Chronic myeloid leukaemia (CML) is a clonal stem cell disorder characterised by the Philadelphia chromosome. The treatment and outcomes of CML patients have improved with the introduction of tyrosine kinase inhibitors (TKI). Imatinib is associated with complete cytogenetic response (CCR) rate of 71% at 12 months, as documented by large phase 3 clinical trials. I carried out a large population study in the Merseyside, Cheshire and North Wales area, which showed a maximal CCR rate of 65% over 5 years of observation. This suggests there is a higher rate of imatinib failure in a general unselected CML population compared to large clinical studies which have strict exclusion criteria. My population study also confirmed that second generation TKIs can produce high CCR rates in imatinib intolerant/resistant patients. There are a number of mechanisms of imatinib resistance. The hOCT1 transporter has been shown to be an important predictor of response to imatinib treatment. However, it has been suggested there are other drug transporters involved in imatinib transport which may have prognostic significance. This thesis examined the role of SLCO1A2, OCTN1 and OCTN2 in the transport of TKIs. Transfected cell lines expressing high levels of the respective drug transporter were made using the AMAXA nucleofection process. The cell lines with the highest gene expression, as quantified by TaqMan PCR, were then selected and used in radioactive uptake experiments. Imatinib was confirmed to be a substrate for SLCO1A2. However, the mRNA expressions levels of SLCO1A2 did not have any prognostic correlation to outcome. A review of patient co-medication also showed inhibitors of SLCO1A2 had no effect on CCR and major molecular response (MMR) rates in imatinib treated patients. OCTN1 and OCTN2 did not transport imatinib. Nilotinib and dasatinib are not substrates for SLCO1A2, OCTN1 or OCTN2. Drug drug interactions have also been implicated in drug resistance. Imatinib and metformin are actively transported by hOCT1. It was postulated that varying concentrations of metformin could potentially affect the uptake of imatinib by competitive inhibition. A metformin concentration of 768µM was required reduce imatinib uptake by 50%. However, this concentration is much higher than therapeutic metformin levels, therefore these drugs do not interact at therapeutic concentrations. This thesis shows imatinib is an effective treatment in CML but the CCR rate is lower than in published phase 3 trials. SLCO1A2 transports imatinib but RNA levels have no prognostic significance. Imatinib and metformin do not interact at normal therapeutic concentrations.

When digital imaging and image processing methods are applied to clinical diagnostic and prognostic needs, the methods can be seen to increase human understanding and provide objective measurements. Most current clinical applications are limited to providing subjective information to healthcare professionals rather than providing objective measures. This Thesis provides detail of methods and systems that have been developed both for objective and subjective microscopy applications. A system framework is presented that provides a base for the development of microscopy imaging systems. This practical framework is based on currently available hardware and developed with standard software development tools. Image processing methods are applied to counter optical limitations of the bright field microscope, automating the system and allowing for unsupervised image capture and analysis. Current literature provides evidence that 3D visualisation has provided increased insight and application in many clinical areas. There have been recent advancements in the use of 3D visualisation for the study of soft tissue structures, but its clinical application within histology remains limited. Methods and applications have been researched and further developed which allow for the 3D reconstruction and visualisation of soft tissue structures using microtomed serial histological sections specimens. A system has been developed suitable for this need is presented giving considerations to image capture, data registration and 3D visualisation, requirements. The developed system has been used to explore and increase 3D insight on clinical samples. The area of automated objective image quantification of microscope slides presents the allure of providing objective methods replacing existing objective and subjective methods, increasing accuracy and rsducinq manual burden. One such existing objective test is DNA Image Ploidy which seeks to characterise cancer by the measurement of DNA content within individual cell nuclei, an accepted but manually burdensome method. The main novelty of the work completed lies in the development of an automated system for DNA Image Ploidy measurement, combining methods for automatic specimen focus, segmentation, parametric extraction and the implementation of an automated cell type classification system. A consideration for any clinical image processing system is the correct sampling of the tissue under study. VVhile the image capture requirements for both objective systems and subjective systems are similar there is also an important link between the 3D structures of the tissue. 3D understanding can aid in decisions regarding the sampling criteria of objective tests for as although many tests are completed in the 2D realm the clinical samples are 3D objects. Cancers such as Prostate and Breast cancer are known to be multi-focal, with areas of seeming physically, independent areas of disease within a single site. It is not possible to understand the true 3D nature of the samples using 2D micro-tomed sections in isolation from each other. The 3D systems described in this report provide a platform of the exploration of the true multi focal nature of disease soft tissue structures allowing for the sampling criteria of objective tests such as DNA Image Ploidy to be correctly set. For the Automated DNA Image Ploidy and the 3D reconstruction and visualisation systems, clinical review has been completed to test the increased insights provided. Datasets which have been reconstructed from microtomed serial sections and visualised with the developed 3D system area presented. For the automated DNA Image Ploidy system, the developed system is compared with the existing manual method to qualify the quality of data capture, operational speed and correctness of nuclei classification. Conclusions are presented for the work that has been completed and discussion given as to future areas of research that could be undertaken, extending the areas of study, increasing both clinical insight and practical application.

