An absence of dystrophin in muscle has a massive impact throughout muscle development, and Duchene Muscular Dystrophy (DMD) is one of the consequences. The disruption of the dystrophin-glycoprotein complex (DGC) is caused by a mutation in the dmd gene, which effects muscle integrity, resulting in progressive muscle degeneration and weakness. In this study, dfd13 (dystrophin-deficient) and C2C12 (non-dystrophic) myoblasts were cultured in low mitogen conditions for 10 days to induce differentiation; however, dfdl3 myoblasts did not achieve terminal differentiation. It has been suggested that Pax7 may play a major role during myogenesis, therefore its expression pattern and transport protein were examined for any impairments. It was established that Pax7 localises in the cytoplasm of dystrophindeficient myoblasts and high expression is retained during differentiation. Colocalisation of Pax7 with subcellular markers analysis indicated that Pax7 is synthesised during the proliferative state. Pax7 was shown to possess a nuclear location signal and KPNA2 was suggested as escort protein for Pax7 translocation into the nucleus. The PTEN-PI3K/Akt signalling pathway was investigated and protein synthesis regulation and Fox03 was found to be impaired. Autophagy related genes were found to be highly expressed; however, LC3 lipidation and autophagy flux showed a reduction upon differentiation, indicating defective autophagy. The contribution of PTEN overexpression was assessed in relation to endoplasmic reticulum (ER) stress and activation of the unfolding protein response (UPR). It was established that a reduction in ER stress and changes to UPR activation lead to apoptosis. Finally, minidystrophintransfection of both types of myoblasts was utilised to examine the effect, especially in dystrophin-deficient myoblasts. Minidystrophin improved protein synthesis activation and increased autophagy (increased LC3 lipidation), suggesting that minidystrophin ameliorates dystrophic events at the level of autophagosome formation. To conclude, destabilisation of the plasma membrane owing to a dystrophin mutation causes cell signalling alterations which minidystrophin restoration can partly improve.

\(Escherichia\) \(coli\) encounters nitrosative stress from various sources. It was shown in a previous study that a possible role for Hcp in the nitrosative stress response. The focus of this study was to determine the function of Hcp. The growth of the \(hcp\) mutant lacking all known NO reductases was inhibited by various sources of nitrosative stres. The growth defect was complemented by native Hcp protein, but not by mutated Hcp protein with a disrupted hybrid cluster. The role of Hcp was shown to protect \(E\). \(coli\) from nitrosative stress, and the hybrid cluster is critical for its function. Direct interaction between Hcp and its oxidoreductase Hcr was demonstrated. The \(hcp\)\(^+\)\(hcr\) strain showed that it was still resistant to nitrosative stress. Possibly alternative oxidoreductase of Hcp exists in \(E\). \(coli\). Gas analysis of the headspace of the anaerobic cultures showed that when treated with NO, the Hcp\(^+\) strain lacking all known NO reductases was still capable of reducing sub-micro molar NO into N\(_2\)O, while the further deletion of Hcp completely abolished NO reduction. This provides the first in vivo evidence that Hcp is a high affinity, low capacity NO reductase in \(E\). \(coli\).

Platelets play a crucial role in haemostasis by preventing bleeding at sites of vascular injury. Inherited or acquired platelet defects can impair haemostasis resulting in bleeding symptoms of varying severity ranging from mild to excessive which can be life threatening. Diagnosis of mild platelet-based bleeding disorders is challenging due to the absence of a gold standard technique and their variable bleeding symptoms and bleeding phenotypes observed in healthy individual as well as other haemostatic disorders. The work in this thesis built on the previous studies in the genotyping and platelet phenotyping project allowing further characterization of inherited platelet function defects in individuals with mild bleeding disorders. Platelet aggregation and secretion in samples from 206 patients were investigated during the course of this thesis and were categorised on the basis of the observed defects. Surprisingly, in over a half of these patients, an ex vivo platelet function defect was not found. The genetic investigation of selected cases using whole exome sequencing identified mutations in number of genes previously known to be critical in platelet biology. This thesis also focused on evaluation of three other platelet techniques by comparison with lumi-aggregometry to assess their overall potential in detecting platelet function defects. Further studies are still needed to further assess the potential of these techniques before they can be applied in routine clinical diagnosis.

