Shock is defined as inadequate delivery or utilization of oxygen by the body tissues. Currently measured cardiorespiratory variables however, indicate decompensation only when patients become (near) shocked. Belated intervention often fails to reverse injury, leading to organ dysfunction and failure. Precise monitoring of the adequacy of tissue perfusion thus represents a major deficiency in clinical practice, particularly in the critically ill. As mitochondria utilize >90% of the oxygen consumed by the body, predominantly for ATP production, there is an obvious logic in targeting this organelle for monitoring the adequacy of tissue perfusion. Within mitochondria, NADH transfers electrons from the Krebs’ cycle to Complex I of the electron transport chain. In doing so, NADH is oxidized to NAD+. A rise in NADH:NAD+ ratio (redox state) will occur with a downstream block in the chain, e.g. due to lack of oxygen. As NADH (but not NAD+) fluoresces in response to UV light excitation (340nm), with an intensity relating to its concentration, and as most of the NADH signal represents changes in mitochondrial NADH, this property may be utilized for non-invasive assessment of tissue hypoperfusion. I validated the technique in vitro, and investigated its utility in rat shock models. During graded or abrupt decreases in the constituent parts of tissue oxygen delivery (cardiac output, haemoglobin and oxyhaemoglobin saturation), as well as during resuscitation, I assessed the relationship between skeletal muscle NADH fluorescence intensity, organ perfusion and oxygenation. I compared these against measures of global haemodynamics and tissue perfusion routinely measured in critically ill patients. With each graded insult, NADH fluorescence demonstrated increases reflecting severity of the insult, with improvements upon resuscitation. A persisting rise in NADH fluorescence >50% above baseline foretold death within the following 30-45 minutes, in advance of the other monitored variables. My results indicate that NADH fluorescence may be used for monitoring tissue hypoperfusion in shock states, and as a guide to the timing and adequacy of therapeutic interventions.

Background: Coronary artery disease is set to be the world’s leading cause of mortality by 2020. Hence novel treatment strategies are urgently required to protect the human myocardium against ischemia-reperfusion injury. This thesis examines the role of the mitochondrial permeability transition pore (MPTP) as a novel target for myocardial protection by interventions applied solely at the time of reperfusion, which can protect the human heart against lethal reperfusion injury. Methods and Results: Using human atrial tissue (harvested at the time of routine cardiac bypass surgery), subjected to simulated ischemia and reperfusion injury model, we have demonstrated that the opening of the MPTP at the time of reperfusion is a critical determinant of cardiomyocyte death. We also show that inhibiting MPTP opening, by administering known pharmacological inhibitors of MPTP at the onset of reperfusion, is cardio-protective. Using experimental models in adult human atrial trabeculae, we demonstrate that inhibiting MPTP opening at the time of reperfusion improves myocardial contractile function. Also using human atrial cardiomyocytes we demonstrate that inhibiting MPTP opening at the time of reperfusion improves cellular viability. Finally using the human atrial cardiomyocyte model for inducing and detecting the MPTP opening, we demonstrate the opening of MPTP and also the inhibitory effect of known MPTP inhibitors on MPTP opening. Conclusion: We find that MPTP opening does occur in the human atrial cardiomyocyte following ischemia-reperfusion injury, and that inhibiting the opening of the MPTP at the time of reperfusion, provides a potential target for human myocardial protection, when the intervention is applied at the time of reperfusion. Therefore, interventions, which target and inhibit MPTP opening, at the time of reperfusion, can protect the myocardium from lethal reperfusion injury and may improve morbidity and mortality from coronary artery disease. This is useful in the clinical settings of ischemia-reperfusion injury such as thrombolysis following an acute myocardial infarction, heart surgery and percutaneous transluminal coronary angioplasty.

