Urinary pharmacokinetic methods have been introduced to identify the relative lung and systemic availability of inhaled drugs but have not been extended to corticosteroids. The main aims were to validate the urinary pharmacokinetic methodology when applied to inhaled beclometasone dipropionate (BDP), demonstrate the usefulness of the method and compare its indices to the in-vitro characteristics of the emitted dose. A simple and sensitive LC-MS method for quantifying BDP and its metabolites in methanol (for in-vitro studies) and urine samples was identified and validated in accordance with the FDA and ICH guidelines. The accuracy, precision, and recovery of the method were within acceptable limits (±15%). Twelve healthy volunteers completed the in-vivo urinary pharmacokinetic validation of the methodology to determine the relative lung bioavailability of inhaled beclometasone following inhalation. Twelve healthy volunteers received randomised doses, separated by >7 days, of 2000μg BDP solution with (OralC) and without (Oral) 5g oral charcoal, ten 100μg inhalations from a Qvar® Easi breathe metered dose inhaler (pMDI) with (QvarC) and without (Qvar) oral charcoal and eight 250μg inhalations from a Clenil® pMDI (Clenil). Subjects provided urine samples at 0, 0.5, 1, 2, 3, 5, 8, 12, and 24 hours post study dose. Urinary concentrations of BDP and its metabolites, 17-beclometasone monopropionate (BMP) and beclometasone (BOH) were measured. No BDP, BMP, or BOH was detected in any samples post OralC dosing. Post oral dosing, no BDP was detected in any of the urine samples and no BMP or BOH was excreted in the first 30 minutes. Significantly more (p<0.001) BDP, BMP and BOH was excreted in the first 30 minutes and cumulative 24 urinary excretions post Qvar and Clenil compared to Oral. Using 30 minute urinary excretion the mean ratio (90% confidence interval) for Qvar compared to Clenil was 231.4 (209.6, 255.7). The results confirm that the relative lung and systemic bioavailability can be identified from urinary excretion of BDP and its metabolites over the first 30 minutes and 24 hours respectively. The 2-fold difference between Qvar and Clenil is consistent with related clinical and pharmacokinetic studies. The low inter and intra-subject variability of the study confirms the reproducibility of this method. When compared to the in-vitro aerodynamics characteristics of the emitted dose, using standard compendial methods, the in-vivo indices showed a relationship to the fine particle dose (FPD) and the emitted dose (ED), respectively. The application of this urinary pharmacokinetic method was demonstrated in further studies to compare the effect of different spacers and different washing methods on the in-vivo drug delivery post inhalation from Clenil and Qvar inhalers in healthy volunteers. In addition, the in-vitro aerodynamic particle size distribution of the same inhalation methods has been investigated using the Andersen Cascade Impactor according to the standard compendial methodology. Urinary excretion, using 24 hour excretion, revealed that relative bioavailability to the body was reduced with spacers for both inhalers. There was no increase in the relative lung bioavailability when Qvar was used with spacers. When Clenil was attached to a spacer (either AeroChamber or Volumatic) the relative lung bioavailability was significantly greater only if the spacers were not rinsed after washing with detergents. Consistent with the above study there were correlations between the in-vivo urinary indices and the in-vitro characteristics of the emitted dose. The thesis highlights the extension of the urinary pharmacokinetic method to inhaled beclometasone dipropionate and provides further evidence of in-vitro in-vivo correlations between the urinary methodology and the aerodynamic characteristics of the emitted dose.

