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Aortic valve stenosis (AVS) is the progressive calcification and loss-of-function of the aortic valve (AV). Left untreated this leads to left-ventricular failure and ultimately death. To date, no medical treatment for AVS exists with only invasive surgery as a treatment option. Central to the limited progress made towards developing novel treatments is our lack of understanding as to how AVS initiates and the environmental changes which occur within the AV. The aim of this study was to investigate hypoxia as a key contributing factor towards the progression of AVS. My hypothesis was that hypoxia would act as a stimulus of angiogenesis in the human aortic valve tissue and stimulate AVS progression. I performed in vitro techniques upon primary human aortic valve interstitial cells (VICs) cultured from valves obtained from patients undergoing valve replacement surgery. Immunohistochemistry was performed on formalin- fixed paraffin embedded human AV tissue which was eitherstenotic (diseased) and non-stenotic (healthy) from patients to to identify morphological and expression changes which occur during the progression of AVS. VICs which were isolated from stenotic aortic valves were placed in a hypoxic environment (1% O2, 5% CO2). These cells demonstrated the ability to proliferate at an increased rate compared to their normoxic control. This result occurred alongside a reduction in cAMP signalling in hypoxic stenotic VIC. Hypertrophy of the AV is central to the progression of AVS. These data may delineate the cause of AV hypertrophy and highlight hypoxia as a key process in the initiation of AVS. The neovascularisation of the AV is also critical to the progression of end-stage AVS. As hypoxia is a canonical stimulus of angiogenesis, the growth of nascent blood vessels from pre-existing vessels, I investigated the role of hypoxia in this process in the AV. Soluble fms-like tyrosine kinase 1 (sFlt-1) is the soluble isoform of the vascular endothelial growth factor (VEGF) receptor 1. It can bind the canonical driver of angiogenesis, VEGF-A, and sequester signalling, thus is an antiangiogenic mediator. VICs from stenotic valves incubated in hypoxia for 24 hours demonstrated increased expression of VEGF-A. Immunohistochemistry of sFlt1 within aortic valves showed a significant decrease in expression in AVs compared to non-stenotic controls. Incubation within hypoxia also stimulated a decrease inprotein expression of sFlt-1 in stenotic VICs. siRNA knockdown of sFlt-1 in VICs resulted in increased in vitro angiogenesis in a conditioned media assay. Furthermore, the addition of recombinant sFlt-1 to either endothelial cells or ex vivo human aortic valve tissue in Matrigel was able to inhibit tubule formation and sprouting of angiogenesis respectively. Together these data demonstrate the ability of hypoxia to drive angiogenesis in the AV. Furthermore, sFlt-1 is highlighted as a critical factor in the maintenance of AV avascularity and as a potential therapeutic target to inhibit angiogenesis in the AV. G-protein coupled receptors (GPCRs) are the target of ~40% of FDA approved drugs. I performed a microarray, which compared the mRNA expression of 355 GPCRs between non-stenotic VICs and stenotic VICs. This array highlighted interesting receptors, which demonstrated increased expression in stenotic VICs or even are only expressed in stenotic VICs. Their success in being targeted in other diseases make GPCRs an exciting source of targets in the search of a novel treatment for AVS. In conclusion, I demonstrate the potentially important role of hypoxia in the initiation and progression of AV by stimulating VIC proliferation and angiogenesis in the AV. These results demonstrate a unique mechanism of disease progression in the aortic valve, which could potentially be targeted for the treatment of AVS. As we seek the first medical treatments for this disease, it is vital that we continue to enhance our understanding of the biology of the aortic valve and the progression of AVS.

