This thesis concerns investigations of novel pressurised metered dose inhaler (pMDI) formulations containing tiotropium (Tio) in association with a secondary particulate (SP). A number of formulation and hardware variables were studied using in vitro methods to determine their influence on the performance of these novel formulations. Initial studies indicated that Tio was practically insoluble in HFA propellants and its solubility was not increased under raised moisture levels during long-term stability tests. Formulations with L-leucine (Leu) or lactose (Lac) as SP’s were investigated in Tio:SP ratios ranging from 1:2.5 to 1:25 and with different SP sieve fractions from < 20 μm to < 63 μm. Many formulations demonstrated improved aerosol characteristics compared with Tio alone, particularly in through life performance (TLP). The inclusion of fine SP’s (< 20 μm) was found to significantly improve dose uniformity, fine particle fraction (FPF) and fine particle dose (FPD). Tio:Lac formulated in HFA 227 resulted in slightly greater FPF and FPD but also a higher mass median aerodynamic diameter (MMAD) than when formulated in HFA 134a. With respect to the hardware parameters investigated, smaller actuator orifices (in the range 0.25-0.46 mm) and lower valve volume (25 μl instead of 50 μl) were generally associated with significantly increased FPF and reduced MMAD, whereas a smaller canister volume and fluorocarbon polymerization canisters tended to improve TLP. In comparison with marketed Tio products, comparable FPF’s to Spiriva Handihaler® (41%) and Spiriva Respimat® (53%) were demonstrated with bespoke Tio:Lac and Tio:Leu formulations respectively. The Tio:Leu formulation also had a much lower submicron fraction of Tio than Spiriva Respimat®. This research concerning Tio:SP pMDI formulations has demonstrated the advantages of including a SP to promote drug-SP association in the HFA suspension and promoting particle de-aggregation during propellant atomisation. Further research regarding direct measurement of particle interactions and aerodynamic behavior is warranted.

Strategies for expediting preclinical development of drugs include using alternative (different to clinical) formulations or exposure routes to reduce compound usage. This may alter pharmacokinetics and hence efficacy and/or toxicopathology compared with the final formulation. Three p38 mitogen-activated kinase inhibitors (similar in vitro potency) with different physicochemical properties were administered to rats as dry powder or nebulised aerosols to investigate the influence of changes in particulate form on in vivo endpoints. When rats settled in restraint tubes before aerosol administration, inhaled doses calculated from lung function measurements were consistent with values derived from body weights using a published algorithm. Drug-lung deposition was 12% of the inhaled dose, correlating with the rat deposition fraction (10%) used by US-FDA officials when reviewing regulatory submissions. Drug persistence (GSK-899) in rat lung was associated with increased efficacy (inhibition of lipopolysaccharide-induced inflammation) four hours post challenge, compared with two readily absorbed drugs. However, administration of GSK-899 at a higher dose for 28 days resulted in accumulation of drug and alveolar macrophage aggregates in lungs with infiltration of neutrophils, a consequence of accumulating ‘nuisance particles’ and mild irritancy by GSK-899. GSK-361 (lipophilic; membrane permeable) resulted in lower lung exposure and efficacy, and no lung toxicopathology. Repeating experiments with compounds of different pharmacologies would confirm if these physicochemical properties support general trends for inhaled drugs or represent standalone results for these molecules. Changing aerosol form did not fundamentally change the nature of toxicopathology but presented technical limitations for evaluating the dose response. This thesis demonstrated changes in aerosol form could modulate dose response without changing the nature of toxicopathology. Membrane permeability had a more profound effect on lung clearance and toxicopathology than aqueous solubility. Given technical limitations for drugs of low aqueous solubility, the aerosol form intended for clinical formulations should be used in early preclinical studies.

