Breast Cancer Associated gene 2 (BCA2), is a little-studied E3 ligase that is overexpressed in 56% of all primary breast cancers and has been linked with increased cell proliferation and invasion in vitro. BCA2 has been implicated in EGFR degradation however there is conflicting evidence surrounding its function and effect on receptor biology. This project aimed to elucidate the role of BCA2 in EGFR endocytosis and downregulation and to determine its link with breast cancer survival. Data generated with online mRNA analysis tools indicated that high BCA2 levels were often associated with improved breast cancer prognosis. In silico studies also demonstrated that many genes coexpressed with BCA2 were regulators of membrane trafficking and suggested that BCA2 expression was repressed by HER2/EGFR/Ras signalling. Experimentally, it was shown that siRNA depletion of BCA2 led to increased EGFR protein levels while transient BCA2 overexpression reduced levels of the receptor. It was found that BCA2 overexpressing, EGF stimulated cells demonstrated reduced lysosomal degradation of both receptor and ligand. Associated with this, downstream EGFR signalling in BCA2 overexpressing cells was reduced in magnitude but prolonged in duration and ultimately cell viability was impaired. 3 These findings support a role for BCA2 in the endolysosomal system. In agreement with this it was shown that BCA2 overexpression inhibited the vesicle membrane association of Rab7, a regulator of late endocytosis and reported BCA2 interactor. Transferrin receptor levels and transferrin uptake were unaffected by BCA2 overexpression suggesting trafficking effects may be restricted to EGFR, a distinct class of receptor and/or to later (degradation) stages of endocytosis. This thesis provides a detailed exploration of BCA2 biology and presents evidence of a functional role for the protein in the endocytic regulation of EGFR. The mechanism/s underlying the complex relationship between BCA2 and breast cancer outcome have yet to be fully determined.

Inhalation represents a potentially attractive delivery route for biologics, especially those designed to treat pulmonary diseases such as asthma, cystic fibrosis or lung cancer. Delivery directly to the site of action should increase local concentrations of drug, whilst reducing systemic side effects. However, there is limited knowledge regarding the mechanisms of pulmonary clearance, with gaps in understanding; where molecules are absorbed, the mechanisms involved, regional variability throughout the lung, and how to control pulmonary retention and/or facilitate cellular uptake. The work presented in this thesis details the development of a SPECT/CT imaging protocol to determine the pulmonary retention and tissue redistribution of technetium labelled antibodies and their fragments in vivo, in mice, to begin to address these knowledge gaps. The SPECT/CT imaging method was applied to a whole monoclonal murine immunoglobulin G1 (mlgG1), as well as its Fab and scFv fragments and a small protein (FN3) in order to determine whether diffusion controlled pathways were important in pulmonary antibody clearance. Additionally, the pulmonary retention of mutant mlgG1 with differing binding affinities to the murine neonatal Fc receptor (mFcRn) were assessed in order to determine whether antibody transport across the epithelium occurred via active transcytosis. It was determined that 54.4 ± 0.63 % of the total instilled dose of a whole monoclonal antibody remains in the lung over 24 hrs, with Fab and scFv fragments cleared significantly quicker with 28.7 ± 0.73 % and 34.9 ± 0.85 % respectively of the total instilled dose remaining in the lung at 24hrs. The pulmonary retention of the 11 kDa FN3 protein was also assessed with 21.0 ± 0.65 % remaining in the lungs after 24 hrs. No evidence of build up of any protein was detected in the oesophagus/stomach, suggesting little contribution by mucociliary clearance. Very little build up of whole antibody, Fab or scFv was observed in the liver or kidneys. However, very clear evidence of renal filtration of the 11 kDa fragment was observed. There was no difference in the pulmonary retention of wild type IgG and any of the mFcRn binding mutants. Additional investigation of antibody retention rates in the murine house dustmite (HOM) model of asthma, although not making use of the SPECT/CT method, showed that antibody is cleared more rapidly from the diseased lung than the normal lung. It was also shown that the expression pattern of the mFcRn receptor is the same in the normal and HDM exposed lung and so this increase in clearance rate occurs via passive diffusion controlled processes. This is most likely a result of increased paracellular transport due to disrupted mucosal barrier function. The SPECT/CT imaging method developed has proven to be a simple and reliable method to assess, non-invasively, pulmonary antibody retention in vivo. Overall it appears that antibody transport across the pulmonary epithelium occurs predominantly via diffusion controlled mechanisms, which include both paracellular transport and nonspecific transcytosis by pinocytotic routes. Additionally, in the mouse, neither receptor mediated transport by mFcRn nor mucocillary clearance is important in the pulmonary clearance of antibodies.

