Infantile neuroaxonal dystrophy (INAD) is a debilitating, intractable and ultimately lethal neurodegenerative disorder. It is caused by mutations in the PLA2G6 gene which encodes for phospholipase A2. INAD patients present neurodegeneration-associated symptoms between six months and three years of age. Severe spasticity, progressive cognitive decline, and visual impairment typically result in death during the first decade (Morgan et al, 2006). There is no disease-modifying treatment available and palliative care focuses on quality of life. Therefore, there is an overwhelming need to develop novel therapies to treat INAD patients. To create a landscape of the behavioural and pathological deficits, we aim to first conduct an in-depth characterization of the PLA2G6 mouse model developed by Wada et al (2009). Additionally, we aim to develop an AAV-mediated gene therapy approach for the treatment of INAD and conduct a pre-clinical study in the pla2g6-inad mouse model. The objective is to be able to prevent or ameliorate both the central and peripheral nervous system phenotype and improve the lifespan and/or quality of life of the animal. Recombinant adeno-associated virus serotype 9 vector (AAV9) will be used to deliver the therapeutic human PLA2G6 gene to the neonatal pla2g6-inad mouse. The strong neuron specific synapsin-I promoter will drive the human PLA2G6 gene. The efficacy of different administration routes including intracerebroventricular (ICV), intravenous (IV) and a combination of intracerebroventricular (ICV)/ intravenous (IV) and intracerbroventricular (ICV)/intraperioteneal (IP) will be investigated in the pla2g6-inad mouse model. AAV9-hSyn1-hPLA2G6 gene therapy treated pla2g6-inad mice showed an increased lifespan with the largest improvements observed in the animal cohort that received a combined administration of AAV9-hPLA2G6. The significant increase in lifespan supplemented with significant improvements in behavioural tests validates the potential beneficial use of gene therapy for infantile neuroaxonal dystrophy (INAD).

The self-assembling polymer N-palmitoyl-N-monomethyl-N,N- dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ) has been shown to improve the oral and parenteral delivery of various drugs. GCPQ contains a number of chemical ‘building blocks’ that are amenable to independent modification. The ability to finely control the level of modification for those moieties allows systematic modulation of polymer chemistry to become a tool to understanding the role and relative importance of specific modifications for the process of oral drug delivery. GCPQ polymers with varying molar percentages of palmitoyl (DP) and quaternary ammonium (DQ) groups were synthesized and the effects on physicochemical properties, aggregation, stability and therapeutic applications were examined. Results obtained show that the critical micelle concentration is very low (below 10μM) compared to ≈8mM for sodium dodecyl sulphate and ≈80mM for sodium caprate (C10) and that micelle formation is spontaneous at room temperature. Structural modifications also resulted in a switch from an entropy to an enthalpy driven aggregation at room temperature. The colloidal stability of the synthesized polymers was found to increase with increasing ratio of DQ to DP (QPR) and is greater at acidic pH than at neutral pH. The structural modification also resulted in different morphological characteristics of drug loaded nanoparticles, producing nanocrystals and nano-micelles with varying drug loading capacities. Additionally, in vitro transport studies showed that GCPQ enhances paracellular transport in a graded, modification dependent manner. When compared with C10, GCPQ was found to be a much safer permeation enhancer, potentially more efficient for drug delivery and at least as potent as C10. The mechanism of these effects was found to involve temporary, size selective distortions on the tight junctions which limit the passage of molecules larger than 3.2nm in hydrodynamic radius. Furthermore, it was observed that these modifications did not affect the uptake of encapsulated hydrophobic drugs by transcytosis in an in vivo rodent model.

