Muscle tension recordings were made from earthworm body wall muscle and the action of acetylcholine, cholinomimtics and cholinolytics examined. The effect of cholinergic agents on muscle twitches induced following field stimulation was also investigated. Carbachol was 8 times more active than acetylcholine in the presence of physostigmine. α-Bungarotoxin, d-tubocurarine, gallamine, atropine, mecamylamine and hexamethonium reduced both electrical stimulation of the muscle and the acetylcholine response while β-bungarotoxin reduced the electrically induced twitch but enhanced the acetylcholine contraction. 0.4wM Hemicholinium abolished the electrically induced twitch while having no effect on the acetylcholine response, but at 4.0uiM, did reduce the acetylcholine response. These results provide further evidence for cholinergic excitatory innervation of earthworm body wall muscle. Intracellular recordings were made from identifiable central neurones of Helix aspersa and the action of anthelmintic compounds investigated. The anthelmintics pyrantel, morantel and deacylated amidantel mimicked acetylcholine induced excitation"D" and inhibition"H" had the same ionic mechanism and were blocked by d-tubocurarine. This suggests these compounds interact with acetylcholine receptors on Helix neurones. Levamisole only inhibited the activity. A series of glutamate analogues was tested on Helix neurones which were either excited or inhibited by Lglutamate. The only analogue with clear glutamate-1ike activity was thio-glutamic acid. In normal saline Lglutamate hyperpolarises the membrane potential of cell F-1. This event is chloride mediated and is reversed to a depolarisation followed by hyperpolarisation in low external chloride. This afterhyperpolarisation is reduced in sodium or potassium free saline or following application of strophanthidin, l.O-lOOyM. The local anaesthetics procaine and tetracaine mimicked the "H" and "D" effects of acetylcholine on certain neurones. Tetracaine, O.OlyM, gradually and reversibly reduced both "H" and "D" responses of acetylcholine and the "H" response to dopamine. This provides evidence that local anaesthetics can interact with responses linked to chloride, sodium and potassium ion channels.

Telephone-delivered therapy is often used to deliver support as it can help overcome barriers that may previously have prevented patients with chronic illness from accessing key services. Very little research has looked at the therapeutic relationship during telephone support for people self-managing a chronic illness. the empirical work in this thesis is nested within a randomised controlled trial (RCT) of self-management of chronic dizziness, a condition that can be debilitating with serious consequences. This thesis explored the role of the therapeutic relationship during telephone support for using booklet-based vestibular rehabilitation (VR) to self-manage chronic dizziness. A meta-analysis of telephone delivered therapy for chronic illness was conducted to examine whether or not telephone therapy can affect physical health outcomes. Eight RCTs (1093 patients) were included, and the results found that telephone delivered therapy significantly improved physical health outcomes in people with chronic illness (d = 0.225, 95% Cl = 0.105, 0.344). A qualitative study of people's experiences of self-managing chronic dizziness using booklet-based VR with or without telephone support (n=33) identified themes characterising people's experiences, thoughts and feelings about these models of VR delivery. Findings indicated that participants valued telephone support. Quantitative analysis examining predicators of outcome (n=112) found that the therapeutic relationship predicted change in handicap, and was related to greater enablement, although it was not related to change in dizziness symptoms. A final mixed methods study aimed to evaluate the development of the therapeutic relationship using Roter Interaction Analysis System to examine recorded therapy sessions. This study found patient centredness during therapy to be related to the therapeutic relationship. Exploratory analyses indentified specific features of patient-centredness that may be related to better and worse alliance. A qualitative analysis of high and low patient centred therapy sessions found that high patient centredness sessions were more likely to include general chat, encouragement, reassurance, and therapists were more responsive to participant cues. Low patient centred sessions were more likely to include participant concerns and therapists not responding to participant cues. This thesis indentified a number of potential elements of telephone support that may be important for the development of the therapeutic relationship in patients self-managing dizziness.