Corn Hageman factor inhibitor (CHFI) is a bifunctional serine protease / α-amylase inhibitor protein having 127 residues and a molecular weight of 13.6-kDa. CHFI is selective toward FXIIa without affecting the function of the other coagulation factors. Coagulation FXII is a serine protease recognized to cause kinin generation and blood coagulation, cleaving plasma kallikrein and FXI. Results from FXII-deficient animal models proposed that this protein contributes to stable thrombosis that can cause obstruction of the blood vessels and its subsequent complications such as ischemic stroke. In contrast to other blood coagulation factors, deficiency in FXII is not related with haemorrhage in patients or in animals. These findings propose that specific inhibition of FXII could be an attractive medicine and a new method of anticoagulation to treat or prevent pathological thrombosis that could have a lower risk for bleeding and a safer anticoagulation profile than the currently available anticoagulants. Therefore, the current PhD project aimed at pharmacological investigation into CHFI-FXII interaction and FXII function at molecular level through the following objectives: first, developing an efficient expression and purification system for generating soluble and functional recombinant native type CHFI and establishing an inhibitory activity assay against FXIIa to verify the proper function of the recombinant protein. The second objective was testing the different recombinant variants of CHFI with the desired point mutations guided by a proper prediction study of CHFI-FXII interaction. The third was to investigate into the hypothesis of the tight-binding property of CHFI via different approaches of enzyme inhibition mechanisms and kinetic data analysis. The last objective was to investigate into the function of FXII by examining theeffect of Cys466 and glycosylated peptide remnant from the proline-rich region on the function of the catalytic domain via characterizing the different recombinant variants of the catalytic domain of FXIIa, FXIIc, FXIIac, HISTF-β FXII, and MBP-β-FXIIa. In the current study, an efficient system for soluble expression, single step purification and proper storage of functional, wild type rHIS-GST-CHFI was, for the first time, identified. The fully functional recombinant protein was verified via developing an inhibition test against FXIIa. The established expression, purification and inhibition assays were used as a fundamental guide to both generating and characterizing mutant proteins of interest that were made on the basis of an appropriate docking model of CHFI-FXIIa interaction. For the first time, the current investigation into the question of specificity of CHFI against FXII revealed that the central Arg34 at the very top of the fully exposed region of CHFI inhibition loop play a central role in the inhibition function of CHFI toward FXIIa. In addition, this study identified Trp22 at the N-terminus and Arg43 at the C-terminus of the central inhibition loop as two key interaction residues with FXIIa. It was also observed that, in the preinhibition test, CHFI behaves as a noncompetitive inhibitor. In contrast, it acts as a competitive inhibitor in the acute inhibition test, proposing that CHFI is a competitive inhibitor with slow degree of reversibility due to tightness of binding. Reversibility assay showed that CHFI is an inhibitor with slow degree of dissociation. The tight-binding property of CHFI could be due to a non-active site interaction and or numerous hydrogen bonds between the III key interaction residues and their potential targets on FXIIa. With respect to the investigation into FXII function, It was observed that both Cys340-Cys466 and glycosylated peptide fragment of the proline-rich region have a functional role for the full catalytic activity of FXII protease domain. Cumulatively, the current study identified the key important residues on the exposed surface of CHFI and their potential target residues on the surface of FXIIa that would be highly informative and important factors helping to understand the mechanism of selective and tight binding interaction of CHFI with FXIIa. This project can be considered as an early, necessary approach to design novel, specific and safe anticoagulants for the treatment of thrombosis and its complications.

Thromboembolic disorders are the major cause of death and disability worldwide. Anticoagulants are the mainstay in the prevention and treatment of thromboembolic diseases. However, almost all the currently available anticoagulants cause therapy-related haemorrhages as a side effect. In the recent years, FXIIa was highlighted as an attractive target for the development of new anticoagulant drugs with low rates of therapy-related haemorrhages. In this work, the development of a new class of chemical inhibitors as potent and selective nonpeptidic inhibitors of FXIIa has been described. The structural information of FXIIa is not as prevalent as that of FXa and thrombin. Therefore, the 3D-structure of FXIIa in the active conformation was elaborated by homology studies. Given the structural and functional similarities between FXIIa and FXa, rivaroxaban, a known FXa inhibitor, was used as a starting point for this project. Using a combination of medicinal chemistry and computational chemistry strategies, rivaroxaban analogues with acceptable inhibitory activity against FXIIa were readily identified. However, these early analogues showed poor selectivity profiles. Two important structural features of FXIIa inhibitors have been extracted from iterative make-test cycles, an amine derivative at the P1-position and piperazine derivative at the P4-position. Compound 4-(aminomethyl)-N-({(5S)-2-oxo-3-[4-(piperazin-1-yl)phenyl]-1,3-oxazolidin-5-yl}methyl)benzamide (v8) was synthesised with piperazine at the P4-position and 4-(aminomethyl)benzoyl at the P1-position. It was found that compound v8 inhibited FXIIa activity in a chromogenic biochemical assay with an IC50 value of 0.18 ± 0.1 μM. Interestingly, this compound was found to be 72-fold more potent against FXIIa than FXa. In another set of compounds, compound 4-carbamimidoyl-N-({(5S)-2-oxo-3-[4-(piperazin-1-yl)phenyl]-1,3-oxazolidin-5-yl}methyl)benzamide (z8) was synthesised with a 4-carbamimidoylbenzoyl group at the p1-position and piperazine at the P4-position. This compound was 14-fold more potent against FXIIa than FXa. Also, this compound was 1.5-fold more potent against FXIIa than compound v8, but it was less selective to FXIIa. In a further structural refinement, 4-(aminomethyl)benzoyl was replaced with a 4-carbamoylbenzoyl at the P1-position. This afforded compound N1-({(5S)-2-oxo-3-[4-(piperazin-1-yl)phenyl]-1,3-oxazolidin-5-yl}methyl)benzene-1,4-dicarboxamide (w8) with an IC50 against FXIIa of 0.16 ± 0.05 μM, but with significant improvement in the selectivity factor to FXIIa (selectivity factor = 206) compared to compounds z8 and v8. Docking studies showed that FXa inhibitory activity depends mainly on hydrophobic interactions at the S1- and S4-subpockets. Compounds deprived of the Cl-π interactions at the S1-pocket and/or π-π stacking at the S4-pocket were almost always less potent against FXa than compounds that can make such interactions. However, the inhibitory potency against FXIIa depends mainly on electrostatic interaction in the S1- and S4-pockets especially with Asp189 residue at the bottom of the S1-pocket.

There has been increasing interest in droplet interface bilayers (DIBs) as novel devices for the study of lipid membranes and the development of artificial cell systems. Although DIBs have demonstrated to be useful in a number of laboratory applications, their wider use is hampered by a limited ability to exist untethered and remain mechanically stable beyond controlled laboratory environments. In this thesis, a microfluidic system is developed which enables the facile generation of hydrogel-encapsulated DIB networks which are freestanding and can exist in air, water and oil environments, without compromise to their ability to interface with the surrounding environment. Electrophysiology is employed in order to demonstrate the formation of bilayers between the encapsulated DIBs (eDIBs) and their external environment, achieved via the incorporation of the transmembrane pore α-Hemolysin. The eDIBs produced here are able to form higher-order structures akin to tissues via their assembly and adherence to one another, further demonstrating their potential to act as a chassis for artificial cells. Furthermore, the potential of eDIBs to be used as a platform for membrane studies is demonstrated via their use as a high-throughput array for membrane disruption fluorescence measurements using a plate reader, which makes use of the ability of eDIBs to be generated in large numbers as well as to be mechanically handled and placed in the wells of a 96-well plate. Fluorescence measurements were taken on up to 47 eDIBs simultaneously, and were able to detect bilayer leakage through pores as well as bilayer failure. The above experiments comprise the design, manufacture and use of a novel kind of DIB construct as a chassis for artificial cells and a platform for high-throughput membrane studies. It is proposed that eDIBs may help in realising the unfulfilled potential of DIB networks in applications in healthcare and beyond.

Total joint replacement (TJR) is commonly used for the treatment of end stage arthritis. The use of Poly-methylmethacrylate (PMMA) bone cement is a gold standard TJR, where it is frequently used for local delivery of antibiotics to provide prophylaxis from prosthetic joint infections (PJI). Currently used antibiotic loaded bone cements have many limitations, including burst release which fall below inhibitory levels leading to the selection of antibiotic resistant strains. This study aims to provide a controlled release for antimicrobial agents from bone cement to provide prophylaxis from postsurgical infections. For this purpose, gentamicin and chlorhexidine were loaded alone or in combination on silica nanoparticles surface using layer-by-layer coating technique (LbL). A novel LbL construct was built using hydrolysable and non-hydrolysable polymers. The nanoparticles were characterised by transmission electron microscopy, thermogravimetric analysis, zeta measurement, and drug release in different media. Then, antimicrobial agents LbL coated nanoparticles were incorporated into PMMA cement and the nanocomposite is characterized for drug release, antimicrobial, mechanical, rheological properties and cytocompatibility. The build-up of LbL coating was confirmed by thermogravimetric analysis and zeta measurements. The release of antimicrobial agents was controlled for > 30 days for different drugs used. The nanocomposite drug release profile also continued > 30 days at concentration higher than the commercial formula t ion containing the same amount of antibiotics, where burst release for few days were observed. Moreover, the nanocomposite showed superior antimicrobial inhibit ion for bacterial growth, without adversely affecting the mechanical properties. Different nanocomposites showed cytocompatibility when tested against Saos-2 cells. Techniques from a variety of disciplines were employed in this study and this interdisciplinary approach has allowed many features of PMMA bone cement to be investigated. The developed nanocomposites can have the potential to reduce PJIs, and the newly developed LbL nano-delivery system may have wider application in a variety of biomaterials.