This thesis illustrates the behaviour of cellulose ethers during powder processing, compaction and drug release, as these are frequently employed in the fabrication of compressed hydrophilic matrices. The handling operations can give rise to the electrification of powder particles, which can affect the end product‘s quality. Controlling the parameters which can dictate the quality of compressed matrices is an ambition inherent in the development of pharmaceutical formulations. Thus, the aims and objectives of this thesis were to firstly study the electrostatic, surface adhesion, dissolution and compaction properties of plain polymers and model drugs. Secondly, binary mixtures of fixed drug to polymer ratios were made in order to investigate the effect of polymer concentration and physico-chemical attributes (particle size, chemistry and viscosity) on the tribo-electric charging, surface adhesion (SA), swelling, erosion, drug release kinetics and compaction properties of model drugs. It can be discerned that the both drugs charged negatively, whereas the methylcellulose (MC) and hydroxypropyl methylcellulose (HPMC) particles charged positively. The physico-chemical properties associated with MC and HPMC, such as particle size, chemical heterogeneity and molecular size of cellulose ethers all have a significant effect on charging and adhesion behaviour of plain MC and HPMC particles. Moreover, the concentration, particle size, chemical heterogeneity and molecular size of MC/HPMC all significantly affect the charging and SA propensity of the model drugs studied. The swelling and dissolution results confirm that the extent and rate of swelling, swelling exponent, dissolution rate and drug release kinetic parameters were affected by physico-chemical attributes (concentration, particle size, substitution and viscosity) of MC/HPMC and drug solubility. The mechanism of swelling and drug release was found to be anomalous. However, it inclined towards more diffusion-oriented swelling/drug release with higher MC/HPMC levels, viscosity, Hpo/Meo substitution ratios, drug solubility but smaller MC/MC particle size. The matrix erosion results obtained from newly developed phenol-sulphuric acid assay (PSA) method confirmed that the solubility of the drug, and levels of HPMC in a particular matrix tablet, significantly affect the matrix erosion rate and the results were similar to those determined using the much more labour-intensive gravimetric method. Moreover, the combination of conventional UV drug analysis technique and PSA assay can be used to simultaneously quantify the matrix erosion, polymer dissolution and drug release kinetics in a single set of experiments avoiding the need for separate studies. The compaction results confirmed that the FBP has poor compaction as compare to THP. The particle size, substitution ratios and molecular size of MC/HPMC affect the compaction and consolidation behaviour of plain MC/HPMC compacts. Furthermore, it can be noticed that the concentration and physico-chemical attributes (particle size, chemistry and molecular size) of MC/HPMC have a significant influence on the densification and consolidation process of hydrophilic matrices. In summary, the information obtained can be used in the future to develop and adopt strategies for development and further optimization of compressed hydrophilic matrices.

Tree litter is a key basal resource in temperate deciduous woodlands and streams that drain them. Litter decomposition promotes carbon and nutrient cycling, fueling woodland food webs. Research to date has not thoroughly explored how ongoing environmental changes affect this process. This study used microcosm and field experiments to investigate how multiple stressors (urban pollution, elevated atmospheric CO2 and stream acidification) affected litter chemical composition, invertebrate consumption, and terrestrial and aquatic mass loss. Leaf litter chemical composition differed between ambient- and elevated-CO2 litters, and between rural and urban litters, but the direction of these responses was complex and differed between experiments. In microcosms, leaf litter consumption by terrestrial and aquatic invertebrate detritivores was species-specific. After exposure to a woodland floor or headwater streams, urban litter broke down faster than rural litter, while CO2 treatment did little to influence mass loss. The abundance, richness and diversity of terrestrial and aquatic invertebrates associated with leaf litter generally declined from 28 to 112 days in the field. Taxon richness and diversity were generally higher in elevated- than ambient-CO2 leaf litter through time, while urban leaf litter had greater diversity than rural litter after 112 days only. Abundance was greater in the circumneutral than the acid stream. Aside from leaf litter, small woody debris was also affected by CO2 treatment: elevated-CO2 twigs had a greater concentration of nitrogen and lignin, and broke down faster than ambient-CO2 twigs on a woodland floor and in headwater streams. This work highlights the complexity of invertebrate- and ecosystem-scale responses to the effects of multiple environmental stressors, with implications for nutrient cycling and food webs. Urban pollution may have a greater influence on litter chemical composition than CO2 treatment, while effects of growth condition may be more important than stream acidity in influencing decay and invertebrate communities.

