This thesis focusses on two models (inactive and active) for fibre-reinforced viscous flows, examples of which may be found in numerous industrial and biological applications. In chapters 2-4 we consider Ericksen's model for a transversely isotropic fluid, which treats suspensions of nonmotile particles as a continuum with an evolving preferred direction; this model describes fibrous materials as diverse as extracellular matrix, textile tufts and cellulose microfibers. Linear stability analyses of transversely isotropic viscous fluid between two rotating co-axial cylinders and two horizontal boundaries of different temperatures are undertaken in chapters 3 and 4 respectively. In both cases, the inclusion of transversely isotropic effects delays the onset of instability. In chapter 5 we describe a framework commonly used to model active suspensions, which has been applied to suspensions of self-propelling bacteria, algae and sperm, and artificial swimmers. Through linking this model for an active suspension with that for a transversely isotropic fluid, we identify previously neglected components of the stress tensor that significantly alter the rheology. In chapter 6 we examine the linear stability of isotropic and nearly-aligned suspensions of elongated particles, before giving a summary of our findings in chapter 7.

Detailed steroid hormone profiling of human follicular fluid has paved the way for research into the modulation of human sperm by physiological concentrations of follicular fluid steroid hormones. Synthetic human follicular fluid (shFF), a novel steroid hormone analogue of human follicular fluid, based upon local data, was prepared consisting of 14 different steroid hormones including progesterone. Exposure of human spermatozoa ( > 2000 cells) to shFF stimulus at physiological and standard laboratory temperatures resulted in a rapid biphasic elevation in [Ca2+]i characterised by an initial transient Ca2+ influx immediately followed by a sustained elevation of [Ca2+]i for the duration of shFF exposure. A significant increase in the percentage of acrosome-reacted spermatozoa was observed in shFF-treated sperm (P < 0.05) however, this was significantly lower than the % AR in spermatozoa treated with progesterone alone (P < 0.01). With regards to shFF-induced sperm kinesis, a significant reduction in selected sperm motility parameters was observed 5 minutes post-incubation with shFF (P < 0.05). The study of shFF-induced chemotaxis revealed a chemokinetic effect characterised by a significant inhibition of sperm migration up a gradient of shFF (P < 0.05), possibly due to ‘hyperactivated trapping’. We conclude that the high concentration of progesterone (13.5µM) present in the shFF mixture is likely to be responsible for the biphasic sperm [Ca2+]i influx characteristic of a progesterone stimulus. However, the data obtained from the sperm kinesis and AR experiments leads us to hypothesize that the other steroid hormones present in the shFF mixture exert antagonistic effects on progesterone-mediated physiological responses in human spermatozoa.

The process of angiogenesis in which new blood vessels form from pre-existing vessels, can be intensively studied through the use of in vitro and in vivo models. The in vitro co-culture tube formation assay is used to assess the ability of endothelial cells to develop into three dimensional tubular structures which mimics the growth of capillaries. Different fluorescent labelling techniques were developed and utilised alongside confocal microscopy to visualise endothelial tubulogenesis and investigate the mechanisms of lumenogenesis. Imaging the actin cytoskeletal organisation by expressing the lifeact peptide conjugated to fluorescent proteins revealed that Factin fibres outline lumens within endothelial tubules and enabled clear visualisation of filopodia formation. Further studies presented in this thesis aimed to develop, test and evaluate computational tools for analysing endothelial sprouting from fluorescently labelled spheroids generated using the in vitro hanging drop spheroid assay and quantify blood vessel formation in the in vivo zebrafish model. The results confirmed that both analysis tools were able to rapidly quantify a wide range of angiogenic images and generated comparable results to frequently used manual methods. The developed computational analysis tools are user friendly and can be used to assess the effects of inhibitor compounds and silencing vascular related genes.

Mycobacterium tuberculosis, the etiological agent of tuberculosis, has a unique cell envelope which accounts for its unusual low permeability and contributes to resistance against common antibiotics. The mycobacterial cell wall consists of a cross-linked network of peptidoglycan (PG) in which some of the muramic acid residues are adorned with a complex polysaccharide, arabinogalactan (AG), via a unique α-L-rhamnopyranose–(1→3)-α-D-GlcNAc-(1→P) linker unit. Whilst the cytoplasmic steps of mycobacterial cell wall biosynthesis have been largely delineated, the molecular processes that govern the flux of PG intermediates and the mechanism by which PG and AG pathways converge has remained elusive. We identified key conserved serine/threonine residues of MurC, as potential candidates for phospho-regulation by the cognate protein kinase, PknA. Pseudo-phosphorylated MurC mutants exhibited differential enzyme activity, suggesting that M. tuberculosis is capable of tight control of PG biosynthesis through phosphorylation of MurC. In addition, we have identified Lcp1, a mycobacterial orthologue of the LytR-CpsA-Psr (LCP) family of proteins found in Gram-positive bacteria, responsible for ligating cell wall teichoic acids to PG. We demonstrate that lcp1 is an essential gene required for cell viability and show that recombinant Lcp1 is a phosphotransferase capable of ligating AG to PG in a cell free radiolabelled assay.