Introduction: Critical illness survivors demonstrate skeletal muscle wasting with associated functional impairment. I prospectively characterised this process, and defined the pathogenic roles of altered protein synthesis and degradation. Methods: Critically ill patients (n=63, 59% male, age 54.7±18.0 years, APACHE II score 23.5±6.5) were recruited <24 hours following intensive care admission. Muscle loss trajectory was determined through serial ultrasound measurement of rectus femoris cross-sectional area (RFCSA) and, in a subset, quantification of myofibre area (FibreCSA) and protein/DNA ratio. Histopathological analysis was performed. Muscle protein synthesis and breakdown rates were determined and respective signalling pathways examined. Results: RFCSA decreased significantly, (-17.7±12.1%, [p<0.001]), underestimating muscle loss determined by FibreCSA (-10.3±10.9% vs.-17.5±30.2%, p=0.31), or protein/DNA ratio (-10.3±10.9% vs. -29.5±41.5%, p=0.03). Fall in RFCSA was greater in multi- than single-organ failure (-21.5±10.5% vs. - 7.2± 9.7%, p <0.0001), even by day 3 (-8.7±16.3% vs. -1.8± 9.6%, p<0.01). Myofibre necrosis occurred in >50% (20/37) of subjects. Protein synthesis was depressed to levels observed in fasted controls (0.035±0.018%/hr vs. 0.039±0.011%/hr, p=0.57), and increased by day 7 (0.076±0.066%/hr, p=0.03) to levels associated with fed controls (0.065+0.018%/hr, p=0.30,) independent of nutritional load. Protein breakdown remained elevated throughout (8.5±5.7 to 10.6±5.7mmol phe/min/IBW, p=0.4).Principal component analysis of intracellular signalling supported a programme of increased breakdown (r=-0.83, p=0.005) and depressed synthesis (r=-.69, p=0.041). Conclusions: Early rapid skeletal muscle wasting occurs in critical illness, is greatest in those with multi-organ failure, and results from suppression of protein synthesis and increases in catabolism. These effects are independent of feeding and are commonly associated with myonecrosis.

The aim of this thesis was to develop and validate High Resolution Anorectal Manometry (HRAM) in order to overcome the limitations of Conventional vector volume manometry so to maximise clinical utility. A fully automated analysis method of HRAM and conventional manometry was developed and validated. HRAM was validated by demonstrating that its overall repeatability is equivalent to the current gold standard that is station pull through vector volume manometry. Normality was defined for high resolution manometry and conventional manometry. A normogram was developed for accurate interpretation of high resolution manometry during attempted balloon expulsion. Normal sphincter pressure morphology was defined using HRAM and vector volume manometry using novel display techniques. Sphincter morphology was quantified and comparison made between sexes. Using HRAM we found that constipated patients tended to increase their rectal pressure by a smaller amount than healthy volunteers. There was a poor agreement between HRAM and MR proctography in the assessment of the constipated patient. Resting pressure measured using conventional manometry was better than HRAM at distinguishing patients with passive incontinence from healthy volunteers. HRAM was marginally superior to conventional manometry in its ability to distinguish patients with urge incontinence from those with a normally functioning anorectum. In conclusion HRAM analysis was developed and the technique validated using a testretest repeatability study. Normality was defined quantitatively and qualitatively using novel display methods. Description was made of HRAM in incontinence and constipation and comparison made with conventional manometry and MR proctography.

Radiotherapy and chemo-radiotherapy increasingly often achieve cure in gynaecological and prostate cancer, but radiation enteritis/proctitis remains a problem in acute phases of treatment in more than 70% of patients. In most cases this resolves rapidly, but in at least 5% of patients it persists or re-presents as chronic radiation damage. This may be progressive and is associated with diarrhoea, rectal bleeding, incontinence, and poor quality of life. It has engaged surprisingly little attention. Standard management during radiotherapy includes a low fibre diet. Although this may reduce some acute symptoms it also deprives the bowel of fibre-derived short chain fatty acids which (particularly butyrate) are its essential nutrients. We hypothesised that possible advantages of fibre avoidance are outweighed by less restrained damage to the mucosa, and perhaps by a longer-term insult which in some patients leads to chronic radiation bowel disease. The proposed work explores the provision of short chain fatty acids to the bowel and the importance of short chain fatty acids in angiogenesis. Our meta-analysis has shown that probiotics may have a role in prevention of radiation-induced sequelae. We have been able to encapsulate short chain fatty acids in alginate beads and have successfully used these beads to deliver short chain fatty acids to the chick chorio-allantoic membrane assay model; the beads have the potential for future colon-specific delivery in man. Our results have shown that propionate may increase angiogenesis. The study on effect of Androgen Deprivation Therapy (ADT) on gut microbiota has shown that Androgen Deprivation Therapy before radiotherapy in patients undergoing radiotherapy for prostate cancer does not have a dramatic effect on their microbiota. Our trial with fructooligosaccharide is an ongoing trial where fructooligosaccharide is given prophylactically in a blinded, placebo-controlled fashion to patients undergoing pelvic irradiation for gynaecological and prostate malignancy. It will serve as a pilot study for the postulated influence of fructooligosaccharide on late-stage chronic radiation disease.

