G protein-coupled receptors (GPCRs) are membrane proteins responsible for myriad physiological functions. Around a third of drugs on the market already target GPCRs. However, the emphasis on developing GPCR assays to screen compound libraries for potential GPCR-interacting drugs is still very strong. One such cell-based assay was developed at GlaxoSmithKline (GSK) by Dowell and Brown [1, 2]. This assay uses a cheap, rapidly growing unicellular organism – baker’s yeast. In this organism the pheromone signalling pathway is one of only two GPCR-activated pathways. Yeast engineering done at GSK in 2000 produced MMY strains lacking the native yeast Ste2 GPCR and other key proteins, together with the integration of chimeric yeast/mammalian G protein alpha subunits thereby allowing mammalian GPCRs to couple to the yeast signalling machinery [2]. A significant limitation of the assay has been that some pharmacologically-important receptors cannot be tested using the current MMY strains and protocols. The aim of this study was to optimise the functional expression of adrenergic receptors that had previously been difficult to express and/or characterise pharmacologically in these yeast strains. The work described here examined both the MMY strains themselves and the expression constructs, asking the question how these features might contribute to a robust assay readout for two GPCR classes; the α- and β-adrenoceptors. The origin of the instability of some of the MMY strains was traced to the experimental strategy originally used to delete the URA3 marker gene and a stable MMY strain was generated as a proof-of-principle. A systematic comparison between different expression vector designs identified that the addition of the mating factor α (MF-α) leader sequence to the amino-terminus of the β1- and β2-adrenoceptors is required to produce a pharmacological response in the yeast assay. Notably, this permitted reproducible functional expression of the β2-adrenoceptor, which had not been previously possible at GSK, despite being published in the scientific literature. These improvements allowed for the pharmacological characterisation of a panel of interacting β-AR ligands, yielding data that has shed new light on ligand bias. Finally, a cholesterol-expressing yeast engineering strategy was designed to specifically investigate the role of plasma membrane composition in GPCR functional studies. The insights gained from the experiments described this thesis should allow for improved testing of other GPCRs in the drug development pipeline.

SP-B and SP-C are two pulmonary surfactant proteins. They were isolated in the tear film in 2006 (Lukovic et al. 2006). The molecular mechanism governing lipid spreading in the tear film is not well understood and, along with their production in yeast (as an alternative to animal derived material) provide the motives for this research. To investigate the involvement of SP-B and SP-C in lipid spreading, SP-B and SP-C have been produced as recombinant proteins in Pichia pastoris yeast. SP-B and SP-C are cleaved from their proproteins to produce mature active proteins and both forms were produced recombinantly. The proteins were extracted with detergents, polymers and organic solvents to determine the best method of protein isolation and purification. Purified proteins were subsequently tested for surface activity using a Langmuir trough; proSP-C10 and SP-B demonstrated surface tension activity. The mechanism of SP-B and SP-C was examined through generation of ab initio structural models. The behaviour of SP-B and SP-C was observed in membranes based on phospholipid compositions surrounding the SP-B and SP-C in the yeast plasma membrane using mass spectrometry analysis. Computational modelling then demonstrated that SP-B induces lipid curvature, whilst SP-C may help to stabilise lipid vesicles by forming a molecular bridge between the membrane and vesicle. SP-B is thought to help remodel the lung lipid layer on compression through the formation of vesicles that are connected to the membrane by SP-C.