This study investigated cyclodextrin-based formulations for the delivery of fisetin (a flavonoid) and erlotinib (a tyrosine kinase inhibitor) to the lung. In the first part of the study, complexation of fisetin with β-cyclodextrin (β-CD) and its derivatives, namely hydroxypropyl-β-cyclodextrin (HP-β-CD) and sulfobutylether-β-cyclodextrin (SBE-β-CD), was conducted. Complexation efficiency between fisetin and the cyclodextrins was in the order: SBE-β-CD > HP-β-CD > β-CD. Utilisation of 20%v/v ethanol during complexation with SBE-β-CD improved the solubilisation of fisetin 5.9-fold compared to using water alone. Spray-drying of the fisetin-SBE-β-CD complex from 20%v/v ethanol feed solution, produced a powder with a 2-fold increase in the fine particle fraction (FPF) compared to the spray-dried powder produced from a feed solution containing water alone, when characterised using the Next Generation Impactor (NGI). Addition of 20%w/w leucine into the powder produced from the ethanolic feed solution further improved the FPF by 2.3-fold compared to the powder without leucine. The preparation showed an unchanged cytotoxic activity of fisetin against the human lung adenocarcinoma (A549) cell line, when compared to fisetin solution. In the second part of the study, combinations of fisetin with three tyrosine kinase inhibitors (i.e. erlotinib, gefitinib, crizotinib) were evaluated for their cytotoxic activity against the A549 cell line. Combination of erlotinib and fisetin at 1: 2 molar ratio, showed the highest synergism in cell killing, when analysed using the median effect principle method. This combination was then used to form a complex with SBE-β-CD. Further improvement in the solubility of erlotinib and fisetin, was achieved with the addition of 50%v/v ethanol during complexation, compared to using water alone. The complex solution was lyophilised and reconstituted into a 3-times more concentrated preparation, prior to nebulisation into the NGI. The preparation showed a suitable aerosol size for inhalation of both drugs. In conclusion, cyclodextrin-based formulations in the form of a dry powder inhalation and nebuliser solution, showed promise for pulmonary delivery of fisetin and its combination with erlotinib, respectively, in the treatment of non-small cell lung cancer (NSCLC).

GABAergic synapse formation involves the establishment of specific cellcell contacts between the presynaptic GABAergic neurones and their postsynaptic targets. Postsynaptic GABAA receptors (GABAARs) themselves have been shown to play a direct structural role in the initiation of functional synapses (Fuchs et al., 2013). Yet, characterisation of the molecular mechanisms underlying their structural involvement remains sparse. As the large N-terminal extracellular domains (ECDs) of the GABAAR subunits reside within the synaptic cleft, we hypothesised that these domains participate in trans-synaptic interactions with cleft-spanning presynaptic proteins. To investigate our hypothesis, a baculovirus/Sf9 cell expression system was used to express and purify the ECDs of the α1, β2 and γ2 GABAAR subunits. These ECDs were utilised in proteomics and mass spectrometry experiments to identify candidate trans-synaptic interacting proteins. To dissect the structural role(s) played by individual GABAAR subunits in synapse formation, a co-culture model system incorporating GABAergic medium spiny neurones and HEK293 cells expressing different combinations of GABAARs was developed. The results indicated that the γ2 subunit was necessary for contact formation and that its 'synaptogenic' effects were influenced by the subtype of α and β GABAAR subunits that were incorporated within the receptor. To elucidate whether the synaptogenic effects of the GABAAR subunits were directly mediated by their ECDs, the purified α1, β2 and γ2 ECDs were added to HEK293-MSN co-cultures at 14 Day in vitro (DIV). Contact formation was reduced in the presence of each of the exogenous ECDs. In parallel, the ECDs were added to pure cultures of MSNs to analyse their structural effects on GABAAR cluster size and number. The results demonstrated that synapsespecific effects were observed for each ECD. In summary, the synaptogenic effects of GABAARs depend on their individual subunit composition, with the ECDs structurally contributing towards the initial cell-cell contacts.

The rising demand for foodbanks and increasing hospital admissions as a result of 'malnutrition' are a reflection of growing food insecurity in the United Kingdom (UK). The adverse impact of this on dietary quality, health and general wellbeing is a growing public health concern. This programme of research aimed to explore why people use foodbanks, the factors that influence their quality of diet, and some of the ways to improve the diet of foodbank users. Mixed-methods were used incorporating interviews with foodbank users (N=18) and personnel (N=12) from 10 London foodbanks. Furthermore, foodbank (N=270) and Advice Centre (AC) (N=245) users from three London boroughs were surveyed about what led to their referral to foodbank, and the relationship of food insecurity, social support, competing expenditure and access to cooking or chilled storage facilities to their dietary quality. Interview data suggested that income crisis degraded the diet of foodbank users who used extreme coping strategies to maintain food sufficiency, in the face of competing expenditures, lack of social support and access to cooking and chilled food storage. Interviews with foodbank personnel suggest that future interventions should consider using foodbank as a point of contact to improve the diet of its users. This would involve meeting the needs of both foodbank users and volunteers, as well as working with agencies across other sectors such as policymakers and community members. Survey data showed that compared to local people attending AC, foodbank users had poorer dietary quality. This can be seen by a greater proportion of participants classified as having 'not good' overall dietary patterns, and lower consumptions of 'healthy' foods (e.g. oily fish, fruit, vegetables) (P < 0.001). Furthermore, compared to AC users, foodbank users were more likely to experience severe food insecurity and greater financial strains. They were also more likely to experience adverse life events, lower social support, and had greater competing expenses and difficulties accessing cooking facilities. Of these, the adverse effect of income crisis on users' quality of diet (P < 0.001) was mediated by food insecurity.

