GPCRs are the largest family of proteins in the human genome and a target for huge numbers of therapeutic drugs. However, the role of skeletal muscle in the action of these drugs is unclear. Given the unique importance of GPCR signalling in terms of glucose and fatty acid turnover in other tissues, it would be anticipated that GPCR identified to influence metabolism in these tissues might well be expressed in skeletal muscle. This study investigated the expression of genes encoding GPCRs in skeletal muscle and in cultured preparations thereof. In particular, this study focussed on the expression and signalling of adenosine receptors, a2-adrenoceptor, P2Y receptors and CBI cannabinoid receptors and the impact of CBI receptor modulation upon insulin signalling in rat primary skeletal muscle cells. All experiments in this work looked at GPCR expression and their signalling; with either tissues or cultured cells from rats. These experiments included: 1. Transcriptional profiling of skeletal muscle tissue in Wistar rats for GPCRs and proteins in associated signalling pathways. 2. Signalling of GPCRs (adenosine, a2A-adrenoceptor, P2y) in rat primary skeletal muscle cells. 3. Cannabinoid signalling pathways and cross-talk with insulin signalling. 4. CBI cannabinoid receptor antagonist/inverse agonist/agonist treatment of rat primary skeletal muscle cells. Expression of example members of the three major G protein coupling GPCR families was observed in rat skeletal muscle tissue. mRNA encoding Gs- (A2Aadenosine receptor, P2-adrenoceptor), Gi- (AI adenosine receptor, (l2A-adrenoceptor), and Gq-coupled (P2Y 1. P2Y2 and P2Y6 receptors) receptors were detected using gene microarray (Agilent, all ranked <10220 out of 41090). QRT-PCR (Taqman) identified (l2A-adrenoceptor and CBI cannabinoid receptor mRNA expression at low level similar across myoblasts, myotubes and skeletal muscle tissue. Functional responses to example members of the three major G protein coupling families of GPCR were also observed in rat primary skeletal muscle preparations. First, treatment of myotubes with the non-selective adenosine receptor agonist NECA elicited increases in cAMP, which were inhibited in the presence of the A2Badenosine receptorselective antagonist, PSB603. In contrast, the A2A-selective agonist, CGS21680 failed to evoke a significant cAMP elevation in myotubes. Second, neither basal nor forskolinevoked elevation of cAMP was altered in the presence of the Ar-selective agonist, SENBA. Third, the (l2-adrenoceptor agonist UK14304 inhibited forskolin-evoked cAMP levels, however, rauwolscine did not prevent this effect. Treatment with UK14304 also increased phosphorylation of ERK1/2; these responses, however, were inhibited by rauwolscine. In addition, rauwolscine in the absence of other ligands also inhibited ERK phosphorylation. Fourth, ATP and UTP, P2Y receptor agonists, elevated intracellular calcium ion levels in myoblasts. Although expression of mRNA for CBI cannabinoid receptors was detected in myoblasts, myotubes and skeletal muscle tissue, forskolin-evoked elevation of cAMP was unaltered in the presence of the CBI receptor-selective agonist ACEA or the antagonist/inverse agonist rimonabant in cultured myotubes. AICAR-stimulated AMPactivated protein kinase activity was also unaltered by ACEA. However, treatment with ACEA increased activation ofERK1I2 and p38 mitogen-activated protein kinases; these responses were significantly inhibited by rimonabant. Insulin treatment of myotubes increased the activation (phosphorylation) of AKT/protein kinase B, glycogen synthase kinase 3(1 and ~, ERK1I2 and p38 MAP kinases; however, pre-treatment with ACEA for 24 hours failed to alter these responses. In conclusion, these studies indicate expression and functional responses to select members of the three major G protein coupling families of GPCR in rat skeletal muscle preparations. These findings also provided evidence for expression of functionally active CB) cannabinoid receptors in skeletal muscle. However, they fail to support previous reports suggesting an interaction between insulin and CB) receptor signalling in these cells. The impact of CB) receptor function in skeletal muscle should be the subject of further investigation.