Natural products are an important source for drug discovery. At present there is a resurgent interest in pharmacognosy as a platform for new combinations of active principles to provide highly potent and low-cost medications to treat a growing population with an increasing longevity. This product studied phytochemical interactions in Artemisia annua plant extracts using anti-plasmodium and anti-proliferation assays to identify interactions with potential therapeutic implications. To enable the study a rapid tandem quadrupole mass spectrometry (TQD) method was developed for metabolites in the plant and the validation indices showed the method to be robust, quick, sensitive and adequate for a range of applications.

A crucial and challenging aspect of the drug development process is the requirement to measure the distribution of a pharmaceutical compound and its metabolites in tissue. Industry-standard methods used to look at total localisation of drug-related material are limited due to their dependence on labels. These labelled techniques can have difficulty in distinguishing between the drug of interest and its metabolites. Imaging mass spectrometry is a technique that has the potential to spatially distinguish between drug and metabolites, due to its high chemical specificity and sensitivity. A number of imaging mass spectrometry approaches have been described for localisation of drug compounds in tissue, most notably matrix-assisted laser desorption/ionisation (MALDI) imaging, which can provide data complementary to existing imaging techniques. Two imaging mass spectrometry approaches have been evaluated and compared for use in the localisation of a range of drug compounds in target tissues. The techniques used were MALDI imaging and a recently described electrospray ionisation-based technique, liquid extraction surface analysis (LESA). Both techniques have been successfully used for the detection of drug compounds in dosed tissue sections. A major challenge associated with imaging techniques is the required selectivity of the experiment for the compound of interest, due to the complex nature of tissue sections. Combining the shape-selective method of ion mobility separation with MS/MS fragmentation has been shown to improve the selectivity of both imaging approaches for the compound of interest. Results obtained using LESA-MS have demonstrated the suitability of this technique as a rapid and sensitive profiling technique for the detection of drugs and metabolites in tissue, but with a lower achievable spatial resolution than MALDI imaging. Higher spatial resolution was achieved with MALDI imaging; however data acquisition times were longer and required higher dosing levels for successful detection of drug compounds in tissue. A biological application of MALDI imaging was also evaluated. Mobility-enabled MALDI imaging was used to assess differences in the localisation of important adenine nucleotides between control and metabolically stressed mouse brain sections. Tissue fixation methods were evaluated to overcome rapid post-mortem degradation of adenine nucleotides such that biologically relevant localisation images can be obtained. These studies highlight the crucial importance of appropriate biological sample preparation in MALDI imaging experiments.

The widespread use of antibiotics has played a significant role in the emergence of resistant bacteria. It is of great interest and need to develop novel, effective and safe antimicrobial therapeutics. The biosynthesis of bacterial cell wall peptidoglycan is an intricate process that has become a popular target for antibiotics. Lytic protein E of Bacteriophage ΦX174 was found to inhibit peptidoglycan biosynthesis via an unknown interaction with integral protein MraY. Genetic studies have revealed that E-mediated lysis is dependent on the interaction between Phe288 of MraY and the transmembrane segment of protein E. We have constructed an α-helical model for the predicted transmembrane interactions between protein E and MraY and shown that favourable interactions can be formed between Phe288 and the RWXXW motif of protein E. In this thesis, analogues of the RWXXW motif were synthesised in solution and via solid phase peptide synthesis using 2-chlorotrityl chloride resin as the polymeric support. The inhibitory activity of these analogues was determined on a continuous fluorescence assay against membrane bound MraY. Inhibition studies on site-directed mutants of E. coli MraY were also conducted. Testing the inhibitory activity of RWXXW analogues provided compelling information on the importance of protein E residues for the inhibition of MraY. Peptides which contained a tryptophan residue were especially good inhibitors of MraY presumably due to their interaction with Phe288. Mutation of Phe288 caused a dramatic decrease or complete loss to the inhibitory activity of peptides containing an aromatic residue. Some analogues also contained antibacterial activity across multiple strains of bacteria including E. coli, B. subtilis and P. putida with MIC values as low as 8μg/mL. To confirm if MraY was the target enzyme, E. coli cells overexpressing MraY were treated with RWXXW analogues. An increase in the MIC of RWXXW analogues signified that the MraY was the lysis target. In the course of the project, we noticed that members of the UPA class of natural products contained some structural features that are also found in the RWXXW motif. These natural products were tested for activity against site-directed mutants of E. coli MraY. Results showed that Phe288 plays some role in the inhibition of MraY by pacidamycin. This work identifies a promising target for the development of novel antimicrobial agents that is located on the outer face of the cytoplasmic membrane.