Symmetry breaking of the left-right body axis is a crucial step in development for many vertebrate species. In many this is initiated with a directional cilia-driven fluid flow in the organising structure. This work focuses on the mouse and the zebrafish organising structures, the node and Kupffer's vesicle, wherein cilia perform a tilted rotation producing an asymmetric flow. Using singularities of Stokes flow, slender body theory and the boundary integral equation, a computational model of flow in the mouse node for a range of cilia configurations simulating developmental stages is developed and run on the University of Birmingham's cluster, BlueBEAR. The results show the emergence of a directional flow as the cilia tilt increases. To model the Kupffer's vesicle the regularised boundary integral equation is used with a mesh representation of the entire domain to investigate potential cilia mechanisms that produce the observed flow as there is not a consensus. The results show that a combination of the experimental observations could be a sufficient mechanism. This model is expanded using observations of cilia with two rotation frequencies which are incorporated by allowing such cilia to ‘wobble’. This wobble accentuates the asymmetric flow in wildtype embryos and diminishes it in mutant embryos. All of these results agree well with experiment suggesting that vertebrates develop a combination of rotation mechanisms in their organising structures before an appropriate symmetry breaking flow is established.

In the last 15 years, the increased use of high throughput biology techniques such as genome-wide gene expression profiling, fitness profiling and protein interactomics has led to the generation of an extraordinary amount of data. The abundance of such diverse data has proven to be an essential foundation for understanding the complexities of molecular mechanisms and underlying pathways within a biological system. This thesis demonstrates the capabilities and applications of using biological networks to extrapolate biological information from the wealth of data available in the yeast species Saccharomyces cerevisiae and Schizosaccharomyces pombe. This study marks the first time a mutual information based network inference approach has been applied to a set of specific genome-wide expression and fitness compendia. In particular, this work has generated hypotheses in S. pombe that have led to a deeper understanding of the relationship between ribosomal proteins and energy metabolism, a recently discovered pathway termed riboneogenesis. Experimental validation of this hypothesis has led to new theories on the role of energy metabolism enzymes in controlling ribosome biogenesis in S. pombe, including the novel finding that fructose-1, 6-bisphosphatase (FBP1) may have roles in both gluconeogenesis and riboneogenesis. This thesis also demonstrates how the use of multi-level data allows for comprehensive insight into nuclear functions of the S. pombe nonsense-mediated mRNA decay protein, UPF1. This study provides substantial evidence demonstrating the role of UPF1 in DNA replication. The applicability of fitness data in identifying targets of metal and metalloid toxicity in S. cerevisiae has also been investigated.

Chiral recognition, determination of enantiomeric excess and the separation of enantiomers are challenging problems for the chemist. This work has as its aim the design and syntheses of new electrochemical chiral receptors for neutral molecules. All the receptors prepared contain a ferrocene group as electroactive reporting unit. The differences among the receptors mainly relate to the binding site and the chiral group. The first type of receptor, presented in Chapter 2, consists of chiral ferrocene containing boronic acids that have been used to electrochemically sense aromatic and aliphatic chiral and achiral diols. The electrochemical determination of the enantiomeric excess of a mixture of two enantiomers of Binol performed with one of these boronic acids represents a new advance in supramolecular chiral sensing. In Chapter 3 the synthesis of ferrocene-containing chiral macrocycles of different sizes is described. The binding site is a cavity featuring a diamidopyridine moiety, with the chirality introduced through a Binol unit. These receptors showed low interaction with achiral cyclic ureas and chiral carboxylic acids. Chapter 4 describes the study of self-assembled monolayers onto gold surfaces. The monolayers are formed by ferrocene-containing amides of lipoic (thioctic) acid and, for the first time, isolipoic acid. The studies indicate that isolipoic acid could be an attractive anchor group for SAM formation when strong control over the chirality of the monolayer is required.

With intensive and global usage, flame retardants (FRs) have played critical roles in the prevention of fires for decades. However, there are increasing concerns about the potential adverse effects of these chemicals due to the well documented environmental and human exposures to FRs. To date, relatively little is known about the molecular mechanisms of the potential toxic effects of human exposure to FRs. In this study, microarray-based transcriptomics and direct injection mass spectrometry based metabolomics were employed to investigate the molecular responses of human lung cancer cells (A549) and human hepatoma cells (HepG2/C3A) exposed to a range of sub-lethal concentrations of hexabromocyclododecane (HBCD), tris (1, 3-dichloro-2-propyl) phosphate (TDCIPP) and a mixture of FRs at equivalent concentrations to those found in typical household dust. Combined with the quantification of FRs levels in cells after exposure, this work using the non-targeted capabilities of multi-omics approaches has revealed that at the concentrations investigated, and which are relevant to human exposures, significant molecular perturbations are not induced by exposure to the FRs under study. The results from this thesis are beneficial for both understanding the potential mechanisms of effects of human exposure to FRs and for future risk assessment of these chemicals.