Dengue virus (DENV) is a health burden responsible of 50-100 million new cases and 22,000 deaths per year and its four serotypes are worryingly spreading out of the endemic regions. Current therapy is symptomatic, making antiviral research on DENV an unmet need. Vaccine development is more challenging than expected, so the development of anti-DENV drugs is particularly important for infection management. DENV is a positive sense single stranded RNA virus that replicates within cells exploiting both host and viral enzymes to replicate. Based on the hypothesis that DENV infection can be stopped with the inhibition of one or more of the enzymes that are fundamental for its replication, the aims of the studies reported in this PhD thesis were to: identify novel targets to combat DENV infection, generate new basic knowledge and discover potential novel chemical leads exploiting those targets. Novel approaches combining molecular modelling techniques, classical Medicinal Chemistry approaches, chemical synthesis and in& vitro assays were applied to four essential viral-encoded proteins: the capsid (C), the NS3 NTPase/helicase (NS3hel), the NS5 methyltransferase (NS5 MTase) and the NS5 RNA-dependent RNA polymerase (NS5 RdRp). Novel understanding of the 3'-5' translocation mechanism of NS3hel along the RNA has been hypothesised, increasing awareness about DENV-encoded proteins. Important knowledge on the mode of action of promising antiviral compounds has been acquired, for example that ST-148 stabilises C protein-protein interactions and that published N-sulphonylanthranilic acid RdRp inhibitors bind to a unique allosteric site. Novel promising DENV inhibitor scaffolds have also been developed and the chemical synthesis of one of them has been described, showing that the adopted drug discovery approaches are suitable starting points for the development of anti-DENV medicines. The results obtained represent a significant contribution to DENV research in increasing basic knowledge and in identifying good chemical leads for future work.

Myocardial infarction is a medical emergency and needs an immediate treatment with reperfusion therapy. But, reperfusion itself causes damage to the myocardium by overproduction of reactive oxygen species (ROS). These excessive ROS generated during reperfusion lead to oxidative stress in the myocardium and damage to the cardiomyocytes. Reperfusion injury has been suggested to be responsible for up to 50% of the final infarct size of the myocardium and this resultant infarct size is highly related with the development of chronic heart failure. Although both the superoxide anion and hydrogen peroxide are elevated following myocardial infarction, the levels of their endogenous scavenger enzymes, superoxide dismutase and catalase, decrease even more. Catalase is a major antioxidant enzyme in the body’s endogenous antioxidant defence systems and this enzyme is considered to be responsible for most of the peroxidase activities in the cardiomyocytes. Delivery of active proteins such as catalase to the myocardium is challenging as they can easily be inactivated by proteases, aggregation and natural inhibitors. Delivery of therapeutic proteins using polymeric nanoparticles can offer an attractive approach. No studies on evaluation of the cardio-protective effect of catalase-loaded nanoparticles in conserving cardiomyocytes from the oxidative stress induced by reperfusion injury have been reported yet. Poly(lactic-co-glycolic acid) (PLGA) is a US Food & Drug Administration (US FDA)-approved biocompatible and biodegradable polymer, and this polymer was used in this study to prepare catalase-loaded nanoparticles. In the double emulsion method of fabricating protein-loaded nanoparticles, emulsification process induces denaturation of proteins. No reports have showed the effects of trehalose and bovine serum albumin on the encapsulation of catalase into PLGA nanoparticles and comparison of their effects yet. This study showed that trehalose and bovine serum albumin added to the initial aqueous phase as a stabilizer of the catalase activity were observed to be effective in reducing loss of enzymatic activity of catalase during emulsification steps of the enzyme encapsulation process in the double emulsion method. Surprisingly, unlike BSA-stabilized catalase-loaded PLGA nanoparticles, trehalose-stabilized nanoparticles showed very low enzymatic activity which was comparable to the catalase activity of the unstabilized nanoparticles. Freeze-drying is a useful technique to stabilize the nanoparticles and proteins. But, the process of freeze-drying itself exerts stresses resulting in aggregation of the nanoparticles and denaturation of the proteins. Cryoprotectants are added to protect the nanoparticulate systems from the damage induced by the freeze-drying process. Trehalose is a well-known cryoprotectant. No studies on the cryoprotective effect of trehalose in reducing the loss of enzymatic activity of the encapsulated catalase upon freeze-drying of the catalase-loaded PLGA nanoparticles have been reported yet. In this study, trehalose added as a cryoprotectant minimized the loss of enzymatic activity of the encapsulated catalase of catalase-loaded nanoparticles in a dose-dependent manner. Cryoprotected catalase-loaded nanoparticles were found to be effective in reducing oxidative stress-induced damage in a widely recognized hydrogen peroxide-induced cellular model of oxidative stress in HL-1 cardiomyocytes. This protective response was dose-dependent. The nanoparticles also demonstrated efficacy in reducing apoptosis that plays an important role in myocardial cell death during reperfusion of the myocardium and is linked with the development of heart failure following myocardial infarction. Structural study of the encapsulated catalase of catalase-loaded nanoparticles was performed by Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy and fluorescence spectroscopy which showed that trehalose could reduce the encapsulated enzyme’s structural alterations induced by freeze-drying of the nanoparticles. The promising cardioprotective effect demonstrated by the cryoprotected catalase-loaded PLGA nanoparticles in rescuing cardiomyocytes from oxidative stress, makes this catalase-loaded nanoparticle a valuable candidate for the treatment of reperfusion injury and warrants further investigation including animal trials.