Aseptic loosening, as a consequence of an extended inflammatory reaction induced by wear particles, remains the most common complication of total joint replacement (TJR), representing a major problem for the long-term success and survival of prostheses. Despite it is high incidence, in the last decade any therapeutic approach has been found to treat or avoid aseptic loosening, leaving revision as the only effective treatment for this condition. The local delivery of anti-inflammatory drugs to modulate wear-induced inflammation has been regarded as a potential therapeutic approach to avoid aseptic loosening. In this work, an anti-inflammatory drug-eluting implant model system was developed and characterised. The model system was obtained by attaching DEX to functionalised-TiO2 particles, through different synthetic routes: i) by covalently binding DEX to carboxyl-functionalised particles (amino or mercapto routes) or ii) by coating amino-functionalised particles using Layerby- Layer (LbL) technique. The chemical and physical properties of DEXloaded functionalised TiO2 particles have been determined and the release profiles investigated. Depending on the synthetic route, the DEX release period can vary from hours (amino, mercapto routes) to 3 weeks (LbL route). The model system was then tested for its cytotoxic and anti-inflammatory properties in a rapid and reproducible in vitro mouse macrophage-like cellular model, by utilizing murine RAW 264.7 cells. In this model lipopolysaccharide (LPS) was utilized to activate the Raw macrophages, resulting in the secretion of pro-inflammatory cytokines, including nitric oxide (NO) and tumour necrosis factor alpha (TNF-α), the suppression of which was utilized to investigate the anti-inflammatory effect of DEX released from functionalised-TiO2 particles. In vitro studies showed that DEX decreased LPS-induced NO and TNF-α production at non-cytotoxic concentrations, where DEX released from LbL particles showed the most effective suppression of inflammation for at least 2 weeks. Collectively, these findings show that the model system developed can be a potential therapeutic approach to avoid wear-debris induced aseptic loosening of TJR.

This thesis contrasts vaccine innovation efforts in the cases of poliomyelitis and HIV/AIDS. It addresses the question of why some fields of human endeavour can be seen to yield positive change more quickly than others. The thesis develops a perspective that views innovation as a cumulative learning process. It employs the notion of a ‘testing regime' to draw attention to the role of testing in driving this carefully managed learning process during the development of vaccines. Repeated testing, under conditions that are varied using instruments and skill, generates knowledge that is reliable and robust for technological purposes. Governance is needed to co-ordinate this process of testing to ensure the resulting knowledge growth is shared and cumulative. This lens is used to explore the creation of intermediate conditions, the development of instrumentalities, and the role of governance in vaccine innovation processes. The thesis uses the notion of ‘social visions' to explore how attention directed to poliomyelitis contrasted with neglect and apathy afforded to AIDS in its early manifestations. Shared, rather than competing, visions are found to play a significant role in setting the vaccine innovation process in motion. However, the thesis finds that key pathogenic features of the virus and certain ethical and safety stances make learning and the accumulation of technological knowledge inherently difficult. Importantly, the thesis finds policy measures can mitigate or exacerbate these learning challenges considerably. Whilst greater market support and increased research funding tend to be positive contributions to vaccine development, this research shows they are only part of what is needed to take ideas through to innovation. The empirical evidence gathered in this thesis, when viewed through the testing regime lens, suggests that science and innovation are distinct activities but their inter-relationships can be enhanced with the development of an infrastructure focussed on nurturing skills, fostering the use of new techniques, encouraging the development of new instruments, and implementing governance measures to co-ordinate testing efforts and resources.

