Phosphoinositides are lipid signals that control cellular processes and are particularly closely associated with the control of membrane trafficking. PtdIns(3,5) \(\char{cmmi10}{0x50}\)\(_2\) is the most recently identified phosphoinositide and was previously recognised as controlling events in the late endocytic system between the late endosome and the vacuole/lysosome. Primarily associated with retrograde trafficking from the vacuole/lysosome to the late endosome/MVB, PtdIns(3,5) \(\char{cmmi10}{0x50}\)\(_2\) is generated by the kinase Fab1p (PIKfyve in animals). In mammalian cells, PtdIns(3,5) \(\char{cmmi10}{0x50}\)\(_2\) has also been implicated in control of ill-defined trafficking pathways close to the Golgi; for example, the recycling of mannose-6-phosphate receptor (M6R) back to the Golgi and also the trafficking of some types of ion and nutrient channels from the Golgi to the cell surface. This thesis describes attempts to study putative PtdIns(3,5) \(\char{cmmi10}{0x50}\)\(_2\) dependent trafficking in the early endocytic system of \(\char{cmmi10}{0x53}\). \(\char{cmmi10}{0x63}\)\(\char{cmmi10}{0x65}\)\(\char{cmmi10}{0x72}\)\(\char{cmmi10}{0x65}\)\(\char{cmmi10}{0x76}\)\(\char{cmmi10}{0x69}\)\(\char{cmmi10}{0x73}\)\(\char{cmmi10}{0x69}\)\(\char{cmmi10}{0x61}\)\(\char{cmmi10}{0x65}\) using two model proteins; the recycling of Vps10p from late endosome to Golgi and of Chs3p from recycling endosome to Golgi.

The introduction of electron capture dissociation mass spectrometry in 1998 has provided a unique technique for the analysis of peptides and proteins, especially for the identification and localisation of posttranslational modifications. Despite many successes debate continues on the radical-based mechanism of ECD. This thesis explores ECD behaviour in a wide range of PTMs with the intention of furthering our knowledge of the radical-based mechanism. Studies were undertaken on the effect of 3-nitrotyrosine, which is an electron withdrawing group, on ECD. The presence of nitration dramatically decreases peptide backbone sequence coverage but results in the presence of abundant small neutral losses. The key finding is the insight provided into the hierarchy of the various proposed ECD mechanisms. ECD of cysteine bound modifications is shown to result in the fragmentation of the sulfur-modification bond and backbone sequence coverage is highly diminished when analysing S-nitrosopeptides. ECD behaviour of hydrogen-deficient radical peptides is highly dependent on gas-phase peptide structure, with electron capture typically resulting in an increase in charge-reduced precursor intensity. Comparisons of the intermolecular phospho-guanidinium bond strengths between phospho-serine, -threonine and -tyrosine were undertaken. ECD of these complexes results in the retention of the noncovalent bond allowing backbone sequence coverage.

A method for developing an optical mapping system to quantify electrical activation and repolarisation in murine left atria was created. The spread of activation is important in understanding the mechanisms for the rhythm of the heart in healthy and diseased states as cardiovascular disease is the leading cause of death worldwide. The activation spread was recorded using a novel 2nd generation high resolution (128x2048 pixels) CMOS camera with the voltage sensitive dye di-4-ANEPPS. Algorithms for automatic quantification of action potential duration and conduction velocities were implemented in MATLAB. Optical mapping results were validated against monophasic action potentials and microelectrode measurements showing comparable duration measurements. A genetic mouse model of atrial fibrillation was used (Pitx2c\(^+\)\(^/\)\(^-\)) and was found to have a shorter action potential duration in the left atrium compared to wild-type mice. The results showed a preferential antiarrhythmic effect of the sodium channel blocker, flecainide, to the left atrium of Pitx2c\(^+\)\(^/\)\(^-\) mice. A second mouse model was used to mimic arrhythmogenic right ventricular cardiomyopathy (plako\(^+\)\(^/\)\(^-\)). No significant changes were witnessed in young sedentary cohorts at baseline and flecainide slowed conduction in both WT and plako\(^+\)\(^/\)\(^-\). In endurance trained mice, a prolongation of the effective refractory period was seen after flecainide treatment. Plako\(^+\)\(^/\)\(^-\) sedentary mice treated with dihydrotestosterone showed a prolongation in action potential duration.

Electron transfer is a key part of many chemical, biological and physical processes, that is commonly studied by electrochemical methods, which give insight into reaction mechanisms but no structural information. It is necessary to combine electroanalysis with another technique to gain essential knowledge of metal-ligand bond length and oxidation states. X-ray absorption spectroscopy (XAS) can provide these data for species in dilute solution and, if combined with electrochemistry, could potentially provide powerful insight into electron transfer reactions. This dissertation describes the development and application of techniques for the study of electrochemical intermediates by XAS. Chapters 2 and 3 introduce the theory and practice of electrochemistry and spectroscopy with emphasis on XAS. Chapter 4 describes the development of variable-temperature spectroelectrolysis cells for the study of electrochemical intermediates. In Chapter 5, the electrochemical behaviour of Cp\( \ast \)Rh(CO)\(_2\), is investigated as an organometallic compound representative of the redox chemistry studied in this thesis. Chapter 6 describes a new approach to the study of electrochemical intermediates in which a miniature electrolysis cell is combined with a microdispenser so that electrochemical intermediates can be generated and then dispensed, quenched at low temperature prior to study by XAS. Chapter 7 contains final conclusions.

