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Osteoarthritis (OA) is the most common form of arthritis and one of the leading causes of disability globally. There are no disease modifying treatments, and for end stage disease, total joint replacement is the only therapeutic option. There is a major unmet clinical need for OA patients. Compared to peripheral blood and inflammatory arthritis (IA), there has been less research into the cellular mechanisms and inflammatory processes involved in the synovial tissue of OA. A barrier to this research has been difficulty in isolating cells from tissues at the site of disease activity. I aimed to identify and compare the cellular phenotype, functional capacity and gene expression of synovial tissue mononuclear cell subsets in OA and IA. After assessing an array of published tissue digestion protocols, I developed a standardised synovial tissue digestion protocol allowing the isolation of multiple cell subsets with high yields, preserved cellular antigens and high cell viability. A multi-colour flow cytometry panel was established to allow the accurate identification and fluorescence-activated cell sorting of cDC2 dendritic cells, CD14+ monocytes, HLA-DR+CD14+ macrophages and CD4+ T cells. Computational analysis of flow cytometry data sets demonstrated greater cellular infiltrate in IA, but an increased proportion of macrophages in OA. However, this proportion was not seen in all OA patients. Owing to their increased but varied proportion, and previously described role in OA pathogenesis, I conducted an in-depth analyses of these cells. OA macrophages had higher expression of surface proteins associated with activation such as CD206, FOLR2 and CD86. Functionality of isolated macrophages was retained, demonstrated by their high phagocytic activity. Next generation RNA-sequencing was performed to better understand their function and heterogeneity in OA synovial tissue. Two distinctive OA endotypes were proposed based on functional gene signatures. One gene signature comprised of cell cycle and proliferation mechanisms, whilst the other consisted of cartilage and tissue development. Despite its central role in arthritic processes, the role of the synovium has remained obscure. This optimised digestion protocol now allows the interrogation of individual cell subsets. Analysis of synovial macrophages using this protocol has demonstrated distinct endotypes within OA patients. Improved investigation into the pathogenesis of OA, especially in the context of OA subtypes, can now be conducted. This could ultimately lead to the modification of treatment strategies for OA patients.

Social marketing is the adaptation of commercial marketing principles, tools and approaches to programmes designed to influence the voluntary behaviour of target audiences to improve their personal welfare and that of society of which they are a part. The idea that marketing tools and techniques could be used to promote social good and help address social problems was developed in the late 1960s and early 1970s, when it was argued that marketing is relevant to all organisations with "customers". According to this early view of social marketing, commercial approaches, such as the 4Ps (product, price, place, promotion) framework, could be used to influence individuals. This view places responsibility for change on individuals and largely ignores the systematic and environmental influences on behaviours. This critical review is underpinned by the need for a new perspective on social marketing, recognising the complexities of behaviour change and focusing on human beings within a systems/socio-ecological context. It starts by challenging the traditional view of social marketing and the applicability of models such as the 4Ps. Subsequent papers build upon this by demonstrating behaviour change is dependent on relationships, emotional connections, community cohesion and communications between human beings. A new behavioural change framework was developed and tested to demonstrate how this approach could be applied in practice. This model was developed further to include a unique view of 'midstream social marketing', which proposes service staff, and the interactions they have with clients and other stakeholders, is of critical importance for behaviour change. Since the relevant paper was published, midstream social marketing features regularly in journal papers and conferences. This critical review also adds to social marketing knowledge tlu·ough the adaptation and application of the socio-ecological model to the discipline. The author's research shows how the resilience construct -the ability of individuals and communities to cope with adversity -can be used to integrate upstream (policy level), midstrean1 (organisational and community level) and downstream (individual level) social marketing approaches, underpinned by socio-ecological models of behaviour. This extends the more common use of resilience in the context of ecological sustainability or response to disasters. For the first time, this thesis demonstrates how social marketers can use developmental psychology and social work theory to shift their thinking away from targeting individual behaviour problems (smoking, dangerous drinking and unhealthy eating etc.) towards working with young people, families and communities to build long-term resilience to a range of risk factors.