The effectiveness of combined use of capillary electrophoresis (CE) and X-ray crystallography in fragment-based drug discovery has been investigated. Using Heat Shock Protein 90α (Hsp90α) as a test system, CE fragment hits were validated by X-ray crystallography with the added benefit of structural information. The crystal structure of one of the fragment hits in complex with Hsp90α ATPase domain has shown that even weakly binding fragments can cause conformational change of macromolecules upon binding. Another investigation was focused on identifying stabilisers of the tetrameric plasma protein transthyretin (TTR), which can cause amyloidosis when its monomers are misfolded. Two methodologically distinct CE assays were developed to detect TTR binding fragments. 129 fragments were screened using one method and 15 of the 16 initial hits were tested positive by the other (hit rate = 11.6%). Structure activity relationships were built up from analogue screening on two chemically different fragment hits using both CE assays. In parallel, ligand-protein interactions were characterised by X-ray crystallography to assist hit-to-lead development. The CE assays and structural data yielded a detailed perspective on the binding of heterocyclic rings to TTR. Aromaticity and hydrophobicity are favoured in the binding sites but not basicity. Moreover, Ser117 hydrogen bonding with heterocyclic nitrogen atoms was frequently observed. Through the combined use of two biophysical techniques and testing of fewer than 200 compounds, a novel TTR binding scaffold was discovered. The scaffold is likely to be active in vivo as it binds to TTR in neat plasma (IC50 = 34.45μM). It has high ligand efficiency (0.60) with an ITC Kd of 260.42nM and 15 non-hydrogen atoms. Use of CE and X-ray crystallography provides a powerful combination worth considering in early stage drug discovery.

An unmet demand for high resolution 3D imaging modalities providing enhanced soft tissue contrast exists in a number of biomedical disciplines. X-ray phase contrast (PC) methods can provide a solution: contrast is driven by the refractive index decrement from unity (RID) rather than the absorption term, the former being much larger than the latter for low absorbing materials and energies typically used for biomedical imaging. However, most existing PC methods suff er from drawbacks a ffecting their implementation outside specialised facilities such as synchrotrons and therefore their applicability to biomedical research. The Edge Illumination (EI) PC method has the potential to overcome or at least mitigate most of these drawbacks. Its major strengths are its simple setup, compatibility with laboratory-based x-ray sources and potential for low-dose imaging. Until now, however, EI had not been implemented in computed tomography (CT) mode. The work presented in this thesis bridges this gap. Practical and theoretical requirements for the CT extension were identifi ed, and experimental EI-CT setups were implemented both at a synchrotron and in a standard laboratory. It was shown that EI-CT allows the reconstruction of maps of a number of physical quantities, including the RID and the absorption term. Quantitative imaging via the reconstruction of the RID was demonstrated to be feasible, and to be limited only by physical eff ects not accounted for in the phase retrieval model (namely coherence) and, to some extent, polychromaticity. An optimal sampling scheme was derived for laboratory-based EI-CT. This was demonstrated to be compatible with low-dose scans, which is important for in vivo imaging. The dose such a system would deliver to a small animals (e.g. mice) was estimated, and found to be within acceptable limits. Moreover, new scientifi c areas that can benefi t from the method were identi fied (e.g. regenerative medicine), and the fi rst ever PC-CT images of acellular organ sca ffolds are presented.