Therapeutics based on nucleic acids offer the possibility of specific and potent treatment of diseases. However, the core obstacle is the ability to deliver these nucleic acid drugs safely and efficiently. In our laboratories we have formulated the nucleic acids: plasmid DNA and short interfering RNA in a synthetic delivery system that facilitates cellular delivery. The delivery system (4PPA) was synthesised, characterised with overall 10% modification level and complexed electrostatically with β-galactosidase plasmid DNA and siRNA-ITCH, in phosphate buffer (pH = 6.0). The resulting complexes were sized, their zeta potential measured and imaged using scanning electron microscopy and transmission electron microscopy. The complexes were further characterised for binding stability and nucleic acid protection using gel electrophoresis. The in vitro transfection efficiency and cell cytotoxicity of the polyplexes were determined in MIA PaCa-2 and A-431 cells. Cellular uptake of a fluorescently labelled nucleic acid was also investigated using flow cytometry and confocal microscopy. Additionally, in vivo studies were conducted to obtain comprehensive data on the behaviour of polyplex formulations following an intranasal brain delivery. Polyplex nanoparticles were 150–350 nm in diameter (polydispersity < 0.5), with a zeta potential of 10–20 mV (n=3) and were spherical in shape. The polymer delivered nucleic acids to the cells. Efficient β-galactosidase up-regulation and Itchy E3 Ubiquitin protein ligase (ITCH) down-regulation were achieved in vitro. The new polymer was as efficient as Lipofectamine® 2000 and 3rd Generation poly(propylenimine) dendrimer in transfecting, but more than several hundred times less toxic. In vivo, 4PPA successfully delivered siRNA-ITCH to the CNS of the olfactory bulb and brain tissue and knocked-down ITCH expression with twice the efficiency of siRNA-ITCH alone. A novel, biocompatible delivery system that provides efficient gene and siRNA transferring vehicles was thus synthesised, achieving good in vitro and in vivo transfection/ down-regulation.

Carcinogenesis is a complex process which requires a number of modifications to the genome in order to progress to tumor formation. Reactive oxygen species (ROS) have been identified as one of the causes of these mutations. The cellular response to ROS is to upregulate the production of an array of detoxifying enzymes. The transcription factors Nrf2 (nuclear factor erythroid 2-p45 related factor 2) and the AhR (arylhydrocarbon receptor) are modulators of antioxidant response element and xenobiotic response element regulated genes respectively. These proteins control biological responses to a range of redox, electrophilic and non-endogenous compounds by controlling the expression of overlapping groups of proteins involved in phase I and II metabolism, redox control and anti-inflammatory activity. In the context of cancer cells, activation of these pathways has been associated with cytoprotective activity in the case of Nrf2 and various activities including cytotoxic activity in the case of the AhR. The ligands that activate or inhibit these transcription factors partially overlap and there is cross-talk between the two signalling pathways. A chemical biology approach was explored to elaborate series of heterocyclic inducers of Nrf2 and the AhR with a focus on flavone-like compounds (flavones, flavanols, flavanones, chalcones) and flavone isosteres (2-phenylbenzothiazole, 2-phenylbenzothiophenes and 3,5-diphenylisoxazolines). The aims were to characterise the behaviour of these isosteric compounds in relation to their Nrf2 inducing activity and/or AhR-dependent cytotoxicity. Structural modifications that develop structure-activity relationships and modify drug-like or drug delivery properties were invistigated. The synthesis of selected compounds and the initial biological studies are presented in this thesis.