Clostridium difficile, a Gram positive, anaerobic, spore-forming bacterium is the commonest cause of hospital acquired infection in the UK. The organism initiates infection through spore formation and attachment, germination in the gut and then the production of two potent cytotoxins; toxins A and B. While the contribution of toxins A and B to infection is beyond dispute the relative importance of each toxin is a subject of debate. Thus diagnostic assays capable of rapidly detecting the presence of both toxins are needed. To develop such an assay we first characterised the structure of C. difficile spores to better understand their role in pathogenicity and adherence to organic and inorganic surfaces. Following attachment the spore germinates and the resulting vegetative bacteria express toxins. To facilitate the development of an assay capable of detecting both toxins, we employed a bioinformatics based approach which identified highly conserved nucleotide sequences within regions of each toxin which we hypothesised were under strict selective pressure. The specificity of the probes identified was confirmed using a panel of 58 clinical C. difficile isolates, related Clostridium isolates, non-related species and human gut metagenomic DNA samples. Selected probes were incorporated into a metal enhanced fluorescent assay platform and their ability to detect the organism in various organic backgrounds was determined. We were able to detect as few as 10 bacteria in 500 μl of human faecal material within 40 seconds, suggesting that this approach has the potential to be developed into a commercial assay. To support the development of this assay we sought to develop an insect infection model using the worm Manduca sexta. Our inability to initiate infection, inspite of the fact that bioinformatic analysis revealed the presence of genes with homology to known insect virulence factors, suggests that C. difficile may have potential evolutionary association to invertebrates.

Osteosarcoma (OS) is the most common bone cancer in dogs. It is biologically aggressive and <20% survive >2 years with standard therapy. Hence, new approaches must be considered. TP53 is altered in ~50% of human and canine cancers, including OS, making it a candidate for targeted suicide gene therapy strategies. Canine OS is considered to be a good model for human OS. The aims of this study were to:  examine the site incidence of canine OS retrieved from Glasgow University Veterinary School (GUVS) histology database;  perform TP53 mutational analysis in canine OS cases diagnosed at GUVS;  investigate delivery of exogenous wild-type canine TP53 into D17, CMT3, CMT7 and CMT8 canine OS cell lines;  design and construct vectors for a TP53-targeted suicide gene strategy, which can selectively target canine OS cells containing accumulated TP53, and initially analyse using Dual-Luciferase® reporter assays (DLR);  perform suicide gene/prodrug assays using nitroreductase (NTR) in combination with CB1954 or nitrofurazone (NFZ) in several canine cell lines;  replace luciferase with NTR in vectors for TP53-targeted suicide gene strategy, and with CB1954, determine if survival of CMT7 cells possessing accumulated TP53 are reduced, in comparison to D17 cells, containing wild-type TP53. OS were most commonly found in appendicular areas, followed by axial and extraskeletal sites; this agrees with published findings. No TP53 mutations were found in 7 biopsies removed from 4 dogs, 5 were OS, due to analysis of a small sample number, but still fits within published data. TP53 expression did not have a significant negative effect on canine OS cell growth. Contrasting results have been shown in canine and human OS cells. Luciferase expression levels following transfection with designed constructs were higher in CMT7 cells, than in D17 cells. Similar results were shown in NTR/CB1954 assays as reductions in cell survival only occurred in CMT7 cells but not in D17 cells. NFZ was not suitable as a prodrug for NTR in canine cells as there were no differences in cell survival with cells not expressing NTR. Hence, the TP53-targeted suicide gene therapy strategy appears to selectively reduce survival of canine OS cells possessing accumulated TP53, warranting further investigation as a treatment modality for OS, in both dogs and humans.