The surface roughness growth of electrodeposited copper films was studied with a particular focus on mass transport effects and the influence of millimolar quantities of halide additives on the surface roughness scaling, the surface morphology and microstructure.

The effects of mechanised forest harvesting on soil physical properties in the UK employing the shortwood system of extraction have, until now, remained largely unknown. In addition, whilst the use of designated extraction routes armoured with logging residues (brash mats) has the potential to reduce soil disturbance, the efficacy of this and other preventative measures, was uncertain. Throughout 1998 and 1999, field trials were undertaken at upland mechanised forest harvesting sites in north-east England and south-west Scotland to (a); investigate the degree and nature of disturbance on selected forest soils and (b), examine the efficacy of ground protection measures. Soil density and soil strength (soil penetration resistance) measured directly beneath the machine wheel tracks on gleyed mineral and deep peat soils (considered previously to be at risk of severe disturbance) exhibited only minor changes despite high levels of trafficking. This was ascribed to (a); the protective effect of the brash mats (b), the slow rates of densification associated with saturated fine textured mineral soils (in a confined state) and (c), the inherent strength and elastic recovery of overlying fibrous peat soils (retained in situ as a result of the brash mats). A marked, though statistically insignificant, reduction in the saturated hydraulic conductivity of the mineral soils directly beneath the machine wheel tracks was measured, with implications for the hydrological regime of the organic soil layers (considered to represent the tree rooting zone). The lateral distribution of soil impacts, (trafficked soil penetration resistance), measured both between and adjacent to the machine wheel tracks, suggested that as much as 50% of the site area (based on the total area of brashed extraction routes) may be subject to some degree of soil disturbance. The implications for site productivity of all measured changes in soil structural properties were discussed in relation to previously reported soil thresholds known to limit root and tree growth. In recognition of the increasing mechanisation of forest harvesting operations, quantitative guidelines were developed for use at an operational level. These included; (a) a provisional pre-harvest soil sensitivity analyses based on observations made during this study in combination with fundamental soil mechanical theory and (b), a means of maximising the efficacy of brash mats based on forest character, ground conditions and machine specification. The need for further research into the effects of machine harvesting on long term site productivity, and role of restock cultivation in ameliorating soil disturbance on a wider range of soils, is emphasised.

Studies of phenomena classed as part of the New Age Movement have become increasingly common in recent years. This Thesis develops a new perspective on these through the use of anthropological and sociological analysis of structures of social power and the contextualisation of symbols and discourse. Research was based on a two and a half year ethnographic study of a network in Nottinghamshire, Britain, which included a meditation group and a spiritual fair. Spirit possession was seen as particularly important to these, in the forms of channelling and mediumship. The concept of nonformative spirituality was used to delineate the network as lacking enduring leadership and authority, such that participants' experiences varied within groups and practices. Thus, the network was not seen as part of a movement, but as a collection of informal groups linked through people's practices. Theory of bodily performance, with a critical analysis of the sociology of knowledge, was used to interpret the four sorts of practices in the network: channelling, meditation, holistic health therapies and divination. By paying attention to people's spiritual biographies, their careers of seekership were understood to develop through dissonant experiences. Nonformative spirituality was compared with those more formative groups which it drew upon, such as spiritualism, the Anthroposophical Society and occult study groups, thus providing a broader picture of its place in contemporary Britain.