Pulmonary arterial hypertension (PAH) is a progressive and fatal vascular disease that is more prevalent in women than men. The underlying pathology of the disease involves various factors, including genetic risk (i.e. bone morphogenetic protein receptor type II (BMPR-II) mutations) as well as the influence of hormones such as estrogen. Among the frontline treatments for PAH is prostacyclin therapy; however, the short half-life and associated problems with the need for continuous intravenous administration of synthetic prostacyclin have led to the development of newer analogues such as treprostinil. These have the advantages of a longer half-life and the possibility of subcutaneous and inhaled administration. Mortality rates for PAH are still high despite advancements in treatment, with male survival rates remaining lower than females. The BMPR-II signalling pathway may underlie some of the sex disparity that exists in incidence of PAH. However, patient responses to treatments for PAH have also demonstrated sex-specific effects. A key aim of this study was to identify the influence that sex may have on the actions of treprostinil with in-vivo and in-vitro models of PH. The ability to target treatment to specific sub-cohorts of PH is important to maximise the therapeutic effect of treprostinil. A greater understanding of how the effects of treprostinil are mediated could assist this objective. To examine any potential influence of sex on the effects of treprostinil, we examined the chronic hypoxic model of pulmonary hypertension (PH). Female and male rats were dosed with sub-cutaneously implanted pellets releasing treprostinil at 100ng/kg/min or 400ng/kg/min. Under hypoxic conditions both male and female rats had increases in right ventricular systolic pressure (RVSP), right ventricular hypertrophy (RVH) and pulmonary artery remodelling. 100ng/kg/min of treprostinil partially reversed RVSP, RVH and pulmonary artery remodelling in female hypoxic rats but not in male rats. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed that a possible mechanism of treprostinil was increasing BMPR-II signalling, specifically Id1 and Id3 (inhibitor of DNA binding protein 1/3). This was only observed in female hypoxic rats. Despite no difference in terminal plasma levels of treprostinil, hypoxic males remained unaffected by treprostinil at 100ng/kg/min. However, 400ng/kg/min of treprostinil led to slightly greater decreases in RVSP, RVH and remodelling indices in female rats, also it partially reversed RVSP, RVH and remodelling in male rats. Taqman qRT-PCR of the prostaglandin receptors demonstrated an increase in prostaglandin E2 receptor 2 (EP2) under hypoxic conditions with 100ng/kg/min and 400ng/kg/min treprostinil treatment, specific to female rats. Also under hypoxic conditions female and male rats had significant increases in mRNA expression of potassium two pore domain channel subfamily K (KCNK3). To translate clinical relevance from the in-vivo findings, the influence of sex on treprostinil was investigated in human pulmonary artery smooth muscle cells (hPASMCs) taken from non-PAH (control) and PAH patients. In female control hPASMCs FBS (fetal bovine serum) induced proliferation was partially ablated by treprostinil; this effect was only observed in male control hPASMCs at the highest treprostinil concentration (10µM). In patient hPASMCs, treprostinil had a similar effect in reducing FBS-induced proliferation in both female and males. The addition of a low dose (30nM) of endothelin-1 (ET-1) increased the anti-proliferative effect of treprostinil, specifically, in female control hPASMCs. The addition of a dual endothelin receptor antagonist (SB-217242) partially reduced the anti-proliferative effect of treprostinil in combination with ET-1. Taqman qRT-PCR and western blot analysis demonstrated no difference between sexes or hPASMC groups in the expression of the prostaglandin receptors. Using receptor specific antagonists, it was determined that the anti-proliferative actions of treprostinil in PAH patient hPASMCs were partially mediated via the EP2 receptor. However, in female control hPASMCs, the IP receptor was primarily responsible for this effect. BMPR-II signalling was investigated to ascertain its role in the anti-proliferative effects of treprostinil. Taqman qRT-PCR indicated treprostinil (100nM and 1µM) induced increases in Id1 and Id3 mRNA in female control hPASMCs, this did not occur in male control