Breast cancer is the most common female cancer in Scotland, in common with many Western countries. This thesis aimed to analyse changes in the incidence and molecular epidemiology of breast cancer in Scotland. Part 1 concentrated on epidemiological research, with data derived from various agencies, and Part 2 on a laboratory project aimed at looking at changes in the molecular profile of breast cancers in two cohorts of patients in Glasgow. The period between 1987 and 1994 in which coverage of the country by the breast screening programme gradually increased was expected to raise incidence rates as seen in studies in Scandinavia and elsewhere; after 1994, incidence rates should have returned to normal in women aged 55-64, with incidence in women aged 50-54 remaining slightly above pre-existing rates. An observed/expected analysis of breast cancer incidence rates after 1994 was performed; this showed a 58% increase in rates in women aged 50-54 above that which would have been expected had the trends continued as expected in the absence of screening. In 55-59 year olds and 60-64 year olds there were 42% and 40% increases, respectively, above expected rates. Reproductive risk factors such as low parity and late age at first pregnancy are important risk factors in breast cancer. Reproductive risk factors are likely to affect the ‘birth-cohort’ incidence of breast cancer but the temporal effects of breast screening make this difficult to interpret. Breast cancer incidence in Scotland by year of birth was examined using a Lexis diagram. In women aged 50-54, 55-59 and 60-64, breast cancer incidence rates increased by birth cohort during the presence of the prevalent round of screening, a finding which is likely to have been due to detection of large numbers of asymptomatic tumours. However, in women who were offered screening after the prevalent round, incidence continued to rise with successive birth year, suggesting a contribution from risk factors. This is the first study of birth cohort incidence of breast cancer and its relation to screening (published in Breast Cancer Research and Treatment). The contribution of screening and risk factors to breast cancer incidence in Scotland was also assessed. A small rise in screening uptake between 1990 and 2001 and an increase in standardised detection ratio may indicate that screening improvements could be contributing to increasing incidence. The number of first pregnancies to women in Scotland aged 35-59 has risen from several hundred in 1976 to 2000 in 2001. A plot of completed family size in Scotland against maternal birth year shows that a steadily declining trend has been developing since the 1935 birth cohort. Based on data from the Scottish Health Surveys, the percentage of women with a BMI of over 25 has increased from 47.2% to 57.3% between 1995 and 2003. Mean BMI in women has increased from 25.7 to 26.9 over the same period. It is likely that the observed changes have contributed to changes in breast cancer incidence in Scotland. Using prescription and population data, the prevalence of HRT use in women aged 40-64 in Scotland was estimated; this estimated prevalence has increased from 13.8% in 1993 to 17% in 2001. It is difficult to know if this small increase in prevalence of HRT could have influenced breast cancer epidemiology. A study of breast cancer incidence by deprivation quintile showed that breast cancer incidence between 1991 and 2000 rose in all quintiles. Interaction analysis suggested that breast cancer incidence is rising to the same extent in deprived and affluent women. The risk factor analyses above were also applied to women of different socioeconomic standing (the results were published in Breast Cancer Research and Treatment). A laboratory project was carried out to assess whether increasing survival from breast cancer could be a result of changing molecular epidemiology. This project was an comparison of the prevalence of breast cancers which were ER, PR and Her2 positive and of different grades in two cohorts of Glasgow patients, from 1984-86 and 1996-1997. The application of current molecular techniques to stored tissue aimed to improve the quality of data compared to previous studies based on clinical databases using heterogeneous techniques. There were significant differences in grade distribution of tumours in the two cohorts (p=0.009) with fewer grade 1 and more grade 3 tumours in the second cohort. Further study showed the grade difference to be exerted by the tumours in screened women in the second cohort with there being no difference in grade between symptomatic patients in the two groups. 