Background: Craniofacial morphology has been reported to be highly heritable, but little is known about which genetic variants influence normal facial variation in the general population. Aim: To identify facial variation and explore phenotype-genotype associations in a 15-year-old population (2514 females and 2233 males). Subjects and Methods: The subjects involved in this study were recruited from the Avon Longitudinal Study of Parents and Children (ALSPAC). Three-dimensional (3D) facial images were obtained for each subject using two high-resolution Konica Minolta laser scanners. Twenty-one reproducible facial soft tissue landmarks and one constructed mid-endocanthion point (men) were identified and their coordinates were recorded. The 3D facial images were registered using Procrustes analysis (with and without scaling). Principal Component Analysis (PCA) was then employed to identify independent groups ‘principal components, PCs’ of correlated landmark coordinates that represent key facial features contributing to normal facial variation. A novel surface-based method of facial averaging was employed to visualize facial variation. Facial parameters (distances, angles, and ratios) were also generated using facial landmarks. Sex prediction based on facial parameters was explored using discriminant function analysis. A discovery-phase genome-wide association analysis (GWAS) was carried out for 2,185 ALSPAC subjects and replication was undertaken in a further 1,622 ALSPAC individuals. Results: 14 (unscaled) and 17 (scaled) PCs were identified explaining 82% of the total variance in facial form and shape. 250 facial parameters were derived (90 distances, 118 angles, 42 ratios). 24 facial parameters were found to provide sex prediction efficiency of over 70%, 23 of these parameters are distances that describe variation in face height, nose width, and prominence of various facial structures. 54 distances associated with previous reported high heritability and the 14 (unscaled) PCs were included in the discovery-phase GWAS. Four genetic associations with the distances were identified in the discovery analysis, and one of these, the association between the common ‘intronic’ SNP (rs7559271) in PAX3 gene on chromosome (2) and the nasion to mid-endocanthion 3D distance (n-men) was replicated strongly (p = 4 x 10-7). PAX3 gene encodes a transcription factor that plays crucial role in fetal development including craniofacial bones. PAX3 contains two DNA-binding domains, a paired-box domain and a homeodomain. The protein made from PAX3 gene directs the activity of other genes that signal neural crest cells to form specialized tissues such as craniofacial bones. PAX3 different mutations may lead to non-functional PAX3 polypeptides and destroy the ability of the PAX3 proteins to bind to DNA and regulate the activity of other genes to form bones and other specific tissues. Conclusions: The variation in facial form and shape can be accurately quantified and visualized as a multidimensional statistical continuum with respect to the principal components. The derived PCs may be useful to identify and classify faces according to a scale of normality. A strong genetic association was identified between the common SNP (rs7559271) in PAX3 gene on chromosome (2) and the nasion to mid-endocanthion 3D distance (n-men). Variation in this distance leads to nasal bridge prominence.

In recent years, there has been an increased emphasis to treat patients with parenteral nutrition (PN) at home in an attempt to reduce costs and improve clinical outcomes. This increased interest in home parenteral nutrition (HPN) has stimulated researchers to investigate potential sources of instability. One of the more unstable groups in PN is micronutrients, which can be divided into two groups: vitamins and trace elements. This thesis investigates the effects of artificial light sources (cool white, warm white and UVA) on the physicoP chemical stability of vitamins. Vitamins were chemically analysed using a novel stability indicating HPLC assay that could quantify five waterPsoluble and three fatPsoluble vitamins simultaneously in one run. Samples were physically analysed by visual analysis, microscope analysis, laser diffraction, pH and osmolality. Initial experiments investigated the physicoPchemical stability of vitamins exposed to artificial light sources over a period of 24 hours. In cool and warm white light there was approximately a 20% loss of riboflavin and 10% loss of retinol. In UVA light there was approximately a 20% loss of retinol. All other analysed vitamins were stable over the time period to these artificial light sources. Further experiments investigated these conditions following 6 days of storage between 2P8 C. These experiments revealed similar results in the three types of artificial light source. v The protective effects of lipid emulsions on retinol were then investigated in containers and administration sets. Samples containing lipid emulsions in syringes and administration sets had a statistically significant increase in retinol stability. Nevertheless, degradation in excess of 10% still occurred in these groups. The protective mechanism of lipid emulsions was primarily though to be a result of light obscuration. However, soybean oil (SBO), a clear liquid, provided unexpected obscuration of UVA light suggesting it may reflect or absorb damaging rays thereby improving retinol stability.