Recent published work indicates a role for pre-slaughter dietary vitamin D supplementation to promote post mortem meat tenderization in cattle (Montgomery et al., 2000; Foote et al., 2004; Montogmery et al., 2004), pigs (Wilborn et al., 2004) and sheep (Wiegand et al., 2001; Boleman et al., 2004). The hypothesis being that vitamin D supplementation at supra-nutritional levels is able to cause increases in the calcium status of the animals, increasing the activity of the calcium-dependant proteolytic enzymes, the calpains, which are responsible for meat tenderization (Koohmaraie & Geesink, 2006). Muscle fibre type is a variable factor in muscle and is related to meat quality (Klont et al., 1998). Vitamin D has been suggested to play a role in regulating skeletal muscle function through the creation of vitamin D receptor knockout mice models (Endo et al., 2003) and observations that muscle weakness and falling risk in vitamin D deficient patients is linked to a loss of fast muscle fibres (Aniansson et al., 1986; Larsson et al., 1979; Sorenson et al., 1979; Sato et al., 2005). This thesis investigated two vitamin D pre-slaughter diet regimes on their effects on meat quality of the most economically important cut of the carcass, the longissimus dorsi (LD) muscle (Molina et al., 2005) and the expression of the calpain system. Trial 1 fed sheep vitamin D at 2.0 X 10[superscript]6 IU/day for four days prior to slaughter and found that this had no effect on shear force of LD chops. Calcium levels were unchanged but mRNA levels of calpain I and II were increased 3.7 and 10% respectively (P=0.099 and P=0.014) but there was no effect on calpastatin mRNA nor changes in the calpain system at the protein level. Trial 2 fed sheep the same dose of vitamin D for 7 days with an additional calcium bolus, resulting in a 10% increase in calcium concentrations of both serum and LD. Toughness of LD chops was increased (P<0.01), there was no effect on mRNA of the calpain system but there was an increase in the protein levels of calpain II and calpastatin by 16 and 17% respectively (P=0.05 and P=0.087). A microarray study of rat primary myoblasts treated with 1,25(OH)[subscript]2D[subscript]3 for 24 hours highlighted a number of responsive genes significantly up and down regulated 1.5 fold or more (P<0.05). Pathway analysis identified novel targets of 1,25(OH)[subscript]2D[subscript]3 with a possible relationship to muscle growth and function; these included C/EBPβ metallothionein 2A and the MAPK, ERK. Three muscle cell strains, the rat primary muscle cells, L6 Aston and C2C12, were assessed for myosin heavy chain (MHC) gene expression using semi-quantitative PCR and western blotting analysis. The muscle cell line demonstrating the broadest range of MHC genes relevant to mature muscle tissue was used for the final experiments; this was the C2C12 cell line demonstrating expression of the slow MHC 1/β, an isoform which was absent or showed much lower expression in the other cells. C2C12 cells treated with 1α(OH)D[subscript]3 for 48 hours at varying stages of development responded in changes in myogenic regulatory factors (MRFs), MHCs and novel target gene expression. Real time PCR analysis of C2C12 cells treated with the active vitamin D metabolite 1α(OH)D[subscript]3 affirmed C/EBPβ mRNA expression to be upregulated (P<0.001) and MAPK ERK 1/2 phosphorylation to be down regulated (P<0.001) by 1α(OH)D[subscript]3 in muscle cells. The effect of 1α(OH)D[subscript]3 in myoblasts was reduce proliferation and promote differentiation, as myotubes formed the effect of 1α(OH)D[subscript]3 was to promote MHC gene expression of an intermediate oxidative fibre type, increasing expression of MHC 1/β and 2A, decreasing MHC 2B. In conclusion, there is no apparent benefit of a pre-slaughter dietary vitamin D feeding regime on meat quality, but the active metabolites of vitamin D, 1α(OH)D[subscript]3 and 1,25(OH)[subscript]2D[subscript]3, exert changes in gene expression and MAPK signalling which are likely to affect muscle growth and fibre type, and is of relevance in terms of both meat quality and muscle function in the elderly.