64.2% of the tumours in cohort 1 and 71.5% of the tumours in cohort 2 were ER positive (p=0.042); this is also likely to be a clinically significant difference. The difference between the cohorts appeared to be exerted by high percentage of screen-detected tumours in cohort 2 being ER positive; however this finding still supports a theory of changing biology. 44.9% of the tumours in cohort 1 and 49.9% of tumours in cohort 2 were PR positive (p=0.181). 21.5% of tumours in cohort 1 and 20.6% of tumours in cohort 2 were Her-2 positive; this was not a significant difference. An increase in ER positivity was seen in all age groups in the study, though multivariate analysis did suggest a contribution from a higher number of women over 60 in the more recent cohort. Kaplan-Meier analysis showed survival to be higher in the second cohort than the first. There was a significant difference in survival between ER positive and negative patients. Cox’s regression was performed; as expected this showed a multifactorial contribution to increases in survival in these cohorts rather than it being entirely due to changes in ER status. However the changes in ER status shown in a population of Glasgow patients over time may mean that the results of clinical trials carried out in many years ago need to be interpreted with caution when applying them to the women of today. The results of this project were published in the British Journal of Cancer. Overall, the epidemiological studies within this thesis shed an important new light on the factors contributing to breast cancer incidence in Scotland, with a major finding being a significant association between birth cohort and breast cancer incidence suggesting a significant impact being made by reproductive risk factors. This hypothesis is supported by the analysis of risk factor trends in Scotland undertaken within the thesis. The laboratory study has shown a significant lowering in grade and increase in ER positive status of tumours in a cohort of Glasgow women over time; while the changes are statistically explainable by known effects of a breast screening programme on tumour detection they could still represent a true change in biology. The results of all the studies contained in the thesis could have significant implications for future health service planning.

Abstract Background: Pulmonary hypertension (PH) is a rare condition that can occur as a primary disease process, Idiopathic Pulmonary Hypertension (IPH) or secondary to other disorders. In Familial IPH mutations have been identified in the bone morphogenetic protein receptor II gene (BMPRII) (chromosome 2q32-31) a member of the Transforming Growth Factor  (TGF) (Lane et al, 2000). Despite the mutation being present in all cells, vascular wall remodelling is only seen in the pulmonary circulation with marked thickening of the intima and neointimal formation, muscularisation of small-generation resistance vessels and thickening of the adventitial layer together with increased ECM deposition. Similar appearances are noted in the pulmonary circulation’s response to hypoxia. for this projectProlonged exposure of the pulmonary circulation to hypoxia results in vasoconstriction and subsequent vascular wall remodelling. The hypothesis of this work is that the pulmonary circulation’s response to hypoxia may be partially explained by the existence of differences exist in cell signalling pathways in between adventitial fibroblasts from pulmonary and systemic arteries in HPAF. Studies from the Scottish Pulmonary Vascular (SPVU) Laboratory have shown that pulmonary arterial fibroblasts (PAFB) in bovine and rat models of acute hypoxic exposure preferentially proliferate to hypoxia, whereas systemic arterial fibroblasts (SAFB) do not , that the stress mitogen activated protein kinase p38 MAPK is consistently activated in PAFB exposed to acute hypoxia, and is constitutively upregulated in PAFB cultured from rats exposed to chronic hypoxia (Welsh et al, 1998; Welsh et al; 2001). This response to hypoxic exposure has been shown to be dependent on p38 MAPK activity, as use of SB203580 can block the hypoxia-mediated proliferative response to acute hypoxia (Scott et al, 1998; Welsh et al, 2001). Aims and methods: We wished to establish whether the pro-proliferative response of PAFB to acute hypoxic exposure previously noted in bovine and rat models could also be demonstrated in a human model. We wished to establish a role for both classic MAPK and stress MAPKs in hypoxia-mediated PAFB proliferation. We also wished to examine the role of hypoxia inducible factor 1 (HIF1) in human arterial fibroblast responses to acute hypoxia. There is a body of literature that documents cross talk between p38 MAPK and the Bone Morphogenetic Protein (BMPR) signalling pathways. We wished to establish whether Smad proteins (involved in the downstream signalling cascade from