Human African trypanosomiasis and leishmaniasis are caused by parasites belonging to the genera Trypanosoma and Leishmania, respectively. Significant numbers of people are affected by these diseases worldwide, which are fatal if untreated. Animals can also be infected, posing agricultural and economic hindrances, especially in poor countries. Although chemotherapy can be used for treatment, many problems are associated with it, including drug toxicity, resistance, lack of guaranteed supply, and high treatment cost. Therefore, there is an urgent need for new treatment approaches. Here, we aim to examine the in vitro efficacy of curcumin and phosphonium compounds against these parasites, assay their toxicity to human kidney cells in vitro, and investigate the mechanism of antiparasite activity of curcumin. The Alamar blue assay was used to test 158 curcumin analogues against Leishmania major promastigotes and Leishmania mexicana promastigotes and axenic amastigotes to obtain in vitro EC50 values. Many curcumin compounds such as AS-HK122 and AS-HK126 exhibited anti-leishmanial activities similar to or better than the current clinical drug pentamidine. Similarly, EC50 values of 83 phosphonium compounds against Trypanosoma brucei brucei bloodstream forms were determined. More than 20% of the tested compounds were found to be more active than the standard veterinary drug diminazene aceturate. Multi-drug resistant strains were used to determine that there is no cross-resistance between the tested compounds and the diamidine or melaminophenyl arsenical classes of trypanocides. Structure activity relationship (SAR) analysis revealed that mono-O-demethylated curcumin compounds showed 10-fold higher activity against the parasites than curcumin. The addition of one or two pentyl pyridinium (C10H15N) groups on specific positions of the aromatic ring also increased the activity of these compounds. Furthermore, curcumin compounds with an isoxazole ring instead of the diketo motif showed higher activity and the lowest EC50 values. Similarly, pentyl bromide (OC5H10Br) substitutions on the phenyl rings improved the antiparasitic activity. Curcuminoids with trienone linkers showed increased antiparasitic activity against all parasites tested. Eighty-three phosphonium analogues were tested against T. brucei brucei. SAR analysis indicated that the bulky substituents surrounding the bisphosphonium cations led to strong antiparasitic activity while the nature of the linker had less effect on the activity. Some monophosphonium analogues registered the lowest EC50 values of all the phosphonium compounds. The toxicity of the curcumin and phosphonium analogues to HEK cells was analysed in vitro. All curcumin and phosphonium compounds demonstrated lower toxicity to HEK cells than to the parasites. Of the 83 phosphonium compounds, 60 displayed >200-fold in vitro selectivity index (SI). We also investigated the mode of antiparasitic activity of curcumin compounds. Preliminary toxicity tests had revealed that AS-HK014 caused rapid depletion of glutathione content in rat hepatocytes. Therefore, we tested AS-HK014 activity in the presence of different concentrations of L-glutathione, and AS-HK014 activity was found to decrease with increased L-glutathione concentrations, strongly suggesting that glutathione reacted with the active compound. Indeed, a chemical adduct was observed between the two compounds and identified through mass spectrometry. A trypanosome cell line (TA014) adapted to AS-HK014 was produced. TA014 and wild-type T. brucei brucei were treated with AS-HK014 and compared with each other and with untreated controls. The glutathione and trypanothione levels were lower in the treated WT cells than in the untreated cells. However, there was no change in the glutamate, ornithine, or spermidine levels, providing no evidence for the inhibition of trypanothione synthesis, suggesting that the effect is probably not metabolic but chemical. AS-HK014 did not significantly affect thiol levels in TA014; this might reflect a higher level of trypanothione synthesis through increased glutathione synthetase (GS) and/or γ-glutamylcysteine synthetase (γ-GCS) expression. Therefore, we analysed the protein levels using western blotting, and sequenced the encoding genes in both WT and TA014 to identify any mutations in the open reading frames (ORFs). However, we found no changes in the GS and γ-GCS protein levels in resistant trypanosomes and no mutations were found in the GS and γ-GCS ORFs. It is clear that the resistance is to the reactive enone motif of AS-HK014 rather than to curcumin and curcuminoids in general, since TA014 only displayed resistance to AS-HK014 analogues bearing the enone motif while sensitivity to curcumin remained unchanged, confirming that this motif is responsible for the higher activity of AS-HK014 compared to curcumin. The effects of bisphosphonium analogues on T. brucei brucei bloodstream forms were investigated to identify the target. All tested analogues rapidly reduced the T. brucei