5q-spinal muscular atrophy (SMA) is a rare, autosomal recessive, progressive, and lethal neuromuscular disease, most frequently of paediatric onset, caused by the loss of function of the survival of motor neuron (SMN1) gene. SMN1 codes for the SMN protein, whose best characterised role supports the biogenesis of the spliceosome and efficient splicing of pre- messenger RNAs. Twenty years of drug development have led to approval of three therapies in SMA, two of which are splice-switching therapies targeting the SMN2 paralog and disease- modifying gene to SMN1. The ceiling effect of splice-switching therapies as well as issues associated with the specialised delivery of oligonucleotide therapeutics in neurological use calls for research towards the identification and functional exploration of new therapeutic targets beyond SMN2 pre-mRNA. In this work, I explore three avenues for the identification of new therapeutic targets in SMA, using transcriptomics data in motor neurons (MN) and in skeletal muscle. First, I study time- and treatment- dependent genome-wide transcriptomic changes in neuronal tissues in a target-agnostic way and develop a proof-of-concept meta-analysis of all published human RNAseq data in neuronal tissues. In addition, I report a microarray dataset in laser-captured microdissected motor neurons from a severe (Taiwanese) mouse model and provide the first demonstration of neuronal transcriptome-wide efficacy of an intravenously administered splice-switching therapy. Secondly, I investigate the changes in the transcription of a rare subpopulation of RNAs, circular RNAs (circRNA), derived from the SMN1/2 loci. I show changes of expression of the most expressed SMN-derived circRNA, C2A-2B-3-4, in SMA fibroblasts, and provide computational evidence for their putative biological role. Finally, I design and develop two distinct pharmacological approaches to down-regulate FoxO3, a transcription factor transactivating the transcription of atrophy-inducing genes. I identify 8b2-(A) as a promising candidate for further in vitro development. This work provides new insights in the neurodegenerative processes in MN by tackling the first meta-analysis of published neuronal transcriptomics datasets in SMA, revealing methodological gaps in the study of circular RNAs in a high-isoform context, and identifying novel groups of genes dysregulated in early post-natal MN development in SMA. The identified targets can be further explored for therapeutic development in SMA and in other neurodegenerative and muscle- wasting conditions.

Extracellular vesicles (EVs) are membranous nanoparticles released by cells that are important mediators of intercellular communication via transfer of their cargoes to recipient cells. For this reason, EVs have been harnessed as a novel targeted therapeutic delivery platform. However, EV research faces numerous challenges that need to be addressed if their potential as therapeutic devices is to be fully realised. EV heterogeneity is arguably the most crucial challenge to be addressed. EVs differ in size, biogenesis, and composition, and these characteristic differences have been linked to functional differences which may impact EV therapeutic activity. Here, functionally heterogeneous EV populations were identified among size-based and subclonal EV populations. EVs were characterised revealing differences in marker and functional protein expression, including integrins, cell adhesion proteins and EV marker proteins. These differences may explain functional differences between EV subpopulations and could aid selective purification of therapeutically advantageous EVs. Furthermore, the subclonal nature of the analysed EV populations also indicated that EV heterogeneity might be linked to cellular heterogeneity. Separately, changes in EV protein expression were documented among EVs harvested at different time points, highlighting another critical consideration for EV therapeutic development. Advancement of in vivo EV biodistribution and purification techniques was also sought to improve EV therapeutic development and characterisation. While requiring further experimentation, EV DNA barcoding, a technique that labels EVs with synthetic DNA identifiers, showed potential as a novel EV biodistribution and therapeutic siRNA delivery tool. Additionally, Capto Core binding chromatography showed significant improvement over size exclusion chromatography for purification of high concentrations of EVs from high cell density cultures. Overall, this research offers insights into EV heterogeneity and novel techniques to aid therapeutics EV production. It also highlights the importance of understanding EV biology and overcoming the challenges associated with EV research to improve EV therapeutic development.