BMPR) might play a role in human pulmonary and systemic arterial fibroblast proliferation to acute hypoxia. Following approval from the local Ethics Committee, PAFB were harvested from patients undergoing lobectomy for the treatment of lung cancer. Left internal mammary arteries (SAFB) were harvested from patients undergoing coronary artery bypass grafting. Cells from systemic and pulmonary arterial fibroblasts were grown in conditions of normoxia or acute hypoxia (PO2 35 mmHg ~ 5% O2). Cellular proliferation was assessed using [3H]Thymidine uptake as a surrogate. p38, p44/p42 - ERK1/2 and JNK MAPKs and Smad protein activity was assessed using Western Blotting Techniques with the use of appropriate primary and secondary antibodies and Chemiluminescence to detect the presence of protein. p38 MAPK isoform activity was assessed using Catch and Release® immunophoresis techniques. Findings and conclusions: We demonstrated that acute hypoxic exposure results in human PAFB proliferation, associated with increased p44/p42 – ERK 1/2 MAPK activity, but dependent on p38 MAPK  activity. We also found that the p38 MAPK  isoform was expressed in human PAFB following hypoxic exposure but this did not appear to be involved in the hypoxia-mediated proliferative response. p38 MAPK  activity appeared to occur in a bi-phasic pattern with peaks of activity at t = 6 and 16 hours, the second peak was found to be responsible for the hypoxia-mediated proliferation seen in these cells in agreement with previous work from the SPVU laboratory (Scott et al, 1998; Welsh et al., 2001). The second peak in p38 MAPK  activity was synchronous with peak HIF1 activity (between t = 8 –16 hours). We demonstrated that HIF1 activity can be abrogated by pre-incubation of human PAFB with SB203580 suggesting a mechanistic link between p38 MAPK  activation and HIF1 in a human model of acute hypoxic exposure. We have also demonstrated that that BMPR2-associated Smad 1, 5 and 8 activation is increased in hypoxic human SAFB, suggestive of the activation of an anti-proliferative pathway in these cells that is not associated with p38 MAPK activity. To our knowledge this is the first demonstration of an active response in SAFB to acute hypoxic exposure that involves the active upregulation of an anti-proliferative pathway in these cells. In addition we have demonstrated that in hypoxic pulmonary arterial fibroblasts phospho Smad 1, 5 and 8 expression is reduced (suggestive of the down-regulation of an anti-proliferative pathway) and can be further abrogated by pre-incubation with SB203580. This suggests that in SAFB Smad 1, 5 and 8 activation occurs independent of p38 MAPK activation while in PAFB, p38 MAPK activity augments Smad 1, 5 and 8 activation.

Feline immunodeficiency virus (FIV) is an important veterinary pathogen with comparative significance because of its similarities to its human counterpart HIV. Since FIV is the only non-primate lentivirus which induces AIDS-like symptoms in its natural host, it serves as a valuable animal model for both prophylactic and therapeutic studies of HIV. It is accepted that the induction of neutralising antibodies (NAbs) is a key element in the control of lentiviral infection, since T-cell based vaccines alone failed to prevent infection in most experimental animal model systems. In this project a robust and reproducible in vitro neutralisation assay was developed and optimised, permitting the assessment of the NAb response in naturally infected cats and with the potential to evaluate candidate vaccines. It was demonstrated that, in general, primary FIV strains in the UK belong to subtype A, and therefore the development of a regional, subtype A-specific, FIV vaccine could be considered for use in the UK. The identification of a neutralisation resistant isolate of FIV led to the finding that a linear neutralisation determinant was located within the V5 region of Env and mutations in this region may lead to immune evasion in vivo. In addition, a second neutralisation determinant was identified in the C3/V4 region of Env. Finally, it was observed that a small proportion of naturally infected cats generated NAbs against FIV. Of these, only a very small proportion of the cats had antibodies with the potential to cross neutralise strains within the same subtype as the homologous isolate. Nonetheless, a plasma sample from a single cat was identified that neutralised all strains tested, including strains from different subtypes and geographical regions. It is likely that studies of the homologous isolate that induced the broad NAb response may be capable of inducing a similar broad response in vaccinated cats. Such a finding would have important implications for the design of potential novel lentiviral immunogens.

