Current day nanotechnologies do not match the nanoscale systems that maintain biological cells for their complexity, dynamism or programmability. As such, perhaps more advanced nanotechnologies could be developed by sourcing biological components and employing biological fabrication techniques. In a first step towards bridging this gap, the work here demonstrates that filamentous viruses can be used to engineer nanostructured laser dyes. As a starting point, the suitability of M13 filamentous bacteriophage as a viral scaffold for fabricating laser dyes was assessed. Subsequently, laser systems with gain media composed of M13 phage labelled with fluorescein dyes exhibited both threshold behaviour and spectral linewidth narrowing, which are the two characteristic properties of lasers. In these novel biological gain media, the bulk distribution of the dyes is inhomogeneous but the nanoscale arrangement of the scaffolded dyes and their dielectric microenvironment are highly ordered and tunable. Adopting the design principles at the core of the synthetic biology movement, biological substrates can be chemical and genetically programmed to create enhanced substrates for active nanodevices. Here, the phage was re-programmed to display an exposed thiol group at the surface of the phage. These cys-M13 substrates had interesting bulk properties and could be labelled with spin labels to create “spin phage”. In addition, a recombinant tobacco mosaic virus-like particle (rTMV) was designed, cloned and expressed. The substrates do not have a life cycle in E. coli, so they represent a new, highly programmable material for engineering laser dyes and active media. As an application, the feasibility of using the laser dyes developed here to engineer lasing detection probes was explored. The realisation of a lasing detection probe would be a landmark development in in vitro diagnostics, since they would have exhibit a digital response to excitation and would have a narrow emission linewidth, amongst other advantages.

Weibel‐Palade bodies (WPBs) are endothelial‐specific, rod‐shape secretory organelles that, upon stimulation, exocytose to release strings of the adhesive glycoprotein von Willebrand Factor (VWF). These strings are required to initiate haemostasis by recruiting platelets to sites of vascular injury. Additionally, WPB exocytosis results in translocation of the transmembrane protein P‐selectin to the cell surface, promoting leukocyte recruitment to inflamed tissue. WPB formation and regulated secretion is therefore central to endothelial function. This thesis describes the development of a high‐content RNAi screen aimed at identifying novel regulators of these processes, as well as the subsequent validation and characterisation of one candidate gene, G protein‐coupled receptor (GPCR) kinase 2 (GRK2). In human umbilical vein endothelial cells (HUVECs), GRK2‐depletion caused a 30% reduction in WPB numbers by increasing steady‐state release of VWF. GRK2 attenuates GPCR‐mediated signalling through phosphorylation of agonist‐occupied receptors. It was therefore hypothesised that this phenotype may reflect an increased sensitivity to stimulus. Indeed, activation of GRK2‐depleted HUVECs with the GPCR agonist histamine resulted in a two‐ and three‐fold increase in VWF secretion and P‐selectin dependent recruitment of THP‐1 cells respectively. Calcium signalling was also enhanced, consistent with impaired receptor desensitisation. These studies have therefore uncovered a novel anti‐inflammatory role for endothelial GRK2. WPB biogenesis at the trans‐Golgi network depends on recruitment of an AP‐1/clathrin coat to support the tubulation of VWF multimers. Preliminary work presented here suggests loss of clathrin heavy chain or light chain B results in the formation of shorter organelles, whilst light chain A depletion promotes the biogenesis of longer granules. Clathrin components may therefore differentially regulate WPB formation. Finally, a preliminary model for WPB membrane retrieval following exocytosis (necessary for the maintenance of cell size homeostasis) is presented. This entails closure of the fusion pore and the subsequent recycling of the internalised membrane by clathrin‐mediated vesiculation.