Spinal muscular atrophy (SMA) is a childhood neurodegenerative disease caused by homozygous mutation of the survival motor-neuron 1 gene (SMN1), resulting in decreased levels of survivalmotor-neuron (SMN) protein. Although it is a neurodegenerative disease it also has an extensive systemic pathology, including vascular and cardiac defects, as well as hypoxic spinal cord. This thesis focusses on fully mapping the spread of hypoxia beyond the spinal cord, as well as its effects on tissue and cells with low SMN. Hypoxia was mapped using a Taiwanese SMA mouse model. Labelling of 5-day-old Taiwanese SMA pups with pimonidazole and [18F]-FAZA confirmed hypoxia in spinal cord and revealed hypoxia in neural tissue (brain and eyes), skeletomuscular tissue (forelimb, hindlimb and tail) and various organs (spleen, heart, kidney and liver). It also showed that skin and lungs were not-hypoxic. HIF-1 or HIF-2 expression was measured in the hypoxic tissues, as they are the main proteins responsible for the hypoxia response, but there was no significant increase. [18F]-FDG (glucose) uptake, which normally increases as a result of the hypoxia response, was also measured. Both neural tissue and kidney showed upregulation, while heart showed downregulation. Previous studies indicate that the most significant part of the systemic hypoxia response is angiogenesis. Labelling of PECAM -1, an endothelial cell marker, by western blotting showed that most of the hypoxic tissues (brain, spinal cord, kidney, heart, spleen and hindlimb) had reduced vascular density, suggesting that vessel development may be defective. To test angiogenesis, anendothelial in-vitro model was developed from primary culture of Taiwanese SMA mouse aortas. This model showed defects in endothelial tube formation in normoxic conditions compared to cells cultured form controls. The same experiment done in hypoxia showed no significant difference between control and SMA model. A neural model was developed knocking down SMN in NSC-34 motor-neuron like cells. These cultures were used to test the response to hypoxia of low SMN cells. A Flow cytometry protocol was developed to test SMN levels, which made evident that intrapopulation SMN levels vary greatly both in control and SMN knockdown population, resulting in overlap between them. It also showed cell vulnerability to hypoxia (1% O2) correlates with SMN levels, the lower the SMN, the higher chances of death. A different flow cytometry protocol showed that low SMN populations experience cell cycle alteration in hypoxia, connected to G2 and M phases. The low SMN cells also showed abnormal accumulation of lactate inside hypoxic compared to controls. This thesis showed that hypoxia is a widespread stressor in the Taiwanese mouse model of SMA. It also described several defects in angiogenic processes and cellular hypoxia response correlated to SMN decrease in SMA in-vitro cultures. In summary, it emphasizes the importance of systemic treatments, that would also recover SMN levels in blood vessels, and hypoxia monitoring in SMA therapies.

Purpose: Hypoxia in colorectal cancer (CRC) and gastroesophageal cancer (GEC) decreases tumour responsiveness to radio and chemotherapy leading to cancer progression and poor prognosis. This is the first study to utilise [18F]FAZA hypoxia radiotracer in patients with CRC and GEC. Methods: Six patients (mean age 68±8 years, 2 males and 4 females) with CRC and 4 patients diagnosed with GEC (mean age 65 years, 3 males and 1 female) were included in the study. [18F]FAZA was synthesised at the John Mallard Scottish PET Centre. After injection with 370 MBq of [18F]FAZA, PET/CT images with 60 min dynamic scan were acquired. In addition, 15 min static scans 2 hr post injection were performed. 3D PET images were reconstructed iteratively using an ordered subset expectation maximization (OSEM) method and fused to the corresponding low-dose CT images. [18F]FAZA uptake parameters including maximum standard uptake value (SUVmax), tumour-to-muscle ratio (T/M), tumour-to-bowel ratio (T/B) and volume of interest (VOI) were measured. Results: 4 out 6 patients with CRC (66%) showed clear uptake of [18F]FAZA in the primary tumour. The mean tumour SUVmax was 2.2±0.91 (range 1.12 - 3.71). The tumour SUVmax was significantly higher compared with muscle and bowel (t(5) =3.11, P=0.03), (t(5) =3.08, P=0.03), respectively. However, tumour SUVmean didn't differ significantly compared with muscle and bowel (t(5) =2.41 , P=0.06), (t(5) =2.46 , P=0.06) respectively. The mean tumour to muscle ratio (T/M) ratio was 1.89±0.64 (range 1.10 - 2.87), while the mean tumour to normal bowel (T/B) was 1.92±0.64 (range 1.08 - 2.74). However, [18F]FAZA did not accumulate in any of the tumours found in patients with GEC. Conclusions: [18F]FAZA PET/CT imaging is suitable and feasible for detecting CRC hypoxic tumour regions with image quality that can be used in clinical practice.