Zinc (Zn) is an essential plant nutrient. Most plant species have a shoot Zn concentration ([Zn]shoot) <0.1 mg Zn g-1 dry weight (DW), but extensive natural genetic variation occurs. For example, within the Brassicaceae, some Noccaea (Thlaspi) and Arabidopsis species hyperaccumulate [Zn]shoot >10 mg Zn g-1 DW. There is compelling evidence that orthologues of the Arabidopsis thaliana PIB-type Heavy-Metal-Associated domain-containing ATPase 4 (AtHMA4), which transport Zn2+ and other cations, have a major involvement in the Zn hyperaccumulation trait. The aim of this thesis was to study aspects of genetic variation in the Brassicaceae using a comparative genomic approach, focussing primarily on orthologues of AtHMA4 in Noccaea and Brassica. The first major objective was to clone the full genomic sequence of NcHMA4. This locus was successfully sequenced in Noccaea caerulescens Saint Laurent Le Minier. First, a new genomic fosmid library was generated comprising 36,864 clones with 40 kb inserts, giving ~5-fold genomic coverage. Through DNA fingerprinting, Genome Sequencer (GS) FLX 454 sequencing and contig assembly, a single region collinear with AtHMA4 flanking genes was identified. Unlike A. thaliana, four novel tandem HMA4 gene repeats with highly conserved coding regions, but substantially divergent promoter regions, were present. Preliminary evidence indicates cis-regulated high expression, supporting previous expression data for N. caerulescens. Notably, this observation is remarkably consistant with recent findings in A. halleri. In planta analysis of NcHMA4 remains challenging in N. caerulescens due to a vernal obligate lengthy life cycle (7–9 months) and lack of a robust transformation system. To facilitate future analyses, genetically-stable faster cycling M4 lines were therefore created using fast neutron (FN) mutagenesis. Two non vernal obligate lines have been characterised bearing fruit as soon as 92 days after sowing (DAS) and showing no perturbed [Zn]shoot or obvious pleiotropic effects. Future efforts should focus on their efficient transformation to improve future in planta biological understanding. In Brassica, data from previously reported glasshouse and field studies on B. oleracea L. [Zn]shoot were further analysed to test for the presence of HMA4 orthologues in QTL regions. However, large QTL and multiple paralogues have hindered progress. A more efficient Targeting Induced Local Lesions In Genomes (TILLing)-based approach has therefore been pursued in B. rapa during the latter stages of this study. Locus specific allelic variants in a candidate metal transporter gene BraA.CAX1.a have been identified and methods for rapid downstream genotyping (High Resolution Melt (HRM)-based efficient SNP detection technology) and characterisation have been developed successfully. These approaches are now underway for BraA.HMA4 and an additional candidate metal transporter BraA.ESB1. Since A. thaliana knock-outs of ESB1, CAX1 and HMA genes have altered nutritional phenotypes, future studies will focus on their characterisation under contrasting mineral environments. This thesis has pursued a comparative genomics approach. A previously unreported quadruplication and cis-regulation probably contributes to high HMA4 expression in N. caerulescens. Fast cycling Noccaea lines and a robust Brassica genotyping platform were developed. These will become valuable tools for downstream molecular genetic approaches for in planta functional analysis of HMA4 and other transporters to determine their role in regulating mineral accumulation in Brassicaceae. Ultimately, a greater understanding of genetic variation in [Zn]shoot may have downstream application in genetic biofortification or phytoremediation strategies.