hPASMCs. Treprostinil (100nM and 1µM) led to Id3 mRNA increases in female PAH patient hPASMCs, whereas in male PAH patient hPASMCs treprostinil (1µM) led to a significant Id3 mRNA increase. Western blots indicated that Id3 was upregulated by treprostinil (1µM) stimulation in female control and female PAH hPASMCs vs non-stimulated hPASMCs; this effect was not observed in males. The combination of ET-1 and treprostinil did not influence BMPR-II signalling. After 24 hours of treprostinil stimulation increased Id3 mRNA expression was observed in all hPASMCs groups. Treprostinil only increased Id1 mRNA in PAH patient hPASMCs. Although western blots confirmed treprostinil (100nM and 1µM) mediated increases in Id1 protein expression in female control hPASMCs. Treprostinil (100nM and 1µM) increased Id3 protein expression in female control and female PAH patient hPASMCs. Treprostinil (1µM) in combination with ET-1 led to a significant increase in Id3 protein expression in male control hPASMCs. As with 72-hour treprostinil stimulation, BMPR-II signalling was not influenced by the combination of ET-1 and treprostinil in the other hPASMC groups. The increased BMPR-II signalling in female control and female PAH patient hPASMCs at 24 hours led to the investigation of prostaglandin receptors role in activating BMPR-II signalling. After 24-hours of stimulation with treprostinil (100nM), Id protein induction was partially blocked by dual antagonism of the IP and EP2 receptor in both female control and female PAH patient hPASMCs. To summarise these findings, we have identified sex differences in the action of treprostinil in both in-vivo and in-vitro models of PH. Treatment with a low dose (100ng/kg/min) of treprostinil led to a significant reduction in chronic hypoxic induced PH in female rats but not in males. These differences are driven partially by increases in BMPR-II signalling. Treatment with a higher dose (400ng/kg/min) of treprostinil led to significant reductions in chronic hypoxic PH in both female and male rats. In hPASMCs the results demonstrate that treprostinil can induce the Id proteins of the BMPR-II signalling pathway and that this may account for the greater anti-proliferative effect observed in female control hPASMCs. The induction of the Id proteins was found to be partially mediated by activation of the IP and EP2 prostaglandin receptors. The results suggest that sex may influence the beneficial effects of treprostinil in an in-vivo model of PH and in hPASMCs.

Epithelial ovarian cancer is the deadliest gynaecological cancer: most women die within five years of their diagnoses. Moreover, survival of women with ovarian cancer (OC) has not significantly improved in the past decade. Oncolytic viruses (OVs), a new class of anti-cancer agent, infect and replicate selectively within malignant cells, whilst sparing normal cells. OVs also induce profound immune responses, for example disruption of chemokine and cytokine networks, with potential influence on therapeutic effectiveness. On the other hand, Natural Killer cells (NK cells), a key immune population that surveillance against cancers and viruses, may hinder the spread of OVs or promote anti-tumoural effects of OVs. This work investigated the role of innate immune responses, in particular NK cells and interleukin (IL)-17F, on the efficacy of oncolytic adenovirus in OC. I demonstrated that NK cells were activated by adenovirus-infected OC cells. Activated NK cells then augmented oncolytic adenovirus in eliminating OC via contact-dependent interactions between activating NK receptor DNAM-1 and adenovirus-infected malignant cells. In addition, consistent changes in chemokines and cytokines were observed after wild-type and oncolytic adenovirus infection. In particular, IL-17F, but not IL-17A, was significantly upregulated in different established and primary OC lines after adenovirus infections. Moreover, a range of inflammatory chemokines, including CCL2, CXCL1, CXCL2 and CXCL5, were down-regulated after oncolytic adenovirus infection. This work also revealed the logistical and technical challenges of the use of primary patient materials. I identified that our primary culture method for expanding OC cells was suboptimal. I subsequently evaluated a simple immunohistochemical method to screen for successful primary expansion of malignant cells from OC ascites. I showed that PAX8, but not CK7, was a specific marker of successful ex vivo expansion of HGSOC.

