Transhydrogenase is an enzyme located in the cytoplasmic membrane of bacteria or the inner membrane of animal mitochondria. Using the energy of the proton electrochemical gradient (Δp), transhydrogenase translocates protons across the membrane whilst undergoing its redox reaction, in which hydride ion equivalents are transferred from NADH to NADP+ producing NAD+ and NADPH. Transhydrogenase comprises three components; dI binds NA(H), dIII binds NADP(H) and dII spans the membrane. Transhydrogenase is thought to function by way of a binding-change mechanism, which involves “open” and “occluded” conformations of the enzyme. In the open conformation, nucleotides can readily bind and dissociate from the enzyme but the hydride transfer reaction is blocked. In the occluded conformation, hydride transfer is permitted but the binding and release of nucleotides is blocked. Hydride transfer and proton translocation are coupled. The coupling is not well understood due to the lack of structural information about the membrane-spanning dII component. However, it is believed to involve conformational changes of the enzyme, particularly the dII and dIII components, resulting in the switch between the open and occluded conformations. Enzyme assays and tryptophan fluorescence experiments using apo-dIII in complex with dI revealed two features: Firstly, the binding of NADP(H) to dIII is very slow and is probably limited by the conversion from the occluded to the open conformation. Since the switch between the occluded and open conformations is thought to be central in the coupling of hydride transfer and proton translocation, the results presented here give an insight into the binding-change mechanism of transhydrogenase. Secondly, NAD(H) is able to slowly bind into the NADP(H)-binding site of dIII (the “wrong” site). This brought into question the specificity of the dIII component of transhydrogenase for NADP(H). The significance and likelihood of NAD(H) binding to dIII in the intact enzyme in the living cell are discussed.

Lathyrus species are well placed to meet the increasing global demand for food and feed, at the time of climate change, provided that the problem of the neurotoxins is solved. Conservation and sustainable use of the genetic resources of Lathyrus is of significant importance to allow the regain of interest in Lathyrus species in world. A comprehensive global database of Lathyrus species originating from the Mediterranean Basin, Caucasus, Central and West Asia is developed using accessions in major genebanks and information from eight herbaria in Europe. This information allowed to determine gaps in ex situ collections, mainly for wild relatives of cultivated species, and to identify appropriate sites for in situ conservation, mainly in the Fertile Crescent region. Core subsets were identified and the Focused Identification of Germplasm Strategy (FIGS) was used to derive a subset for heat and drought tolerance. This study used morphological characters and AFLP markers to better understand the taxonomic classification of different Lathyrus sections and species and to gain insights on the phylogenetic relationships among them. A Field Guide for Lathyrus L. species of the Mediterranean Basin and Caucasus, Central and West Asia is produced, to ease their identification by non-professional taxonomists.

This thesis is concerned with speciation and population genetics in the plant genus Silene (section Elisanthe). The introductory chapter is a literature review covering characteristics of the species studied, and the current literature on their evolutionary dynamics and population genetics. The second and third chapters cover techniques used in all experiments, such as DNA extraction, sequencing and genotyping protocols, and explain the rationale behind the initial experimental design. The fourth chapter focuses on the multi-locus analysis of autosomal gene sequences from S. latifolia and S. dioica. The relationship between the two species was investigated using various analyses such as isolation modeling and admixture analysis providing estimates of evolutionary distance and extent of historical gene flow. The maintenance of the species despite frequent hybridization at present-day hybrid zones is discussed. The fifth chapter discusses S. diclinis, a rare endemic found only in Valencia, Spain. The nature of population structuring and the evolutionary history of this species were investigated using a multilocus approach incorporating individuals from S. diclinis populations. The causes of the restricted distribution and low population size of this species is discussed The concluding chapter discusses how the species evolved from a common ancestor amidst changing climatic and environmental conditions.

Since the human genome project was completed in 2003, extraordinary progress has been made in the field of genomics with the development of new sequencing technologies and the widespread introduction of next generation sequencing (NGS). The application of NGS initiated a new era in genomics by massively increasing the number and diversity of the sequenced genomes at lower cost. Human Molecular Genetics has greatly benefited from the use of NGS-based strategies to identify human disease genes. In this thesis, I investigated the application of genetic techniques to investigate the molecular basis of autosomal recessively inherited disorders of unknown etiology. A range of disease phenotypes, including oligodontia and fetal akinesia/multiple pterygium syndrome (FA/MPS), were investigated in patient cohorts that included many cases with parental consanguinity. Using an autozygosity linkage analysis-based approach and Sanger sequencing of candidate genes resulted in the identification germline RYR1 mutations in FA/MPS. Subsequently, using exome sequencing techniques, the molecular basis of FA/MPS was further elucidated by the identification of germline mutations in RYR1, NEB, CHRNG, CHRNA1 and TPM2. The application of NGS in genetically heterogeneous disorders such as fetal akinesia/multiple pterygium syndrome can enable better and less expensive molecular diagnostic services aimed at specific mutation spectra, though more extensive sequencing can lead to the identification of larger numbers of variants of uncertain significance.