Breast cancer is the most common cancer in the UK, but despite recent encouraging increases in survival rates, is still the second most common cause of cancer death in women in the UK. To try to reduce systemic toxicity during treatment of cancer patients, a plethora of targeted therapies are in various stages of development. PARP inhibitors have been shown to be particularly effective in BRCA-deficient cells, making them a contender as a personalised therapy. One of the challenges for targeted therapies is that of resistance, which limits the extent of benefit to the patient. The work described in this study continues previous work within our laboratory, investigating the PARP inhibitor olaparib in a conditional mouse model of BRCA2-mutated human breast cancer. The data presented here establish a correlation between histological tumour type and response to olaparib therapy, with poor responders classified exclusively as mesenchymal-like metaplastic spindle cell carcinomas (MSCC). This suggests that further patient stratification is required when deciding on whether this therapy may be suitable, and may explain why not all patients with BRCAmutated breast cancer have benefitted from olaparib therapy in current clinical trials. Investigation of olaparib resistance in this study indicated that several currently proposed mechanisms of resistance were not pertinent to the Brca2/p53 model, hence novel mechanisms were sought. Histopathological analysis of resistant tumours showed that the majority were MSCCs, representing a significant change in the proportion compared to an untreated cohort. Other resistant tumour types had epithelial morphology, but showed an increase in expression in some mesenchymal-like genes compared to untreated cohorts, suggesting that mesenchymal features may be important in causing resistance to olaparib. A similar tumour model, incorporating the additional deletion of E-cadherin, was used to investigate whether lack of this protein in tumours affected response and resistance to olaparib therapy. Loss of Cdh1 led to an increase in invasive ductal carcinomas of no special type (IDC-NST) and the absence of MSCCs, suggesting that genetic loss of expression does not drive the formation of mesenchymal-like tumours. Correlating with this, loss of E-cadherin did not drive epithelial-to-mesenchymal transition in these tumours and had no effect on response to olaparib therapy or resistance to the inhibitor. Taken together, the data presented in this thesis suggest that MSCCs have an intrinsic resistance to olaparib therapy, and tumours which initially respond to olaparib therapy harness or acquire certain mesenchymal characteristics in order to develop resistance during treatment.

Chronic obstructive pulmonary disease (COPD) is an umbrella term that encompasses chronic bronchitis, emphysema and airway obstruction. COPD patients are also prone to acute exacerbations (AECOPD) caused primarily by viral and bacterial infection, which leads to an increase in inflammation, a worsening of symptoms and can lead to death. There is an unmet clinical to better understand and treat AECOPD as well as COPD in general, but this is hindered by unreliable animal models of COPD and AECOPD. The aim of this thesis was to establish an animal model of COPD that could be exacerbated by an infectious agent. Firstly an LPS model of COPD was established in the guinea pig, which resulted in a macrophage and neutrophil inflammatory profile, emphysematous changes, a decrease in lung function and partial steroid insensitivity that could be partially reversed with low dose theophylline. Human parainfluenza 3 virus failed to cause any infection in the guinea pig, so a model of AECOPD could not be established in this model. A chronic cigarette smoke model in the mouse was established, which again demonstrated a similar phenotype to COPD. This model was able to be exacerbated by the bacteria nontypeable Haemophilus influenza (NTHi) with increases in neutrophils and the neutrophil chemoattractant CXCL1. However, it was also observed that while NTHi could exacerbate the model, responses to NTHi in cigarette smoke challenged mice compared to sham challenged animals were impaired, with significant decreases in CXCL8, TNF-α, IFN-γ and IL-10. This impairment was also observed in monocyte derived macrophages (MDMs) challenged with cigarette smoke extract (CSE) with significant impairment of Il-1β, while chronic LPS challenge also impaired Il-6 and phagocytosis. The data in this thesis highlights a possible increase in steroid responses by low dose theophylline in an LPS model in the guinea pig. It has also demonstrated chronic cigarette smoke exposure in the mouse can be exacerbated by NTHi, however the inflammatory response is impaired compared to sham challenged animals suggesting that cigarette smoke impairs the innate immune response. MDMs also demonstrated an impaired response to NTHi after CSE or LPS challenge.

Irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD) are both chronic relapsing intestinal disorders. Their symptom profiles overlap in terms of abdominal discomfort and altered bowel habit. Meta-­‐analysis of patients with IBD demonstrates that 25-­‐46% of those in clinical remission have symptoms compatible with IBS. These patients report lower quality of life scores compared to their asymptomatic counterparts. There is uncertainty as to the cause of these symptoms, and concern for the influence they may exert on clinical management. The work described in this thesis investigated the nature of IBS-­‐type symptoms occurring in patients with IBD, examined potential diagnostic tools to distinguish between the respective conditions, and conducted a therapeutic trial for the management of functional symptoms in this setting. IBS-­‐type symptoms were observed to occur more commonly in female IBD patients, were associated with high anxiety levels, and occurred in patients with no active inflammation as confirmed by a normal faecal calprotectin level. These findings are characteristic of irritable bowel syndrome, and suggest that this disorder may cause persistent symptoms during IBD remission. Two potential biomarkers of IBS were investigated. The first explored a hypothesis that IBS may be a systemic condition caused by the absorption of toxic metabolites produced by the bacterial fermentation of dietary carbohydrates. This mechanism would potentially explain both the gastrointestinal and the systemic symptoms that are observed in patients with IBS. It was proposed that toxic metabolites may covalently modify albumin in patients with IBS, however on investigation of this theory there was no significant difference observed between the plasma samples of IBS patients, IBD patients and healthy controls. The presence of systemic symptoms in patients with IBS and IBD was associated with higher anxiety levels. Cognitive function was also assessed as a potential biomarker of IBS following anecdotal reports that IBS patients experience impaired concentration. However no significant difference between IBS patients, IBD patients, and healthy controls was identified. Concurrent mood disorders, in particular depression, were associated with impaired performance of specific tasks in patients with IBD. A randomised-­‐controlled trial of a mindfulness-­‐based psychological intervention was performed in IBD patients with IBS-­‐type symptoms or high perceived stress levels. Sub-­‐group analysis demonstrated a significant improvement in quality of life in the intervention group in those patients who were experiencing IBS-­‐type symptoms. Overall, these findings support the theory that IBS can cause persistent symptoms in IBD patients who are in remission. However, until the molecular mechanisms underlying IBS are identified and reliable biomarkers are developed, a systematic diagnostic approach is required to evaluate these patients. IBS-­‐type symptoms in IBD patients represent a therapeutic target to improve quality of life and further trials of psychological intervention, medication and dietary modification are required.

The survival of microorganisms on surfaces is well documented, potentially acting as a reservoir for the dissemination of healthcare-associated infections (HCAIs). Antimicrobial surfaces aim to control surface bioburden and lower HCAI rates. The existing antimicrobial surface efficacy test (JIS Z 2801) is an initial screening test; however, its set up (35°C, >90% relative humidity (RH)) bears little relationship to conditions in practice. This study aimed to develop new surface efficacy tests using wet and dried microbial inocula, reflecting conditions within a healthcare setting. Changes in surface RH, temperature and bioburden were measured over one year at a hospital, allowing realistic parameters to be set for the new tests. Wet and dry inocula tests were developed and validated to mimic aerosol deposition and dry-touch contamination on surfaces, respectively. Aerosols of S. aureus, A. baumannii and B. subtilis spores and dry inocula of S. aureus and A. baumannii were tested against copper alloys and control stainless steel surfaces. Surviving bacteria were enumerated after varying contact times, and under in-use and JIS Z 2801 test conditions. FACS experiments were conducted to understand the mechanism of action of copper against dried microbial inocula. Wet inoculum testing showed copper alloys presented significantly reduced activity against S. aureus aerosols at in-use conditions (>4 log10 after 60 min) compared to JIS Z 2801 test conditions (>4 log10 after 30 min). A >4 log10 reduction in A. baumannii was observed within 30 min but copper alloys were not sporicidal at in-use conditions. Dry inoculum testing showed a <2 log10 reduction in S. aureus and A. baumannii after 24 h at in-use conditions with potential mechanisms of action including; membrane damage, DNA damage and arrested cellular respiration. The new tests developed provide realistic, second-tier tests to the JIS Z 2801. Copper was antimicrobial against both wet and dry inocula but was overall more efficacious against a wet inoculum, which suggests a liquid interface enhanced antimicrobial activity. It is recommended that antimicrobial surfaces are tested under in-use conditions against both wet and dry inocula to confidently predict their performance in practice.