In recent years tumour hypoxia has been extensively investigated, mainly because it is a source of resistance to the common radio and chemo therapies. In fact, the low levels and heterogeneous distribution of oxygen in hypoxic microenvironment render ionizing radiation ineffective in treating cell proliferation. Furthermore, a low oxygen concentration promotes the activation of HIF-1 transcription factor, which favours the development of a more malignant and resistant cancer cell phenotype often associated with poor prognosis. Positron Emission Tomography (PET) imaging is a valuable diagnostic tool for investigating hypoxia in vivo by means of radiotracers, which incorporates both a radioisotope and a hypoxia-sensitive function. The aim of this multidisciplinary project was to develop small libraries of radiolabelled compounds starting from the biological and chemical features of the two gold standard hypoxia PET tracers [18F]FMISO and [18F]FAZA as well as those of the promising new tracer [18F]HX4. These new radiocompounds display the following peculiar structural characteristics: a 2-nitroimidazole hypoxia-sensitive moiety, different spacers to modulate steric constraint, lipophilicity and metabolic stability and a fluorinated aldopentose sugar as prosthetic group (e.g. [18F]FDR). Two series of compounds were designed and developed based on the conjugation method used to introduce the prosthetic group, namely the oxime bond formation and the thiazolidine ring closure. Six radiotracers belonging to the oxime-derivatives series were tested in vitro on MCF7 breast cancer cell lines in hypoxic conditions and a lead radiocompound incorporating a cyclopropyl group was identified. This new hypoxia tracer showed a better kinetic profile than both [18F]FMISO and [18F]FAZA in MCF7 cancer cell lines and comparable uptake values on a panel of different cancer cell lines, up to 120 min post administration at 1% of O2. These promising results will pave the way for futures in vivo studies.

The reduction in O2peak at altitude is well documented. Maximal exercise in hypoxia is accelerated through a reduction in O2 supply with contributions from central and peripheral origins of fatigue. Changes in cerebral and muscle oxygenation have not been well characterised during incremental exercise in hypoxia. It is possible attainment of O2peak is driven by the oxygenation profile of these tissues whilst changes in molecular biomarkers of endothelial function could provide some insight into the mechanisms driving systemic and regional O2 delivery and vascular hypoxic sensing capabilities. The first study of this thesis examined the impact of acute hypoxia (FIO2 = 0.12) on the cerebral and muscle oxygenation response to incremental cycling exercise using NIRS (n = 14; age: 23 ± 5yr; height: 1.80 ± 0.07m; weight: 84 ± 8kg). The profiles were characterised at equivalent relative and absolute exercise intensities and molecular blood-borne markers of O2 sensing and function were measured before and immediately after maximal exercise for changes in oxidative stress (A• and 3-NT), NO metabolites (NOx, NO3•, NO2• and RSNO) and cell adhesion molecules (sICAM-1 and sVCAM-1). The key observations from this study were: 1) O2peak decreased by 22% and the magnitude of cerebral and muscle deoxygenation (↓O2Hb and ↑HHb) was greater in hypoxia, 2) the slope for the relative HHb response was similar between conditions whereas there was an accelerated slope across the absolute workloads in hypoxia implying cycling performance was driven by a premature attainment of maximal O2 extraction capacity of the muscle, 3) there was no evidence suggesting cerebral O2 metabolism was impaired in hypoxia however since SaO2 was 78 ± 4% at PPO it is possible the reduction in systemic O2 delivery could have influenced central fatigue, 4) there was a tendency for a rightward shift in the cerebral THb profile in hypoxia and although muscle THb peaked at 80% PPO in both trials, the response also tended to be lower in hypoxia, 5) there was no change in oxidative stress markers and NOx after exercise, 6) RSNO increased and NO2• decreased after maximal exercise. The decline in NO2• was attenuated in hypoxia possibly due to a blunted NO2•-HHb-NO pathway and may explain the systemic hypoperfusion response, 7) The increase in sICAM-1 and sVCAM-1 after exercise was augmented in normoxia, 8) Only when normoxia and hypoxia data was pooled was there a correlation between sVCAM-1 pre-post exercise and O2peak. Intermittent hypoxia (IH) may be used to improve the efficiency of exercise training and as a pre-acclimatisation strategy prior to high altitude ascent. The purpose of the second study was to evaluate the efficacy of a 10 day IH regime