Gene expression is compartmentalised in eukaryotes due to the nuclear envelope separating the nuclear processes of transcription and pre-mRNA processing from cytoplasmic translation. While ribosomes are synthesised in the nucleus, it is understood that a number of mechanisms keep them inactive until they reach the cytoplasm, where they mature to become translation-competent. However, this consensus view is being challenged by a growing body of evidence in support of nuclear translation. A newly developed technique, known as ribopuromycylation (RPM), had reported the presence of puromycin-bound nascent peptides on immobilised ribosomes in the nuclei of human cells. I investigated whether this method could be used, combined with chromatin immunoprecipitation, to determine whether nuclear ribosomes can cotranscriptionally translate nascent transcripts in Schizosaccharomyces pombe. Surprisingly, I discovered that, in contrast to that reported in the original study, immobilising ribosomes with translation elongation inhibitors does not lead to retention of puromycylated peptides on ribosomes in either S. pombe, Drosophila melanogaster or HeLa cells. However, I show here preliminary data which suggest that despite puromycylated peptides being released from the ribosome, puromycin immunostaining might still be used to visualise the sub­ cellular localisation of ribosomes inS. pombe, along with other approaches which I also describe.

The grape is arguably one of the oldest cultivated products in human history and the analysis of its main product, wine, reveals clues to trade and associations of previous civilizations. In ancient times, wine was stored in clay amphorae, which, if not properly sealed with resin or pitch allowed the wine to wick into clay matrices, dry, and polymerize producing insoluble, intractable materials that may remain within the matrix for several thousand years. Presently, identification of wine residue is based upon the extraction of these polymeric materials from the ceramic matrix and analyzing/identifying the chemical fingerprints. Two main biomarkers have historically been employed for the identification of wine residue: tartaric and syringic acids. In some cases, the presence of one of these biomarkers has been designated as the confirmatory signature of wine often leading to false positives as amphorae were re-used in antiquity. Herein, a novel approach utilizing metabolomics has been applied to archaeological objects in order to further mine possible biomarkers for a more accurate assessment of the original foodstuff. An untargeted metabolic profiling method was combined with a targeted analytical method resulting in the successful validation of eight representative biomarkers in two separate archaeological sites.

Duchenne muscular dystrophy (DMD) is an X chromosome-linked disease caused by the absence of the sarcolemmal protein dystrophin. The skeletal muscles of DMD have disrupted dystrophin-glycoprotein complex (DGC) and impaired sarcolemma integrity. In this study, we show that clonally derived dystrophin-deficient myoblasts PD50A are differentiation impaired. Coculture with osteoblasts improves the differentiation efficiency of PD50A myoblasts. We also establish that supplementation of combinations of IGF-1/IGF-2, IGF-1/LIF and IGF- 2/LIF in cultured PD50A myoblasts ameliorates the differentiation impairment. We establish that there are elevated levels of Cav-3 and Cav-1 proteins in dystrophin-deficient myoblasts and mdx mouse embryos and that Cav-3 and Cav-1 form heterooligomers in adult skeletal muscles. We show that overexpression of Pax7 suppresses Cav-3 in dystrophin-deficient myoblasts. Using a genetic mouse model (mdx/cav3\(^{+/-}\)) embryo we further establish that immunohistochemistry staining of Cav-1 and Cav-3 coincides with the mouse heart development. The DGC of skeletal muscles plays a role in signal transduction and mechanical response. Here we show that AKT/mTOR and IGF-2/p57\(^{kip2}\) (but not ERK) signalling pathways are upregulated in dystrophin-deficient myoblasts and mouse embryos. Using atomic force microscope we show that Cav-1 helps maintain the stiffness of dystrophindeficient myotubes while Cav-3 help maintain that of dystrophin-deficient myoblasts. This study suggests that Cav-1 and Cav-3 have both compensatory and compromising roles in mdx.