Over the last 20 years it has been realised that neonates, infants and children experience pain and considerable stress responses to surgical and medical procedures which are harmful and cause fear, anxiety and distress(Walker, 2008). This thesis will describe a body of work published since 1992 whose aim has been to improve several aspects of pain management for children in terms of both efficacy and safety. The studies encompass research into the four main classes of analgesics used in paediatric clinical practice, namely local anaesthetics, opioids, non-steroidal anti-inflammatory drugs (NSAIDs) and paracetamol. In addition, control of the stress responses to tracheal intubation and to surgery has been studied with the availability of newer potent short-acting opioid agents and the anaesthetic agent propofol. The total body of work described covers 41 peer reviewed publications with 14 index papers selected for more detailed consideration. Local anaesthetics Several studies included in this thesis demonstrate the efficacy and safety of local anaesthetics in children. The optimum dose of the amide local anaesthetic, lignocaine, was determined for preventing pain on intravenous injection of propofol in children(Cameron et al., 1992) and this resulted in the widespread adoption of propofol as an induction agent. Several studies of propofol in children were conducted and this led to the development of more accurate computer-controlled delivery for maintenance of anaesthesia in children down to age 1 year(Morton et al., 1988, Marsh et al., 1990, Morton, 1990a, Marsh et al., 1991, Doyle et al., 1993c, Runcie et al., 1993, Morton, 1998b, Varveris and Morton, 2002). Topical amethocaine (as a gel and as a phase-change patch) was evaluated in children(Doyle et al., 1993a, Lawson et al., 1995, Lawson and Morton, 1998) and found to have a significantly more rapid onset of action than EMLA cream. This gel is now widely used in the UK. For nerve block, the efficacy and safety of fascia iliaca compartment block in children was demonstrated(Doyle et al., 1997) and the additional safety margin provided by adding the vasoconstrictor adrenaline to the local anaesthetic solution was proved by very low peak plasma concentrations of local anaesthetic. This was also demonstrated for caudal epidural blockade in infants(Hansen et al., 2001). New amide local anaesthetics were introduced in the last decade and ropivacaine was shown to be safe and effective for caudal epidural blockade in children(Ivani et al., 1998a). A collaboration with Strathclyde University led to the development of a new micro-assay method for measurement of local anaesthetics in small volumes of plasma with applicability to neonatal age groups of patients where ethically allowable blood sampling volumes are very small(Stumpe et al., 2000). Opioids The technique of patient-controlled analgesia was studied in children with an open feasibility trial in 1990(Lawrie et al., 1990) using conventional electronic syringe pumps and a further innovative study of a disposable elastomeric reservoir device in 1992(Irwin et al., 1992). The optimum regimen for PCA in children was determined by a series of studies(Doyle et al., 1994a, Doyle et al., 1993d, Doyle et al., 1994c, Munro et al., 2002) and a subsequent trial demonstrated that PCA could be delivered by the subcutaneous route(Doyle et al., 1994b). A further collaboration with Strathclyde produced a microassay method for morphine and metabolites(Watson et al., 1995). These studies showed that PCA is very