Urinary tract infections (UTIs) affect significant proportions of the population, particularly prevalent in elderly patients. Up to 40 % of healthcare associated infections are UTIs and 80% of those are associated with catheter use. CA-UTIs significantly effect patient’s health and are frequently associated with substantial morbidity and mortality. Furthermore, the financial implications are enormous as the high complication rate arising from catheterisation requires significant time and cost. In the UK, more than a million cases are reported each year accounting for annual economic burden of approximately £125 million. The aim of this study was to develop novel antimicrobial bioactive glasses that can be used to prevent and treat catheter associated urinary tract infections. This was achieved by assessing the antimicrobial efficacy of increasing cobalt or zinc content (1, 3, 5 and 10 mol %) in phosphate based glass system (P2O5-Na2O-CaO). Glass compositions with two metal oxides (cobalt, copper or zinc) were also studied to determine synergistic combinations against a panel of clinically relevant microorganisms. A decrease in the dissolution rates of cobalt glasses was seen with increasing cobalt content, however an increase in dissolution rate was observed with higher zinc content (5% and 10%). A strong antimicrobial activity was exhibited by 5 and 10 mol % cobalt or zinc doped glasses which was not only time dependent but also strain specific. Moreover, combinations such as Co/Cu, Co/Zn and Cu/Zn showed synergism against E. coli and S. aureus. Whilst a strong antimicrobial activity was seen, the cytotoxic studies demonstrated decrease in cell viability of mammalian cells when exposed to glasses directly and their dissolution products. Nevertheless, cobalt and/or zinc doped phosphate based glasses could potentially be used as a cartridge in drainage bags or to coat catheters with a decreasing amount to prevent cytotoxic effects.

The work presented in this thesis is motivated by the ultimate goal of realizing a fully integrated quantum optical circuit (IQOC), based on a III-V semiconductor, specifically gallium arsenide (GaAs), in a planar architecture with embedded indium arsenide (InAs) quantum dots as single photon sources. Technological challenges involved with achieving a scalable quantum photonic circuit are addressed through the design, development and testing of controllable on-chip nano-photonic elements, such as nanobeam photonic crystal filters and electro-mechanical actuators. The research into both of these types of devices presented here represents the first work of this kind that has been carried out in the LDSD group at the University of Sheffield. The majority of the measurements that have been undertaken and which are presented here are of an optical spectroscopic nature. An on-chip optical filter based on a one-dimensional photonic crystal structure has been modelled and demonstrated experimentally. Such devices can be integrated with other circuit elements in order to achieve a purely electrically driven IQOC. Tuning the resonant wavelength of the device in order to attain control over the filtering parameters has also been investigated. Control over the splitting ratio of an on-chip optical beam splitter operating at the single photon level has been achieved through an electro-mechanical cantilever based system for the first time on the GaAs platform. This technology, which can be used for switching and phase shifting, now paves the way towards the physical realization of reconfigurable IQOCs. Other more efficient and versatile electro-mechanical systems that could be used to provide greater control over a variety of optical circuit elements, such as filters and beam splitters, have also been investigated experimentally. Comb-drive actuators, which are well established on silicon based platforms, have been developed for use in the GaAs based quantum optical architecture.

No description available.

