The ability of Shigella flexineri to spread within and between epithelial cells is essential for Shigella infection causing bacillary dysentery. This is a particular problem in the developing world. The movement of Shigella within the host cell requires the accumulation of actin at one pole of the bacterium and the protein VirG is responsible for this function. While the C-terminal domain (β-domain) of VirG is integrated into the outer membrane of Shigella, the N-Terminal domain (α-domain) is exposed on the surface of the bacterium. The β-domain acts as autotransporter of the α-domain. The exposed α-domain has multiple binding partners including N-WASP, Vinculin and IcsB that are required for infection in man and cell to cell spread. To understand the molecular basis of VirG’s activity, it is first necessary to produce the protein in quantity; this study investigates the expression of VirG α-domain in E.coli. The optimum construct corresponded to residues 58-506 of VirG expressed in Rossetta-gami cells.

The local structure evolution as a function of composition and temperature, and the resulting electrical conductivities were studied in samples with general composition (Yb1-xNdx)2Zr2O7, using Reverse Monte Carlo (RMC) modelling of total neutron scattering data and Impedance Spectroscopy. At low neodymium content (0.00 ≤ x ≤ 0.50), this system crystallizes in the fluorite structure with space group Fm3̄m, where-as at high neodymium content (0.75 ≤ x ≤ 1.00), a pyrochlore structure with space group Fd3̄m is observed. For compositions with high neodymium content prepared at lower temperatures, a fluorite structure is seen, which shows gradual evolution of pyrochlore ordering with increasing calcination temperature. Cation anti-site disorder was present throughout the compositional range. Total conductivity increases with increasing Nd content, up to a maximum at x = 0.75 and then decreases. The conductivity of these oxides is of the order of 10-4 S cm−1 at 700 °C. Variable temperature neutron powder diffraction experiments were carried out on the (Nd1-xCax)2Zr2O7-x (0.10 ≤ x ≤ 0.50) system, to investigate the local structural evolution as a function of composition and temperature. The entire compositional range studied exhibits a single phase with a pyrochlore-type structure. The variable temperature study showed that the pyrochlore structure is maintained throughout the temperature range studied. No cation anti-site disorder was present in any of the compositions, over the temperature range studied. Total conductivity increases with increasing Ca content, up to a maximum at x = 0.30 and then decreases. The conductivity of these oxides is of the order of 10-3 S cm−1 at 700 °C. Structural evolution in oxidising and reducing atmospheres was investigated in cerium zirconate, Ce2Zr2O7+δ. A number of transformations were revealed, depending on the atmosphere (O2 vs CO) and temperature. Upon heating the pyrochlore phase in flowing O2, three distinct phases were observed, with transitions at ca. 140 C and 410 C. At around 140 C, a previously un-identified phase formed, which was fitted with a triclinic model that contained 10 Ce and 10 Zr sites, each with a distorted cubic coordination. Above 410 C, the triclinic phase transformed to a cubic phase, corresponding to the κ-phase (space group P213). RMC modelling of the local structure of the -phase, revealed an ordered, pyrochlore-like cation sub-lattice, with a disordered anion sub-lattice. Upon re-heating the κ-phase in CO, the pyrochlore phase re-appeared at around 260 °C. At 290 °C, the pyrochlore phase was phase pure.

Multi-substituted optically pure cyclopropanes are important motifs present in many agrochemicals, pharmaceuticals and materials employed in manifold applications. Their synthesis is challenging due to both the strained conformation and the need to control both the relative and absolute stereochemistry. This thesis describes an investigation of the scope of the Wadsworth-Emmons cyclopropanation, highlighting it as a potential efficient methodology for diastereoselective and enantiospecific synthesis of these valuable ring systems. Chapter 1 is an introduction to the cyclopropanation protocols and is split in two subsections. The first is a description of the currently most exploited synthetic pathways to cyclopropanes, with analysis of their substrate scope and critical analysis of their limitations as compared to potential of the Wadsworth-Emmons cyclopropanation (Sections 1.1 to 1.3). The latter section consists of a review on the history and the evolution of this procedure and introduces the preconditions for the development of the project (Sections 1.4 to 1.17). Chapter 2 describes the results and discussion, introducing the use of novel alkyl-substituted triethyl phosphonoacetates to yield cyclopropyl-esters containing quaternary stereocentres. The high yields and the excellent trans-diastereoselectivity values obtained (proved by X-ray crystallography) allowed the proposal of a suggested reaction mechanism, which is supported by the subsequent experiments carried out in the presence of other functional groups, e.g. other carbonyls, nitriles, hetero-aromatics, substituted phenyl rings. This study was further extended to examine the effect of fluorine atom, due to its importance in biologically active environments. The study on the stereochemistry of the Wadsworth-Emmons cyclopropanation has been strongly supported by a range of X-ray crystal structures of the cyclopropane products. The chapter ends with an empirical set of guidelines, helpful for the design of successful cyclopropanations. Chapter 3, describes the experimental methods in full as well as full characterisation of the products obtained. Literature references, noted throughout the thesis, are listed in the final chapter.