β-Arrestins are essential adaptors for G protein-coupled receptor (GPCR) trafficking. Evolutionary ancestors of the β-arrestins – dubbed α-arrestins – are present in yeast/fungi and, similar to β-arrestins, recognise cargo proteins and mediate their intracellular trafficking. Mammalian α-arrestins include five largely uncharacterised arrestin domain-containing (ARRDC1-5) proteins that display a predicted arrestin structure; the current study focuses on human ARRDC2. Confocal microscopy of exogenous, fluorescent protein-tagged ARRDC2 in U2OS cells in combination with compartment-specific markers indicated that ARRDC2 is dynamically distributed throughout the plasma membrane and endocytic system, predominantly to late endosomes/lysosomes. Anti-ARRDC2 immunostaining in several primary cell lines broadly supported this conclusion. ARRDC2 contains two proline-rich (PPxY) motifs that in other α-arrestins have been reported to mediate interactions with WW domain-containing NEDD4 family E3 ubiquitin ligases. Coimmunoprecipitation indicated that ARRDC2 is able to interact with several NEDD4 E3s via its PPxY motifs, and confocal microscopy suggested that this interaction may influence the subcellular targeting of the ligases. Ubiquitination of ARRDC2 was detected by coimmunoprecipitation, although this modification was independent of ARRDC2 interaction with NEDD4 E3s. ARRDC2 colocalised with agonist-stimulated, internalised GPCRs (β2-adrenergic receptor (β2AR) and δ-opioid receptor (δOR)) and colocalisation analysis indicated that this involved compartmental redistribution of ARRDC2 to receptor-containing early/recycling endosomes, suggesting a specific effect. Interaction of ARRDC2 with δOR was detected using coimmunoprecipitation, and confocal analysis suggested that ARRDC2 may influence δOR and β2AR intracellular trafficking. ARRDC2 was also found to oligomerise with itself and the β-arrestins. Confocal microscopy showed that ARRDC2 overexpression can induce the redistribution of β-arrestin1 to ARRDC2-positive vesicles, and a punctate bimolecular fluorescence complementation (BiFC) signal was detected between ARRDC2 and β-arrestin2. From this, it is speculated that α-/β-arrestins may function cooperatively or competitively to mediate discrete GPCR sorting events in the endocytic pathway.

This thesis investigates structural and dynamic properties of drug recognition mechanisms to duplex and quadruplex DNA using primarily high field NMR techniques and molecular dynamics simulations. The mechanism of co-operative binding of Hoechst 33258 to the DNA minor groove of duplexes that contain two binding sites such as d(CTTTTGCAAAAG)2, d(GAAAAGCTTTC)2 and d(CTTTTGGCCAAAAG)2 has been studied. NMR and other titration techniques have evidenced co-operative binding and no detection of an intermediate 1:1 complex. High-resolution NMR structure determination showed no evidence of direct contact between Hoechst 33258 molecules or DNA structure deformation that would facilitate co-operativity, Molecular dynamics simulations based on NMR data, allowed us to calculate thermodynamic quantities of the two binding events, and lead us to conclude that ligand binding can induce changes in DNA conformational flexibility in sites of the structure distant from the binding site and result in more favourable second ligand binding. The results highlight the general importance of flexibility in determining the properties of ligand-DNA interactions. The relative importance of ligand isohelicity and phasing in DNA minor groove has been investigated by studying the structure and dynamics of the 1:1 complex of Hoechst IO-d(GCAAATTTGC)2. The results suggest that DNA sequence-dependent structure and flexibility have significant role for the strong binding of Hoechst 10 to the duplex. The formation, stability, structure and dynamics of the d(TTAGGGT)4 quadruplex structure, which contains the human telomeric repeat TTAGGG, have been studied. Characteristic features of the quadruplex structure were determined and this information was used for understanding drug-quadruplex interactions. The complex of the fluorinated polycyclic methylacridinium cation RHPS4, lead compound for telomerase inhibition, with the d(TTAGGGT)4 quadruplex structure has been investigated. RHPS4 forms a stable G-quadruplex complex by endstacking externally to the a-tetrads of the Apa and Gp'T steps. This study presents detailed properties of the complex and provides further information for lead optimisation studies.

Epidemiological associations between birthweight and cardiovascular disease in adult life are supported by rodent experiments showing that undernutrition in fetal life programmes adult blood pressure. In rats, the feeding of a maternal low protein(MLP) diet during gestation programmes hypertension. Given interest in the mechanistic role of epigenetic modification of gene expression in programming, this study aimed to assess the potential for a nutritional insult to impact across several generations. Pregnant female Wistar (F0) rats were fed a control (n=10) or MLP diet(n=10) throughout gestation. At delivery all animals were fed the same standard laboratory chow diet. At approximately 10 weeks of age, F1 generation offspring were mated to produce a second generation (F2) without any further dietary change. The same procedure was adhered to, to produce the F3 generation. Physiological analysis confirmed F1 generation MLP exposed offspring exhibited raised (P<0.001)systolic blood pressure and reduced nephron number (P<0.001) compared with controls. Raised blood pressure and reduced nephron number were also noted in the F2 generation (P<0.001) and this intergenerational transmission occurred via both the maternal and paternal lines, No effect was noted in the F3 generation. Microarray analysis highlighted a number of genes that were differentially expressed however upon RT-PCR analysis results were not significant. DNA methylation analysis noted in a trend towards hypomethylation in MLP exposed rats and their offspring as described in previous studies. In conclusion, data within this thesis shows for the first time, that fetal protein restriction may play a critical role in determining blood pressure and overall disease risk in a subsequent generation. It is clear from the data that both males and females can transmit their phenotype to a subsequent generation. This finding suggests that maternal diet can influence the nature of epigenetic markers in germ line cells.

