A proteomic screen to identify novel SUMO (small ubiquitin-related modifier) substrates in budding yeast Saccharomyces cerevisiae revealed the TATA-binding protein (TBP) as a candidate. TBP is a ubiquitous transcription factor required for transcription by the three eukaryotic RNA polymerases. TBP was confirmed to be SUMOylated in vivo using affinity purification techniques. Lysine (K) residues in the N terminus were confirmed to be the SUMO-accepting sites, suggesting the existence of a 'SUMO region' in the N-terminal domain of TBP. Among the six lysine residues identified, K47 was a strong SUMOylation site. I generated TBP SUMO-deficient mutants to interrogate the functional role of TBP SUMOylation. The mutants showed much higher protein stability. Additionally, chromatin immunoprecipitation (ChIP) analysis revealed that the occupancy of TBP SUMO mutants at promoters of both constitutive and inducible genes were much lower than TBP wildtype, with a lower recruitment efficiency. Cross-linking kinetic (CLK) analysis further proved that the lower promoter occupancy of the SUMO mutants was caused by lower promoter dynamics. This was supported by fluorescence recovery after photobleaching (FRAP) assays, which showed a larger immobile population for the TBP SUMO-deficient mutants. The lower promoter dynamics is expected to lead to lower transcription plasticity and/or aberrant transcriptional output. Consistent with this, cells expressing either type of TBP mutant exhibit significant defects during sporulation, which is a cellular process involving genome-wide transcription reprogramming.

The aim of this thesis was to develop a novel behavioural technique to disrupt parietal function in order to induce top-down cortical modulation of low-level brain structures, namely the brainstem mediated vestibulo- ocular reflex and the early visual cortex. The premise of the technique was based upon using stimuli that engaged overlapping neuronal networks. To this end, we employed a technique that involved concurrent vestibular activation and viewing of bistable perceptual visual stimuli or performing visualised spatial attention tasks. The thesis presents data that shows the ability of this technique to induce a handedness related cortical modulation of the vestibulo-ocular reflex and modulation of the early visual cortex. Subsequently we applied trans-cranial direct stimulation to directly disrupt parietal inter-hemispheric balance in order to induce an asymmetrical modulation of the VOR and propose a revised computational model for vestibular processing. The results from these experiments present the first behavioural demonstration that vestibular cortical processing is strongly lateralised to the non-dominant hemisphere. We propose that this technique developed and validated in this thesis can be used to further probe and investigate cognitive parietal function such as numerical cognition and human decision making.

Obesity is increasingly recognised as a major health threat in the developed world, with more than 120 million people worldwide classified as clinically obese. Increased weight causes increased morbidity and mortality due to its association with cardiovascular disease, diabetes and certain cancers. Bariatric surgery is currently the most efficacious treatment for morbid obesity and has the best long-term outcomes. Bariatric surgery is not without risks. Some of the early risks include postoperative bleeding, anastomotic leaks, and venous thromboembolism. Late complications include marginal ulcer formation, nutritional deficiencies and small bowel obstruction. The latter may be caused by internal hernia formation. In this thesis, an analysis of the causes of small bowel obstruction after laparoscopic Roux-en-Y gastric bypass (LRYGB) is presented, looking specifically at internal hernia formation. A detailed account of the presentation and radiological findings of internal hernia following laparoscopic gastric bypass is provided. The impact of altering surgical technique on the occurrence of internal hernia is analysed: an Observational Clinical Human Reliability Assessment (OCHRA) tool was used for root cause analysis of internal hernia following gastric bypass and in the final study, the employment of a new technique demonstrated significant reduction in the incidence of internal hernia.