Accurate chromosome segregation during meiosis requires the reciprocal exchange of DNA between homologous chromosomes, via a process called homologous recombination, resulting in the formation of crossovers (COs). This process begins with the formation of programmed DNA double-strand breaks (DSBs). Certain genomic loci, called hotspots, are more likely than others to produce DSBs. This is thought to be determined by various factors, which include post-translational histone modifications, such as H3K4 trimethylation. The histone methyl-transferase AtSDG2 is largely responsible for the deposition of this histone mark. This research shows that CO frequency and distribution are altered in an \(Atsdg2\) background. Study of a mutant allele of a gene which codes for a subunit of a histone-acetyl transferase complex, called AtMRG2, revealed a strongly reduced fertility phenotype and failure to produce DSBs. Further study revealed that the defects were due to mutation to the \(AtPRD3\) gene, known to be essential for DSB formation, and that the mutation to \(AtMRG2\) was not responsible. During meiosis, homologous recombination takes place in the context of specific structural arrangement of DNA organised as an array of loops emanating out from a proteinaceous axis, a major component of which is AtASY1. My studies demonstrate that the dynamics of AtASY1 are affected by mutation to AtPCH2, an AAA+ ATPase, and that formation of the synaptonemal complex is perturbed. \(Atpch2\)mutants initiate DSB formation and CO designation normally, but defects occur in CO maturation, causing a reduced CO frequency and formation of univalent chromosomes at metaphase I. Finally, the effects of temperature on the structure of meiotic chromosomes and homologous recombination were studied by cytological analysis of Col 0 and different meiotic mutants subjected to a range of temperatures for the duration of meiosis. I have demonstrated that certain mutations, such as \(Atsdg2\) and \(Atpch2\), confer some resistance to the effects of high (32˚C) temperature treatment, which causes various meiotic defects in Col 0.

CD248 is a pericyte-associated, mesenchymal stem cell (MSC) marker that is highly expressed during embryological life. This expression is down regulated during development, becoming restricted on lymphoid stroma to the capsule, but reappearing during inflammation, as well as in a number of disease states (Lax et al., 2007). CD248 has been shown to play a role in controlling the differentiation ofMSC to osteoblasts, both in vitro and in vivo, achieving this effect by modulating PDGFRsignalling, as treatment with the PDGFRinhibitor imatinib mesylate phenocopies the effects seen in the CD248·;. mouse (Naylor et al., 2012). Here we present evidence that CD248 is involved in the differentiation of MSC, via PDGFRsignalling, into lymphoid stroma progenitors both in vitro and in vivo. In adult mice expression of CD248 is detected on FDCs following immunisation. Using CD248·1- mice, we observe that FDC networks in CD248·1- mice do not form normally and lack the reticular, dendrite-like structure typical ofFDCs. This defect associates with a reduction in the functionality of the germinal centres. Embryonic development of lymph node stroma occurs in a stepwise manner with progressive upregulation of VCAM and ICAM on resident mesenchyme. In the adult stroma, recent work has established links between different stromal cell subtypes; Jarjour eta/. (2014) used a fate mapping technique to discover that marginal reticular cells are able to differentiate to follicular dendritic cells in response to immune challenge. Contrasting evidence shows that FDC in the spleen derive from ubiquitous perivascular precursors, likely to be pericytes (Krautler et al., 2012).

RNA polymerase (RNAP) \(\sigma\) factor must recognise and bind to specific DNA elements, usually AT-rich, in order to initiate transcription. At AT-rich regulatory regions or with more than one \(\sigma\) factor binding site; RNAP has to distinguish between different targets to initiate transcription correctly. At two regulatory regions: i) cbpA regulatory DNA with overlapping binding sites for \(\sigma\)70 and 38 associated RNAP and ii) regulatory region for ehxCABD operon with AT content of 71 %, I examined how correct RNAP binding is ensured. For cbpA regulatory region it was found that the shared promoter spacer region played a key role. I identified a location in spacer region that differently affected overlapping cbpA promoters. The base change at this position is sensed by \(\sigma\)70 side chain R451. Alterations in spacer sequence modulate conformation, making it easier, or more difficult, for R451-DNA interactions. Using tethered particle motion analysis, DNA compaction properties of cbpA gene product; CbpA was measured. ehxCABD regulatory region contains many sequences resembling \(\sigma\) factor binding elements. RNAP is capable of binding to the correct promoter elements in this region only in the presence of a chromosome folding protein, H-NS which binds AT-rich DNA. H-NS “coats” ehxCABD regulatory region and enables specific RNAP binding. Finally, many intragenic promoters within ehxCABD operon were identified. We thus propose that H-NS plays a role in silencing this pervasive intragenic transcription.