brucei mitochondrial membrane potential Ψm and decreased the intracellular ATP level after one hour of incubation, suggesting that the compounds may be targeting the mitochondria. The intracellular Ca2+ levels increased gradually after eight hours, suggesting that the damaged mitochondria are unable to retain the stored Ca2+ as their membrane potential dissipates. We also studied the trypanosome cell cycle after incubating the parasites with bisphosphonium compounds. The cell cycle defects became apparent after eight hours of incubation: DNA synthesis could not be initiated, leading to a dramatic reduction of cells in the S phase. This result was also confirmed by fluorescence microscopic assessment of DNA configuration. After eight hours of incubation with the bisphosphonium compound CD38, the number of 2K1N cells significantly decreased as compared with the control. There may be a causal relationship between mitochondrial damage and cell cycle defects. Transmission electron microscopy images of the cells obtained after 12 h of exposure to CD38 also revealed the presence of mitochondrial damage. We tested whether bisphosphonium compounds can induce programmed cell death in trypanosomes. A TUNEL assay was used to detecting DNA fragmentation; the results showed increased DNA fragmentation after 24-h treatment with two different bisphosphonium compounds, CD38 and EFpI7. This result indicates is consistent with apoptosis occurring in treated cells but there was no evidence suggesting that bisphosophonium-induced cell death in trypanosomes is dependent on new protein synthesis. In conclusion, curcumin and phosphonium analogues exhibit promising antiparasitic activity, and some analogues could be optimised for in vivo evaluation. Further investigations on the site of action of phosphonium compounds in the mitochondrion are in progress.

Adenoviruses are the most commonly used vectors for clinical gene therapy applications, accounting for 24% of all clinical trials to date, the majority of which are based on Ad serotype 5 (Ad5). However, the high prevalence of neutralising antibodies and a range of “off target” interactions result in liver sequestration, hepatic transduction and decreased circulation times. Such interactions include Kupffer cell uptake and binding to blood components such as erythrocytes, platelets, complement and coagulation factors. Recent studies have shown that hepatocyte transduction by Ad5 is mediated by a high-affinity interaction between coagulation factor X (FX) and the Ad5 major capsid protein hexon, with FX bridging the virus to heparan sulphate proteoglycans (HSPGs) on the cell surface. This thesis has focused on gaining a greater understanding of the Ad5:FX pathway and potential strategies for its manipulation. FX, a key component of the blood coagulation system, is a zymogen of a vitamin K-dependent serine protease that is primarily synthesised in the liver and circulates in the bloodstream at 8-10 μg/ml. It is composed of a light chain consisting of a domain rich in γ-carboxylated glutamic acid (Gla) residues, two epidermal growth factor-like domains and a serine protease (SP) heavy chain. The Gla domain of FX binds to the virion by docking in the cup formed by each hexon trimer, whilst the SP domain tethers the Ad5:FX complex to the hepatocyte surface through binding HSPGs. Previously, it was demonstrated that pharmacological blockade of the heparin-binding proexosite (HBPE) in the SP domain prevents FX-mediated cell binding. Here, the specific residues of FX which mediate Ad5 attachment to HSPGs were identified. Employing mutagenesis techniques each of the seven basic residues R93, K96, R125, R165, K169, K236 and R240 that were previously shown to bind heparin, were converted to alanine. This mutated FX was termed “SP mutant”. Stable cell lines were generated to constitutively produce the wild-type and SP mutant rFX protein in the presence of vitamin K. The conditioned media was affinity purified using a FX specific mouse monoclonal antibody 4G3 coupled to sepharose. The rFX proteins were quantified by ELISA, had the predicted molecular weight of 59 kDa and were biologically active, as shown by conversion to FXa in the presence of tissue factor and FVIIa. Surface plasmon resonance (SPR) analysis demonstrated the SP mutations had no effect on FX-specific binding to the Ad5 hexon. However the proexosite mutations ablated FX-mediated Ad5 cell surface binding, internalisation, cytosolic transport and gene transfer as shown by confocal microscopy, qPCR and quantification of transgene expression. Assessing the involvement of rFX with single (R125A) and double (R93A_K96A, R165A_K169A and K236A_R240A) point mutations in the SP domain, indicated the residues exhibit different levels of contribution to Ad5:FX complex binding to HSPGs. The seven SP mutations also inhibited FX-mediated Ad5 binding to mouse liver sections ex vivo. Taken together, this study uncovered that basic residues within the HBPE of FX have a fundamental role in Ad5:FX complex engagement