Spinal muscular atrophy (SMA) is a rare monogenic disease of mostly paediatric onset, affecting approximately 1:8,000 - 10,000 live births. If untreated, it leads to progressive muscle weakness and paralysis, due to degeneration of lower α-motor neurons in the spinal cord and brainstem. Three therapies are currently approved for SMA and while effective, they are extremely costly and not without limitations. It is becoming apparent that with SMA individuals living longer, new therapies, alone and/or in combination with the ones already on the market, are needed to improve quality of life and ensure sustained benefit for patients. Epigenetic mechanisms have been implicated in several neuroprotection and neurodegeneration processes as well as SMA itself. The aim of this project is to identify novel epigenetic mechanisms involved in the regulation of SMN with the ultimate goal to identify new therapeutic targets. To this end, I have screened a library of epigenetic probes from the Structural Genomic Consortium, for their ability to modulate SMN2 in SMA patient-derived fibroblasts. Lead molecules have been further validated in the SMA mouse model. Among these molecules, I have identified MS023, an inhibitor of type I protein arginine methyltransferases (PRMTs), and IOX2, an inhibitor of prolyl hydroxylase domain 2 (PHD2), and characterized MS023 in depth. MS023 is able to increase the levels of SMN protein in vitro and in vivo, via a splice switching mechanism and improve survival and weights in a severe mouse model of SMA. Furthermore, the combinatorial treatment of oral MS023 and the market-approved antisense oligonucleotide nusinersen, is able to prolong the lifespan of severe SMA mice to a greater extent than the oligonucleotide treatment alone. MS023 alters the methylation pattern of hnRNPA1, the main negative regulator of exon 7 inclusion, weakening its binding to SMN2 pre-mRNA. Transcriptomic analysis in spinal cords of SMA mice identified correction of immune-related pathways by the combinatorial treatment but not nusinersen alone. These results provide a new insight into epigenetic regulation of SMN and PRMT function in splicing and justify moving forward with MS023 preclinical development as an add-on therapy for SMA.

Extracellular vesicles (EVs) are cell-derived, membranous nanoparticles released by cells that facilitate the intercellular transfer of proteins and nucleic acids. This intrinsic delivery property of EVs, together with their low immunogenicity and potential for repeat administration, makes them promising delivery vehicles for novel therapeutics. The loading of cargo molecules into EVs constitutes one of the main challenges limiting clinical translation of EV-based therapies. Post-translational modification (PTM) of proteins with ubiquitin-like protein 3 (UBL3) has been previously implicated in the sorting of specific proteins into EVs. Initially, we assessed the utility of UBL3 as an EV-scaffold to enhance EV-loading of Cas9/sgRNA complexes by fusing Cas9 to UBL3 (to mimic the UBL3 PTM). This approach was shown to be successful in terms of the loading of Cas9 and sgRNA into the EVs, although no functional activity of the complexes was observed in the recipient cells after EV transfer. PTMs such as myristylation, ubiquitination, and prenylation have also been shown to play a role in EV-protein sorting. We were motivated to identify additional PTMs (and other sequence features) associated with EV loading that may be exploited for therapeutic purposes. To this end, we developed a novel bioinformatics analysis: integrated proteomics and feature annotation analysis (IPFA) that enabled the identification of EV-enriched annotated PTMs, protein motifs, and domains by combining protein feature annotations from UniProt with experimentally-observed EV-enriched proteins observed in proteomics data. This analysis identified several promising features, of which N-glycosylation was selected as the most promising PTM associated with EV protein enrichment. Specifically, the majority of N-glycosylation-annotated transmembrane proteins were EV-enriched, and nearly half of all EV-enriched transmembrane proteins were annotated as being N-glycosylated. Candidate protein features that were identified with the IPFA analysis were then experimentally assessed for their capacity to mediate functional cargo protein delivery. PTTG1P (Pituitary tumor-transforming gene 1 protein-interacting protein), a single-pass transmembrane protein with two annotated N-glycosyation sites, was selected as a putative EV-sorting scaffold. PTTG1IP was found to be highly enriched in EVs in an N-glycosylation dependent manner. Highly efficient functional delivery of Cre recombinase to reporter cells and mouse xenograft tumors was accomplished using PTTG1IP as an EV scaffold. Indeed, cargo molecules fused to PTTG1IP scaffolds were successfully delivered to recipient cell cultures with improved efficiency relative to the commonly used CD63-based scaffold. The PTTG1IP platform was further utilized for successful Cas9 and Cas9/sgRNA complex delivery to reporter cells. Several PTTG1IP variants with improved properties for therapeutic delivery were also developed (i.e., reduced size, enhanced loading potential, and enhanced intrinsic cleavage for cargo release), demonstrating the potential for further engineering PTTG1IP beyond its native human amino acid sequence. To achieve functional delivery, PTTG1IP-cargo fusion constructs were designed to include self-cleaving sequences (i.e., an intein from Mycobacterium tuberculosis or an intrinsic cleavage sequence derived from PTTG1IP itself) to facilitate cargo release from the EV scaffold. Furthermore, co-expression of the fusogenic protein VSVG (vesicular stomatitis virus glycoprotein) was utilized to enhance cellular uptake and promote endosomal escape. In summary, PTTG1IP was identified as a suitable EV-loading scaffold using IPFA, and its ability to efficiently package and deliver protein and protein/RNA complexes was demonstrated in both cell cultures and in vivo. Notably, PTTG1IP EV-sorting was shown to be dependent on its N-glycosylation PTMs, providing a validation of the robustness of the IPFA approach. The PTTG1IP-based platform offers significant advantages over other EV-loading strategies, (such as favorable membrane topology, the potential for further engineering, and functional delivery capability), which will enable the development of improved EV-based therapeutics.

