations of incubation. Study 1 uses clinical PA isolates with a known genotype and phenotype (mucoid / non-mucoid) and Study 2 uses a selection of the same PA isolates cultured under biofilm and planktonic conditions. Study 4 investigates if HCN is an in-vivo marker of PA infection in children with cystic fibrosis (CF). It is a 2 year observational study of 233 children with CF who are free from PA infection. A breath sample for HCN analysis is collected each time they attend the out-patient clinic. Exhaled breath HCN concentrations are then compared to routine microbiology sample results. The breath samples for this study are collected in sampling bags. In preparation for this, Study 3 identifies the most appropriate bag type as well as the maximum duration of storage and the need for sample warming prior to analysis. Some healthy adults produce HCN in their oral cavity and therefore mouth-exhaled HCN alone cannot be used as marker of PA infection. Study 5 investigates nose-exhaled HCN as a marker of chronic PA infection in adults with CF. A recent study has shown that Burkholderia Cepacia Complex (BCC) produces cyanide when cultured under biofilm but not planktonic conditions. Study 6 measures the HCN released into the gas phase by in-vitro cultures of BCC as well as HCN concentration in the breath of patients with chronic BCC infection.

No description available.

Streptococcus pneumoniae is a common commensal of the human upper respiratory tract flora living peacefully without causing any harm to the host in normal conditions. S. pneumoniae can cause serious life threatening diseases in certain circumstances and usually affects children, the elderly and immunocompromised people. S. pneumoniae is a highly transformable pathogen and newly emerging antibiotic resistant strains are becoming commoner. Developing multi-drug resistant strains is a serious threat to the community, and the available drug treatment and vaccines do not provide full protection. Some newer strategies such as harnessing the immunological response to S. pneumoniae must be adopted to overcome this invasive pathogen. The human immune system has evolved in a way to successfully detect, isolate and eliminate invading pathogens. It provides a generalised and rapid response during invasion of infectious agents and is usually enough to stop infections. Normally immunological and inflammatory responses against invading pathogens and foreign antigens are under strict control. However, they are potentially dangerous and may cause autoimmune diseases. Autophagy is an emerging pathway associated with the innate immune system. It has an important role in infection control and maintains a fine balance in inflammatory responses to protect the host from harmful effects. Autophagy is basically a homeostatic pathway for degradation of unwanted protein aggregates at a cellular level, but has an important role in innate immunity. Autophagy-related (Atg) proteins have a crucial role in the body’s immune system and take part in innate and adaptive immunity. This pathway is considered to prevent the body’s immune system from attacking self-tissues and suppression of the auto-immune inflammatory responses. In this thesis I present that infection with S. pneumoniae strain D39 WT and its pneumolysin deficient counter-part D39 ΔPly induces autophagy in primary murine bone marrow derived macrophages and human neutrophils in-vitro and in- vivo. We confirmed autophagy by a classical marker protein LC3 through immunofluorescence and western blot. The associated inflammasome activation in S. pneumoniae infection has an inhibitory effect on autophagy induction as was observed using WT and pneumolysin deficient strains. Similarly inflammasome inhibition with pharmacological and genetic methods up-regulates autophagy in S. pneumoniae infection. I also present here that autophagy is associated with phagocytosis and intracellular killing of S. pneumoniae and both these pathways are used as innate immune mechanisms for clearing infection. Previous research links autophagy and phagocytosis, and the phagocytosed microbe is targeted to the lysosome for degradation and killing. Our findings here in this thesis demonstrate that these pathways are also influenced by the virulence factors of S. pneumoniae. Pneumolysin have some inhibitory effects on autophagy induction and phagocytosis which may be a direct effect or indirectly through the inflammasome activation. Next, I present here a novel extracellular killing pathway in human neutrophils, the neutrophil extracellular traps generation or NETosis. S. pneumoniae infection induces NET generation, that is autophagy-dependent and can be inhibited by blocking autophagy pharmacologically or genetically. NET generation is morphologically the same in S. pneumoniae D39 WT and D39 ΔPly but pneumolysin helps in pathogen escape from NET entrapment which is a novel finding and needs further exploration. I present here the role of different pattern recognition receptors in S. pneumoniae induced autophagy signalling. S. pneumoniae infection induces autophagy independent of TRIF, MyD88, TLR4, TLR2 and NOD2 pathways. P38MAP kinase was also explored and has no association with autophagy induction in S. pneumonia infection. Autophagy induction in S. pneumoniae infection may be associated with some unknown signalling pathway which needs further exploration.

During pregnancy, the maternal immune system protects the allogeneic foetus from rejection. At the same time the mother maintains immunity defences against potential pathogens. This study aimed to identify whether immunological receptors associated with antigen presentation and strong inflammatory responses play a role in fetal-maternal tolerance. One such receptor is CD74; a membrane-bound protein involved in HLA class II- mediated antigen presentation and a macrophage migration inhibitory factor (MIF) receptor. CD44 is the signalling component of the MIF-CD74 receptor complex. The expression of CD74, MIF and CD44 was studied in the human trophoblast-derived cell lines JEG-3 and ACH-3P by RT-PCR, flow cytometry, Western blot, immunoprecipitation and fluorescence microscopy. Results obtained showed that untreated JEG-3 and ACH-3P cells did not express CD74 mRNA. By contrast, CD74 mRNA was upregulated in response to IFN-γ or LPS in these cell lineages. Slight upregulation of CD74 was observed following exposure of cells to 500 IU/ml IFN-γ for 12 hr. However, after 5 µg/ml LPS treatment for 4 hr, CD74 was highly upregulated. Results from flow cytometry showed no detectable surface expression of CD74 in the JEG-3 and ACH-3P cell lines even after IFN-γ or LPS treatment. However, IFN-γ and LPS exposure resulted in intracellular expression of CD74 in both cell lines. Western blotting showed the absence of a protein band for CD74 in both cell lines after IFN-γ treatment. However, the 35 kDa isoform of CD74 was detected in JEG-3 and ACH-3P cells treated with LPS. The results of this study indicate that LPS regulates the expression of MIF and CD44 and trophoblast cell proliferation. It was also demonstrated that CD74 positivity significantly increased after incubation with IFN-γ or LPS, and that MIF and CD44 are up-regulated by LPS. No evidence was found for colocalization between CD74 with either CD44 or MIF in JEG-3 and ACH-3P cells using confocal microscopy and coimmunoprecipitation. This indicated that MIF plays an essential role in cell proliferation, whereas both MIF and CD44 play an important role in human pregnancy maintenance. Together, the results suggest that the absence of cell surface expression specific isoforms of CD74 may provide a protective role by minimising inflammatory processes, and thus maximising a healthy pregnancy. In turn, it is reasonable to assume that the overexpression of CD74 in early pregnancy may be related to gestational complications.