with HSPGs at the surface of target cells. This study contributes to the existing knowledge of the FX-mediated Ad5 transduction pathway. Whilst the classical in vitro CAR-mediated Ad5 infection mechanism has been extensively studied, the post-binding events governing FX-mediated Ad5 intracellular transport and gene expression have not been fully characterised. This study employed a panel of small molecule inhibitors of cellular kinases in vitro to investigate cellular and signalling events occurring during FX-mediated Ad5 infection. Blockade of protein kinase A, p38 mitogen-activated protein kinase and phosphatidylinositol 3-kinase significantly hindered efficient Ad5 intracellular trafficking and colocalisation with the microtubule organising centres (MTOC), as shown by confocal microscopy, indicating their fundamental involvement in the pathway. Screening a library of 80 diverse kinase inhibitors for effects on FX-mediated gene transfer, highlighted the compound ER-27319 had the ability to prevent Ad5 transduction in vitro. Previous work reported that ER-27319 acts by binding to the immunoreceptor tyrosine based activation motif (ITAM) of the FcεRI receptor gamma subunit in mast cells to prevent spleen tyrosine kinase (Syk) activation. Here, this compound had no effect on FX-mediated cell binding but substantially disrupted intracellular transport at 3 h in the absence of toxicity. It was postulated that this effect may be due to ER-27319 binding to a viral or cellular ITAM-containing protein involved in viral trafficking. Sequence analysis of the Ad capsid proteome for ITAM-like motifs ((D/E)-x-x-Y-x-x-(L/I)-(xn=6-8)-Y-x-x-(L/I)) identified two motifs on the hexon. However neither followed that reported for the FcεRI gamma subunit, instead of the conventional 6-8 amino acid residues between the two Y-x-x-I/L, the hexon ITAM-like sequences expressed 17 or 22 amino acids. Alternatively the ITAM-containing cellular proteins, ezrin, radixin and moesin (ERM) were investigated. The ERM family are key regulators of the cell cortex, capable of interacting with both the plasma membrane and filamentous actin. However, in the time frame imposed by this study this hypothesis could not be studied in depth, but warrants further research to investigate whether ERM proteins have a novel role in FX-mediated Ad5 intracellular trafficking. A wide range of approaches have been investigated to detarget Ad5 from the liver. In this thesis, a pharmacological strategy to preclude FX-mediated liver gene transfer was implemented. A high throughput screening platform was developed to identify a novel small molecule(s) to manipulate the Ad5:FX infection pathway. In addition to the value of such an agent in the gene therapy setting, it may also have potential to treat life-threatening disseminated Ad infections in immunocompromised individuals. Using a fluorescence and cell-based in vitro high throughput assay 10,240 small molecules were screened using the Pharmacological Diversity Drug-like Set library. Initial screening identified 288 compounds that reduced FX-mediated Ad5 gene transfer by > 75% without causing toxicity. Upon further analysis, three compounds, T5424837, T5550585 and T5660138 were identified as consistently ablating Ad5 transduction both in the absence and presence of FX and all had IC50 values < 5.5 μM. These compounds did not directly interfere with Ad5 binding to FX, instead they primarily caused a post-binding stage block of the Ad infection pathway and all affected optimal virus trafficking to the MTOC, as demonstrated by SPR, flow cytometry and confocal microscopy. The candidate molecules have common structural features and fall into the “one pharmacophore” model. Focused mini-libraries were generated relating to these molecules and structure-activity relationship analysis was performed. In vitro screening of the analogues revealed novel hits with similar or improved activity, thereby further validating the initial hits and pharmacophore model. Six compounds, T5550585, its analogue T5572402, T5660138, its analogue T5660136, T5424837 and its analogue T5677956 were tested in vivo. 10 μM T5660138 substantially reduced Ad5 liver accumulation 48 h post-injection and, in addition to its closely related analogue T5660136, significantly reduced transgene expression at 48 h post-intravenous administration of a high viral dose (1 x 1011 vp/mouse). Therefore, this study identifies novel small molecule inhibitors of circulating Ad5 infection. Through investigation and manipulation of Ad5 interactions with host proteins the work presented here, increases the understanding of the key in vivo Ad5:FX tropism determining pathway. In summary, in this thesis the mechanism of FX-mediated Ad5 complex binding to hepatocytes was dissected and potent inhibitors of this important Ad5 infectivity pathway both in vitro and in vivo were identified. This data may contribute to the optimisation of Ad vectors for gene therapy applications and potentially the advancement of anti-adenoviral drug development.