Wingless (Wg), the major Drosophila Wnt, contributes to patterning, growth and cell survival during development. Wg is produced in a stripe at the dorsal-ventral boundary of the wing imaginal disc, a pseudostratified epithelium. Whole mount staining of permeabilised discs reveal that the Wg protein is tightly localized in the apical region of secreting cells. By contrast, extracellular Wg is barely detectable at the apical surface. Instead, extracellular Wg is mostly found at the basolateral surface of secreting, as well as surrounding, cells. These observations suggest that, in secreting cells, apically produced Wg traffics to the basolateral surface for release and gradient formation. This possibility has not been formally investigated. Nevertheless, specific regulators of Wg secretion have been identified. For example, Wntless (Wls)/Evenness interrupted (Evi) binds Wg in the ER and transports it to the plasma membrane. Without Evi, Wg accumulates within the secretory pathway. To test the transcytosis model, I have designed means of tracking Evi and Wg in secreting cells, using classical secretory and endocytic markers as guideposts. I have constructed tagged versions of Wg and Evi, which rescue wg or evi mutants when expressed at an endogenous level. In one set of experiments, I have produced a step of Wg expression and fixed discs at subsequent time points. With this approach I have determined that Wg moves from its apical production site towards the basolateral surface. This was confirmed with experiments utilising a temperature-sensitive dynamin mutant (shibirets) to control endocytosis. With this genetic tool, I have obtained data suggesting that Evi too transits through the apical surface of expressing cells before progressing basally. Unlike Wg, Evi is not released at the basolateral surface. I suggest that instead, it is recycled to replenish the secretory pathway, where it can escort more Wg to the apical surface.

Dynamics of proteins are increasingly recognised as key features as they can contribute to the function of the protein. Structural dynamics manifest as protein folding, protein domain movement and small allosteric responses. Hence, investigating and understanding atomistic motions in proteins to elucidate their implication in protein function mark a crucial paradigm shift from a structure-function relation to a structure-dynamics-function relation. The human histone deacetylase 8 is a key hydrolase in gene regulation and has been identified as a drug target for the treatment of several cancers. I used molecular dynamics simulations to propose a mechanism by which dynamic loop interactions can influence the activity of the human histone deacetylase 8. Subsequently I substantiated this hypothesis by using experimental techniques such as biochemical assays and single point mutations. Furthermore, I studied the structure and dynamics of the histone deacetylase 8 using nuclear magnetic resonance techniques. The proposed mechanism of loop interaction yields a mechanistic rationale for phenomena that could not be explained on a molecular level before.

PcrA is a DNA helicase involved in unwinding plasmi ds as a part of a complex in asymmetric rolling - circle replication of certain plasmids carrying antibiotic resistance genes. PcrA translocates on single stranded DNA by coupling ATP hydrolysis to movement on DNA. Initiator protein, RepD is required to nick supercoiled plasmid site - specifically and open an ssDNA stretch that PcrA can bind. The presence of RepD is needed throughout plasmid unwinding to maintain processivity. Using fluorescent - based techniques, PcrA helicase mechanistic functions and interactions with different components of the replication complex were examined at the ensemble and single molecule level. The development of ensemble techniques included labelling of PcrA with environment sensitive fluorophores such as the coumarin derivative, MDCC. Using MDCC - PcrA(K138C) a transloc ation assay was developed, which determined that PcrA translocates two times faster on dC (500 bases s -1) than on dT oligos (240 bases s -1). MDCC - PcrA(E449C) was used to investigate the PcrA and RepD interaction. The signal observed with this and other labelled PcrA mutants indicated that the 2B subdomain of PcrA is likely to be interaction site between PcrA and RepD . MDCC - PcrA(E449C) enabled the determination of PcrA kin etics with the initiation complex and showed that ATP - PcrA binds differently to PcrA to RepD - DNA complex as compared to apo PcrA , possibly indicating tha t ATP binding stabilises PcrA in a specific conformation when binding to RepD - DNA. A single - molecule a ssay was developed to study individual surface - bound PcrA helicases unwinding full length plasmids by total internal reflection fluorescence microscop y . PcrA unwinds plasmid with the average rate of 40 - 50 bp s - 1 showing 10 - fold variation between the indivi dual helicases. The experiments indicated that the unwinding starts almost immediately after addition of ATP . The single molecule experiments with fluorophore - labelled biotinylated PcrA so clear indication that PcrA unwinds plasmids as a monomer with proce ssivity over 3000 base pairs. The comparison of the ssDNA translocation rate and dsDN A unwinding rates show ed a significant difference between the two forms of translocation, which indicate d PcrA to be a passive helicase. This means that PcrA is n o t active ly destabilising the hydrogen bonding between DNA bases but taking advantage of the thermal fluctuation between the base pairs and so leading to separation of component DNA strands.