efficacious and safe for perioperative pain conrol in children from age 5 years upwards and this technique is now in routine use worldwide(Walker, 2008, APAGBI, 2008, Morton, 2007, Lonnqvist and Morton, 2005b). NSAIDs and Paracetamol Following the demonstration of the utility of PCA in children, the technique was used to assess the analgesic efficacy of the NSAID diclofenac and paracetamol in children(Morton and O'Brien, 1999). This showed diclofenac to be particularly efficacious in producing a 40% morphine-sparing effect in children. An innovative study of NSAID eye drops showed them to be as effective as local anaesthetic eye drops for providing analgesia after strabismus surgery in children(Morton et al., 1997). Dosing regimens for paracetamol have evolved in the last decade based on better information on developmental pharmacokinetics and elucidation of the mechanism of action(Arana et al., 2001, Ottani et al., 2006, Pickering et al., 2006, Anderson and Palmer, 2006). There is renewed interest in this decade with the availability of new IV formulations of this old drug. In 1999(Hansen et al., 1999) we contributed to the PK data for paracetamol in neonates and infants which was subsequently used by authors from New Zealand to determine the population PK parameters in this young age group(Anderson and Palmer, 2006). We collated the knowledge on dosing regimens in 2001 in a review(Arana et al., 2001) which has informed the current recommendations in the BNFC. A further collaboration with Strathclyde University led to the development of a microassay for paracetamol and its metabolites from blood spots which has been taken up by Medecins Sans Frontieres as a possible method to use in the field in developing countries(Oliveira et al., 2002). The morphine-sparing efficacy of paracetamol was shown to be less than that due to diclofenac in the study mentioned above under NSAIDs(Morton and O'Brien, 1999). Controlling the stress response Noxious stimuli produce a stress response. A series of studies has shown that using short acting opioids, tracheal intubation could be safely performed without the aid of muscle relaxant drugs in children(Steyn et al., 1994, O'Brien et al., 1998, Robinson et al., 1998). This technique is now widely practiced. Two studies explored methods to reduce the stress response to open heart surgery with cardiopulmonary bypass, one of the most potent surgical stressors. Propofol anaesthesia was shown to significantly ameliorate this response(Laycock et al., 1992) and the newer opioid remifentanil was shown to be as efficacious as the older drug fentanyl for this purpose(Bell et al., 2004). Audit, guidelines and protocols Two major analgesic techniques have been audited in large national projects looking at the risk of epidural infusions in children(Llewellyn and Moriarty, 2007) and opioid infusion techniques in children (Morton, 2008c) and the results show these techniques to be of comparable safety. The evidence from the past 20 years has recently been synthesised into a clinical guideline for management of postoperative and procedural pain in children which has highlighted good practice based on high quality evidence but also revealed a paucity of evidence in some fields(APAGBI, 2008). Guidelines for safer paediatric procedural sedation practice is also described(SIGN, 2004, Playfor et al., 2006). The implementation of guidelines relies on the development of a local protocol and the evolution of the acute pain relief service protocol in Glasgow is described.(Morton, 2008a)