The first project presented here was generated around 1 calcitriol (1,25D₃), and novel low-calcemic analogues (VDAs). The hypothesis of this study was that VDAs may be effective in Diffuse Large B cell Lymphoma. It has been demonstrated that 1,25D₃ and certain VDAs displayed moderate cytotoxic and pro-apoptotic actions upon DLBCL cells. Additionally, 1,25D₃ and VDAs used in a combination with antidepressant clomipramine displayed concentration anti-stimulatory actions upon activated normal B-cells. The second part of the study is focused around a novel immunomodulator CX1001, data from which is currently confidential. The third project has been built around GPR61. I have demonstrated that myc-tagged GPR61 is expressed in the cell membrane and is subject to N-glycosylation. The N12S mutant of GPR61, which is not subject to N-glycosylation, is also expressed on the cell surface. Treating the cells with tunicamycin reduces cell membrane expression of non-mutated and mutated proteins. Furthermore, GPR61 mRNA and protein are expressed in white blood cells with significantly increased level of expression of the protein in Th17 cells.

G-protein-coupled receptors (GPCRs) are major pharmaceutical drug targets due to their crucial role in cell signalling. The neurohypophysial peptide hormones [Arg⁸] vasopressin (AVP) and oxytocin (OT) signal through a subfamily of GPCRs, comprising the AVP receptors (V₁a R, V₁bR and V₂R) and the OT receptor (OTR). The aim of this work was to understand the molecular basis of receptor activation by defining exact contacts between agonist and receptor. Molecular modelling suggested that two conserved negatively-charged residues may be important for agonist binding and receptor activation. Interactions between agonists and the human AVP/OT receptors were probed using a combination of site-directed mutagenesis of the receptors and ligands incorporating modifications at specific points. The wild-type (WT) and mutant receptors were expressed in HEK 293T cells and pharmacologically characterised with respect to ligand binding, receptor activation, cell surface expression and ligand-induced internalisation when stimulated by endogenous, or modified, ligands. Mutual exchange of functional groups revealed direct interactions between AVP/OT/[Arg⁸] vasotocin (AVT; a chimera of AVP and OT) and the human AVP/OT receptors required for receptor activation. These studies advance current understanding of the molecular basis of receptor activation and have the potential to facilitate future rational drug design.

In this thesis, thermodynamic-based mathematical modelling is combined with experimental in vitro extracellular flux analysis in order to assess drug redox cycling and cellular bioenergetics. It is widely accepted that pharmacological activity of certain classes of drugs (e.g. anticancer, antimalarial) is related to their ability to accept one or two electrons. However, pharmacological activity via redox cycling is an understated mechanism of toxicity associated with many classes of drugs. In particular, oxidative stress as a result of redox cycling plays a pivotal role in the cause of cardiac toxicity. For example, doxorubicin is an anti-neoplastic drug used to treat cancer. It has strong links to redox cycling-induced cardiac toxicity associated directly with elevated levels of reactive oxygen species (ROS) and oxidative stress within the mitochondria. The underlying mechanisms of redox cycling is very difficult to elucidate, due to the fleeting existence of the radical species. However, assessment of such cellular bioenergetics can be ameliorated with the aid of computational assistance. In chapter 2 the development of a novel thermodynamic-based in silico model of doxorubicin redox cycling is described, which is parameterized using data from in vitro extracellular flux analysis. The model is used to simulate mitochondrial-specific ROS, with its outputs confirmed against in vitro data. Chapter 3 describes construction of a pH-dependent thermodynamic model of hepatic glycolytic flux, used to determine the role of the monocarboxylate transporter 1 flux during extracellular acidification. Finally, chapter 4 describes a thermodynamic-based in silico model of mitochondrial bioenergetics, capable of simulating oxygen consumption rates of a cohort of in vitro human primary hepatocyte data. The model is finally used to simulate perturbations in key bioenergetic variables and reaction fluxes, illustrating the resulting changes on mitochondrial pH, membrane potential and subsequent oxygen consumption rates.