Despite various efforts to undertake substantial democratic reform in the extractive sector in Indonesia, a full democratic governance process has process never been entirely realised. Therefore, by deploying depoliticisation as the overarching framework, focusing on contextual ignorance, denial of the pluralism and antagonism and impediment of participation, this study investigates the Extractive Industry Transparency Initiative (EITI) in Indonesia between 2010-2018. It shows that the shifts of global and domestic political landscape are the main drivers of the localisation of transparency. The study finds that EITI Indonesia acts as an anti-politics "machine" by partially recognising the local context. However, the civil society organisations (CSOs) persistently try to repoliticise and make use the EITI for demanding more information. Furthermore, the deliberative processes of the multi-stakeholder group (MSG) also indicate a complex process of the pursuit and prevention of issues onto EITI's public agenda. Along with such dynamic process of governmental depoliticisation, this study also finds that societal depoliticisation in Indonesia's extractive sector works through the participation of intermediaries and the primacy of technical solutions. This study provides conceptual contributions that are: (1) a stipulative definition of depoliticisation which reconciles narrow and extensive definitions; and, (2) reconsidering the contingency of depoliticisation but emphasising that the contingency is dynamic and highly dependent on the circumstances in which the process has taken place. Empirically, it claims that: (1) the centrality of transparency in creating its own contestation: transparency is deployed as a tool of knowledge-based power struggle by continuously "testing the boundaries" of information disclosure and making use the MSG for scaling up CSOs' leverage; and, (2), that local context does matter. New democratic spaces, waves of transparency and the rise of civil society stimulate the repoliticisation of EITI while, at the same time, actors' cultural and historical parameters and the nature and scale of extractive commodities limit the extent of the contestation process itself.

18α(H)-Oleanane is a biomarker that is proposed to be derived from β-amyrin, which is produced by angiosperm plants. As such, the presence of 18α(H)-oleanane is often used as an indicator that the petroleum source is of Cretaceous age or younger. Pre-Cretaceous occurrences of 18α(H)-oleanane are rare but have been identified. 18α(H)-Oleanane, as well as an aromatic degradation product of β-amyrin, 1,2,7-trimethylnaphthalene, have been tentatively identified in the Chesterian Limestone in the Anadarko Basin in northwest Oklahoma. The presence of these compounds suggests the presence of either angiosperms themselves or another plant group capable of synthesizing precursors of oleanane and oleanane-type compounds in the depositional environment. The present study analyzed 26 source rocks from the Chesterian Limestone and 14 oil samples from other Mississippian Limestones in the Anadarko Basin. The observed tentative distribution of 18α(H)-oleanane and 1,2,7-trimethylnaphthalene in the Chesterian Limestone are presented and possible explanations for the presence of these compounds are discussed. Unusual distributions of 17α(H)-diahopanes and 9,15-dimethyl-25,27-bisnorhopanes (another series of rearranged hopanes) are also observed in the source rock samples. Both series of rearranged hopanes closely co-vary with each other, which is likely because of their similar structures and suggests that they have similar formation processes. The rearranged hopanes are thermally more stable than the regular hopanes and are known to increase in concentration with increasing maturity. The tricyclic terpanes are also thermally more stable than the regular hopanes and are observed in unusually high abundances in the study area and even dominate the regular hopanes in three source rock samples and in six oil samples from the study area. The corresponding 17α(H)-diahopane/17α(H)-hopane values suggest that thermal maturity plays a role in the increased abundance of the tricyclic terpanes. The distributions of rearranged hopanes and tricyclic terpanes suggests that the oils in the Mississippian Limestones in the Anadarko Basin are not sourced from the underlying Woodford Shale. This is further confirmed by the observed presence and distribution of head-to-head isoprenoids in both the source rock and the oil samples used in the present study.

