Primary focal hyperhidrosis (PFHH) is a medical condition characterised by overactivity of the eccrine sweat glands, primarily occurring on palmar, plantar and axillary regions. PFHH can have a significant adverse impact on a patient’s quality of life. Multiple intradermal injections of a commercial formulation of botulinum toxin A (BTX A) (Botox®) is the most effective non-surgical treatment currently licensed in the UK for cases of severe PFHH. Although effective, intradermal BTX A injections are associated with considerable pain and discomfort for the patient and are time-consuming for the administering clinician. This study aims to evaluate the potential of using pocketed microneedle devices for minimally invasive intradermal delivery of BTX A, as a liquid formulation, into human skin. Pocketed microneedles, metallic 700 μm-long needles containing a cavity within the needle shaft, were selected as an appropriate and relatively untested intradermal delivery device. Pocketed microneedle devices (PMDs) were liquid loaded by immersion into a ‘Botox® like’ formulation that mimicked the composition of the commercial Botox® formulation, with the exception of BTX A, which was replaced by the model macromolecular protein β-galactosidase (~465 kDa). A water-soluble dye was also included to enable visualisation. Microneedles were assessed for loading uniformity by light microscopy and the formulation residency time was evaluated by monitoring evaporation using a digital camera. The microneedle loading capacity was determined using an established quantitative assay for β-galactosidase. Studies using excised human breast skin, maintained in organ culture, examined delivery of the model β-galactosidase from liquid loaded PMDs and the time-dependent diffusion of the protein within the dermal tissue. A more clinically representative model of BTX A, formaldehyde inactivated BTX A, i.e., botulinum toxoid, was used to determine the deposition pattern of the therapeutic within the skin. Following skin delivery the toxoid was detected by immnohistochemical staining and fluorescence imaging, following its conjugation to an appropriate fluorophore. Immersion of the PMD into a ‘Botox® like’ formulation resulted in successful uptake and retention of the model protein solution. Quantitative studies indicated that nanogram quantities (~100 ng/microneedle array) of the β-galactosidase model can be loaded and retained on individual microneedles, in a liquid state. These results suggest that the loading capacity of the microneedle device is appropriate for therapeutic botulinum toxin formulations, although loading uniformity will need to be addressed. Histological analysis revealed effective delivery of the model β-galactosidase from a PMD to the epidermal and the dermal layers of the skin. Rapid and extensive diffusion of the protein within the deeper dermis was also demonstrated. Further, immunohistochemical and fluorescence studies indicated effective PMD loading and successful delivery of botulinum toxoid to the dermis of human skin. These data suggest that it should be possible for BTX A to access its therapeutic target (the eccrine sweat glands) following delivery via PMDs. This study has demonstrated for the first time that pocketed microneedles represent a viable, minimally invasive alternative, for the intradermal delivery of botulinum toxin A (Botox®). Future pre-clinical and clinical studies are now required to test and optimize a microneedle-based delivery system that is most suited to clinical practice.

There are many questions associated with the understanding of the origin and regulation of bioluminescence. A key question underlying this thesis was why coelenterazine evolved as the most common marine luciferin. Another problem addressed was the coelenterazine source of the bioluminescent hydroid Obelia, which is unknown. Coelenterazine is an integral part of Obelia’s photoprotein, triggered by Ca2+. The thesis also investigated the uncertainty over how Ca2+ enters Obelia’s photocytes. Obelia is considered to have four species, but they have been misidentified. Green fluorescent protein (GFP) acts as a fluor in Obelia producing green light. The selective advantage of this green bioluminescence requires confirmation. Results showed that Obelia geniculata cultures rapidly lost their bioluminescence, indicating Obelia requires a dietary supply of coelenterazine. Adding coelenterazine to Obelia briefly restored its bioluminescence. Levels of obelin dropped in Obelia geniculata cultures. Obelia longissima had a lower level of coelenterazine and bioluminescence than Obelia geniculata. Coelenterazine was detected in species living on Obelia and, three non luminous species of copepod. A range of species were identified in zooplankton, as possible coelenterazine sources. Ratios of obelin to apoobelin changed in older colonies. The problem of misidentification of Obelia species was solved by utilising the fluorescent patterns of Obelia’s photocytes. GFP was recorded for the first time in the hydrotheca and tentacles of Obelia dichotoma. Fluorescence maxima for Obelia geniculata and Obelia dichotoma iv were different. GFP in Obelia was found to photobleach far slower than GFP in EGFP. This supports the hypothesis that Obelia has a molecular mechanism which protects its GFP from photobleaching. Light emission from Obelia geniculata was different from Obelia longissima. This suggests that the mode of entry of Ca2+ into the photocytes of these two species is different. To investigate the exact pathway by which initial stimulation of Obelia causes Ca2+ to enter the photocytes, experiments were conducted using K+ channel blockers. The potassium ion channel blockers tetraethyl ammonium chloride and 4-aminopyridine both produced a bioluminescent response in Obelia geniculata. This suggested that the pathway included K+channels. In a polar solvent coelenterazine produced low chemiluminescence, which increased with increasing luciferin concentration. Human albumin and BSA increased this effect. This supported the solvent cage hypothesis that bioluminescent proteins originally evolved as primitive oxygenase enzymes.