Aneuploidy (extra or missing individual chromosomes) is the leading cause of miscarriage, embryo wastage and in-vitro fertilization (IVF) failure. Aneuploidy increases with maternal age and is widespread in human preimplantation embryos. Thus, aneuploidy screening before implantation during an IVF cycle (preimplantation genetic screening or PGS), to increase pregnancy rates and decreasing miscarriage rates, is also widespread. Despite this, PGS faces challenges in terms of both biological and technical limitations that may impede its full potential. Biologically, the phenomenon of chromosomal mosaicism (the presence of two or more cell lines - typically, one aneuploid and one euploid) may lead to false positives or false negatives, and the discard or transfer of euploid or aneuploid embryos, respectively. Technically, it is uncertain whether diagnosis on the biopsied piece is representative of the remaining embryo. Because these dilemmas it is unknown if PGS will only benefit a few selected groups of patients or potentially the entire IVF patient population. In a series of published works, this thesis demonstrates a significant contribution to field of preimplantation genetics, provides insight into technical and biological limitations of PGS, and into the etiology of aneuploidy and mosaicism. Specifically, I introduce a novel technique to "map" chromosomal mosaicism, by reconstructing a virtual image of the blastocyst with the approximate location of individual cells and their corresponding chromosomal makeup. I also demonstrate the ability of PGS to be performed on blastocysts that were previously frozen; thus, blastocysts have to be thawed/warmed, biopsied, vitrified and rewarmed prior to use. From a clinical standpoint, I present evidence of the differences in PGS outcomes between day 5 and day 6 blastocysts: The data suggests that day 6 blastocysts are less likely to be euploid than day 5 blastocysts. Furthermore, day 6 euploid blastocysts exhibit similar pregnancy and implantation rates when compared to their day 5 counterparts. I also published on a study examining differences in PGS outcomes in those patients that are defined as "presumed fertile" as opposed to those that are "infertile". Another study examined pregnancy and implantation rates between two competing platforms, quantitative polymerase chain reaction (qPCR) and array comparative genomic hybridization (aCGH). I also examined the pregnancy rates of poor quality embryos on day 6 that would have been discarded. From a biological standpoint, I examined the mechanisms through which embryos diagnosed as aneuploid on day 3 could develop to a euploid blastocyst, demonstrating that euploid blastocysts can develop from aneuploid cleavage stage embryos. I also demonstrated differences in aneuploidy rates between polar, mural, and a piece defined as "mid" trophectoderm, and blastocysts diagnosed as aneuploid may not reflect the chromosomal constitution of the whole embryo proper. This work herein presented provides a deeper understanding of the technical limitations of PGS and into the etiology of the chromosomal basis of early human development.

The long term aim of the work is to study non-equilibrium and symmetry-breaking phenomena for a Bose-Einstein condensate in time-dependent optical traps. The work presented in this thesis is mostly devoted to the design, construction and testing of the apparatus, in which condensation was successfully achieved. The first part of this thesis describes the design and construction of a hybrid setup for the production of a Bose-Einstein condensate (BEC) of 87 Rb in the |F = 2, m F = +2 > state. The vacuum system is composed of a first chamber, where a low-velocity intense source (LVIS) of atoms is produced to provide a pre-cooled atomic beam to the science chamber, where the magneto-optical trap (MOT) is produced, trapping more than 3 x 10 8 atoms at a temperature of roughly 400 μK. Forced radio- frequency evaporation is performed in a quadrupole magnetic trap and the atoms are transferred to a hybrid far-detuned optical trap in a crossed-beams configuration; evaporation in the optical potential leads to reliable production of a BEC every 50 s, composed of 10^5 atoms at a temperature of 10-30 nK. To ensure fine control on the optical potential depth and high intensity and pointing stability, a stabilisation system has been developed and characterised. The second part of this thesis describes the realisation of a 1D optical lattice, produced by two phase-locked laser beams emitting at 780 nm, with a red detuning of approximately 3.5 GHz, in counter-propagating configuration. Oscillating forces obtained by phase-modulating the optical potential are applied.

The possibility of revealing the presence and identifying the nature of electrically conductive targets is of central interest in many fields, including security, medicine, industry, archaeology and geophysics. In many applications, these targets are electromagnetically shielded by other external materials and thus cannot be directly accessed and detected. Hence, material interrogation techniques are required that allow penetration through the shielding materials, in order for the targets to be identified. Electromagnetic interrogation techniques represent a powerful solution to this challenge, as they enable penetration through conductive shields. Two resonant electromagnetic induction imaging (EII) methods, based on the use of LCR circuits, were developed in this research work. These proof-of-principle EII methods were based, respectively, on position-resolved-measurements of resonant frequency and Q-factor shifts, which occurred as a consequence of eddy current induction inside the conductive targets to be detected. The proposed techniques were applied to 2D imaging of conductive targets (having conductivities σ ranging from 0.54 to 59.77 MSm-1), both unshielded and shielded by an aluminium shield (1.5-mm-thick). The experimental results achieved in the first part of this work highlighted a limitation in the LCR resonant circuits used for EII investigations, linked to the Q-factor low absolute 5 values (between 7 and 23). Therefore, investigations were conducted, leading to the implementation of an improved version of the EII system, based on active bandpass filters. The sensitivity of the novel EII system was found to be larger by a factor of 3.5 compared to the previously used LCR-based system, when both systems were used for imaging copper. Additionally, the new system allowed achieving images with higher contrast. The research work reported in this thesis led to establishing a proof-of-principle method for EII of conductive samples, also in the interesting scenario where the samples were shielded by conductive shields preventing them from being directly accessed.