β-Arrestins are multifunctional signalling adaptors, facilitating and regulating many signalling pathways in a variety of sub-cellular locales. This thesis work describes a novel role for β-arrestin 1 at the Golgi where it acts as a homeostatic regulator of Golgi morphology and function. β-Arrestin 1 knock-out MEFs have a more dispersed Golgi morphology when compared to WT MEFs, a difference in Golgi morphology that cannot be explained by differences in cell size or the cytoskeleton. Consistent with this observation, the overexpression of β-arrestin 1 in MEFs and HT29 cells causes Golgi compaction, suggesting a regulatory role for β-arrestin 1 at the Golgi. Furthermore, immunofluorescence experiments confirm that endogenous β-arrestin 1 is localised to this organelle. Mutants of β-arrestin 1 with characterised disruptions in protein binding abilities were expressed in HT29 cells and Golgi morphology was assessed by immunofluorescence. A mutant of β-arrestin 1 previously shown to lack the ability to bind AP2 failed to induce the compact Golgi phenotype associated with wild-type β-arrestin 1 expression. We confirmed by co-immunoprecipitation that β-arrestin 1 binds to AP1 suggesting that the association of β-arrestin 1 with AP1 is required for maintenance of a tightly packed Golgi structure. Conversely, the expression of a previously reported non-Src binding mutant produced an excessively compact Golgi. Inhibition of ERK, but not Src, activity recapitulated this phenotype suggesting this mutant may be defective in both Src and ERK binding, and implicating ERK in β-arrestin 1-mediated Golgi dispersal. Trafficking of the ts045-VSV-G protein is also perturbed when these β-arrestin 1 mutants are expressed, suggesting a functional role for β-arrestin 1 at the Golgi. β-Arrestin 1/AP1 complex formation is potentiated by active Src and serves to negatively regulate anterograde trafficking in addition to preventing Golgi dispersal. Conversely, β-arrestin 1 mediates ERK activation to facilitate anterograde trafficking and promote Golgi dispersal. β-Arrestin 1 thus likely plays a homeostatic role at the Golgi maintaining Golgi structure in the face of highly variable membrane flux through this organelle.

Blood coagulation occurs through a cascade of enzymes and cofactors that produces a fibrin clot, while otherwise maintaining haemostasis. The 11 human coagulation factors (FG, FII-FXIII) are associated with thousands of mutations. Variations caused by these mutations are the basis for evolutionary consequences and several of these mutations are also associated with clinically diagnosable bleeding disorders. Trailing the success of sequencing and diagnostic methods, increasing numbers of mutations are being compiled in order to present a more detailed patient-specific description of the disease. This thesis describes an interactive locus-specific database system in which the mutations in patients with haemophilia (A and B) are presented in searchable formats, and viewed in comparisons against protein structures and sequence alignments. The approach used within these databases are set up in a way that they could be extended and combined with a database management system to design a mutation database for the other bleeding disorders of the coagulation system. Our main focus was to correlate the protein sequence and structure with their functional interactions, also keeping in mind the evolutionary selective pressure at the mutation sites. We evaluated the strength of selective pressures on the 11 coagulation factors during vertebrate evolution, and compared these with human mutations in FVIII, FIX and FXI. We have showed that the coagulation system in vertebrates was under strong selective pressures, perhaps to adapt against blood-invading pathogens. It was concluded that when a site was under positive selection, it was less likely to be associated with disease-causing mutations. In contrast, sites under negative selection were more likely to be associated with disease-causing mutations and be destabilizing. The scientific design of the databases with improved understanding of evolutionary changes will lead to a more comprehensive comparative understanding of the genetic factors that influence bleeding risk thus providing optimised genetic services.

Replacement heart valves offer substantial benefits to the patients with severe valvular heart disease. However, current prosthetic heart valves are still unable to meet the needs for more durable tissue valves and less thrombogenic mechanical valves. The aim of this research was to develop and evaluate a new generation heart valves made from a new shape-memory nitinol scaffold and a novel nanocomposite material known as polyhedral oligomeric silsesquioxanes poly(carbonate−urea) urethane (POSS-PCU). During the course of this research, the biomechanical properties of POSS-PCU nanocomposite were evaluated and compared to those of the materials currently used to fabricate bioprosthetic valves. Moreover, POSS-PCU nanocomposite was chemically engineered to improve its oxidative stability and minimise biodegradation. A novel artificial aortic valve was devised and transferred from a stented design into a semi-stented concept and then a retrievable and repositionable valve suitable for transcatheter implantation. The proposed surgical and transcatheter valves were manufactured by an innovative automated dip-coating technique. An industrial-scale automated dipping mechanism was designed to improve the reproducibility of polymeric valve leaflets with no need to suturing or adhesives. The POSS-PCU valves were assessed in vitro for their hydrodynamic performance in a test setup complying with ISO 5840 standards and were shown to have an advanced function compared to clinically available prosthetic valves. The transcatheter POSS-PCU valves (Triskele valves) were then subjected to preliminary acute animal study in ovine model and successfully implanted, retrieved and repositioned in orthotopic position with excellent valve function. The promising results from the in vitro functional tests and the preliminary in vivo study encourage the use of POSS-PCU nanocomposite material and superelastic nitinol wire-frame to develop a new generation transcatheter heart valves which can overcome the main limitations experienced with current solutions. Although further durability and long-term animal studies are required which are currently under investigation.