The production of antibiotics, antifungal, enzymes and anti-tumoral agents of economical importance in Streptomyces lividans occurs during the copperdependant morphological switch step of its distinct lifecyle. However, copper can be toxic to the cell if it is not well regulated, affecting copper homeostasis. The regulation of the concentrations of copper is performed by CsoR, a Cu(I)-metalloregulator of the CsoR/RcnR family, on upon Cu(I) binding, it dissociates from its own csoR regulon. This event leads to Cu(I) to be trafficked outside the cytosol via a CopZ chaperoning system. Although Cu(I)-bound structures of CsoR/RcnR family members have been solved, its still unclear how CsoR dissociates from DNA upon Cu(I) binding and how promiscuous its metal ion binding site is, i.e., if it other metals bind and trigger a similar allosteric response as Cu(I) does. Through a structural and kinetic approach, these questions were explored on this work, in order to give insights at atomic and mechanistic level in this metalloregulator family. A novel CsoR structure at pH 6 revealed a striking quasi-Cu(I) bound state, which provides important information on how CsoR may bind to DNA. A mechanism of metal binding to Cu(I) and a non-cognate metal, Ni(II) is proposed, with novel insights on metal selectivity and specificity in this poorly understood family of bacterial metalloregulators.

Direct Infusion (DI) Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) is becoming a popular measurement platform in metabolomics. This thesis aims to advance the data processing and analysis pipeline of the DI FT-ICR based metabolomics, and broaden its applicability to a clinical research. To meet the first objective, the issue of missing data that occur in a final data matrix containing metabolite relative abundances measured for each sample analysed, is addressed. The nature of these data and their effect on the subsequent data analyses are investigated. Eight common and/or easily accessible missing data estimation algorithms are examined and a three stage approach is proposed to aid the identification of the optimal one. Finally, a novel survival analysis approach is introduced and assessed as an alternative way of missing data treatment prior univariate analysis. To address the second objective, DI FT-ICR MS based metabolomics is assessed in terms of its applicability to research investigating metabolomic changes occurring in liver grafts throughout the human orthotopic liver transplantation (OLT). The feasibility of this approach to a clinical setting is validated and its potential to provide a wealth of novel metabolic information associated with OLT is demonstrated.

The research presented here constitutes the latest advances in the use of the Antarctic bivalve mollusc Aequiyoldia eightsii as a sclerochronological proxy for Antarctic coastal waters. A. eightsii has the potential to provide annually-resolved records for the Antarctic shallow waters beyond the beginning of the instrumental record. A comprehensive study of A. eightsii shell growth was carried out, which highlighted two ontogenetic trends: i) negative exponential and ii) a quasi-cyclic trend of a period of nine years, which may relate to allocation of energetic resources. Using crossmatching techniques, four chronologies from adjacent locations near Rothera Station (West Antarctic Peninsula, WAP) and an additional chronology from historical samples collected near Signy Station (South Orkney Islands, SOIs) were developed. Additionally further work on a pre-existing chronology from the SOIs is presented. Instrumental records and climatic indices were analysed to determine environmental variability and the factors controlling shell growth. Seawater temperature and fast-ice duration seem to be the main environmental drivers of A. eightsii shell growth. Shell growth of South Cove (WAP) specimens seem to better reflect to environmental conditions recorded in the Rothera Biological Time Series. Stable oxygen and carbon isotope (δ18O and δ13C) records were developed from shell carbonate material; these showed sub- and inter-annual variability and all specimens showed similar trends in δ18Oshell and δ13Cshell, with the exception of a deep-collected specimen of the SOIs, which showed much reduced interannual variability and a stronger δ13Cshell negative trend with ontogenetic age. Additionally, stable isotope fractionation depending anatomical part of the shell (anterior, ventral and posterior) was studied, which showed intra-increment variability in δ18Oshell and δ13Cshell. The present work constitutes a comprehensive calibration of A. eightsii as a sclerochronological proxy for Antarctic shallow coastal waters, which will help to expand our understanding of climate trends in the region.

The main objective of this research was to understand phenomena occurring during food digestion and nutrients absorption in the small intestine from an engineering perspective. Intestinal flow and mixing processes were simulated using a dynamic in vitro Small Intestine Model (SIM). Of particular interest was to study the effect that mixing and food formulation has on glucose absorption and starch hydrolysis. Results showed the effect of segmentation motion on nutrient delivery to the intestinal wall as a consequence of changes in the mass transfer coefficient. This is most likely due to the increased mixing in the SIM. Experiments of starch digestion with and without the presence of guar gum have shown that viscous fibres reduce the rate of starch digestion and glucose absorption by impairing mixing and reducing diffusion within the fluid. Similarly, use of particulate systems demonstrated a significant effect on the delivery rates. Flow visualization techniques used for studying flow paths in the SIM showed that this in vitro model reproduces the characteristic flow events and mixing found in the small intestine in vivo. This research provides insights into the role of mixing on enhancing mass transfer on the course of digestion-absorption processes and also the action of viscous polysaccharides on the delay of glucose absorption in the small intestine. The end findings resulted in a better understanding of the factors which control the development of new functional food that could be applied both in academia and industry.

