Today antenatal ultrasound is a necessary tool for screening and diagnosis of pregnancy. It is extensively used for fetal chromosomal risk assessment, fetal anatomy and biometry, placental localization and fetal head presentation in singleton and multiple pregnancies. It is essential for intrauterine invasive procedures and for the management of growth restricted fetuses. Ultrasound during labor or "Intrapartum Ultrasound" has been explored widely in the last decade for the assessment of fetal presentation, fetal heart beat localization and more recently in advanced research topics such as monitoring the progress of labor and prediction of mode of delivery. The overall concept of this thesis was to describe new methods of assessment of parameters of labor by ultrasound. For this purpose, we performed an observational study investigating the use of a novel method for assessing cervical dilatation by ultrasound during labor; a novel method for monitoring the progress of labor "sonopartogram"; assessment of caput succedaneum by transperineal ultrasound and furthermore to analyze the parameters of labor by ultrasound in prediction of mode of delivery. In addition to the above methods we will give an insight on the current established methods for assessment of the progress of labor and will compare these methods to ultrasound. We applied innovative use of technology such as transperineal ultrasound in monitoring labor progress. Obstetrics has the opportunity to develop into an objectively guided skill. Assessment of fetal head descent no longer needs to rely on an imaginary line drawn between the ischial spines; cervical dilatation could be measured accurately using simple two-dimensional (2D) ultrasound. Monitoring the progress of labor has the opportunity to be performed solely by the means of 11 ultrasound. Labor characteristics such as head descent, head rotation, caput succedaneum, cervical dilatation when measured by ultrasound could predict the outcome of delivery (i.e. vaginal or caesarean delivery) in women with prolonged labor.

Side effects from medicines can have considerable negative impact on peoples' daily lives. As a result of an aging UK population and attendant multi-morbidity, an increasing number of medicines are being prescribed for patients, leading to increased risk of unintended side effects. The aim of this study was to explore experiences and opinions of patients and the public in identifying and managing side effects from medicines. It also sought to develop a novel causality scale for use by patients to assess suspected side effects. A mixed methods approach with four phases was selected. In Phase One surveys were distributed in pharmacies to gather information on patients' experiences of side effects and recruit potential interviewees for the following phase (935 surveys distributed; 230 returned). In-depth interviews were conducted in Phase Two with 15 people who had experienced side effects. These explored their opinions and experiences and informed Phase Three. This phase developed and validated a side effects assessment tool for patients' use (SE-PAST). The validation consisted of two strands, initial validation (by 31 assessors) followed by online validation (273 completed responses). In Phase Four 2285 patient reports to the Yellow Card Scheme were examined to learn about experiences of side effects, to investigate the value of patient reports to pharmacovigilance and to compare experiences of Yellow Card reporters to the public. This study provided novel insights into the strategies employed by patients to identify and manage their side effects. Patients seeking side effect information used a variety of information sources and the findings suggest that a key aspect of source selection may be a hierarchy of source characteristics. The strategies used to manage side effects varied, including both cognitive and behavioural responses such as non-adherence and consultation with healthcare professionals. The findings suggest that these strategies were influenced by a range of factors including established health beliefs; previous experience of side effects and cognitive biases. Areas of similarity and difference were identified between Yellow Card reporters and the general public. There was evidence of patterns in the causative drugs, the type and impacts of effects between those who report side effects and the wider public; however there was a difference in coping strategies between these groups, with non-adherence being more prevalent among Yellow Card reporters. Most on-line users of the SE-PAST agreed it would encourage them to report their side effect or talk to a healthcare professional about it. The thesis provides a unique and insightful perspective on patients' personal experiences of side effects, with implications for policy and practice. It has established that side effects can have noteworthy impacts with prolonged consequences on many aspects of patients' lives.

Each biomolecule in a living organism needs to adopt a specific threedimensional conformation to function properly. Function itself is usually achieved by specific interactions between biomolecular units. Structural knowledge at atomic level of biomolecules and their interaction is important to understand the mechanisms leading to biological response and to develop strategies to interfere with them when necessary. Antibodies are molecules of the immune system playing an ever more prominent role in basic research as well as in the biotechnology and pharmaceutical sectors. Characterizing their region of interaction with other proteins (epitopes) is useful for purposes ranging from molecular biology research to vaccine design. During my PhD studies I used a combination of solution NMR mapping, molecular biology and computational docking to provide a structural and biophysical characterization of new neutralizing antibodies from Dengue virus recovered subjects, comparing the binding of the same antibody to the four Dengue serotypes and the binding of different antibodies to the same serotype. We were able to rationally mutate an antibody to first alter its selectivity for different viral strains and then increase its neutralization by ~40 folds. For the first time, this was achieved without the availability of an x-ray structure. In a second sub-project, I investigated the interaction of the chemokine CXCL12 with the chromatin-associated protein HMGB1, confirming their direct interaction (only proposed but never proved before) and providing a structural explanation for the HMGB1 dependent increase of CXCL12 cellular activity. High profile publications resulted from the two above projects. The above mentioned projects relied heavily on solution NMR spectroscopy, which is ideally suited to the atomic level characterization of intermolecular interfaces and, as a consequence, to antibody epitope discovery. Having provided a residue-level description of a protein-protein interface by NMR, we subsequently used this experimental information to guide and validate computational docking experiments aimed at providing a three dimensional structure of the protein-protein (or antibody-protein) complex of interest. In collaboration with other members of my research group I validated the use of NMR and computational simulations to study antibody-antigen interactions, publishing two reviews in collaboration with other members of my research group.