Existing methodologies for biomolecular detection are limited in several key areas. Heterogeneous assays struggle with various wash steps which can prolong assay time, while other assays require costly reagents, lack mobility and can be highly complex in nature. This project demonstrates how a bio-nano particle in the form of M13 bacteriophage (M13) can be used for the basis of a novel homogeneous immunoassay which incorporates the use of linear dichroism spectroscopy (LD). M13 has a high aspect ratio which allows it to align easily in shear flow, this in turn generates a large LD signal. This property of M13 has been manipulated for use in a new in-vitro diagnostic technique. Existing M13 production yields are much lower than those required for this assay. A new method was developed which increased the yield 10 fold. Chemical modifications were made by covalently attaching chromophores, this enabled the M13 LD signal to be visualised in the visible region and develops the potential for multiplexing. By chemically modifying M13 with chromophores and antibodies it was possible to create an assay capable of detecting 10⁵ cells/mL of Escherichia coli O157. This is 100 times more sensitive than the M13 based assay developed by Pacheco-Gomez et al. (2012). The assay was reassembled to detect small molecules and was found to have a sensitivity of 0.01 mM. The assays presented form a sensitive, specific, fast diagnostic tool capable of detecting pathogens and small molecules. It offers significant improvements over existing methods, and could act as a platform in developing a multimodal detection system.

Drug toxicities represent a major problem in drug discovery and development; therefore there is a push to develop new technologies to detect these early on. In this thesis I investigated the utility of zebrafish embryos and larvae in evaluating the biological activity of novel compounds and developed new methods for assaying the potential toxic effects of drugs in vivo. An electrocardiogram (ECG) recording set-up for zebrafish embryos and larvae was developed to assay drug-induced cardiotoxicity. The set-up was validated by testing drugs known to induce cardiotoxicity in humans in zebrafish larvae. The results obtained were in agreement with those documented in humans demonstrating the utility of the zebrafish larva in detecting drug-induced cardiotoxicity. The zebrafish embryo was also found to be a useful model for probing the biological activity of novel and marketed compounds providing an insight into the relationship between chemical properties and biological effects. Additionally, the assessment of the anti-inflammatory activity of a set of reference drugs revealed that the zebrafish larva also presents a promising model for therapeutic drug screens. Overall, the results described in this thesis show that the zebrafish presents an effective, reliable and rapid model for assessing the biological activity of drugs in vivo.

The mammalian gut microbiota is an essential factor in intestinal function and thus overall health. In the post genomic era, culture independent studies into the gut microbiota, particularly that of humans have allowed great leaps forward in knowledge of a once cryptic ecosystem. Furthermore, recent advances in sequencing technologies have allowed acceleration and broadening of work in this research field. Despite this, the canine gut microbiome has remained relatively uncharacterised. This work investigates the faecal microbiota of a diverse multi-breed and multi-location group of 79 dogs, by amplifying and sequencing the 16S rDNA from these dogs using both Sanger sequencing of clone libraries and high throughput pyrosequencing. A robust census of the canine faecal microbiota was undertaken. The most abundant genera were the Bacteroides, Prevotella, Cetobacterium, Fusobacterium, Sutterella and Megamonas. A limited core microbiome was defined in 90% of the study population; this represented less than 0.5% of richness but more than 37.4% of abundance. Influences of host sex, diet and age were investigated but were found not significant. Some evidence was found for breed associated richness differences, most marked in Labrador retrievers and miniature Schnauzers. Furthermore, the microbiota of the Labradors appeared to cluster separately from the other breeds.