This work aims to address the need for more durable electrocatalysts with lower precious metal content for proton exchange membrane fuel cells (PEMFCs), through the development of novel electrocatalyst materials and preparation routes. In this work, 'Nafion-Pt/C' electrocatalysts have been derived from ionomer-stabilised Pt nanoparticles synthesised via a novel, wet-chemical route that offers unprecedented control over the formation of the Pt-ionomer interface, with a view towards maximising the utilisation of the electrocatalyst. Nafion-Pt/C electrocatalysts have been characterised using ex-situ electrochemical techniques, and single-cell PEMFC testing to determine their activity and selectivity towards the oxygen reduction reaction (ORR), and to compare their utilisation and durability with commercially-available electrocatalysts. Nafion-Pt/C catalysts with agglomerated Pt particles exhibited a twofold improvement in durability vs. commercial catalysts, whilst offering similar ORR activities. Their enhanced durability was attributed to inhibition of Pt particle growth mechanisms by a passivating layer of Nafion introduced during the synthesis of Nafion-stabilised colloidal Pt. The second part of this work investigated methods for the synthesis of bimetallic nanoparticles consisting of an early transition-metal core (Ti) enclosed in a Pt shell, expected to offer higher intrinsic activity towards oxygen reduction than Pt alone, whilst being less prone to degradation than other alloys of Pt such as Pt-Ni, Pt-Co and Pt-Fe.

Hollow-fibre liquid-phase microextraction (HF-LPME) was introduced in 1999 as a miniaturised version of liquid-liquid extraction (LLE) in order to reduce the consumption of organic solvents and offer an environmentally-friendly approach to extraction procedures. Since then, several studies have been published in the field of forensic and clinical toxicology applying the technique to a broad range of analytes; however more studies are necessary regarding its applicability to bioanalyses. The principle of HF-LPME is the extraction of analytes across a thin supported liquid membrane within the walls of a hollow fibre from a donor phase (DP) into an acceptor phase (AP). It is an extraction technique that encompasses several parameters that require optimisation for an efficient method; this is most effectively achieved by utilising a design of experiment (DoE) approach rather than the conventional one-factor-at-a-time (OFAT) approach. The main aim of this work is to further investigate the applicability of HF-LPME to the fields of forensic and clinical toxicology by developing and validating methods to extract various drugs from different biological matrices. Complex matrices, such as whole blood, are commonly used in forensic toxicology. Considering that not many studies have been performed on the application of HF-LPME to whole blood (only 10 up to the present day), this is an aspect that requires further investigation. For this, a fast, accurate and precise 3-phase HF-LPME method followed by LC-MS/MS analysis was developed and validated to simultaneously quantify 5 NBOMe drugs in human whole blood. NBOMe drugs are a group of substances part of the so-called “novel psychoactive substances” (NPS); drugs that have been emerging with increasing frequency over the last few years. NBOMes are associated to deaths as the causa mortis, and due to their high potency, these drugs are normally abused in micrograms. For that reason, the HF-LPME method developed had to present high sensitivity (LOD of pg/mL). The aim of the second part of this project was to challenge HF-LPME further by developing and validating methods to assess the potential application of HF-LPME in multi-drug analyses. Urine was selected as biological matrix, and the group of chosen analytes were 14 anti-hypertensive drugs and their metabolites with very different physical-chemical properties. HF-LPME has never been applied to such a broad spectrum of substances in previous bioanalytical studies. These drugs were divided into two groups (acidic and basic/neutral), and a total of four extraction methods (two for each group of analytes) were developed and optimised using chemometrics (DoE) then analysed by LC-MS/MS. Two of these methods were liquid-liquid extraction (LLE) methods that were developed and validated to be used as reference to which the two HF-LPME methods were compared. The LLE methods were sensitive, accurate, precise, and valid for application to real case samples. The HF-LPME methods presented some limitations due to the lack of isotopically-labelled analogues of each specific analyte as internal standards (IS); for non-exhaustive methods the use of these IS should be adopted as standard practise. Real urine samples from genuine patients were extracted using all 4 methods followed by LC-MS/MS analysis. By applying the methods to real case samples, it was possible to define that the HF-LPME methods were suitable for qualitative screening of urine to determine the level of compliance of patients under anti-hypertensive pharmacotherapy. However, for quantification of the drugs applying HF-LPME, further development is required to incorporate the use of isotopically labelled analogues. This study proved that HF-LPME is a potential asset not only for forensic but also for clinical toxicology. It can be a very powerful tool which, mainly due to its green-chemistry approach and pre-concentration capabilities, which allows direct injection into the analytical instrument, could potentially become a more used technique in the future. However, the analyst should be careful when developing HF-LPME methods, to bear in mind its limitations so that methods that are fit-for-purpose can be developed.