Droplet microfluidics is a new approach for chemical and biological analysis. Discrete nano-litre droplets ensure chemical reactions occur quickly without cross-talk, allowing samples to be processed without dispersion. Droplet microfluidics is effectively a digital sample processing platform enabling sample droplets to be handled in a continuous serial format. This thesis describes a method for in situ compartmentalization of biological and chemical samples after separation using Slipchip technology. Isoelectric focusing (IEF) was used to separate biomolecules within the Slipchip. The device was used to compartmentalise the IEF separated samples into micro-droplets. This approach solves the compartmentalization challenge faced by current IEF systems. The digitised sample droplets can be collected serially or in parallel. In serial collection, droplets are collected in tubing that maintains their spatial sequence. For parallel collection, droplets are collected with a multi-pipette. Separated samples were assayed by gel electrophoresis on an Agilent Bioanalyzer. Separated samples were also postprocessed on-chip by mixing with pH indicator droplets for calibration of droplet pH. Such continuous-flow/micro-droplet conversion methods have the potential of hyphenating different separation techniques for performing advanced analysis of complex samples. One disadvantage with droplet microfluidics is the very small dimensions of the sample meaning that classical absorption assays are difficult. Therefore, a high sensitivity absorption-based optical method called Cavity Ring Down Spectroscopy (CRDS) was developed and evaluated. The integration of CRDS with microfluidic devices was explored. Microfabricated cylindrical lenses were used to increase the light coupling efficiency within a chip, but results showed that light losses in the system were too high to enable the effective use of CRDS for analysis.

Porphyrins, a substance class that can be found in diverse materials including green leaves and red blood cells, have been studied extensively over many years due to their potential industrial applications, e.g. in making optical electronic devices, in artificial photosynthesis or in sensors. In contrast, DNA has only recently been found to be a good scaffold for the construction of functional molecules. Combining the chemical properties of porphyrins and DNA could open the door to the production of multiporphyrin arrays using DNA as a scaffold; such materials could have multiple applications, an example being molecular electronic devices. The work described in this thesis reports on further investigations of the synthesis of the porphyrin‐nucleotide and its incorporation into DNA sequences, in order to study the structural, chemical and electronic properties of the porphyrin‐modified DNA. The analytical results, obtained from employing a variety of techniques such as UV‐vis and fluorescence spectroscopy, UV‐vis and fluorescence melting studies, circular dichroism spectroscopy and EPR (electron paramagnetic resonance) spectroscopy showed electronic interaction between porphyrins stacked on DNA. Metallation with zinc or copper of different porphyrins (namely diphenyl porphyrins and tetraphenyl porphyrins) was also successfully achieved, after they had been attached onto DNA. Based on EPR measurements, evidence was found for intermolecular stacking of the porphyrin‐DNA which leads to self‐assembled higher order structures. The EPR investigation also demonstrated that different oxidation states of metals held inside porphyrin‐DNA could be monitored. Using CD spectroscopy based on a synchrotron light source, the first measurements were made of DNA in the far UV region (< 200 nm). All the results obtained in this work and elsewhere during the past few years show that DNA based materials are highly promising for future applications in many areas of science, but especially in electronics and health care.

P.aeruginosa is one of the most important pathogens in nosocomial infections and fails to respond to standard treatment, particularly in the case of patients subjected to prolong antibiotic treatment. To generate a more comprehensive understanding of the failure of antimicrobial treatment, focusing especially on the adaptive resistance could be the key area that the bacterium develops in this phenomenon. Most studies on antibiotic resistance in P.aeruginosa have focused upon genotypic studies. This study set out to develop an in vitro model to examine the effect of continual exposure of P.aeruginosa PA01 to the antibiotics studied. Experiments were initially conducted to consider the factors that having a significant influence on antibiotic susceptibility using a novel fluorescence based assay (OxoPlate® system). P.aeruginosa was subjected to the action of tobramycin, amikacin and colistin under various environmental factors. The results of the in vitro analysis showed that, from among the three antibiotics used, amikacin was the antibiotic where resistance was most readily developed. From these results, chemostat studies were designed to examine prolonged exposure of the antibiotic to planktonic cells. Chemostat cultures were exposed to amikacin at sub-inhibitory concentrations using Evans defined synthetic medium at different dilution rates (D) under glucose limitation. Both cultures grown at 0.025h-1 and 0.06h-1 developed the following characteristics i. low-level amikacin resistance, which exhibited an increase in the MIC 4-fold. ii. a clear development of phenotypic resistance and this resistance was not acquired as evidenced by the loss of resistance on culture into fresh medium lacking antibiotic. iii. adaptive resistance to amikacin conferred low-level resistance to other aminoglycosides such as tobramycin and antibiotics with different modes of action such as colistin. Low oxygen availability was seen in the cultures grown at 0.099 h-1 and 0.125 h-1, which lead to i. the appearance of the so called “persister” phenotype. These persisters are sub populations of cells that showed a reduction in bacterial cell size as evidenced from the flow cytometry output as well as being slow growing and resistant ii. extracellular polymeric fibrils were produced in the cells derived after 72h incubation time. In all cases, continual exposure resulted in phenotypically distinct mucoid and non- mucoid colony morphotypes, which were clearly observed on amikacin-free nutrient agar. Some of these selected morphotypes showed from the MIC and MBC data a high-level resistance to the antibiotic when left without antibiotics. The biological responses resulting from these studies offer valuable clues underlying unsuccessful treatment. Conducting experiments using robust systems renders this project extremely novel in the field of microbiology and this will contribute to the development of viable treatment options and ultimately the reduction of the emergence of antibiotic resistance.