Crucial biological processes are often dictated by the action of proteins, including cell growth, intercellular communication and apoptosis. The behaviour and function of proteins, and other important bio-macromolecules, is inherently linked to the 3D con-formation of the polypeptide and the range of dynamics within the structure. The rapidly developing tool, solid-state NMR, is uniquely placed to structurally probe large, non-crystalline macromolecules, such as proteins, and provide data at atomic-level resolution. Amyloid diseases, such as Alzheimer's disease and Parkinson's disease, are linked to neuronal damage caused by toxic species which occur through misfolding and aggregation of naturally expressed proteins. Aggregation of the 36-43 residue protein amyloid-beta (A ) is thought to be involved in the pathology of Alzheimer's disease. In this thesis, solid-state NMR is used to obtain 1 H,15 N and 13 C chemical shifts for U-[13 C,15 N] A 1-40 aggregates formed in the presence of copper, a metal found in abnormally high concentrations in amyloidogenic plaques in Alzheimer's-disease-afflicted patients' brains via post-mortem examination. A suite of 2D13 C-13 C DARR and15 N-13 C DCP experiments alongside 2D and 3D X-1 H,15 N-1 H-1 H and13 C-15 N-1 H. Inverse Detection experiments employed at 100 kHz MAS frequency is employed to obtain chemical shift values. TALOS-N provides torsion angle restraints and secondary chemical shift analysis is performed to identify turn and -sheet regions. The results are compared to previously published models, and indicate the sample is similar to a brain-derived fibrillar structure. 2D13 C-13 C DARR experiments are also performed on selectively labelled samples of A 1-42 with cysteine replacements at locations A21 and A30. This cross-linking mechanism between C21 and C30 has been shown to stabilise an oligomeric form, thought to be the most toxic, on the aggregatory pathway to fibrils. The labels allow detailed characterisation of the turn region between residues 22 and 29. The incorporation of the oligomers into lipid membranes was studied by 1D31 P NMR, specifically to investigate the effect of the oligomers on the stability of the membranes and the effect of cholesterol and curcumin, a potentially therapeutic compound currently of high interest, on this de-stabilisation. Work with collaborators has enabled the oligomeric form to be modelled and the results indicate these toxic oligomers form a hexamer barrel structure, with the hydrophobic sidechains contained within the barrel. The secondary structure of the oligomer is shown to contain 3 or 4 -sheets; with an extended -sheet region between K16-V24, 1 or 2 -sheets between N27 and V36, and then a short -sheet between V39 and A42. A method for inter-molecular –sheet interaction in the hexamer is postulated. A method for further aggregation from this oligomeric form to a protofibrillar form has also been suggested, thus enabling a claim to be made for an on-pathway nature for this oligomeric form.