Current technologies focus on the use of single drug delivery carriers delivering a single drug species, or multiple species, in a compromisingly loaded or damaged state. Here we aim to develop controlled liposome (vesicle) clusters as potential multi-drug carriers for applications in nanomedicine. Specifically; applications include but are not limited to, combinational therapeutics and for the simultaneous delivery of prodrug and complementary activating enzyme. To this end, we are utilising a platform where different liposomes can be connected through DNA linkers, in hope to deliver multiple species in close proximity whilst keeping the individual cargos separate. Liposome clusters can be formed using complementary lipid-DNA conjugates integrated into a vesicle s surface. To ensure strong directional liposome bridging, a patch of localised DNA allows the assembly of size-limited clusters through the directionality of the adhesive interactions. Studies have localised DNA through phase coexistence, mixing saturated (DPPC) and unsaturated (DOPC) lipids with cholesterol, forming lo patches of saturated lipid surrounded by a ld phase of unsaturated lipids. However, these studies displayed poor saturated lipid-DNA partitioning to the lo ordered patches, allowing a lack of specificity in directional interactions between functionalised domains. To enhance saturated lipid-DNA lo partitioning, 10 mole percent (mol%) cardiolipin (CL) was added. CL increases the free energy required for a saturated lipid to insert into the ld phase, increasing DNA partition coefficient to the lo phase by an order of magnitude. Here, a new four component phase diagram incorporating 10 mol% CL was plotted using liposomes with diameters of ~100 nm, an appropriate particle magnitude required for cellular uptake. Förster Resonance Energy Transfer (FRET) was implemented to map out the four component phase diagram as vesicle size was below optical microscopy limits. Results showed similar phase topology to the analogous three component diagram with the addition of potential lo-HII phase coexistence. Using the new phase diagram in conjunction with lipid-DNA conjugates, dynamic light scattering (DLS) displayed size limited tethered clusters < 200 nm in diameter could be achieved. This Indicated 10 mol% CL and phase diagram position limits the average number of tethered vesicles to single figures. Moreover, DNA-lipid bridging stab off analysis, where aggregates were shown to be stable at homeostatic temperature, pH and salt concentration. Furthermore, through the implementation of DNA i-motifs, small clusters were shown to be able to disassemble at pH 3, forming ~single vesicles, and to reassemble when the pH was raised to pH 7.4.

No description available.