Receptor tyrosine kinase (RTK) signalling is frequently deregulated in cancers, developmental syndromes and metabolic diseases. The fibroblast growth factor (FGF) receptor (FGFR) family of RTKs have pleiotropic roles in development, metabolism and tissue homeostasis. A decade of deep sequencing studies has led to the discovery of somatic cancer-associated alterations in FGFRs including point mutations, gene fusions and amplifications. Missense substitutions appear most frequently in FGFR3 and FGFR2, some of which have been implicated as oncogenic drivers. This has led to rapid progress in anti-FGFR therapies. This work compares and analyses twenty five reported FGFR kinase domain (KD) variants using FGFR3 as a model. Kinase activity assays were developed to screen missense substitutions in vitro using purified FGFR3 KD proteins. It was found that hyperactive mutations did not necessarily occur frequently and conversely, frequently occurring mutation 'hotspots' were not necessarily kinase hyperactive. Cellular models of FGFR mutations suggested that hyperactive mutants activated signalling but did not always transform cells. Investigations into the ligand binding characteristics of a subset of activating mutants revealed mutation specific signatures of binding properties. However, there appeared to be no link between KD activity and stability. These results reinforce the importance of screening clinical tumours prior to treatment to provide better data-driven therapies for patients. Endocrine FGFR signalling was also explored in this work. Klotho family proteins bind to specific FGFs and FGFRs to signal through endocrine signalling complexes regulating diverse metabolic activities. This work developed and optimised methods to express and purify components of these binary and ternary signalling complexes. Preliminary biophysical experiments were then performed to begin to understand the protein-protein interactions involved in complex formation. This will inform the rational design of therapies already in development for targeting deregulated endocrine FGFR signalling in various metabolic disorders and cancer.

Over recent years the sensitivity of alkali-metal vapour magnetometers has been demonstrated to surpass that of even Superconducting Quantum Interference Devices (SQUIDs), the current commercial gold standard in laboratory weak- field magnetometry sensing. Here we present a proof-of-principle approach to building an RF atomic magnetometer which is robust, portable, tunable, non-invasive and operable at room temperature in an unshielded environment. In view of these characteristics, we discuss the potential application of alkali-metal magnetometry in imaging concealed objects, non-destructive evaluation of the structural integrity of metallic objects (e.g. pipelines and aircraft), and detection of rotating motors. We present a cost-effective approach to operating an atomic magnetometer in a Magnetic Induction Tomography (MIT) modality, to non-invasively map the conductivity of conductive objects concealed by conductive materials remotely and in real time. This is achieved by measuring the secondary eld in the subject due to eddy currents circulating as a result of application of a tunable radio-frequency oscillating eld, which overcomes the bandwidth and sensitivity limitations of using coils for sensing as in conventional MIT. In addition, we demonstrate the use of the atomic magnetometer for the remote detection of DC and AC electric motors with an improved response compared with a commercial fluxgate magnetometer in the sub 50 Hz regime (particularly detection down to 15 Hz). Its capability for non-invasive measurement through concrete walls is established, with potential for use in industrial monitoring and detection of illicit activity. Finally, the possibility of detection of submerged targets or for the atomic magnetometer to be mounted on submarine vehicles was explored. Promising results were obtained, but further investigation is required in this environment to establish this as a viable marine detector.