The seasonal variation of anammox is yet to be comprehensively studied, unlike denitrification, the more traditional sink for fixed nitrogen. A seasonal study of anammox, denitrification and benthic oxygen consumption using the revised isotope pairing technique is presented in Chapter 2. Experimental temperature and NO3- concentration were kept constant throughout so that the capacity of the sediment for anammox could be estimated. Similar seasonal variations in the rates of anammox, denitrification and oxygen consumption suggest that anammox is controlled by the availability of organic carbon. Furthermore the effect of tidal inundation by overlying water rich in NO3- was investigated by measuring rates of anammox, denitrification and oxygen consumption at three tidal elevations throughout the year. A significant relationship between anammox and denitrification was established at each tidal elevation, which increased in strength as length of inundation decreased. To complement this seasonal study, additional experiments were undertaken, which are described in Chapter 3, to determine how anammox, denitrification and sediment metabolism responds to variations in experimental NO3- concentration and temperature. There were significant increases in rates of anammox, denitrification and sediment metabolism with temperature until 20oC when rates of anammox began to reduce. Furthermore there was significant variation in the response of all three processes to temperature in samples collected at different dates, which suggested that reduced bioavailability of organic carbon in the winter months was limiting the response to temperature. In addition to exploring how inorganic N is cycled in estuarine sediments, the ability of estuarine sediments to oxidize urea via nitrite was examined using 15N and 13C labelled substrates. Results, which are presented in Chapter 4, indicate that urea added to anaerobic sediment slurries was rapidly hydrolysed to ammonium before being oxidized via the anammox pathway.

Two of the most rapid drivers of evolution are hybridisation and polyploidisation. Hybridisation allows the rapid introduction of novel genetic material, potentially much faster than mutations, but this process is impeded by reproductive barriers between species. Differencesinploidylevelcanformsuchabarrier. Hybridisationaswellaspolyploidyareknown to occur frequently in the plant kingdom, including the genus Betula, which is investigated in this thesis. Three species of the Betula genus that exist in the United Kingdom are studied here: B. nana (dwarf birch), B. pendula (silver birch), and B. pubescens (downy birch). They differ in ploidy: B. nana and B. pendula are diploid and B. pubescens is a tetraploid. HybridisationandgeneflowbetweenthesethreespecieswasanalysedbyusingaRAD-seq dataset derived from 196 wild individuals. It was found that introgression acts unidirectionally from the diploid into the tetraploid species and that there is a cline of introgression between the north and south of the UK. This result suggests a range shift of the species from different distributions in the past. Gene flow from B. nana to B. pubescens could be a neutral or even maladaptive consequence of their past species distributions. Alternatively, it could be an adaptive process: alleles from B. nana could be helping B. pubescens to adapt to harsher, more northerly populations. To gain a preliminary understanding of the possible effects of introgression, the loci in close linkage to RAD tags introgressed from B. nana into B. pubescens were investigated and their putative function inferred by comparing their homologs in related species. To enhance the analyses, a draft whole genome sequence assembly of a B. nana individual was improved with long read data generated by PacBio sequencing, as well as the addition of RNA-seq data. This produced a more contiguous and complete reference sequence, enabling a closer look at more genes in linkage to the RAD tags.