In nature, several known proteins are glycosylated, and carbohydrate-cell receptor interactions mediate a plethora of key biological events, e.g. parasitisation and immune responses. Cell membranes display carbohydrate-binding proteins (lectins), which are able to selectively recognise specific sugar-ligands, decipher sugar-encoded instructions, and convert them into downstream biological processes. To overcome low binding affinities, which are typically observed for simple monosaccharides, protein-binding sugars are typically displayed within large multivalent ligands, where biological systems rely on multivalent interactions as a way to enhance selectivity and binding avidity. Glycoliposomes, phospholipid vesicles coated with sugars, can be designed to exploit this phenomenon through the presentation of multiple glycosylated ligands for actively targeting specific receptors, enhancing uptake of nanomedicines into target cells, or both. Among all the nanocarriers, liposomes are an advantageous strategy for drug delivery for their biocompatibility and high drug encapsulation efficiency. Functionalised liposomes have been developed over the last 30 years to optimise these nanocarriers by improving stability, circulation time and targeting. This thesis work focussed on the use of glycosylated liposomal systems for the treatment of intracellular bacterial infection in cells presenting lectin endocytic receptors. Antimicrobial resistance is one of the current main global healthcare challenges. In particular, antimicrobial resistance in Salmonella Enterica serotypes is responsible for 16 million cases of typhoid fever, 94 million cases of gastroenteritis and 600.000 deaths worldwide. Salmonella is able to survive in intracellular compartments of macrophages. Mannose Receptor (MR, CD206) is a potential access gate to Salmonella infected macrophages which could be selectively targeted with mannosylate-decorated liposomes. In this work, we present the design, the development and investigation of liposomal systems to deliver antimicrobials into Salmonella-infected macrophages. In order to investigate and identify the key structural parameters for efficient delivery of glycotargeted liposomes to selected cell targets (MR+ macrophages), two sets of monovalent glycoligands and two sets of multivalent polyglycosides - synthetic lipid-terminated glycopolymers - bearing a range of membrane-inserting anchors were synthesised. These synthesised membrane-inserting glycoligands have been used to formulate glycosylated liposomes with different glycosylation patterns and lipid composition through the Bangham method. Concanavalin A – a carbohydrate-binding protein – has been initially utilised as model protein target to study the surface presentation of the carbohydrate ligands. Firstly, the effect of lipid composition on the rate of liposomal clustering mediated by Concanavalin A (Con A) model lectin has been established. Our results showed that the binding properties of glycoliposomes are affected by the nature of both lipid constituents and carbohydrate ligands. Next, the uptake of glycosylated liposomes was investigated in salmonella infected macrophage-like cells. This in vitro infection model was used to evaluate the effect of different glycosylation patterns on the liposomal surface on mannose receptor (MR, CD206) targeting efficacy. Liposomes coated with mannose-containing glycopolymer significantly enhanced uptake compared to uncoated liposome control, and showed higher gentamicin delivery, resulting in reduction in internal infection.