Lung cancer is the most common cancer killer worldwide. One of the major reasons for failing to cure this malignancy is the high rate of metastasis. Hence, understanding the mechanisms by which these cells metastasise is required to improve patients' survival. Using the non-small cell lung cancer cell line A549 we optimised a 3D Invasion assay to enable a robotised high-throughput siRNA screen of large gene libraries. Despite thorough optimisation a pilot screen for the Phosphatome highlighted multiple issues with our set-up. Further optimisation work was conducted to improve the resolving power of the assay. However, as no clear explanation for the poor reproducibility could be uncovered, work began on a potential modulator of cell motility identified during a previous motility screen of the kinome. Microtubule affinity regulating kinases 4 (MARK4) is one of four members of the MARK family. A previously performed kinome siRNA screen for modulators of cell migration revealed that depletion of MARK4 reduced A549 cell migration. We validated this observation and additionally found that MARK4 silencing inhibited cell invasion through 3D collagen matrices. MARK4 depletion markedly changed cell structure, as exemplified by an increase tubulin network area. Follow-on experiments revealed an altered speed of microtubule polymerisation in MARK4-silenced cells. We observed that MARK4 downregulation promoted resistance to the chemotherapeutic agents Paciltaxel and Cisplatin, an effect potentially linked to a reduced proliferation in MARK4-silenced cells. MARK4 overexpression in NSCLC cells increased cell motility but did not impact the cell area or resistance to chemotherapy. A targeted siRNA cell motility screen of a selection of proposed MARK4 interacting proteins enabled us to connect MARK4 with Protein Phosphatase 2A and GSK3. Although further investigation is required into the signalling pathways upstream and downstream of MARK4, this work identified novel functions for this kinase and highlighted potential mechanisms underlying its effects.

The research reported in this thesis investigated the neural systems involved in auditory attention, and in particular how these may differ from those recruited for visual attention. One current leading theory of attentional control postulates that a single frontoparietal network (the 'dorsal attention network' or DAN) subserves top-down attention to all sensory modalities. However, there is an abundance of published evidence which contradicts this claim (which is discussed herein). This thesis reports the results of three studies. In the first study, I investigated auditory attention whilst controlling for crossmodal and executive factors which may have confounded the interpretation of previous studies. In the second study, I investigated whether another crossmodal factor, the control of eye movements, may also have contributed to the controversy regarding auditory attention. Lastly, I investigated whether some regions of the brain contain multiple overlapping signals, a finding which could explain how a cortical region might display 'amodal' properties, and participate in multiple cognitive functions simultaneously. As a whole, this thesis provides evidence that the DAN is a predominantly visuospatial attention network whose recruitment during auditory attention reflects indirect crossmodal mechanisms rather than the direct modulation of auditory information. In addition, this thesis provides evidence that a candidate frontotemporal network, which links executive regions of the prefrontal cortex with temporal auditory association areas, subserves top-down attention to non-spatial auditory features.

The primary objective of this thesis was to establish quantitative measurements of normal fetal brain tissue across gestation using diffusion magnetic resonance imaging (MRI); the secondary aim was to compare diffusion metrics in fetuses with normal brain development to those with isolated ventriculomegaly (VM), congenital heart disease and in infants born preterm. Fetal diffusion weighted imaging (DWI) was optimised, and a motion-corrected diffusion tensor imaging (DTI) technique was utilized to produce apparent diffusion coefficient (ADC) and fractional anisotropy (FA) values across gestation in normal fetal cohorts. Tract-based spatial statistics (TBSS) was utilised to analyse DTI in neonates with isolated VM compared to controls. Diffusion and volumetric MR data in preterm infants were analysed using an objective segmentation approach to characterise ex utero neurodevelopment, and to establish the effects of perinatal clinical factors on brain development. Normative ADC values were established in the fetal brain (n=52) across a large gestational age range. Increased ADC values were found in fetuses (n=24) and neonates (n=22) with isolated VM compared to controls; decreased FA was also demonstrated in neonates with VM. In preterm neonates (n=208), white and deep grey matter exhibited significantly increasing FA, and decreasing ADC, axial and radial diffusivity measures with increasing age at scan. DTI measures in the cortex significantly decreased with increasing age at scan; volume measures increased in all brain regions. Clinical factors including respiratory support and age at birth affected regional DTI and volumetric measures in preterm infants. FA values from a normal fetal cohort using motion-corrected DTI (n=26) were compared to preterm neonates (n=32) and significant differences were found. This thesis produced normal fetal diffusion data comparable to that produced in neonates. Quantifiable MR techniques can be used to explore the relationship between in utero and ex utero brain development and study alterations of normal fetal maturation.