Irritable bowel syndrome is one of the most common problems reported to general practitioners and gastroenterologists. It has been shown that many of the gut and systemic symptoms are due to lactose sensitivity, the sugar found mainly in milk. Undigested carbohydrates and other foods absorbed by the small intestine reach the bacteria in the large intestine. There is little oxygen here, the bacteria metabolise these to produce gases such as hydrogen and methane, and a variety of small organic metabolites such as methylglyoxal. These metabolites are absorbed into the bloodstream and can affect tissues around the body. The overall aim of this thesis was to investigate the potential role of the bacterial metabolic toxin hypothesis in the development of diabetes. To specifically investigate the ability of these toxins to covalently modify proteins and to investigate the biological activity of these modified proteins on glucose uptake and cell differentiation. Albumin and insulin have been shown to exhibit mono-oxygenase activity demonstrated by coelenterazine chemiluminescence. It was heat denaturable, demonstrated saturable substrate characteristics, was inhibited or activated by cations (Fe2+, Fe3+, Zn2+ and Ca2+) known to bind to these proteins and was inhibited by drugs that are known to bind to Sudlow’s site I on albumin. The inhibition of albumin catalysed coelenterazine chemiluminescence observed in the presence of drugs that are known to bind to Sudlow’s site I on albumin proposes that this is also the coelenterazine binding site. Molecular 3D modelling confirmed that coelenterazine binds to this site. Methylglyoxal covalently modified these proteins resulting in reduced biological activity. Tetraethylammonium significantly inhibited 3T3-L1 cell differentiation in the presence of insulin. However, methylglyoxal and tetrandrine did not significantly inhibit 3T3-L1 cell differentiation. The results in this thesis support the hypothesis that bacterial metabolic toxins can covalently modify proteins and alter their biological activity.

Both excitable and non-excitable cells possess plasma membrane ion channels and evidence has accumulated over the last 30 or so years that these channels perhaps play key roles in the cell life and death. This Thesis investigated the characteristics and putative functions of one class of potassium channel, the BK channel in osteoblast-like cells and primary osteoblasts from human, rat and mouse. The properties and functions were defined in vitro using a combination of patch-clamp, reverse transcription-polymerase chain reaction (RT-PCR) and functional assays for cell growth and mineralisation. RT-PCR showed the presence of KCNMA1, KCNMB1, KCNMB2, KCNMB3 and KCNMB4, the gene for BK channel α, β1, β2, β3 and β4 subunits respectively. The channel was voltage-dependent with a mean unitary conductance of 315 pS in cell-attached patches, a conductance of 124 pS in excised outside-out and 151 pS in inside-out patches. The channel was blocked by TEA (0.3 mM), TBuA (1 mM), TPeA (1-10 μM), THeA (1-3 μM), tetrandrine (5-30 μM) and paxilline (10 μM) and was activated by isopimaric acid (20 μM). Notably iberiotoxin (IbTX) (90 nM) only blocked a proportion of the channels tested (2/5). Osteoblast-like MG63 cell number changed in response to BK channel modulators. It increased significantly with TEA and tetrandrine at low concentrations (1 mM, 3 μM respectively), and reduced at high concentrations (>10 mM, >10 μM respectively). It was not affected by IbTX (20-300 nM) or slotoxin (300 nM). The increase in cell number by TEA was blocked by isopimaric acid. In addition, TPeA and THeA caused a decrease of osteoblast-like SaOS2 cell mineralisation at the concentrations (3 and 0.3 μM, respectively) increased MG63 cell numbers. The BK channel has a distinctive pharmacology and represents a new target for therapeutic strategies in modulating osteoblast proliferation.