This thesis explores the potential of chiral plasmonic nanostructures for the ultrasensitive detection of protein structure. These nanostructures support the generation of fields with enhanced chirality relative to circularly polarised light and are an extremely incisive probe of protein structure. In chapter 4 we introduce a nanopatterned Au film (Templated Plasmonic Substrate, TPS) fabricated using a high through-put injection moulding technique which is a viable alternative to expensive lithographically fabricated nanostructures. The optical and chiroptical properties of TPS nanostructures are found to be highly dependent on the coupling between the electric and magnetic modes of the constituent solid and inverse structures. Significantly, refractive index based measurements of strongly coupled TPSs display a similar sensitivity to protein structure as previous lithographic nanostructures. We subsequently endeavour to improve the sensing properties of TPS nanostructures by developing a high through-put nanoscale chemical functionalisation technique. This process involves a chemical protection/deprotection strategy. The protection step generates a self-assembled monolayer (SAM) of a thermally responsive polymer on the TPS surface which inhibits protein binding. The deprotection step exploits the presence of nanolocalised thermal gradients in the water surrounding the TPS upon irradiation with an 8ns pulsed laser to modify the SAM conformation on surfaces with high net chirality. This allows binding of biomaterial in these regions and subsequently enhances the TPS sensitivity levels. In chapter 6 an alternative method for the detection of protein structure using TPS nanostructures is introduced. This technique relies on mediation of the electric/magnetic coupling in the TPS by the adsorbed protein. This phenomenon is probed through both linear reflectance and nonlinear second harmonic generation (SHG) measurements. Detection of protein structure using this method does not require the presence of fields of enhanced chirality whilst it is also sensitive to a larger array of secondary structure motifs than the measurements in chapters 4 and 5. Finally, a preliminary investigation into the detection of mesoscale biological structure is presented. Sensitivity to the mesoscale helical pitch of insulin amyloid fibrils is displayed through the asymmetry in the circular dichroism (CD) of lithographic gammadions of varying thickness upon adsorption of insulin amyloid fibril spherulites and fragmented fibrils. The proposed model for this sensitivity to the helical pitch relies on the vertical height of the nanostructures relative to this structural property as well as the binding orientation of the fibrils.

This thesis explores two distinct parts of mitochondrial physiology: the role of mitochondria in generation of reactive oxygen species (ROS) and mitochondrial morphology and dynamics within cells. The first area of research is covered in Chapters 1-8. Mitochondrial biofunctionality and ROS production are discussed in Chapter 1, followed by the strategy of targeting bioactive compounds to mitochondria by linking them to lipophilic triphenylphosphonium cations (TPP) (Chapter 2). ROS sensors relevant to the research are reviewed in Chapter 3. Chapter 4 presents design and synthesis of novel probes for superoxide detection in mitochondria (MitoNeo-D), cytosol (Neo-D) and extracellular environment (ExCellNeo-D). The results of biological validation of MitoNeo-D and Neo-D performed in the MRC MBU in Cambridge are presented in Chapter 5. A dicationic hydrogen peroxide sensor that utilizes in situ click chemistry is discussed in Chapter 6. Preliminary work on the synthesis of mitochondria-targeted superoxide generators, which led to the development of mitochondria-targeted analogue of paraquat, MitoPQ, is presented in Chapter 7. A set of bifunctional probes (BCN-Mal, BCN-E-BCN and Mito-iTag) for assessing the redox states of protein thiols is discussed in Chapter 8 along with their biological validation. The second part of the thesis is aimed at the study of mitochondrial morphology and dynamics and is presented in Chapters 9-11. Chapter 9 provides background on the classes of fluorophores relevant to the research, the phenomenon of fluorescence quenching and the principle of photoactivation with examples of photoactivatable fluorophores. Next, the background on mitochondrial morphology and heterogeneity is presented in Chapter 10, followed by the ways of imaging and tracking mitochondria within cells by conventional fluorophores and by photoactivatable fluorophores exploiting super-resolution microscopy. Chapter 11 presents the design and synthesis of four photoactivatable fluorophores for mitochondrial tracking, MitoPhotoRhod110, MitoPhotoNIR, Photo-E+, MitoPhoto-E+, along with results of biological validation of MitoPhotoNIR. The results and discussion concludes with Chapter 12, which is a summary and suggestions for future work, followed by the chemistry experimental procedures (Chapter 13), materials and methods for biological experiments (Chapter 14) and references.

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