Wound healing is a vital process, ensuring re-establishment of homeostasis and protection from pathogens. A few regions exhibit privileged healing, such as buccal mucosa, repairing very rapidly. In contrast, wound healing can be impaired, creating chronic wounds. Connexins (Cx) are a group of proteins that form gap junctions, enabling direct communication between the cytoplasm of connected cells. They play a pivotal role in normal skin wound healing. Cx43 is involved in cell proliferation and migration, but the roles of Cx26 and Cx30 are less well understood. Connexin expression changes in the privileged healing of buccal mucosa have not previously been determined. Cx26, Cx30 and Cx43 were highly expressed throughout the mucosa epithelium, but became rapidly down-regulated at the wound edge. This is in contrast to skin, where Cx26 and Cx30 are expressed at low levels, but increase at the wound edge, showing that the connexin dynamics differ between the tissues. Cx26, Cx30 and Cx43 expression also differs in chronic wounds compared to normal skin. They are expressed at abnormally high levels throughout the epidermis, and Cx43 is highly expressed in the dermis. It is thought that bacterial infection is involved in chronic wound development and healing impairment. Staphylococcus aureus is found in 90% of chronic wounds, where it forms a biofilm. I found that in vitro S.aureus biofilm exotoxins caused senescence in fibroblasts, impaired migration, and reduced Cx43 expression. The bacteria themselves reduced Cx43 expression, which increased their internalisation into fibroblasts. Furthermore, loss of Cx43 expression resulted in increased toxicity of S.aureus infection. In vivo S.aureus infection impaired healing, and resulted in epidermal hyperplasia and connexin up-regulation, mimicking the aetiology of chronic wounds. Connexin expression differs in buccal mucosa and S.aureus infected wounds compared to normal skin during healing, but their role in the healing status of these tissues is still poorly understood.

Pre-replicative complexes (pre-RCs), containing the helicase Mcm2-7, are assembled on origins of replication during G1 phase of the cell cycle. This ‘licenses’ origins for subsequent activation during S-phase. The loading of the Mcm2-7 complex requires ATP hydrolysis and the licensing factors ORC, Cdc6 and Cdt1, and results in the assembly of a head-to-head double hexamer of Mcm2-7 bound around duplex DNA. To understand how the Mcm2-7 complex is loaded into a double hexamer, we need a better understanding of the stoichiometry and positioning of licensing factors relative to each other during pre-RC assembly. To address this, I used a tagging and immunoaffinity purification strategy. For this purpose, I generated purified protein preparations where subunits of the licensing proteins were fused to either a 9x Myc or a 3x FLAG tag. These proteins were tested for their ability to support loading of the Mcm2-7 complex in vitro. I used the tagged proteins in an established in vitro pre-RC assembly assay coupled with an immunoaffinity purification approach. I found that in the absence of ATP hydrolysis, one molecule each of ORC, Cdc6 and Cdt1 recruit a single Mcm2- 7 hexamer to origin DNA. Using an ATPase mutant, I showed that ATP hydrolysis by Cdc6 is not required for Mcm2-7 double hexamer formation. I found that a conserved C-terminal region of Mcm3 is critical for Mcm2-7 recruitment to ORCCdc6- DNA. Mutations in this C-terminal domain were lethal in vivo and inhibited Mcm2-7 loading onto origin DNA in vitro. I used the tagged proteins coupled with crosslinking and denaturing immunoaffinity purifications and found that Mcm3 interacts with Orc2 and Cdc6 during Mcm2-7/Cdt1 recruitment to ORC-Cdc6-DNA. The results of this thesis suggest that Mcm2-7 is recruited to origin DNA via Mcm3 interaction with Orc2 and Cdc6 and that the Mcm2-7 hexamers are loaded in a sequential manner.