My aim was to acclimate calli of Medicago truncatula to be NaCl tolerant and discover the extent to which various traits could act as markers of improved NaCl tolerance in vitro. I developed a new tissue culture protocol incorporating multi-step up acclimation (Phase I) followed by two rounds of culture ± NaCl (Phases II and III) over 0 to 350 mM exogenous NaCl. Following Phases I, II and III, calli were tolerant at 150 mM and embyrogenically competent at 100 mM NaCl. Remarkably, in Phase III, calli grew faster in the 100 and 150 mM NaCl treatments compared with the 0 mM controls. There were positive and negative linear relationships between Na+ and K+ uptake and exogenous NaCl concentration, respectively that intercepted each other at 160 mM suggesting a Na+/K+ homeostasis at this NaCl concentration. Proline level and vacuolar size peaked at 100/150 mM whilst highest osmolarity and lowest water content were at 250/350 mM NaCl. The concentration of water soluble sugars was positively related to 0 to 250 mM NaCl. Flow cytomerty was used to examine the extent to which cells underwent to endoreduplication. From 50 to 250 mM NaCl, cells were observed in G1 (2C), G2 (4C) or a state of endopolyploidy (8C). Hence, callus growth and embryogenesis occurred regardless of endoreduplication. Expression of genes linked to growth (WEE1), in vitro embryogenesis (SERK), salt tolerance (SOS1), proline synthesis (P5CS) and ploidy level (CCS52 and WEE1) peaked at 100/150 mM NaCl. Hence, these genes and various physiological traits except sugar levels, served as useful markers for NaCl tolerance. To my knowledge, this is the first report of multi-step acclimation conferring salt tolerance in leaf-derived calli of Medicago truncatula. A pilot study of drought tolerance, mimicked by polyethylene (PEG), demonstrated tolerance to 10% PEG increases in gene expression of WEE1, with proline and sugar accumulation.

Saprotrophic fungi are key regulators of nutrient cycling and re-distribution within woodland ecosystems. They are the primary agents of wood and leaf litter decomposition, and their hyphal networks, which grow throughout the soil-litter interface, represent highly dynamic channels through which nutrients are readily distributed. These networks also represent the primary resource for a huge diversity of mycophagous soil fauna. This study uses soil microcosms to investigate and compares the potential of soil invertebrates, representing the Isopoda, Myriapoda, Acari, Collembola, Tubificida (Enchytraeidae) and Nematoda, to influence mycelial emergence, morphology, extracellular enzyme production and wood decomposition by cord-forming basidiomycetes. While all invertebrates disrupted mycelial growth to some extent, macrofauna (woodlice and millipedes) generally exerted the strongest grazing pressures. By severing thick cords, these larger invertebrates limited mycelial growth and induced the strongest enzymatic responses. In contrast, while the smaller micro- and mesofauna reduced extension rates of some fungal species, their low-intensity grazing also induced compensatory growth responses, stimulating growth of less palatable fungal species. The varying susceptibility of different fungi to grazers also caused grazers to exert selective pressures on fungal communities. By removing entire networks of the most combative fungal species, the woodlouse Oniscus asellus prevented the competitive exclusion of three fungal opponents from soil and wood. By stimulating growth of the less competitive fungal species, the nematode Panagrellus redivivus, also reversed the outcomes of specific mycelial interactions. Via these two opposing mechanisms soil invertebrates are likely to exert top-down control, influencing the community compositions of saprotrophic fungi. Overall, the effects of grazers on mycelial distribution, decomposition and community compositions were strongly specific, suggesting that the factors which influence invertebrate diversity and community compositions will also indirectly affect mycelial growth and functioning in temperate woodland soils.