In molecular electronics, the building material (traditionally elemental semiconductor) is replaced by single molecules or a nanoscale collection of molecules. Key to molecular electronics is the ability to precisely embed molecules into a nano device/structure and to manipulate large numbers of functional devices so they can be built in parallel, with each nano-device precisely located on the electrodes. In this work, the assembly of organic and inorganic nanostructures dispersed in aqueous solutions has been controlled via chemical functionalisation. By combining this bottom-up assembly strategy with traditional top-down lithographic apporaches, the properties of these nanostructures have been investigated via a range of different techniques. The high degree of control on the molecular design through chemical synthesis and the scalability by self-assembly make this approach of interest in the field of molecular electronics. In this regard, this dissertation presents a solution-based assembly method for producing molecular transport junctions employing metallic single-walled carbon nanotubes as nanoelectrodes. On solid substrates, electrical and electronic properties have been investigated by Conducting Atomic Force Microscopy (C-AFM). Furthermore, different strategies for asymmetric junction formation have been explored towards the development of a potential nanoscale Schottky diode. Moreover, various patterning techniques based on shadow evaporation and AFM probe scratching have been investigated for the assembly of 1-D nanostructures. Nanostructures dispersed in solution were organised onto surfaces by means of dielectrophoretic assembly, and their electronic properties was then measured by the means of a probing station. In addition to the aforementioned organic nanostructures, we also report on the dispersion of boron nitride nanotubes (BNNT) by DNA wrapping, followed by the formation of nano-hybrids of boron nitride nanotubes and carbon nanotubes. Previously, researchers have adopted BNNT as a 2D dielectric layer. The work inspires me to adopt boron nitride nanotubes as 1D dielectric materials. The techniques developed in this thesis are of interest for fundamental studies of electron transport in molecules and nanostructures. Addtionally, the approaches developed in this work may facilitate the advancement of new technologies for electronics, including, but not limited to, future circuits based on single-wall carbon/boron nitride nanotubes with specific functionality.

Genomes are constantly challenged by agents that promote DNA damage, with interstrand crosslinks (ICLs) representing a particularly dangerous lesion. Ongoing work in the Wilkinson laboratory aimed at identifying novel agents that target Trypanosoma brucei, the causative agent of African trypanosomiasis, identified several prodrugs that once activated form ICLs in this protozoan parasite. To understand the complexity of ICL repair systems that T. brucei employs to resolve such damage, a variety of null mutant lines were generated that lack activities postulated to fix such lesions. Phenotypic screens using various DNA damaging agents revealed that TbMRE11, TbEXO1, TbCSB, TbCHL1, TbFAN1, TbBRCA2 and TbRAD51 all help to resolve ICLs, implicating components of the homologous recombination, nucleotide excision repair and mismatch repair pathways in resolving this form of damage: This approach demonstrated that components of the translesion synthesis pathway (TbREV2 and TbREV3) do not play a significant role in ICL repair. In many organisms, nucleases belonging to the SNM1/PSO2 family play a key and specific role in the repair of ICLs with this property extending to the T. brucei homologue, TbSNM1. To assess whether there is a functional linkage between the DNA repair factors noted above and TbSNM1, a series of double null mutants were constructed and the susceptibility of these lines to ICL inducing agents determined. Identification of their epistatic/non-epistatic interactions revealed that T. brucei expresses at least two ICL repair systems with one pathway involving the concerted activities of TbSNM1/TbCSB/TbEXO1, that we postulate functions to repair ICLs encountered by the transcriptional machinery, while the other is centred upon TbMRE11/TbFAN1/TbEXO1 that may help resolve lesions which cause stalling of DNA replication forks. By unravelling how T. brucei repairs ICLs, specific inhibitors against key components of these pathways could be developed and used in combination with DNA damaging agents to target trypanosomal infections.

El Niño is a climatic event that can have large-scale impacts on global rainfall patterns, causing severe droughts in some regions and floods in others. The frequency of strong El Niño events is expected to increase in the future under scenarios of climate change. Despite this, the consequences of El Niño-induced droughts for ecological interactions are poorly understood. Here I applied DNA barcoding to assess the diets of frugivorous and insectivorous bats in the dry forest and rainforest of Costa Rica during one of the strongest El Niño on record (2015) and compare it with a non-El Niño year. My data indicated that the mutualistic network structure observed during the El Niño event was similar in both dry forest and rainforest, despite these habitats experiencing droughts and flooding, respectively. However, during the non-El Niño wet season in the dry forest, niche overlap was higher than the El Niño event. Antagonistic networks showed little change in the overall size and diversity of modules of interaction, but there were significant changes in modularity and the position of the nodes between the networks constructed during the El Niño year versus the normal year in dry forest. Additionally, I evaluated the relationship between wing morphology and diet specialization and differentiation of individuals. I observed that individuals of a common insectivorous bat species, Pteronotus mesoamericanus, showed differences in diet that correlated with wing morphology. To conclude, El Niño was associated with similar changes in the organisation of mutualistic networks in both dry and wet forests, as well as with modifications at the node level in antagonistic networks of dry forest. Such changes could have profound impacts for network resilience and the maintenance of interactions and species at both sites over time.