Rhinovirus (RV) infections trigger exacerbations of chronic obstructive pulmonary disease (COPD) exacerbations and may precipitate secondary bacterial infections. Inhaled corticosteroids (ICS) are used commonly in COPD but are relatively ineffective in the context of virus-induced exacerbations and may also increase the risk of pneumonia. We hypothesised that, in a mouse model, ICS would suppress anti-viral and anti-bacterial immune responses leading to alteration of the airway microbiota and secondary bacterial infection following RV-induced exacerbation of COPD. Despite extensive optimisation, we were unable to define a representative mouse model of the deficient anti-viral and anti-bacterial responses that are indicative of human COPD. For this reason, and because of difficulties in measuring the airway microbiota in mice, we employed models of primary RV1B and Streptococcus pneumoniae infection as surrogates for viral exacerbation and bacterial colonisation in COPD. Fluticasone propionate (FP) administration prior to RV1B infection suppressed innate and adaptive immune responses leading to impaired virus control, in a dose dependent manner. This effect was causally related to suppression of type I interferon (IFN) as administration of recombinant IFN-β reconstituted IFN-stimulated gene expression and restored virus control. FP suppressed RV-induced airway inflammation but led to enhanced airway mucin production, effects that were unaltered by recombinant IFN-γ. FP administration also suppressed innate responses to S. pneumoniae including expression of anti-bacterial cytokines and cathelicidin-related anti-microbial peptide. High dose FP increased lung tissue bacterial loads with the opposite effect observed with lower dose FP despite similar anti-inflammatory effects. Our findings demonstrate beneficial anti-inflammatory effects of ICS during virus-induced COPD exacerbations but reveal some previously unrecognised detrimental effects including increased virus replication and enhanced mucin production. Additionally, we show that high dose ICS administration may increase bacterial loads and thus increase pneumonia risk but lower doses may conversely reduce bacterial loads and therefore could be safer in COPD.

Introduction: Obstructive sleep apnoea syndrome (OSAS) is common but remains underdiagnosed and is linked with several disease states and increased risk of mortality. The key symptom, excessive daytime sleepiness, is commonly measured with the Epworth Sleepiness Scale which is not always easily completed by patients. The aim of this thesis is to develop pictorial material for assessing sleepiness and risk of OSAS. Methods: Health literacy was measured in a sample sleep population and the Epworth Sleepiness Scale was investigated for ease of use. Images were developed to translate the Epworth into pictures and the response to pictures of 'driving while sleepy' was investigated in detail. A new tool, the pictorial Sleepiness and Sleep Apnoea Scale, was devised by adding four sleepiness images from the pictorial Epworth to four new images representing 'risk of OSAS'. Evaluations were made in two populations of the tool's potential in predicting those at risk of OSAS. Results: Poor or impaired health literacy was found in 16% of patients attending the sleep clinic. Evaluation of the Epworth Sleepiness Scale found that a third of new patients made quantifiable errors. A preference for the pictorial Epworth Sleepiness Scale was reported by 55% of users and a kappa statistic indicated good agreement between the pictorial and traditional Epworth Sleepiness Scale. Drivers were more inclined to record feeling sleepy if the image in Q8 depicted the sleepy person in the car as a passenger. In a sleep clinic population the pictorial Sleepiness and Sleep Apnoea Scale was slightly better at predicting disease than the Epworth. In a cardiac rehabilitation clinic use of the witnessed apnoea image from the pictorial Sleepiness and Sleep Apnoea Scale, along with the Epworth Sleepiness Scale, helped to identify symptoms suggestive of sleep apnoea in a third of those screened. When investigated with a sleep study, the prevalence of sleep-disordered breathing in this patient group was 14.8%. Conclusion: Pictorial tools for patients with potential obstructive sleep apnoea syndrome have clinical value and can help bridge the gap between poor or impaired health literacy and the material we use to assess sleepiness and likelihood of obstructive sleep apnoea syndrome.