Anti-inflammatory therapies are promising candidates for the prevention of brain injury following prolonged seizures (status epilepticus). Biomarkers for therapy monitoring are needed to translate these recent findings to the clinic. The aim of this thesis was to develop imaging methods that can be used to monitor anti-inflammatory therapies and monitor disease progression following prolonged seizures. In order to achieve these goals, the lithium-pilocarpine model was used as a model of status epilepticus and novel MRI imaging methods were employed. Various imaging approaches including: quantitative, structural, molecular and functional imaging were tested for their possible investigative utility as imaging biomarkers for neuroprotective therapies. Alongside this, two different anti-inflammatory therapies were tested for their effectiveness to alter brain injury following status epilepticus. This thesis demonstrates that molecular imaging has potential to monitor neuroprotective therapies. Surprisingly, there was little evidence that the anti-inflammatory therapies tested here had beneficial effects. However, this thesis shows that employing novel imaging approaches and automated analysis methods can enable accurate in vivo assessment of disease altering therapies.

SOX2 is expressed in the pituitary anlagen, Rathke’s Pouch (RP), from its induction at 9.0dpc. RP progenitors initially expand, later giving rise to all pituitary cell types. As cells differentiate, SOX2 expression is down-regulated, but a population of SOX2+ve cells persists until adulthood. These represent adult stem cells, displaying regenerative potential upon physiological demand. In humans and mice, heterozygous loss-of-function mutations in SOX2/Sox2 are associated with hypopituitarism. It was therefore decided to investigate the role of SOX2 during murine pituitary development. Homozygous null mutations of Sox2 lead to embryo lethality following implantation; consequently conditional strategies were used to delete the gene specifically in RP. Nkx3.1Cre and FoxG1Cre, display different spatio-temporal patterns of activity in RP. The severity in hypoplasia and reduced progenitor cell proliferation correlated with the efficiency and timing of Cre-mediated deletion of Sox2. The expression of the transcription factor SIX6, known for its role in promoting cell proliferation, was downregulated. Conversely expression of the cell cycle inhibitor p27kip1 is up-regulated, in the absence of Sox2. Furthermore, the proliferation defect in Sox2 mutants can be rescued by homozygous loss of p27kip1, demonstrating a genetic interaction. This suggests that SOX2 promotes progenitor cell proliferation in the early RP and may do so, at least in part, by regulating Six6 and p27kip1 expression. Sox2 RP mutants display a disproportionate reduction in melanotroph cell numbers in the intermediate lobe (IL). The Sox2-deleted cells display a downregulation of the melanotroph lineage specifier, PAX7. Consequently, the few differentiated cells present in mutant IL switch from a melanotroph identity to a corticotroph fate, despite these cells never being present in the normal IL. This phenotype was not rescued in p27kip1;Sox2 compound mutants. This suggests that SOX2 plays two independent roles during pituitary development, initially promoting progenitor proliferation and later specifying IL melanotroph cell fate.

A planar HPGe Compton camera for nuclear medicine applications that contains 177 pixels of 4 × 4mm2, of which 25 are at the back detector, is being used to image point sources of Cs137, line sources and clinical-like shape distributed sources. Experimental results are obtained to study the e ffects of energy resolution, position sensitivity, and reconstruction algorithms on camera images. Preamplifi ed pulses are digitized for pulse shape analysis using gamma ray tracking GRT4s data acquisition cards to improve camera performance. Pulse shape analysis includes improving energy resolution of the camera, improving position sensitivity of the camera by using induced charges in neighboring pixels and implementing time coincidence algorithms to select good events. The energy resolution e ffects will be presented for three di fferent energy resolutions at 662 keV; 1.4% by implementing a basic pulse height algorithm, 0.7% with a curve fitting algorithm and 0.3% by implementing the moving window deconvolution algorithm (MWD). By improving the position sensitivity from 2.5mm to 1.25mm, an improvement of 24% in spatial resolution is expected where the currentspatial resolution of a point source 5cm from the camera is 8mm. The results show that the e ects of energy resolution are less important for this camera. Images are reconstructed using back-projection and ITEM (Imaginary Time Expectation Maximization) algorithms. Eff ects of scattering materials on embedded sources are studied. Diff erent source distributions have been studied including point sources, line sources and clinical simulations (baby-heart-like distributed sources).