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.

CML is treated effectively with TKI, however two key problems remain - the insensitivity of CM HSC to TKI and the emergence of TKI-resistant BCR-ABL mutations. BCR-ABL activity is associated with increased proteasome activity and PI are cytotoxic against CML cell lines. We demonstrate that bortezomib is antiproliferative and induces apoptosis in CD34+ cells taken at diagnosis from patients with CP CML cells, with an LD50 below concentrations achieved in vivo. We also demonstrate for the first time that CD34+38- CML cells, representing the TKI-insensitive primitive HSC, are similarly susceptible. Bortezomib is associated with inhibition of proteasome activity, however that of BCR-ABL appears unaffected. Significant synergy is seen when bortezomib and dasatinib are used in combination. We also demonstrate that bortezomib is effective in inhibiting proteasome activity and inducing apoptosis in cell lines expressing BCR-ABL mutations, including T315I. Therefore we believe that bortezomib offers a potential therapeutic option in CML by targeting both TKI-insensitive stem cells and TKI-resistant BCR-ABL mutations.

Despite the scientific, technological and surgical improvements of the past 50 years organ dysfunction following elective paediatric cardiac surgery utilising cardiopulmonary bypass continues to account for increased complications, often leading to a protracted course in hospital with a longer stay in intensive care and the potential for irreversible organ damage long term. Furthermore, paediatric cardiac surgeons are routinely undertaking more complex operations with a shift from palliation to early correction. This has resulted in younger children being subjected to longer periods on the bypass machine with increased effects on vital organs. This thesis describes two clinical studies designed to further assess and characterise peri-operative cardiac, renal and pulmonary function in children undergoing elective cardiac repair at a tertiary referral centre in Scotland, UK. In the first instance a prospective, observational study was undertaken in forty-five children to examine the use of tissue Doppler imaging in the assessment of peri-operative cardiac function, its relationship to myocardial injury and clinical outcome. Tissue Doppler parameters were obtained using a Vivid 7 ultrasound scanner with a 7-MHz probe pre-operatively, on admission to paediatric intensive care and on day one. Myocardial injury was assessed using Troponin-I on the first post-operative day by a commercially available chemiluminescent immunoassay. In twenty children within this group peri-operative renal function was also investigated using standard estimates of glomerular filtration rate, namely creatinine clearance measured by the kinetic Jaffe method during the first and second twelve hour post-operative periods, in comparison to serum creatinine and the novel biomarker cystatin C. Routine plasma retained pre-operatively and on days 0, 1, 2 and 3 post-operatively was used to measure serum cystatin C and creatinine using a particle-enhanced nephelometric immunoassay and the Roche Creatinine Plus enzymatic assay respectively. The association between cystatin C and recorded perfusion parameters including bypass duration, pump flow, haematocrit, oxygen delivery and Troponin-I was investigated. Peri-operative pulmonary function was evaluated through a phase IV, randomised, double-blind, placebo controlled trial. In total, twenty four children were randomised to receive oral sildenafil or equivalent volume placebo four times the day before surgery. Blood samples were collected peri-operatively to measure serum cyclic guanosine monophosphate with a commercially available competitive enzyme immunoassay. Haemodynamic data and echocardiography were acquired at two and twenty four hours post-operatively including pulmonary vascular resistance index and bi-ventricular contractility. Post-operative oxygenation was also determined at the same time by oxygen delivery and oxygenation index. In Chapter 2, peri-operative cardiac function as assessed by tissue Doppler imaging was examined. The results of this study demonstrated that pre-operatively, bi-ventricular systolic function in the study group was reduced compared with normal controls, displaying a significant step-wise decrease with increasing complexity of lesion. This picture persisted post-operatively predominantly in the right ventricle and was significantly associated with the extent of myocardial injury. Impaired peri-operative left ventricular function correlated with clinical outcomes. In Chapter 3, peri-operative renal function as assessed by cystatin C and its association with parameters of perfusion was examined. The results of this study demonstrated that in comparison to serum creatinine, cystatin C had a superior correlation with glomerular filtration rate in the early post-operative period. An elevated level of this biomarker was significantly associated with bypass duration, minimum pump flow and post-operative myocardial injury. Haematocrit was not directly linked to renal dysfunction in this study although evidence of a critical dysoxic threshold within the kidney was suggested indirectly through oxygen delivery calculations. In Chapter 4, peri-operative pulmonary function and vascular reactivity in association with the pre-operative administration of oral sildenafil (0.5mg/kg, six hourly) was examined. The results of this trial demonstrated that compared to placebo, pre-operative sildenafil resulted in modest elevations of serum cyclic guanosine monophosphate, limited effects on pulmonary vascular resistance index, significant reductions in peri-operative bi-ventricular contractility, significant reductions in post-operative oxygen delivery and a trend for increasing ventilatory support. In summary, the current thesis has demonstrated that in children undergoing corrective cardiac surgery peri-operative bi-ventricular function can be accurately assessed by tissue Doppler imaging which to date has had limited use in this patient group. With regards to renal function, cystatin C was shown to be a better estimate of glomerular filtration rate and a more sensitive marker of early renal dysfunction in children after surgery. Furthermore, cystatin C identified a transient post-operative renal impairment, the magnitude of which was associated with duration of bypass, pump flow and myocardial injury. In relation to pulmonary function, this research identified that pre-operative administration of oral sildenafil to children undergoing cardiac surgery produced limited effects on pulmonary vascular resistance but was associated with reduced ventricular contractility and post-operative oxygenation raising significant concerns over its routine clinical use.

• 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.