Since its first use to solve the structure of sodium chloride in 1915 X-ray crystallography has developed significantly to become the premier technique for obtaining 3D structural information of small molecules and macromolecules alike. As the technique continues to develop and focus its attention on weak diffraction from the likes of micro-crystals and poorly packed crystals of membrane proteins and large protein complexes; as well as ultra-high resolution data and weak anomalous signal from native atoms, data quality is becoming more and more important. Data quality is particularly important in the wake of long wavelength macromolecular crystallography (MX) for phasing using anomalous signal from native sulphur and phosphorous atoms in proteins and DNA. This thesis first investigated the use of a new sample handling technique using a humidity controlled stream to preserve macromolecular crystals while excess surrounding solvent is removed (Chapter 2). Following the successful development of this technique the effects of excess surrounding solvent on data quality was assessed when collecting at standard MX X-ray wavelengths (~ 1 Å) and longer X-ray wavelengths (~ 2 Å). Datasets were collected from large populations of control and test crystals at standard and longer wavelengths to allow robust statistical methods to be applied; a practice not widely adopted in method development studies in X-ray crystallography. This made it possible to assess the small differences in data quality in the presence and absence of excess surrounding solvent. The effects of surrounding solvent at longer wavelengths appear to be protein dependent with some proteins tested showing no significant difference and others a significant decrease in data quality at longer wavelengths (Chapter 3). Originally this project aimed to use the new long wavelength in-vacuum MX beamline, I23, at Diamond Light Source UK to carry out phasing experiments using native sulphurs for structure solution. However, the considerable complexity involved in developing in-vacuum MX meant these experiments could not be carried out during the time frame of this thesis. Chapters 4 and 5 outline the production of a novel cancer protein (cancerous inhibitor of protein phosphatase 2A) and two protein targets from the Achromobacter xylosoxidans (Ax) genome intended for sulphur single wavelength anomalous dispersion phasing experiments on I23. Of these proteins the structure of Ax-α/β hydrolase was solved by conventional methods, the structure of which is discussed in Chapter 5. Of the protein crystals used in long wavelength data quality experiments in Chapter 3 the molecular biology of PA3825-EAL, a biofilm regulating protein essential to the swarming ability of Pseudomonas aeruginosa, was investigated further. The crystal structure of PA3825-EAL was solved in the resting, substrate bound and product bound states to high resolution. Comparison of the crystal structures of monomeric and dimeric PA3825-EAL with the inactive dimeric structure of MucR-EAL suggests dimerisation via helix 8 plays a role in inhibition of EAL domains. Prior to this, dimerisation was thought to be an activating factor in EAL domains. The product bound state of PA3825-EAL showed the presence of a previously unreported third metal binding site which may form an essential component of the reaction mechanism of EAL domains. Inability of MucR-EAL to incorporate this third metal due to dimerisation may explain the lack of activity despite possessing the conserved catalytic residues necessary. The fast detector technology and improvements in automated data processing software that allowed diffraction data for large populations of crystals to be collected in Chapters 2 and 3 have also been applied to development of a serial data collection technique. Of 159 datasets collected from 8 crystals of a copper nitrite reductase from Achromobacter cycloclastes, 45 datasets from a single crystal were analysed to observe the reaction mechanism using high resolution crystal structures. X-ray radiolysis initiated the reaction and high resolution data allowed the conversion of nitrite (NO2) to nitric oxide (NO) to be observed in the crystal. Other aspects of the reaction were investigated from the data series including a conserved water chain connecting the copper sites which may act as a proton wire to donate a proton and produce NO. This technique may have wide applications to the study of the reaction mechanisms of other metallo-proteins.