Self-assembled protein nanotubular materials are attractive as putative building blocks for a variety of applications. Knowledge of the three-dimensional structures and the physical properties of these protein nanotubes then becomes a prerequisite for their use in rational materials design. The main purpose of the work presented in this thesis is to investigate both the structural and mechanical properties of protein nanotubes utilizing atomic force microscopy (AFM). Several different protein nanotubes will be used as exemplars to develop AFM methods. AFM is capable of both visualizing and monitoring dynamic processes. Within this thesis, not only could the change in morphology of protein nanotubes be visualized by AFM, but also changes in their mechanical properties were monitored as dynamic processes. For example, changes in the morphology (in chapter 3) and flexibility (in chapter 4) of lysozyme fibrils during fibrillization were investigated. Chapters 4 to 6 describe a range of different methods to obtain the mechanical properties of protein nanotubes: the persistence length method (chapter 4), the adhesive interaction method (chapter 5) and the bending beam method (chapter 6). All of these had their own advantages. However, each method was found only to be suitable for protein nanotubes with elasticities within a defined range. The protein nanotubes investigated by AFM in the thesis included Salmonella flagellar filaments, lysozyme fibrils and diphenylalanine (FF) nanotubes. All of the investigated protein nanotube structures had Young’s moduli lying between that of gelatin and bone. This highlights their potential, in terms of mechanical properties, for a range of applications in drug-delivery systems and tissue-engineering scaffolds. In future, if a database of mechanical properties of protein nanotubes could be built up using the AFM methods developed and utilized within this thesis, the development of the applications of protein nanotubes will be accelerated, as the right protein nanotubes will be selected for appropriate applications.

Background. In vitro studies have shown that ER+ acquired tamoxifen resistant MCF7 breast cancer cell lines can show elevated levels of EGFR expression with an increase in its subsequent signalling pathway(s) and that these are growth inhibited by gefitinib, an EGFR tyrosine kinase inhibitor. This thesis examines the effect of gefitinib on tamoxifen resistant human breast cancer in the clinical setting and in an ‘in-vivo’ mouse model. Patients and Methods. This phase 2 clinical study recruited 54 patients. 28 were oestrogen receptor positive and had progressed on tamoxifen treatment(acquired resistance), the other 26 (48.1%) were oestrogen receptor negative(de novo resistance). Patients were given a loading dose of 1000mg gefitinib on Day 1 and then gefitinib 500mg as a once daily oral dosing until evidence of disease progression. Clinical data were recorded. Sequential tumour biopsies were taken pre-treatment, after 8 weeks therapy and at the development of resistance and analysed immunocytochemically to identify predictive factors for response to treatment and also to see the effect of treatment and resistance on tumour biology, encompassing monitoring steroid receptors, EGFR, HER2 and IGFR, downstream kinases MAPK and AKT, and the proliferation marker Ki67. In parallel with the clinical study, ER+ acquired tamoxifen resistant MCF7 xenografts (TAMR) were grown in nude mice in the presence of tamoxifen and treated with gefitinib 50mg per day orally (designated 3 treatment) or tamoxifen alone (designated control) and monitored for impact on tumour growth. Results. In the phase 2 study gefitinib treatment was well tolerated with an overall clinical benefit rate of 33.3% (n=18/54). Pre-treatment oestrogen receptor positivity was associated with tumour response to gefitinib (p=0.015, longer TTP (p = 0.015), and with clinical benefit (CB) in 53.6 % of the ER+ acquired tamoxifen resistant patients. In contrast, the clinical benefit rate was minimal in the steroid receptor negative patient cohort (11.5%). All patients in this series expressed detectable levels of EGFR, but high pre-treatment levels of EGFR predicted a poorer outcome (p=0.075) Only patients achieving CB had a significant fall in Ki67 staining as measured at 8 weeks versus pretreatment levels (p=0.024), and that Ki67 levels were lower in CB than PD patients at this time. We observed lower levels of EGFR phosphorylation at this time point in some CB patients. Further examination of the CB pts who showed a >10% decline in EGFR phosphorylation revealed decreases in phosphorylation of MAPK and also in Ki67. TAMR xenografts expressed high levels of EGFR as previously observed in vitro. Their growth was significantly inhibited by gefitinib (p=0.039) over the study period while after only 2 weeks of gefitinib treatment tumours showed a decrease in the level of Ki67 staining (p = 0.068). Conclusion. Acquired tamoxifen resistance in vivo both in patients and in a xenograft model appears to be in part mediated through EGFR pathway signalling and this can be blocked and growth inhibited with gefitinib. In ER 4 negative tumours the effects of gefitinib were less striking, suggesting alternative signalling pathways are dominant in promoting their growth despite obvious overexpression of EGFR.