Rho-associated kinase 1 (ROCK1) is a serine/threonine kinase important for the regulation of the cellular cytoskeleton through the induction of actin stress fibres and acto-myosin contractility. The cleavage and subsequent activation of ROCK1 by caspase 3 during apoptosis is believed to cause many morphological phenomena associated with programmed cell death such as dynamic membrane blebbing. I now formally prove the necessity of ROCK1 cleavage for apoptotic blebbing by knocking-in a caspase cleavage resistant mutant of ROCK1 in a genetically modified model. In addition, animals homozygous for non-cleavable ROCK1 demonstrate a phenotype consistent with auto-immune disease suggesting that apoptotic blebbing is important to mediate rapid efferocytosis, which is a rapid phagocytic clearance of the cellular corpse, and thus maintain self-tolerance. Furthermore, apoptotic blebbing is important for the clearance of apoptotic cells and I demonstrate a novel mechanism for ROCK to mediate the release of factors participating in macrophage migration to dying cells. ROCK induced apoptotic blebs and bodies lose membrane integrity prior to secondary necrosis and leak intracellular material. Using quantitative mass spectrometry I identified numerous proteins that were previously unrecognized to be released during apoptosis. The release of protein was found to be impaired following ROCK antagonism with Y27632 which underscores the importance of ROCK activity in apoptotic protein release. One of these proteins, gelsolin, was released following caspase cleavage and encourages macrophage motility towards apoptotic cells. Finally, I now demonstrate that the three nonsynonymous somatic mutations in the ROCK1 gene identified in the Cancer Genome Project lead to elevated kinase activity and drive actin cytoskeleton rearrangements that promote increased motility and decreased adhesion, characteristics of cancer progression. Mapping of the kinase-interacting regions of the carboxy-terminus combined with structural modeling provides insight into how these mutations likely affect the regulation of ROCK1. Consistent with the frequency of ROCK1 mutations in human cancer, these results support the conclusion that there is selective pressure for the ROCK1 gene to acquire ‘driver’ mutations that result in kinase activation.

In this thesis high intensity focused ultrasound (HIFU) is utilized for cancer treatment (thermal mode) and treatment of ischaemic stroke (mechanical mode). These two applications were investigated in vitro and in vivo models. MRI was utilized to monitor the lesions created by HIFU either in thermal or cavitation mode in freshly excised lamb brain tissue in vitro, and in rabbit brain in vivo. Additionally, MRI was used to monitor lesions deep in tissue for both in vitro and in vivo exposures. All three MRI sequences used (T1-W FSE, T2-W FSE and FLAIR) were able to detect lesions. Both thermal and bubbly lesions were best monitored using T1-W FSE with excellent contrast, proving the potential of HIFU to treat reliably tumours in the brain. A HIFU system was also used to assist thrombolysis in cooperation with a thrombolytic drug such as recombinant tissue plasminogen activator (rt-PA) in vitro and in vivo. It was shown that higher intensity results to higher volume of dissolved clot, but there is a limit of the intensity to be used in order to avoid heating of the clot and the surrounding tissue. The goal in this study was to achieve temperature elevation not exceeding 1ºC (called safe temperature). It was found that the larger the beam area the larger the dissolved clot volume. Also, the lower the frequency, the larger the volume of the dissolved clot. The results reported herein point to the use of frequency around 0.5 MHz and pulsing to optimize thrombolysis and skull penetration and at the same time avoiding unwanted heating. Finally, an Acrylonitrile Butadiene Styrene (ABS) phantom skull model was developed in order to evaluate the propagation of ultrasound using a single element transducer. The skull model was appropriately designed so that it has the same attenuation as a human skull. It was demonstrated that using a frequency of 0.5 MHz versus 1 MHz, ultrasound propagation through the phantom skull was higher. Therefore, higher frequency has poor skull penetration and a small beam size at the focus, while low frequencies have better skull penetration but with the risk of reaching the unpredictable effect of cavitation. The developed system has proven to successfully create large lesions in the brain and at the same time, these lesions are successfully monitored with excellent contrast using MRI (T1-W FSE) enabling the accurate determination of the margins of these lesions. The results reported in this study point to the use of frequency around 0.5 MHz and pulsing to optimize thrombolysis and skull penetration and at the same time avoiding unwanted heating. For treating tumours located deep in the brain and for dissolving thrombus causing an acute ischaemic stroke, further extensive clinical studies will be needed before this technology is applied to humans.