Conventional communication networks route data packets in a store-and-forward mode. A router buffers received packets and forwards them intact towards their intended destination. Network Coding (NC), however, generalises this method by allowing the router to perform algebraic operations on the packets before forwarding them. The purpose of NC is to improve the network performance to achieve its maximum capacity also known as max-flow min-cut bound. NC has become very well established in the field of information theory, however, practical implementations in real-world networks is yet to be explored. In this thesis, new implementations of NC are brought forward. The effect of NC on flow error control protocols and queuing over computer networks is investigated by establishing and designing a mathematical and simulation framework. One goal of such investigation is to understand how NC technique can reduce the number of packets required to acknowledge the reception of those sent over the network while error-control schemes are employed. Another goal is to control the network queuing stability by reducing the number of packets required to convey a set of information. A custom-built simulator based on SimEvents® has been developed in order to model several scenarios within this approach. The work in this thesis is divided into two key parts. The objective of the first part is to study the performance of communication networks employing error control protocols when NC is adopted. In particular, two main Automatic Repeat reQuest (ARQ) schemes are invoked, namely the Stop-and-Wait (SW) and Selective Repeat (SR) ARQ. Results show that in unicast point-to point communication, the proposed NC scheme offers an increase in the throughput over traditional SW ARQ between 2.5% and 50.5% at each link, with negligible decoding delay. Additionally, in a Butterfly network, SR ARQ employing NC achieves a throughput gain between 22% and 44% over traditional SR ARQ when the number of incoming links to the intermediate node varies between 2 and 5. Moreover, in an extended Butterfly network, NC offered a throughput increase of up to 48% under an error-free scenario and 50% in the presence of errors. Despite the extensive research on synchronous NC performance in various fields, little has been said about its queuing behaviour. One assumption is that packets are served following a Poisson distribution. The packets from different streams are coded prior to being served and then exit through only one stream. This study determines the arrival distribution that coded packets follow at the serving node. In general this leads to study general queuing systems of type G/M/1. Hence, the objective of the second part of this study is twofold. The study aims to determine the distribution of the coded packets and estimate the waiting time faced by coded packets before their complete serving process. Results show that NC brings a new solution for queuing stability as evidenced by the small waiting time the coded packets spend in the intermediate node queue before serving. This work is further enhanced by studying the server utilization in traditional routing and NC scenarios. NC-based M/M/1 with finite capacity K is also analysed to investigate packet loss probability for both scenarios. Based on the results achieved, the utilization of NC in error-prone and long propagation delay networks is recommended. Additionally, since the work provides an insightful prediction of particular networks queuing behaviour, employing synchronous NC can bring a solution for systems’ stability with packet-controlled sources and limited input buffers.

Methylated amines, such as trimethylamine (TMA) and trimethylamine N-oxide, are nitrogenous compounds that are thought to be ubiquitous in the marine environment. TMA is a product of the anaerobic degradation of quaternary amines, such as glycine betaine and choline. Through a set of complex chemical and biological interactions, methylated amines play a role in regulating the planet’s climate. Microbial degradation of methylated amines is thought to be a sink for these compounds in the marine environment, however some of the key genes and enzymes responsible for the degradation of methylated amines are unknown. Using Ruegeria pomeroyi DSS-3 as the model organism, the key enzymes for the uptake and catabolism of trimethylamine N-oxide were identified and it was discovered that these genes and enzymes are highly expressed in the seawater, as revealed by the re-analysis of a number of recent metatranscriptomic and metaproteomic datasets. Again using R. pomeroyi as the model organism, it was shown that trimethylamine and trimethylamine N-oxide can be oxidised to CO2 to generate reducing equivalents and ATP. The generation of this reducing power results in a number of physiological benefits which are further discussed in detail. It was determined that bacteria possessing trimethylamine monooxygenase, the key enzyme required for the oxidation of TMA could also oxidise the reduced sulfur compound, dimethylsulfide, when supplemented with methylated amines. The ecology of methylated amine-utilising bacteria was investigated using a newly designed primer set targeting the trimethylamine N-oxide demethylase. The results are presented in detail within. The key genes and enzymes essential for the catabolism of the quaternary amine, choline were also discovered, again using R. pomeroyi as the model organism. The occurrence of genes required for the catabolism of choline are widespread among certain groups of marine bacteria known to interaction with eukaryotic biota, suggesting that this compound may be an essential nutrient for these organisms.

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