Sex differences have been identified in both external appearance of faces (e.g. Bulygina et al., 2006; Weston et al., 2007) and the way information about faces is extracted by our brains, that is in face processing (e.g. Tahmasebi et al., 2012; Hampson et al., 2006). The mechanisms leading to the development of such sex differences are not well understood. This thesis explores the role of sex hormones in face development and face processing. Data from two large-scale studies (Saguenay Youth Study and Imagen, with n=1,000 and 2,000, respectively) and four smaller datasets (Cycle-Pill Study, n=20; Pill Study, n=20; First Impression Study, n=120, and Twin Study, n=119) were used to explore the effects of sex and sex hormones on face development (head MR images, MRI-face reconstruction) and face processing (functional MRI data, eye-tracking data). Shape of male and female faces was influenced by both prenatal and pubertal androgens. Facial signature of prenatal androgens, identified by the sex-discordant twin design, was found also in an independent dataset of female adolescents (singletons) and we showed that prenatal androgens, indexed indirectly by the facial signature, were associated with larger brain size. We propose that this facial signature might be used, similarly to digit ratio, as an indirect index of prenatal androgens. Variability in postnatal sex hormones due to the use of oral contraception and the phase of menstrual cycle influenced brain response to faces. Using the same dynamic face stimuli as in the functional magnetic resonance imaging (fMRI) study, we showed that eye-movements scanning the face did not differ between the users and non-users of oral contraception. We conclude that effects of sex hormones can be observed in both the face and the brain and that these effects help us understand sex differences in face shape and face processing. **This version does not contain the previously published journal articles reproduced in the printed thesis (appendices 1-3). For details see p. 188. **

Circular dichroism (CD) is an important technique in the structural characterization of proteins, and especially for secondary structure determination. The CD of proteins can be calculated from first principles using the matrix method, with an accuracy that is almost quantitative for helical proteins. Thus, for proteins of unknown structure, CD calculations and experimental data can be used in conjunction to aid structure analysis. The vacuum-UV region (below 190 nm), where charge-transfer transitions have an influence on the CD spectra, can be accessed using synchrotron radiation circular dichroism (SRCD) spectroscopy. Calculations of the vacuum-UV CD spectra have been performed for 71 proteins, for which experimental SRCD spectra and X-ray crystal structures are available. The theoretical spectra are calculated considering charge-transfer and side chain transitions, which significantly improves the agreement with experiment, raising the Spearman correlation coefficient between the calculated and experimental intensity at 175 nm from 0.12 to 0.79. The influence of the different conformations used for the calculation of charge-transfer transitions is discussed in detail, focussing on the effect in the vacuum-UV. Linear dichroism (LD) provides information on the orientation of molecules but is more challenging to analyze than CD. To aid the interpretation of LD spectra, the calculation of protein LD using the matrix method is established and the results compared to experimental data. The orientations of five prototypical proteins are correctly reproduced by the calculations. Using a simplified approach, matrix method parameter sets for the nucleic bases and naphthalenediimide (NDI) have been created and are used to determine DNA/RNA conformations and to study NDI nanotubes. Finally, to make CD and LD calculations available for the scientific community in an easy-to-use fashion, the web interface DichroCalc is introduced.