Aspergillus species are opportunistic mould pathogens that can cause a wide variety of pulmonary diseases. They are mainly caused by Aspergillus fumigatus (Munoz et al., 2006). Germination of conidia in hosts with a susceptible immune system is the first step in Aspergillus infection, and invasive aspergillosis (IA) is a major cause of mortality in transplant patients. Due to the increased occurrence of IA in high-risk populations, prophylaxis against IA is important. Prophylactic prevention of life threatening infections with drugs is now the preferred clinical strategy for these patients rather than treating the disease after it has become established. Inhaled amphotericin B (AmB) has potential for preventing IA and it has been reported to decrease the incidence of IA in solid organ transplant patients (Arthur et al., 2004). I have generated a new respiratory formulation of AmB (AmB-PMA) with a commercially available high purity, low polydispersity and non-toxic poly methacrylic acid sodium salt (PMA-Na). The chemical synthesis was optimized and showed that AmB-PMA complex was effective against A. fumigatus and was also less toxic than Fungizone in-vitro. The efficacy of AmB-PMA was determined in-vivo in BALB/c and C57BL/6 mice. Mice were infected intranasally with A. fumigatus CEA10 and treated with AmB-PMA by the nasal and nebulisation routes. In this thesis, the optimum routes of in-vivo administration, dosing regimens and treatment frequency were defined. Disease progression in A. fumigatus-infected BALB/c and C57BL/6 mice was monitored by histology, qPCR analysis of chemokine and cytokine responses in mouse lung, colony forming unit (CFU) and qPCR for Aspergillus ribosomal 28S rRNA. The results showed a significant reduction in CFU and DNA fungal load in AmB-PMA infected mice as compared to infected untreated mice. In addition, AmB-PMA could also affect cytokine production by increasing IFN-γ production and reducing MIP-1β, TNF-α and IL-10 as compared to infected untreated mice. However, the increase in IFN-γ production was not statistically significant when compared to infected untreated mice. My thesis demonstrates that a new low-cost AmB based PMA-Na polymer antifungal drug can be successfully given by the aerosol route to immuno-suppressed mice by nebulisation to prevent IA.

COX is the rate-limiting enzyme in the conversion of arachidonic acid to the prostanoids. It is present in humans as two isoforms, COX-1 and COX-2. COX-1 is constitutively expressed and involved in homeostatic functions, while COX-2 expression is mostly limited to sites of inflammation. The precise function of COX isoforms is a subject of debate, particularly with respect to nonsterodial anti-inflammatory drug (NSAID)-induced toxicity. Toll-like receptors (TLRs) trigger the immune response following recognition of pathogen-associated molecular patterns (PAMPs), such as bacterial lipopolysaccharide (LPS), which activates TLR4. The role of COX-1 and COX-2 in responses to different TLRs is incompletely understood. The main focus of my PhD was to investigate COX-1 and COX-2 function in innate immunity, using TLR agonists in mice lacking COX-1 or COX-2. I also studied this in relation to lung inflammation using lung fibroblasts, and isolated pulmonary arteries to examine the effect of prostanoids on pulmonary vascular responses. Lung fibroblasts isolated from COX-2-/- mice were more proliferative than lung fibroblasts from wild-type or COX-1-/- mice. They also released greater amounts of cytokines when stimulated with various TLR agonists. Human lung fibroblasts were particularly sensitive to TLR3 agonists, and cytokine release was enhanced in the presence of NSAIDs. The effect of diclofenac may have been caused by inhibition of COX-related prostaglandin (PG) E2. In in vivo studies, Cox2 gene expression was strongly induced by TLR4 activation in all organs and less so by TLR3 activation, where induction was restricted to the spleen and stomach. Mice lacking COX-2 released higher amounts of anti-viral proteins following TLR3 activation with poly (I:C). This suggests that COX-2-specific NSAIDs may boost the anti-viral response, thus proving beneficial over traditional NSAIDs during viral infection. In the pulmonary vasculature, I found that most prostacyclin drugs were limited by actions on EP3 receptors, but that selective peroxisome proliferator-activated receptor (PPAR)β/δ agonists were active as dilators under all conditions. Deletion of COX-1 or COX-2 affected the ability of mouse pulmonary arteries to release endothelial-derived nitric oxide but not to respond to IP or PPARβ/δ agonists. Finally, activation of adenosine monophosphate kinase (AMPK) had no direct dilator effect on pulmonary vessels. However, preliminary data suggest that pretreatment of vessels with an AMPK activator enhanced (additive) dilation induced by PPARβ/δ, indicating a potential benefit in pulmonary arterial hypertension (PAH). In summary, my in vitro and in vivo experiments using mice lacking COX-1 or COX-2 provide insight into the role of COX in immunity and the pulmonary vasculature.