Molecular recognition refers to the interaction between two or more molecules through complementary noncovalent bonding, for example, via hydrogen bonding, electrostatic interactions, van der Waals forces or hydrophobic forces. Molecular recognition plays an important role in biology and mediates interactions between receptors and ligands, antigens and antibodies, nucleic acids and proteins, proteins and proteins, enzymes and substrates, and nucleic acids with each other. Many cellular processes are governed by a group of proteins acting in a coordinated manner; such complicated mechanisms are closely regulated: changes in the populations of particular complexes or changes in concentrations of the products of protein mediated reactions can switch cells from one state to another (from replication to apoptosis, for example). These small variations in molecular populations are caused by very delicate differences in the thermodynamics or kinetics of reactions. This implies that in order to understand not only biological systems in terms of their molecular components, but also to be able to predict and model system response to stimuli (whether it is a natural substrate or a drug), characterisation of the thermodynamic and kinetic components of the binding process is of paramount importance. This combined computational-experimental project was focused on the development of new computational approaches able to predict the enthalpic component of ligand binding, using quantum mechanics. A concept of ‘theoceptors’ was developed, which are theoretical receptors constructed by computing the optimal geometry of ligands binding in the receptor. This project was supported by AstraZeneca, and it included an industrial placement in the Structural and Biophysical Sciences area, where the experimental data was generated to characterise the thermodynamics and kinetics of binding of a range of ligands to two biological targets, using two experimental techniques, isothermal titration calorimetry and surface plasmon resonance. The findings contribute greatly to the process currently underway of expanding our understanding of the relevance of both of these aspects of biochemistry to drug discovery.

No description available.

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.

Dimethylsulphoniopropionate (DMSP) is a dimethylated sulphur compound that appears to be produced by most marine algae and is a major component of the marine sulphur cycle. The majority of research to date has focused on the production of DMSP and its major breakdown product, the climatically important gas dimethylsulphide (DMS) (collectively DMS/P), by phytoplankton in the open ocean. A number of functions for intracellular DMSP (DMSPi) in phytoplankton have been identified and the cycling of DMS/P appears to be critical for ecosystem function. However, mechanisms for the production and release of DMS/P in the coastal ocean are poorly understood, despite the region’s economic and ecological importance. Coralline algal habitats (e.g. maerl beds, coral reefs, seagrass meadows, kelp forests) are distributed throughout the coastal oceans worldwide. Their three-dimensional structure supports high biodiversity and provides numerous services, generating considerable economic wealth. DMSPi in coralline algae is known to be high, thus coralline algal habitats may be critical components of the coastal sulphur cycle. This research aimed to improve our understanding of the production of DMS/P by coralline algal habitats by investigating (1) natural spatiotemporal variation and (2) the influence of environmental pressures. This was achieved through a number of laboratory and field-based studies, utilising modern and well-established techniques. The first objective of this research was to better understand the photosynthesis of red coralline algae (Chapter 3), as the algal precursor to DMSPi is methionine, a product of photosynthesis. The photosynthetic characteristics of coralline algae exhibited acclimation to changing light conditions (e.g. over a diurnal cycle or between natural and static lighting conditions). Further, for the species tested, coralline algae are often subjected to light-saturating natural conditions, therefore requiring efficient photo-protective mechanisms, which may include DMSPi regulation. On a global scale, DMSPi in coralline algae may decline with latitude, reinforcing the role of DMSPi as an antioxidant (Chapter 4). At smaller spatial scales, DMS/P production, release and recycling mechanisms were apparent in a number of habitat types (Chapter 4). A strong seasonal trend in DMS/P was also observed at a Scottish maerl bed, driven by water temperature and cloud cover (Chapter 5). Annually averaged DMS and DMSP concentrations were 230% and 700% respectively higher than the open ocean, highlighting the potential importance of the coastal ocean in the marine sulphur cycle (Chapter 5). The influence of environmental pressures (decreased salinity, variable pH and grazing) on DMS/P production by coralline algal habitats was examined (Chapters 6 – 8). In agreement with the phytoplankton literature, a chronic, but not acute, reduction in salinity led to a significant decline in coralline algal DMSPi concentrations and a sinking of the surface epithelial cells but no apparent impact on photosynthesis (Chapter 6). In the naturally variable tropical reef environment, calcifying algae continually regulated DMSPi concentrations in response to the diurnal cycling of carbonate saturation state (Chapter 7), suggesting that DMSPi may be enhanced under low pH regimes to compensate for enhanced oxidant production. Under low pH conditions, cracks were observed between the surface epithelial cells of coralline algae, potentially allowing DMSPi to leak from the cells (Chapter 7). In the field, grazing by urchins appeared to facilitate the release of DMS/P from kelp in coralline algal habitats (Chapter 8). In the laboratory, DMSPi in coralline algae increased in response to chemical cues from grazers rather than direct grazing activity, as had been previously proposed. Prior to this research, little information was available on DMS/P concentrations in coralline algal habitats. The marine sulphur cycle may impact climate regulation and ecosystem function on a global scale. This research provides a comprehensive source of information on the importance of coralline algal habitats in the marine sulphur cycle by examining natural variability and potential changes in response to environmental perturbations. This work will form a baseline for continued research in this field, investigating, for example, the impact of multiple stressors on DMS/P production, release and recycling in coastal marine habitats.