An extraordinary diverse components of animal toxins have emerged along with the long-term evolution of animals, which have unique functions that protect them to survive in the wild. Specifically, the granular glands of amphibians contain multiple chemical compounds, among which, peptides are one of the major types of the constituents. This thesis is divided into six chapters. Chapter 1, as general introduction, describes the detailed background on peptide-based therapeutic agents, the promising therapeutic application of animal toxins, and a brief review on anuran skins and their secretions. Chapter 2 presents the materials and methods employed in this project. In chapter 3, a novel bradykinin-related peptide was isolated and identified from the skin secretion of odorrana livida using shotgun cloning and tandem mass spectrometry fragmentation sequencing approach. This peptide exhibited a dose-dependent contractile property on rat bladder and ileum and increased the contraction frequency on rat uterus ex vivo smooth muscle preparations. It also showed vasorelaxant activity on rat tail artery smooth muscle. In addition, the peptide was modified by substituting the penultimate amino acid in the amino terminus from phenylalanine to leucine. Theanalogue completely abolished parental peptide activity, but showed an inhibition effects on bradykinin-induced rat tail artery smooth muscle relaxation. By using specific antagonists for bradykinin B1 and B2 receptors, we found that bradykinin b2receptor is highly likely to be involved in the rat tail artery related effects caused bythis novel bradykinin-related peptide and its analogue. Chapter 4 and 5 are about thediscovery of two pairs of novel antimicrobial peptides belonging to Bombinin and VIBombinin H families, respectively, from the skin secretion of Bombina genus. In chapter 4, the sequence modification was applied on bombinin HL by replacing Lisomer-leucine to D-isomer leucine from the second position of the amino terminus. Both the wild type and modified peptides displayed well-defined α-helical structure in bacterial membrane mimicking environment. BHL-bombinin displayed broad-spectrum bactericidal activity against a wide range of microorganisms, while bombinin H only exhibited a mild bacteriostatic effect on gram positive bacteria. The synergistic antimicrobial effects were observed between BHL-bominin with bombininH and between bombinin H with ampicillin. In addition, haemolytic and cytotoxic examination exhibited a highly synergistic selectivity and low cytotoxicity on mammalian cells of these three peptides. In chapter 5, the sequence modification was employed in the BHK-bombinin by replacing the glutamic acid with lysine at the 23rdposition from amino terminus, which increased the net charge and expanded thenonpolar face of the original peptide. According to the results from in vitro function alanalysis, this modification strategy significantly improved the selectivity index of thepeptide with increased antimicrobial activity and decreased haemolysis activity. The combined antimicrobial evaluation on both natural and modified peptides showed synergistic inhibition activity against both gram positive bacteria and fungi yeast. In summary, this thesis reveals the combined strategy of using a molecular cloning technique and mass spectrometric method for novel host-defensive peptides identification from amphibian skin secretions. In vitro and ex vivo functionalevaluations were subsequently employed which not only bring us a better understanding on the diversity of natural sourced bioactive peptides but also emphasized the research value for characterizing their in depth mechanisms.

In this thesis, the 18F-labelling of new "chemotypes" will be described with the aim of application in pharmaceutically interesting targets. In Chapter 1, a general introduction to the effect of fluorine substituents on molecular properties and reactivity is provided. This includes the application of organofluorine in both medicinal chemistry and positron emission tomography. Chapter 2 describes a novel silver-mediated 18F-labelling of Ar-SCF3, Ar OCF3 and Ar-OCF2H, including [18F]Riluzole, the 18F labelled version of a drug for treatment of amyotrophic lateral sclerosis (ALS). This work demonstrates that AgOTf can induce halogen exchange nucleophilic 18F-fluorination under mild reaction conditions with a wide range of substrates. In addition, the 18F-labelled Ar-SCF3 substrate is further transformed into the [18F]Umemoto reagent in a single step. The Umemoto reagent has been extensively used for electrophilic trifluoromethylation of various functional groups. Therefore the labelling of this reagent could potentially expand the radiochemical space available for PET applications. To augment the utility of this reagent, we developed a late-stage stereoselective trifluoromethylation-thiocyclisation of alkenes using electrophilic trifluoromethylating reagents such as Umemoto reagent or Togni reagent, which will be discussed in Chapter 3. In this process, thiourea acts as both S-nucleophile and CF3 radical initiator; therefore no metal or photoredox catalyst is required. The reaction affords novel trifluoromethylated 2-amino-thiazolines and 2-amino-thiazines, important scaffolds in the development of aspartate beta-secretase enzyme (BACE-1) inhibitors, a therapeutic target for Alzheimer's disease. Chapter 4 investigates the 18F-labelling of a,a-difluoro-a-aryloxyacetic acid, a class of substrate that can serve as a versatile intermediate which can undergo various decarboxylative functionalisation reactions to afford a wide range of novel 18F labelled a fluorinated aryl ethers. Finally, Chapter 5 gives full experimental procedures and characterisation data for all compounds.