• Gastro-oesophageal reflux disease (GORD) is the commonest chronic disease in Western countries. Symptomatic GORD is the strongest risk factor for the development of oesophageal adenocarcinoma with obesity and male sex also linked to the development of neoplasia at this site. Recent decades have seen a significant increase in the incidence of this highly lethal cancer among Western populations with Scotland having the highest recorded incidence worldwide. • Human saliva has a high nitrite content derived from the entero-salivary recirculation of nitrate in our diet which has resulted from the increased use of nitrogenous fertilisers over the past 50-60 years. • The luminal chemistry produced at the gastro-oesophageal junction (GOJ) when swallowed salivary nitrite reacts with gastric acid, and most notably the production of nitric oxide (NO), may explain most of the physiological abnormalities that contribute to the pathogenesis of GORD. NO has been shown to reduce lower oesophageal sphincter (LOS) pressure, impair oesophageal clearance, delay gastric emptying and may be the final mediator of transient lower oesophageal sphincter relaxations (TLOSRs). Previous studies to investigate the role of this luminal chemistry in the pathogenesis of GORD show conflicting results. • In addition to the distal oesophageal acidification produced by traditional trans-sphincteric reflux, previous studies suggest ‘splaying open’ of the distal lower oesophageal sphincter following a meal may expose the gastric cardia and the most distal oesophageal squamous mucosa to the noxious effects of gastric acid. • Although the gastric cardia is an important site of pathology in the upper gastrointestinal tract, it is a complex and poorly understood area. It has been proposed, from autopsy studies, that cardia mucosa itself may be pathological and in fact an ‘acquired cardia’ due to metaplasia of the most distal oesophageal squamous mucosa. • A series of studies were designed to examine the effect of salivary nitrite on post-prandial GORD, gastro-oesophageal function and GOJ morphology in 20 healthy, asymptomatic adult volunteers using high-resolution pH manometry, an isotope gastric emptying breath testing and X-ray localisation of the squamo-columnar junction (SCJ). • Despite an excellent range of salivary nitrite concentrations extending over and above the normal physiological range no effect of salivary nitrite on gastro-oesophageal reflux, function or morphology was demonstrated. However, the studies did confirm, for the first time using high-resolution manometry, that distal opening of the LOS occurs after a meal. • The relationship of age and obesity to the SCJ position relative to the proximal border of the gastro-oesophageal high pressure zone (HPZ) was examined in 15 Helicobacter Pylori negative healthy volunteers. Strong negative correlations were seen between SCJ position relative to the proximal HPZ and increasing age, body mass index (BMI) and waist circumference (WC) respectively. These correlations were stronger in the male sub-group. • In 25 healthy volunteers, parietal cell density was measured from endoscopic biopsies taken from the macroscopic SCJ, 1cm distal to the SCJ, the gastric body and the gastric antrum. Again, a strong negative correlation was seen between increasing age and parietal cell density at the SCJ. This effect was localised to the SCJ and not seen at the other biopsy sites. • Our findings suggest that salivary nitrite does not alter gastro-oesophageal function, the integrity of the gastro-oesophageal barrier or gastro-oesophageal reflux in healthy volunteers. They confirm distal opening of the LOS after meals. The strong negative correlations between age and both SCJ position relative to the proximal HPZ and parietal cell density support the hypothesis of an ‘acquired’ cardia. The development of cardia mucosa may also be linked to obesity, visceral obesity and male sex. • Future work could examine the carcinogenic effect of salivary nitrite and its luminal chemistry but this would require large scale epidemiological research. Further, larger clinical studies are needed to investigate the role of distal opening of the LOS after meals and to improve our understanding of the gastric cardia. Such studies should focus on the role of obesity and posture.