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.

Aims: To fully characterise the RGC-5 cell line and determine whether it would be a suitable substitute for primary RGC cell culture. To optimise a CNTF Nogo-P4 inhibitory assay and establish whether (a) CNTF alone is capable of stimulating RGC neurite outgrowth and survival, or (b) an increase in intracellular cAMP is required for CNTF to be effective. To determine whether recombinant AAV2 viral constructs were capable of producing detectable levels of CNTF in HEK-293 transfected conditioned media. To optimise AAV2-eGFP delivery and establish whether AAV2-CNTF-hrGFP and AAV2-CNTF-shRhoA-hrGFP could promote RGC survival and regeneration after optic nerve crush surgery. Methods: The RGC-5 cell line was characterised using semi-quantitative PCR, sequencing and immunocytochemistry. RGC-5 cells were screened for a selection of neuronal, glial, progenitor, oligodendroglial lineages and cone photoreceptor cell markers to identify the cells origin. Retinal cultures were treated with recombinant CNTF and/or Forskolin to promote RGC neurite outgrowth and survival - this was quantified after 3 d. Retinal wholemounts were prepared to assess GFP transduction and survival after intravitreal delivery of AAV2-eGFP. Axonal regeneration and RGC survival were assessed through histological examination of optic nerves and retinal sections. Results: The RGC-5 cell line predominantly expressed oligodendroglial lineage markers and only weakly expressed \(\beta\)III-Tubulin mRNA. RGC-5 cells did not express mRNA for many of the phenotypic markers of RGC. CNTF was effective at stimulating RGC neurite outgrowth without the need for cAMP elevation - furthermore recombinant CNTF could disinhibit Nogo-P4 treated RGC in vitro. GFP transduction was low when injected alone, however, when administered with Pronase-E there was a significant increase in GFP expression. AAV2-CNTF-hrGFP and AAV2-CNTF-shRhoA-hrGFP did not promote RGC survival or regeneration 23 d post optic nerve crush. Conclusions: RGC-5 cells are not an appropriate substitute for primary retinal cell culture in vitro as they express many of the same markers as oligodendrocyte progenitors. CNTF is capable of stimulating RGC neurite outgrowth without an additional elevation of cAMP. AAV2-mediated GFP expression could be enhanced through the partial digestion of the inner limiting membrane - this seems to be the major obstacle in achieving optimal AAV2 transduction.

Ice cream is a fatty, low satiating food which may increase obesity levels. This thesis aims to understand if it is possible to develop a more satiating product by manipulating formulation and/or processing. First, the effect of two emulsifiers (mono and diglycerides, MDGs and Tween 80) on the thermal behaviour of a bulk fat matrix was investigated. MDGs were shown to possibly enhance ice cream fat destabilisation more than tween 80. This research continued on an ice cream matrix, investigating the effect of different HLB (hydrophilic-lipophilic balance) number emulsifiers (MDGs based). Low and high HLB number emulsifiers (compared with intermediate HLB numbers) led to the formation of a more structured fat network. Moreover, the investigation of different solid fat content (SFC) blends and the aging step showed that 1) the fat network became more structured as the SFC increased (unless this was too high); 2) aging step could be avoided; 3) it is feasible to considerably decrease the SFC without affecting consumers’ response; and 4) SFC had a predominant effect on palatability. Finally, an appetite study allowed answering the research question of this work, demonstrating that formulation manipulation could be a promising way to reduce food intake (i.e. obesity levels).