consisting of 9x 5 min daily exposures of 9.5% O2 breathing followed by equal periods of normoxia on submaximal and maximal cardiorespiratory responses to exercise in hypoxia. Additionally, cerebral and muscle oxygenation was monitored throughout incremental cycling to exhaustion and changes in NO metabolites (NO3•, NO2• and RSNO) and CAMs (sICAM-1 and sVCAM-1) were measured before and immediately after maximal exercise. The key observations from this study were: 1) a tendency for IH to reduce submaximal O2 and increase O2peak in hypoxia, 2) IH increased the muscle THb response to exercise due an increased intercept for both the muscle O2Hb and HHb in the absence of any change in slope, 4) cerebral oxygenation increased (↑O2Hb) at rest and during exercise, 4) the reduction in nitrite was attenuated in the IH group whilst resting sICAM-1 decreased and the pre-post maximal exercise increase in sICAM-1 was augmented after IH. It is concluded that exercise performance in acute hypoxia is driven by the magnitude of hypoxaemia and an accelerated rate of cerebral and muscle deoxygenation. Molecular biomarkers of endothelial function in particular, NO2• and CAMs, are also influenced by hypoxia and may contribute to the reduction in O2peak. IH may be used to improve exercise economy and O2peak in hypoxia by improving cerebral and muscle oxygenation in the absence of any change in central O2 delivery. It is possible a recalibration of mechanisms that affect NO bioactivation could have enhanced vascular hypoxic sensitivity, O2 delivery and adaptation within brain and muscle tissue which ultimately translated to an improved hypoxic exercise performance. These results give motivation for athletes and mountaineers to incorporate an IH strategy prior to athletic performance at altitude.

This thesis is concerned with the chemical control of breathing during exercise in humans. Chapter 1 reviews some of the relevant studies in animals and humans. Chapter 2 describes the experimental apparatus and the technique of dynamic end-tidal forcing performed using a computer-controlled gas-mixing system. Chapter 3 describes a study of the effects of sustained hypoxia on ventilation during steady exercise. The acute ventilatory response to hypoxia (AHR) was increased during exercise as compared with rest, but the magnitude of the subsequent decline in ventilation (HVD), expressed as a fraction of the AHR, was reduced. A simple model of the hypoxic peripheral chemoreflex is proposed, in which the mechanisms underlying AHR and HVD are functionally separate and can be independently modulated by external factors. Chapter 4 assesses changes in peripheral chemoreflex sensitivity to hypoxia in terms of the degree of decline in AHR measured in the resting periods shortly after prior conditioning periods of hypoxia and/or exercise. At rest, a second AHR measured 6 min after a period of sustained hypoxia had declined by 30% as compared with the initial AHR. In contrast, the AHR measured in the resting period after a period of sustained hypoxic exercise was only 11% smaller in magnitude than the AHR measured after a period of euoxic exercise. The results suggest that the degree to which hypoxic sensitivity declines during sustained hypoxia is genuinely attenuated, rather than masked, by exercise. Chapter 5 describes the changes in respiration during prolonged exercise breathing air with and without added CO2. During prolonged poikilocapnic exercise, ventilation remained constant, but metabolic CO2 production, respiratory quotient and end-tidal PCO2 declined; a result which suggests that in man, ventilation can be dissociated from the CO2 flux. During hypercapnic exercise, ventilation progressively increased; this was interpreted as being due to a correction by end-tidal forcing of the natural tendency for end-tidal CO2 to decline, together with an independent effect of CO2 per se on the ventilation. Chapter 6. Electrical muscle stimulation was used as means of inducing non-volitional exercise. Electrically-induced exercise increased the AHR as compared with rest, and with voluntary exercise at matched external work rate. The AHRs during electrical stimulation and voluntary exercise matched to the internal work rate were similar. Chapter 7. Electrical muscle stimulation was used in paraplegic subjects in whom there would be no neural control of exercise. Electrically-induced exercise increased the AHR as compared with rest. When compared with the data from Chapter 6, the results suggest that the observed increase in AHR during normal voluntary exercise can be wholly accounted for by the increase in metabolic CO2 production, or closely related factors. Chapter 8 presents a brief summary of the findings in this thesis.