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.

No description available.

Transketolase is a TPP dependent enzyme that affects the availability of intermediates in both the Calvin cycle and non-oxidative pentose phosphate pathway. Previous studies have indicated that changes to the activity level of transketolase can limit growth and development as well as the production of isoprenoids, starch, amino acids and thiamine. The overall aim of this project was to further advance the understanding of the mechanism linking increased TK activity and thiamine metabolism. Nicotiana tabacum mutants with increased total transketolase activity ~ 2 to 2.5 fold higher than WT plants were shown to have a reduced growth and chlorotic phenotype. In seedlings, these phenotypes were attributed to a reduction in seed thiamine content. Imbibition of TKox seeds in a thiamine solution produced plants that were comparable to WT plants. However, the chlorotic but not growth phenotype was found to return unless the plants underwent irrigation with a thiamine solution indicating that TKox plants are unable to produce sufficient quantities of thiamine to meet demand. Furthermore, the application of deoxy-xylulose-5-phosphate was also found to be able to partially complement the phenotype suggesting that flux from the C3 cycle into the non-mevalonate pathway is being affected. Analysis of thiamine and TPP levels demonstrated that TKox plants were deficient in thiamine but not TPP in the majority of cases. In plants that had begun to flower, TKox lines had reduced thiamine and TPP levels in the 20th fully open leaf compared to the same leaf in WT plants. Furthermore, sampling of leaf tissue from both WT and TKox seedlings at the same developmental stage indicated that high levels of TK protein may lead to the accumulation of TPP in these areas causing a reduction in the levels of thiamine and TPP in the rest of the plant thereby limiting growth and development.

A change in environmental conditions often leads to changes of physiology and biochemical composition of microalgae. Temperature and light intensity are important environmental factors regulating the growth of microalgae. In this study, the elemental and biochemical composition were measured in 2 marine microalgae under different temperatures and light intensities in nutrient replete and deplete conditions. The effect of temperature was observed in the marine haptophyte Emiliania huxleyi (CCMP 1516) at nutrient replete semi-continuous cultures. Triplicate cultures were incubated different temperature from 14 to 22oC and under photon flux densities (PFD) 600 μmol photons m−2 s−1. The growth rate (GR) of E. huxleyi increased with temperature. Cell volume varied with temperature, being about 40% smaller at higher temperature (22oC). Cellular chlorophyll a (chl a), nitrogen, phosphorus, and carbon contents were also lower at 22oC than other temperatures. Protein, total amino acids from free and combined amino acid, and total pigments [mol accessory pigment (mol chl a)-1] were decreased with increasing temperature; however, the opposite response was observed in fatty acids. In addition to the effect of combined temperature and light intensity was investigated in the marine diatom Thalassiosira weissflogii (CCMP 1056) under nutrient-limited semi-continuous cultures. The cultures were incubated at 16 and 26°C and PFD of 50 ± 10 (low light; LL) and 500 ± 10 (high light; HL) μmol photons m−2 s−1. HL incubated-cultures were diluted at 50% day-1 and LL incubated-cultures were diluted at 25% day-1. The GR were largely set by dilution rate (nitrogen limitation), but not by temperature and irradiance. The GR were around 0.72 d-1 in HL placed-cultures and 0.32 d-1 in the LL placed-cultures. Temperature did not affect mean cell size, whereas mean cell size decreased with increased irradiance by 20 to 29 %. Both temperature and irradiance influenced cellular chl a, carbon and chl a specific light absorption. Cellular nitrogen and phosphorus varied with temperature and irradiance. Protein, total amino acid (free and combined amino acid) and total fatty acid increased with increased temperature and irradiance; however, the opposite response was found in carbohydrate. Overall, temperature and light affected elemental and biochemical composition in 2 marine microalgae. Both relationship of the chlorophyll (chl):carbon (C) and RNA:protein ratio and growth rate in E. huxleyi under variable temperature positively supported a bio-optical and growth rate hypothesis respectively. However, the opposite response was found in T. weissflogii. Instead the C:chl and RNA:protein ratio and growth rate in T. weissflogii under variable irradiance positively supported a bio-optical and growth rate hypothesis.

Aggregation of α-synuclein (α-syn) into toxic fibrils is a pathogenic hallmark of Parkinson’s disease (PD). This research aimed to develop peptides capable of inhibiting α-syn aggregation using a semi-rational design combined with a multiplexed intracellular Protein-fragment Complementation Assay (PCA) library screening system. Successfully selected peptides must bind to full length α-syn and lower its toxicity to confer bacterial growth. PCA selected peptides were characterized using several biophysical assays and a cell viability assay. The peptides were screened using library templates based on α-syn71-82 initially and later on the α-syn45-54 region in which many key mutations associated with early onset PD are found. In both cases we targeted the peptide libraries at the wild type protein or again by using mutated versions of α-syn. Results demonstrate that some of those selected peptides had the effect of delaying or even preventing the aggregation process, with others providing more subtle effects in reversing the fully formed amyloid fibrils. PCA peptides selected against 71-82 region of wild type α-syn showed a moderate level of efficacy whereas against mutants, it showed a low level of efficacy in inhibiting amyloid fibril formation. In the final part of the study, the peptide selected against 45-54 region of wild type α-syn was capable of preventing the aggregation and reducing the amyloid cytotoxicity at an equimolar ratio. We have thus demonstrated that the PCA strategy can be used as a generalised method for deriving peptide antagonists of α-syn aggregation, together with a new region; α-syn45-54 as an inhibitor target and produced a peptide inhibitor expected to provide a scaffold for future drug candidates to slow or even prevent the onset of PD.