Recurrent novae are binary systems which undergo outbursts due to a thermonuclear runaway on a timescale of decades. Two systems, U Scorpii and RS Ophiuchi, are the main focus of this thesis having gone into outburst in 2010 and 2006 respectively. Optical and near-IR spectroscopy of U Sco obtained with facilities including the Liverpool Telescope, the Very Large Telescope, and the Southern African Large Telescope, as well as ultra-violet and optical spectroscopy of RS Oph obtained with the Ultra-Violet and Optical Telescope, are presented here. Observations of U Sco obtained in outburst are used to determine the helium abundance of the ejecta, with observations in quiescence used to constrain the nature of the companion star and the accretion disc. Observations of RS Oph are used to produce lightcurves of the decline from maximum to the post-outburst minimum. Analysis of optical and near-IR spectra following the 2010 outburst of U Sco leads to the determination of the reddening and the helium abundance. The reddening is found to be E(B-V) = 0.17 ± 0.14, consistent with previous estimates. The helium abundance, N(He)/N(H), is determined to be 0.122 ± 0.018, lower than previous estimates and suggesting that the companion is not helium rich. The fitting of model stellar atmospheres to observations of U Sco in quiescence, obtained ∼18-30 months after the 2010 outburst, constrains the effective temperature of the companion and indicates that it is a cool F or hot G star. The accretion disc is found to be still in a state of development until at least ∼18 months after outburst, contrary to previous studies. The strength of the Heii 4686 ̊A line in quiescence is found to be consistent with a high mass white dwarf accreting solar-abundance material at a high rate. The He line strengths do not require an overabundance of helium, as has been previously suggested, consistent with the solar abundance of helium found in the ejecta of the 2010 outburst. UVOT observations of RS Oph following the 2006 outburst are used to produce B and u′ lightcurves of the decline to minimum. These lightcurves are used in con- junction with published V and B lightcurves to show the change in the colour of the system throughout the outburst. Spectra of RS Oph show the spectroscopic evolution during the initial decline, plateau, final decline, and post-outburst minimum phases. SALT spectra obtained during the late decline phase of the 2011 outburst of a third recurrent nova, T Pyxidis, are used to derive a helium abundance of N(He)/N(H)= 0.161 ± 0.013, approximately twice the solar value. Very strong [O iii] lines are also detected.

Gemcitabine (2’,2’-difluoro-2’-deoxycytidine, dFdC) is an established chemotherapeutic agent used in the treatment of various carcinomas such as lung, breast, bladder and especially pancreatic cancer. However, its general application and bioavailability is compromised due to both poor cell uptake and rapid metabolism by gut and liver cytidine deaminase (CDA). A 18F-gemcitabine positron emission tomography (PET) probe could enable biodistribution studies and the imaging of gemcitabine pharmacodynamics in vivo. However, the potential and clinical relevance of a 18F-gemcitabine PET probe would have to be evaluated using appropriate PET tumour models. In order to approach the synthetic target molecule 18F-dFdC, proof-of-principle studies on more straightforward synthetic targets including 18F-FAU and 18FFAC (see figure below) were carried out first. An appropriate precursor was synthesised for 2’-stereoselective late-stage radiofluorination based on previously developed conditions. First, the 2’-[18F]fluorinated arabino nucleoside 18F-FAU, which was considered as a rapidly accessible 2’-fluorinated uracil-based dFdC analogue was successfully synthesised in our radiochemical laboratory. Subsequently, this procedure was used as a template method to obtain the cytidine analogue 18F-FAC via a novel synthetic route in moderate radiochemical yield (4.3-5.5%, decay-corrected), high specific activity (1700 mCi/μmol) and purity (98%) after a synthesis time of 168 min.