A novel methacrylate based nanogel system has been designed and developed for drug delivery applications. Methacrylates are optimal tuneable materials in terms of polarity, with combination of hydrophobic and hydrophilic moieties. Synthesis of these nanogels (NGs) was achieved via high dilution radical polymerisation using 2-(tert-butylamino)ethyl methacrylate (tBAEMA) as functional monomer, methacrylic acid (MAA) or ethylene glycol methyl ether methacrylate (EGMMA) as co-monomer and N,N'-methylenebis(acrylamide) (MBA) as cross-linker. Fabricated nanoparticles (NPs) were shown to possess water solubility higher than 2 mg/mL and diameter ranging from 5 to 20 nm (depending on nanogels' composition) as confirmed by either dynamic light scattering (DLS) and transmission electron microscopy (TEM). Moreover, nanogels produced have shown the ability to be employed as Pickering-Ramsden emulsion stabiliser. Their reduced size together with their emulsion capabilities make these nanoparticles a promising system for drug delivery, in particular taking into account skin as administration route. The size is in fact small enough to favour their penetration through the stratum corneum. Furthermore, in the view of their ability to form emulsions, nanogels could be used both as drug carrier and emulsifier in a final pharmaceutical formulation. NGs proved to be able to incorporate both small molecule such as fenoprofen (an anti-inflammatory non-steroidal drug) and big macromolecule such as siRNA. Cytotoxicity and cell metabolism were also evaluated by transfecting normal human dermal fibroblasts (NHDF), keratinocytes (HaCaT) and HeLa cells with nanogels. Data showed that nanoparticles did not affect viability, cells' morphology and adenosine triphosphate (ATP) levels up to high concentration of 100 μg/mL. In addition, preliminary studies indicated the ability of the nanogels to internalise and release their payload inside cells. In conclusion, the results confirmed that this novel system possesses all the desired characteristics to be used as a promising platform for drug delivery.

Antibiotic resistance occurs when bacteria change in response to selective pressures induced by antibiotics, which has become a major concern worldwide and one of the biggest threats to global health. Antibiotic resistance can occur naturally, but the misuse and overuse of antibiotics is accelerating the process. One way to combat this process is to understand the different relationships between microbes, also known as polymicrobial interactions. Bacteria can interact with one another synergistically or antagonistically and understanding the mechanisms behind these interactions can lead to the discovery of new therapeutics or targets to fight and kill pathogenic microbes. The rarely pathogenic Gram-negative bacterium, Alcaligenes faecalis, has previously been shown in our lab as playing an important role in potentially fighting antibiotic and antifungal resistance due to its competitiveness during polymicrobial interaction. Our research has found that A. faecalis kills Bacillus cereus, Staphylococcus aureus, and Candida albicans. This is a unique characteristic as these targets encompass both prokaryotic (bacteria) and eukaryotic (fungi) microbes. These three species are known to cause numerous infections in humans and have increased cases of antibiotic and antifungal resistance. In the present study, we investigated the genetic elements A. faecalis utilizes to inhibit growth when interacting with B. cereus, S. aureus, and C. albicans. Transposon mutagenesis was performed to create a genetic library of A. faecalis loss-of-function mutants. These strains were then screened against all three microorganisms to determine which mutants no longer inhibited growth. The mutants that lacked zones-of-inhibition were sequenced to determine the gene that had been interrupted. BLAST analysis of these sequences identified a MFS transporter, a 2FE-2S iron sulfur binding protein, a mechanosensitive ion channel, and a glucose-6-phosphate isomerase as instrumental in this inhibitory mechanism. Results from this research study can be used to further study polymicrobial interactions and potentially discover new therapeutics to combat antimicrobial resistance.