The main hypothesis of this research project was that optimisation of treatment based on pharmacokinetic principles is on its own a powerful approach in improvement of treatment outcomes. This work therefore focused on optimisation of treatment of cancer based on principles of pharmacokinetics using two main approaches 1) lipophilic prodrug approach to specifically target the intestinal lymphatic system following oral administration and 2) identification of orally bioavailable candidate anticancer agents and biopharmaceutical development to increase the bioavailability for sufficient systemic exposure to the drug. The first approach was the prodrug derivatisation to take advantage of the physiological process of intestinal lymphatic transport in order to deliver anticancer agents to the mesenteric lymph nodes. Similar prodrug approaches have been researched by other groups but the main focus previously was on increasing the overall bioavailability where they mostly used long-chain or triglyceride mimetic prodrug moieties. However, in this project, through a series of stability and chylomicron association studies, it was revealed that activated ester prodrugs are the most suitable forms for yielding high concentrations of active drugs in the mesenteric lymph nodes. It was remarkable that using this novel approach significantly higher concentrations of the active drugs were achievable in the intestinal lymphatics without affecting the systemic exposure. The second approach taken in this PhD project was achieving sufficient systemic exposure of anticancer agents by identification of orally bioavailable candidate and improvement of oral bioavailability by biopharmaceutical development. The candidates with promising pharmacokinetic properties were rank-ordered by application of a rational drug discovery and development approach of integrated in vitro-in silico assessments. Following in vivo confirmation studies, oral bioavailability was further enhanced for a compound that exhibited a double-peak phenomenon. The results of the two approaches indicate that pharmacokinetic optimisation can be useful in development of anticancer agents to improve the treatment outcomes of cancer.

In order to achieve drug delivery via the respiratory route, an understanding of particulate interactions is of vital importance. For successful delivery to the distal airways, an aerodynamic diameter of less than 5 μm must be achieved. However, particles of this size presents a difficult formulation challenge, due to the inherent cohesiveness between particles and adhesion to the device, due to the high surface to volume ratio of such small particles, causing the particles to clump together. This tendency will thereby cause a reduction in dispersion, aerosolisation and device efficiency; for this reason dry powder inhalers (DPIs) invariably fail to achieve a fine particle fraction efficiency above 15%. There are a wide variety of factors which affect particulate interactions including; surface roughness, surface chemistry, particle size or shape and particle mechanical properties. However, these factors are highly interrelated and so previous attempts to investigate their effect on particle adhesion generally have difficulty isolating the impact of each factor. For instance, investigating the effect of morphology on particulate interactions invariably utilise destructive techniques to alter the roughness, which is likely to alter other factors like surface energy and provide limited control for optimisation. With the rise of 3D printing (additive manufacturing) there is now the capability to produce sub- micron morphologies, and so a bottom-up approach to studying the effect of morphology on particulate interactions can be achieved. The aims of this thesis are therefore twofold. Firstly, to identify, optimise and evaluate a suitable additive manufacturing technique to produce well-defined micron scale morphologies appropriate for furthering the understanding of the importance of morphology on particle adhesion. This is a scale which is at least two orders of magnitude improvement on current state of the art 3D inkjet printers. Secondly, to measure the effect on particle adhesion and deposition to these morphologies, both on an individual particle and on a bulk powder basis, allowing elucidation and understanding of the effect of surface roughness on particle adhesion, with a specific focus on respiratory drug delivery. Printing well defined geometries of an appropriate micron scale size range for particle adhesion testing has been achieved, using two photon polymerisation (TPP). TPP is a novel 3D printing technique which as its name suggests involves the curing of usually acrylate containing polymer resins by the absorption of two infra-red photons in the focus of the laser beam. TPP has been shown to produce a sub-diffraction limit lateral resolution of 120 nm. By optimising the printer parameters and experimentation with differing structure fill and input settings the creation of a well- defined curve on a micron scale was achieved. The initial test morphologies comprised of a ridge with a semi-circular top with a diameter of 1 μm, which were shown to be reproducibly printed. These morphologies were then