Ras proteins (H-, K- and N-Ras) are small signalling proteins that function as molecular switches controlling cell proliferation, differentiation and survival. Somatic mutations of the Ras proteins have been linked to the development of sporadic human tumours with K-Ras mutations being the most prevalent. One of the most frequent point mutations found in K-Ras, occurring close to the GTP binding site, is the substitution of glycine 12 for a cysteine residue and accounts for 10-20% of all Ras-driven cancers. Several efforts have been made towards mutant specific targeting of K-RasG12C by taking advantage of the reactive cysteine group. However, to date such approaches have suffered from either a lack of target selectivity or insufficient potency. Cyclohexa-2,5-dienones belong to a class of cross-conjugated Michael acceptors that are able to react with nucleophiles such as thiols and hence have the potential to modify cysteine residues incorporated in target proteins. The aim of this project was to design and synthesise mutant specific inhibitors of K-RasG12C incorporating the cyclohexadienone moiety as the thiol-reactive warhead to covalently modify the mutant cysteine residue of K-Ras and to evaluate their biological activity in vitro. Several novel covalent-binding inhibitors incorporating mono and bis-Michael acceptor functionalities were synthesised and were tested for their inhibitory potential and target selectivity in a cell viability assay, using SW48 colon cancer cell lines expressing wild-type or mutant K-RasG12C. From this study, several compounds that incorporated cyclohexenone motifs were found to induce strong cytotoxic effects with IC50 values below 1 nM in both cell lines indicating high inhibitory potential, but an absence of target selectivity. Compounds incorporating cyclohexadienone moieties exhibited limited cytotoxicity in both cell lines. The cyclohexadienone structure was also explored to synthesise a small subset of compounds as potential inhibitors of Keap1-Nrf2 protein-protein interactions. A linear 8-mer peptide derived from the Keap1 binding motif of Nrf2 was modified with cysteine residues and subsequently cyclised with the cyclohexadienone analogues. These cyclised analogues may find wider application in the development of novel biological probe molecules.

Solid dispersion technology represents a well-established approach for enhancing dissolution and oral bioavailability of BCS Class II drugs. This work explores the use of a solvent-free temperature-controlled centrifugal spinning process as an alternative technique for producing amorphous solid dispersions in the form of drugloaded sucrose microfibres. This technique has promising large scale production capability and microfibres can be prepared with significant dissolution improvements for three BCS Class II model drugs olanzapine, piroxicam and itraconazole, assessed in both sink and non-sink conditions. This was attributed to the amorphous nature, the solubilising capacity of sucrose and the high surface area of the microfibres formed. However, because of the hygroscopic nature of amorphous sucrose, such microfibres were found to rapidly recrystallise when exposed to high moisture environment. Drugloaded microfibres were observed to collapse into a fine powder and a detrimental effect on the dissolution performance was expected as a result of the moistureinduced recrystallisation. Unexpectedly, the solubility advantage was instead fairly preserved if not enhanced in the case of itraconazole, leaving a product with higher physical stability and morphological properties that is less difficult to handle and process into a conventional dosage form compared to the original product. In fact, moisture-treated itraconazole-loaded microfibres were successfully incorporated into tablets and the influence of several tableting process variables on the tablet characteristics were also evaluated. For instance, by increasing the compression force, tablets with different disintegration times and drug potency could be produced and the latter factor was found to significantly affect the drug dissolution performance, especially in terms of degree and extent of drug supersaturation. Overall, these findings suggest that is not only possible to produce a product with high yield, enhanced dissolution performance and physical stability, but also the final product can be easily incorporated into conventional tablets, while preserving the solubility advantage.

Pancreatic cancer is one of the most deadly of all types of cancer, with a yearly incident that equals its mortality. Gemcitabine (Gem) is currently the first-line chemotherapeutic drug used to treat pancreatic cancer. The major deficiencies of Gem therapy are poor cell membrane permeability, short plasma half-life and toxic side effects. In order to improve the pharmacokinetic characteristics and overcome the obstacles to achieve effective drug delivery, a nanoparticulate drug delivery system can be utilised; gold nanoparticles (GNPs) have been investigated as carriers for drug delivery due to their appealing physicochemical and optical properties. This research project concerns the development of a new generation of GNPs for cancer treatment by co-delivering anti-cancer drugs in combination with laser-induced photothermal effects confined at the diseased areas. Gold nanoshells (GNShells) were synthesised with the capability to carry and deliver Gem and exert phototherapeutic properties. Protein repellent thiol capped poly (ethylene glycol) methyl ether methacrylate polymers were synthesised by RAFT polymerisation and used as efficient particle stabilising ligands. Significant stability enhancement was achieved allowing for the co-functionalisation of GNShells with Gem for applications in in vitro assays against pancreatic cancer cells. GNShells mediated strong photothermal effect owing to their strong surface plasmon absorption in the red/NIR region. This property was exploited to enhance the toxicity of Gem using laser light as the external stimulus. The concerted antitumor activity of Gem with the photothermal effect of the GNShells upon irradiation with a continuous wave laser, increase the cellular uptake and efficacy of Gem-loaded GNShells against MiaPaCa-2 cells. Therefore, the proposed nanoformulation might provide an active strategy for synergistic chemo-photothermal combined therapy.