Introduction: Type 2 Diabetes (T2DM) and sleep-related disorders share common risk factors such as obesity; but the interrelationships between T2DM and sleep disorders are not well examined. Aims: In this thesis I aimed to assess: 1. The longitudinal impact of obstructive sleep apnoea (OSA) on micro vascular complications in patient with T2DM. 2. The relationship between sleep quality, sleep duration and adiposity in patients with T2DM Methods: To examine the first aim, I utilized the data collected from a previous project that examined the cross-sectional associations between OSA and micro vascular complications in patients with T2DM and followed up the study participants longitudinally using 1-2-1 interviews and electronic health records. For aim 2, I conducted a crosssectional study in patients with young-onset T2DM who were recruited from Heart of England NHS Foundation Trust and primary care. Result: For Aim 1: Depending on the micro vascular outcome examined, we had approximately 200 patients in the analysis. Patients were followed up for 2.5 years for renal outcomes, and 4-4.5 years for retinopathy and neuropathy outcomes. The prevalence of OSA was 63%. I found that baseline OSA was significantly associated with greater decline of eGFR and greater progression to pre-proliferative and proliferative retinopathy. I also found that OSA was associated with progression to a combined outcome of foot insensitivity or diabetic foot ulceration but this was a non-significant trend (p=0.06). In addition, I found that patients who received and were compliant with continuous positive airway pressure (CPAP) treatment (delivered during routine care) had improvements in heart rate variability parameters by study end. For Aim 2: Poor sleep quality and shorter sleep duration were associated with increased total body fat% after adjustment for potential confounders. Conclusion: I found that OSA plays an important role in the progression of micro vascular complications in patients with T2DM. Whether treatment with CPAP has a favourable impact on micro vascular complications is currently being examined in a randomized controlled trial. I also found that sleep duration and quality are associated with increased adiposity. The direction of this relationship need to be examined in longitudinal studies and interventional trials.

The non-native predatory phytoseiid mite \(Neoseiulus\) \(californicus\) has been found in recent years in UK apple orchards. The aims of this study were to determine whether this mite could establish in the UK and its potential as a biocontrol agent for \(Panonychus\) \(ulmi\). By reviewing the literature and examining specimens of \(N. californicus.\) it was concluded that taxonomic synonymies with \(Amblyseius\) \( californicus.\) \( Amblyseius\) \(chilenensis\) and \(Typhlodromus\) \( mungeri\) could be supported, but those with \(Typhlodromus\) \( marinus\) and \(Neoseiulus\) \(fallacis\) could not. \(Neoseiulus\) \(californicus\) was found in strawberry, hop, blackcurrant and apple plantations in the main fruit growing regions of the UK. Field and laboratory studies showed that \(N.californicus\) possesses the ability to diapause, is a chill tolerant species and can survive winter field conditions in the UK. \(Neoseiulus\) \(californicus\) was found to readily consume both \( Panonychus\) \(ulmi\) and \(Tetranychus\) \(urticae\) and consumed greater numbers of prey than the native phytoseiid \(Typhlodromus\) \(pyri\). Deutonymphs consumed an average of 1.8 and 1.6 immature \(P. ulmi\) stages per day respectively and an average of 2.6 and 1.4 \(T. urticae\) respectively. The total mean development time for \(N. californicus\) was 7.47 days and for \(T. pyri\) was 12.45, feeding on \(P.ulmi\). \(Neoseiulus\) \(californicus\) from USA, Spain and UK displayed differences in measurements of a selection of morphological characteristics, diapause ability (16, 0 and 960/0 diapause respectively), development times (shortest for USA and longest for UK), fecundity (0.82-0.97 eggs per day) and esterase banding patterns, indicating the existence of different detectable strains. In conclusion, \(N. californicus\) was found to be a component of fruit plantation fauna in the UK, has the potential to survive winter field conditions and readily consumes \(P. ulmi\) and \(T.urticae\).

This thesis investigated reconstitution of CMV-specific T-cells in two cohorts of HSCT patients and studied the potential role of Tumour Necrosis Factor Receptor 2 (TNFR2) in regulation of CMV-specific T-cell expansion post HSCT. The first cohort included patients of a randomized phase II trial of adoptive cellular therapy for CMV-specific CD8\(^+\) T-cells. Cellular therapy resulted in earlier and greater expansion of CMV-specific CD8\(^+\) T cells and also reconstitution of CMV-specific CD4\(^+\) and non-infused CMV-specific CD8\(^+\) T-cells. The number of infused therapeutic T-cells and circulating levels of Alemtuzumab were found to influence immunotherapy. Additionally, reconstitution of CMV-specific CD4\(^+\) T-cells was studied using HLA-class II tetramers. CMV-specific CD4\(^+\) T-cell count of >0.7x10\(^3\)/ml was found to protect from recurrent CMV reactivation. One third of specific CD4\(^+\) T-cells were perforin and granzyme-B positive indicating cytotoxic potential, whilst the majority expressed T-bet. Expression of CD57 molecule on CD4\(^+\) T-cells was demonstrated as a potential biomarker of immune response to CMV. Also, distinct cytokine receptor expression patterns in naïve versus memory T-cells were observed. The results showed rapid decrease in IL-6R and increase in expression of TNFR2 after T-cell differentiation from naïve to effector cells and engagement of TNFR2 led to the apoptosis of CMV-specific T-cells.