Optogenetics is a recently established experimental technique that involves the heterologous expression of light sensitive proteins (opsins) in mammalian cells to modulate cell function. One of the most commonly used opsin is the blue light gated depolarising channel, channelrhodopsin-2 (ChR2). Optogenetics involving ChR2 has revolutionised the field of neuroscience by enabling the definition of novel brain circuitries. Optogenetic control of the electromechanical coupling in vascular smooth muscle cells (SMCs) is now emerging as a powerful research tool with potential applications in drug discovery and therapeutics. However, the exact ionic mechanisms involved in this control remain unclear. The overall aim of this thesis was to establish ChR2 for use in vascular optogenetics. The main findings of this thesis are: 1) Blue light activation of ChR2 and the ChR2 variant ChR2(H134R) led to long-lasting and non-inactivating depolarising currents. 2) Transgenic mice expressing ChR2(H134R) selectively in SMCs were generated. Isolated SMCs obtained from these mice demonstrated blue light induced depolarising whole-cell currents. Fine control of artery tone was attained by varying the intensity of the blue light stimulus. This arterial response was sufficient to overcome the melanopsin-mediated light-depended arterial relaxation observed in the presence of contraction-eliciting agonists. 3) Pharmacological analyses revealed that Ca²⁺ entry through voltage-gated Ca2+ channels, and recruitment of plasmalemmal depolarising channels (TMEM16A and TRPM) and intracellular IP₃ receptors were mandatory for the ChR2(H134R)-mediated arterial response to blue light at intensities &LT;~0.1 mW/mm². Light stimuli of greater power evoked a significant Ca²⁺ influx directly through ChR2(H134R) and produced dramatic intracellular alkalinisation of the SMCs. The light intensity range that enables optical control of arterial tone primarily through the recruitment of endogenous channels and without substantial alteration of intracellular pH, was identified. Within this range, mice expressing ChR2(H134R) in SMCs are a powerful experimental model for achieving accurate and tuneable optical voltage-clamp of SMCs and finely-graded control of arterial tone, offering new avenues to the discovery of vasorelaxing drugs. 4) The Cl--selective ChR2 mutant iChloC mediates depolarising currents while also preventing H⁺ or Ca²⁺ fluxes. This mutant was also found to have enhanced sensitivity to blue light. This new optogenetic tool could be a good candidate for an improved mouse model of vascular optogenetics. To conclude, the work presented in this thesis represents an in depth analysis of the use of ChR2(H134R) in vascular optogenetics. The cellular mechanism linking ChR2(H134R) opening with contraction of vascular SMCs was defined. Additionally, the limitations of the current optogenetic mouse model have been identified, and iChloC was shown to resolve these limitations. This study established vascular optogenetics as novel research tool vascular physiology and pharmacology.

The use of chemical probes is a powerful tool that can help to address important biological questions. The study presented in this thesis aims to target reader domains of chromatin-associated proteins with small molecules, in order to provide information on their ligandability, useful to develop - potent chemical probes. This thesis work is divided in three parts. In the first and second part it is shown how structural information obtained by the use of synthetic peptides to study the binding mode of reader domains with their natural binding partner can be combined with fragment screening to gauge future optimization of small molecules. The first section described the disclosure of the binding mode of the H3 histone tail by the PHD zinc finger of BAZ2A. A crystal structure of the complex of BAZ2A with the H3 10-mer peptide identified a helical conformation of H3 upon binding with the PHD. This information coupled with further structural and biophysical analysis led to the identification of a subfamily of PHD characterized by an acidic patch on the helical turn, which is responsible of inducing helicity on H3 tail upon binding. The second part of the work investigated the ligandability of the PHD zinc finger domains of BAZ2A and BAZ2B. Using a combination of biophysical techniques and X-ray crystallography it was probed that it is possible to target these reader domains. Despite the similarities of the two PHDs, comparison of the fragment-bound crystal structures of the two proteins highlighted some differences in the binding mode. The last part of the project describes the several attempts performed in trying to elucidate the histone binding partner of the PHD-BrD tandem of the chromatin-related proteins BAZ1B and TRIM66, both involved in diseases.

The principle aims of this thesis are to investigate the synthesis of layered double hydroxides (LDHs) for use as drug delivery systems, and to explore the potential of these compounds to provide solutions to typical pharmaceutical problems. In Chapter 1, background information on pharmaceutics and drug delivery is given. LDHs are introduced and their structure, synthesis and general applications described. The biomedical and pharmaceutical applications of LDHs are reviewed. Chapter 2 presents the intercalation of the GABAergic drugs gabapentin and pregabalin into an LDH using the coassembly method. The optimum conditions for the restacking process are investigated, an improved method of anhydrous restacking in ethanol at reduced temperatures is described, and a mechanism is suggested. The stabilising effect of LDH intercalation on the drug ions is measured. Chapter 3 explores the potential for LDHs to stabilise and deliver catecholamine drugs levodopa, carbidopa and methyldopa. The ability of a range of LDHs to intercalate and stabilise these drugs against racemisation and oxidative decomposition across a variety of conditions is reported. In Chapter 4, the potential for LDHs to deliver neutral hydrophobic and cationic drugs through the intercalation of cyclodextrin-drug inclusion complexes is investi- gated. A range of partially substituted anionic cyclodextrins are synthesised and intercalated into an LDH. Cyclodextrin-drug inclusion complexes are intercalated, their interlayer orientation is suggested and drug release measured. In Chapter 5, the potential of polymer-LDH composites for drug delivery is explored. A selection of drug intercalated LDHs from Chapters 2 and 3 are encapsulated in several enteric polymers typically used for drug delivery. The release profiles and stabilising effects of the composites are measured. Chapter 6 details the experimental procedures and analytical instruments used throughout this thesis. The Appendices contain supplementary characterisation and analytical data that are referred to in the main text.