varied in a controllable fashion with varying ridge height and spacing between the ridges. A uniform and consistent surface chemistry was created using a plasma polymerised hexane (ppHex) coating. In order to evaluate particulate interactions relevant to pulmonary drug delivery both an understanding of the effect of morphology on both individual particle adhesion and bulk powder deposition in a fluid environment is needed. Individual particle-surface adhesion was achieved by testing the TPP structures against three particle types using single particle colloidal probe microscopy (polystyrene beads diameter 10 μm and 5 μm and a lactose particle designed for inhalation formulations). The analysis of this data provides evidence of a clear trend between particle contact area and adhesion recorded both on the ppHex control and the TPP coated morphologies. The TPP morphologies are shown to locally reduce the overall adhesion, in comparison to the flat substrate. The ridge height is also seen to have a significant effect on particle adhesion, with 5 μm < 3 μm < 1 μm for the polystyrene beads, but 3 μm < 5 μm < 1 μm for the Respitose SV003 lactose particle for all ridge spacings. Varying the ridge spacing produced two differing trends in adhesion to the polystyrene beads. If the particle was unable to penetrate the valleys of the roughness, for the 1 μm high ridges, a significant effect on particle adhesion was seen with 3 μm < 1 μm for the polystyrene beads. In contrast, the 3 μm and 5 μm high ridges showed the opposite trend when the particle is unable to descend between the ridges with 1 μm < 3 μm < 8 μm for the polystyrene beads. Investigation of the bulk powder deposition of the particles on the TPP structures and any subsequent re-entrainment in a fluid environment was then achieved using a novel methodology developed during the course of this work. This combines the use of a standard next generation impactor, which generally is used to separate out a respiratory formulation based on aerodynamic diameter, with the TPP substrates. This shows that ridge height has a significant effect on particle adhesion with 3 μm < 1 μm < 5 μm. In contrast, the different spacings of the ridges were not shown to produce a significant difference in particle deposition. This is likely due to the conflicting effect of asperity spacing on the processes of particle deposition and re-entrainment. This thesis therefore highlights the capability of TPP, to produce well-defined micron scale structures with varying morphologies. It then shows that these can be successfully utilised to provide valuable insight into the effect of surface morphology on particle- surface interactions, specifically; adhesion, deposition and re-entrainment.

Studying the mechanism of action (MoA) of biocides against pathogenic microorganisms is crucial to understand their efficacy and limitations, and to develop more efficient microbicidal formulations. Combining alcohol and zinc has been reported to enhance microbicidal activity, but the reasons for such activity are unknown. This study focuses on the impact of combining ethanol and zinc salt at pH 10.5 against nonenveloped viruses. The study is focused on three different aspects: i) virucidal activity screening of ethanol:zinc combinations against bacteriophages and human viruses; ii) impact of ethanol:zinc combinations on virus structure, particularly the viral capsid and nucleic acid, using Transmission Electron Microscopy (TEM); Atomic Force Microscopy (AFM) and agarose gel DNA electrophoresis and iii) chemical speciation and stability of ethanol:zinc combinations over time. The combination of ethanol with zinc salt was found to be more effective against viruses than control formulations containing sole active ingredients and/or excipients only. Activity test of 40%(w/v) ethanol with 0.1% (w/v) zinc salt with excipients (RB- 002 formulation) against F116 and adenovirus type 2 (AdV2) at 60 min contact time yielded 0.68 ± 0.02 and 5.26 ± 0.10 log10 reduction, respectively. In comparison, 0.1% (w/v) zinc salt only with excipient (RB-002G formulation) showed no virucidal activity against bacteriophage F116 (0.14 ± 0.02 log10 reduction) and AdV2 (0.80 ± 0.12 log10 reduction) in suspension. Differences between activities against bacteriophage MS2 and poliovirus type 1 were similar as the ones found between F116 and AdV2. Formulation containing 40%(w/v) ethanol with 0.1% (w/v) zinc salt produced a range of structural damage to F116 and attP AdV5 indicating possible capsid alteration. Effect of the combined formulation on viral capsid was confirmed with AFM with a possible decreased in virus capsid stiffness and significant virus capsid height reduction over 10 min contact time. F116 DNA damage was detected upon exposure to 40%(w/v) ethanol with 0.1% (w/v) zinc salt with excipients, but no damage was detected on AdV2 DNA through electrophoresis analysis. The alcohol/zinc formulation system at pH 10.5 was shown to have promising virucidal activity against non-enveloped viruses at room temperature following an alteration of the viral capsid, and possible damage to the viral nucleic acid. This study also showed the limitations of using bacteriophage as surrogate for mammalian viruses.