Orthopaedic infections can be polymicrobial existing as a microbiome. Infections often incorporate staphylococcal species, including Staphylococcus aureus. Such infections can lead to life threatening illness and implant failure. Furthermore, biofilm formation on the implant surface can occur, increasing pathogenicity, exacerbating antibiotic resistance and altering antimicrobial mechanism of action. Bacteria change dramatically during the transition to a biofilm growth state: phenotypically; transcriptionally; and metabolically, highlighting the need for research into molecular mechanisms involved in biofilm formation. Metabolomics can provide a tool to analyse metabolic changes which are directly related to the expressed phenotype. Here, we aimed to provide greater understanding of orthopaedic infection caused by S. aureus and biofilm formation on the implant surface. Through metagenome analysis by employing: implant material extraction; DNA extraction; microbial enrichment; and whole genome sequencing, we present a microbiome study of the infected prosthesis to resolve the causative species of orthopaedic hip infection. Results highlight the presence of S. aureus as a primary cause of orthopaedic infection along with Enterococcus faecium and the presence of secondary pathogen Clostridium difficile. Although results were hindered by the presence of host contaminating DNA even after microbial enrichment, conclusions could be made over the potential increased pathogenicity caused by the presence of a secondary pathogen and highlight method and sample preparation considerations when undertaking such a study. Following this finding, studies were focused on an orthopaedic clinical isolate of S. aureus and a metabolome extraction method for staphylococcal biofilms was developed using cell lysis through bead beating and solvent metabolome extraction. The method was found to be reproducible when coupled with liquid chromatography-mass spectrometry (LC-MS) and bioinformatics, allowing for the detection of significant changes in metabolism between planktonic and biofilm cultures to be identified and drug mechanism of actions (MOA) to be studied. Metabolomics results highlight significant changes in a number of metabolic pathways including arginine biosynthesis and purine metabolism between the two cell populations, evidence of S. aureus responding to their changing environment, including oxygen availability and a decrease in pH. Focused investigations on purine metabolism looking for biofilm modulation effects were carried out. Modulation of the S. aureus biofilm phenotype was observed through the addition of exogenous metabolites. Inosine increased biofilm biomass while formycin B, an inosine analogue, showed a dispersal effect and a potential synergistic effect in biofilm dispersal when coupled with gentamycin. Changes in metabolism between planktonic cells and biofilms highlight the requirement for antimicrobial testing to be carried out against planktonic cells and biofilms. Untargeted metabolomics was used to study the MOA of triclosan in S. aureus. The triclosan target and MOA in bacteria has already been characterised, however, questions remain over its effects in bacteria. Although the use of triclosan has come under increasing speculation, its full effects are still largely unknown. Results show that triclosan can induce a cascade of detrimental events in the cell metabolism including significant changes in amino acid metabolism, affecting planktonic cells and biofilms. Results and conclusions provide greater understanding of orthopaedic infections and specifically focus on the S. aureus biofilm, confirming S. aureus as a primary cause of orthopaedic infection and using metabolomic analysis to look at the changing state of metabolism between the different growth states. Metabolomics is a valuable tool for biofilm and drug MOA studies, helping understand orthopaedic infection and implant failure, providing crucial insight into the biochemistry of bacteria for the potential for inferences to be gained, such as the MOA of antimicrobials and the identification of novel metabolic drug targets.