Estimating the minimum PMI (mPMI) based on larval age involves identifying the species, reconstructing the thermal history at a crime scene, and modelling the rate of development. However, few studies take into consideration the mass-generated heat produced by larvae co-existing in an aggregation. These localized increases in temperature are often highlighted in the literature as having an influence on larval development, but there are ongoing difficulties with incorporating this concept into mPMI estimates. This is mostly due to a lack of research on the topic, particularly with controlled laboratory experiments or in natural conditions simulations. The aim of this research was to determine whether heat generation varied in different sized aggregations and, if so, did it influence larval development and behaviour. Various sized aggregations (50-2500 larvae) composed solely of Lucilia sericata (Meigen, 1826)(Diptera: Calliphoridae) larvae were reared in the laboratory at a constant ambient temperature of 22 °C (±1 °C). Data loggers and a thermal imaging camera were used to record mass temperatures throughout the feeding stage of development. Larvae were sampled from these different sized aggregations at set times and had their instar determined and/or their lengths and fresh weights recorded so that developmental rates could be monitored. To investigate the movement of larvae as they fed in an aggregation, individuals were tagged with a fluorescing elastomer. These larvae were easily distinguished from the rest of the cohort, which allowed their positions within the mass to be recorded at regular intervals. The results showed a strong positive relationship between mass size and the amount of heat generated by the aggregation (p<0.001), with temperatures rising as masses increased in size. A minimum mass size of 1200 larvae was required for the local temperature to increase significantly above ambient, with aggregations of 2500 larvae producing temperatures that exceeded ambient by up to 14 °C (± 1.2 °C). Larvae sampled from increasingly large masses showed an accelerated rate of development during the 2nd and 3rd instar. This coincided with when masses were at their warmest. These faster growth rates resulted in larger aggregations entering the post-feeding phase of development an average of 13 hours earlier than smaller, cooler masses. Physical measurements taken from larvae at 70 hours development demonstrated that individuals sampled from larger masses were significantly longer and heavier than those sampled from smaller aggregations (p<0.001). This provided further evidence of faster growth rates. However, when compared to solitary larvae, all mass-reared larvae, regardless of the size of the aggregation, appeared to benefit from a faster rate of development, reduced mortality and larger body sizes at dispersal. Larvae were observed to be in a constant state of motion and continually repositioned themselves within the mass, rotating between the periphery and the centre where they presumably fed. The thesis highlights the need to incorporate mass temperatures into forensic casework when using larval development to estimate the time of death. Larvae sampled from large masses, particularly during the 3rd instar, could appear older than they actually are due to the accelerated rates of development experienced under warmer conditions. If this isn’t taken into consideration then it could result in an overestimation of the mPMI. Future research should focus on identifying how other variables influence heat generation in masses, as well as finding ways to estimate the size of a mass, and hence its thermal history, at a crime scene.

This research aimed to develop new fire retardant rubber formulations, by surveying the existing knowledge base for fire retardant approaches for polyisoprene rubber, characterising unmodified compounds, formulating and studying fire retarded compounds for use within suspension and anti-vibration mounting systems. Materials have been prepared on a bench scale and evaluated for physical properties. Thermal decomposition has been studied using Thermogravimetric Analysis (TGA) in both air and nitrogen. Burning behaviour has been studied using a horizontal burning rate method, Limiting Oxygen Index (LOI) and the cone calorimeter. Basic rubber mixtures were prepared to investigate the interactions between the polymer and additives, under TGA conditions. Zinc oxide was found to have little effect on the polymer decomposition, while silica reduced thermal stability. When decomposed in air, increasing levels of carbon black reduce the rate of mass loss in the polymer. Comparing formulations with different cross-linking types, sulphur without cross-linking increases the heat release in a cone calorimeter; but when low levels of sulphur form efficient cross-links, heat release is suppressed, as also occurs with organic peroxide cross-linking. The effect of carbon black on burning behaviour was compared with inorganic fillers. Within the cone calorimeter, material containing carbon black formed a char-like residue which provides some reduction in the rate heat release, and did not contribute to the fuel load. The use of inorganic fillers yielded more rapid burning behaviour. Any level of carbon black addition gives a reduction in the rates of heat, smoke, CO, and CO2 release, confirming that carbon black had a stabilising effect. Intumescent formulations were prepared using ammonium polyphosphate (APP), pentaerythritol and melamine, and separately using expandable graphite (EG). Within the cone calorimeter both systems yielded a significant reduction in the first peak of heat release rate, but a much higher second peak than for the unmodified compound. This second peak value is associated with the significant levels of intumescence observed. The use of EG gave a greater level of fire retardance compared to the APP formulation. Hydrated fillers, and blends thereof, were investigated; an equal blend of aluminium hydroxide (ATH) and magnesium hydroxide (MH) was found to yield the lowest peak release rate on the cone calorimeter. Hydrated fillers were investigated with synergists proposed in the literature. Little benefit was noted for these additives when used as partial replacements for the filler.