Neuromuscular diseases are a broad group of debilitating disorders that impair muscle functioning. Duchenne muscular dystrophy (DMD) is the most common hereditary form of neuromuscular disease, for which there is currently no cure. DMD is caused by null mutations in the dystrophin gene (DMD), which encodes an essential cytoskeletal protein required for myofibre integrity and function. The leading DMD therapeutic strategy is antisense oligonucleotide-mediated 'exon skipping', however, this requires lifelong repeat administration. Single intervention strategies for the permanent correction of disease-causing mutations in the genomic DNA (such as CRISPR/Cas9, clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) are important avenues for DMD therapeutic development. An AAV9-delivered CRISPR/Cas9 system was employed to excise a disease-causing Dmd exon in the severely affected double knockout (dKO) mouse (lacking dystrophin and its developmental paralogue, utrophin). Dystrophin protein expression was partially restored in the muscle of dKO mice, although no extension in lifespan was observed. Within-fibre patchiness of dystrophin expression was observed in longitudinal skeletal muscle sections, an as of yet unreported consequence of CRISPR-mediated Dmd gene editing in myofibres. This provides an insight into the myonuclear domain structure of dystrophic fibres, and raises concerns about the possible functionality of gene edited myofibres that contain interspersed dystrophin-positive and -negative regions. The development of non-viral therapeutic strategies focused on improving muscle architecture are also of particular interest for neuromuscular diseases. Extracellular vesicles (EVs) are membrane-enclosed nanoparticles involved in natural cell-cell communication, believed to transfer pro-myogenic and anti-fibrotic factors in the muscle niche. Both myotube- and myoblast-derived EVs enhanced myogenic differentiation at lower, rather than higher, doses in recipient C2C12 muscle cell culture. Highly pro-myogenic effects observed in non-vesicular transfer experiments were attributed to the co-purification of insulin within the isolation medium, highlighting important methodological considerations for the EV field. Strategies that enable the specific activation of gene expression by translational modulation are currently lacking. Upstream open reading frames (uORFs) present within the 5ʹ untranslated regions of mRNA transcripts function in cis to repress translation of the downstream primary ORF. The in situ disruption of uORFs would be therapeutically beneficial for upregulating expression of a disease-relevant protein or protective factor. Validation of a panel of bioinformatically predicted uORFs revealed that not all are functional, including two uORFs with relevance to neuromuscular disease (i.e. SMN2 and UTRN for SMA and DMD respectively). A complex interplay of uORF features was found to influence uORF activity, where initiation of uORF translation was essential for functionality. The biophysical properties of poly-amino acid residues in synthetic uORF micropeptides were found to be major determinants of uORF activity. A synthetic poly-acid uORF sequence was highly repressive, and truncations of this sequence resulted in predictable levels of transgene attenuation. Such uORFs could be useful in biotechnology for the development of tunable, inducible, and tissue-specific transgene expression systems. Finally, CRISPR/Cas13b-mediated RNA editing was applied for the upregulation of a reporter transgene by uORF start codon disruption.

Nucleic acid therapeutics allow sequence-based targeting of disease genes, such as the genes involved in spinal muscular atrophy (SMA) pathogenesis. SMA is a neuromuscular disorder caused by mutations in the survival motor neuron 1 gene (SMN1). All patients have at least one copy of a paralog, SMN2, but a C-to-T transition in this gene results in exon 7 skipping in a majority of transcripts. As a result, only 10 to 20 percent of SMN2 transcripts encode the fully functional SMN protein. Nusinersen, an FDA-approved therapeutic for SMA, is an antisense oligonucleotide (ASO) that promotes exon 7 inclusion in the SMN2 transcript. The ceiling effect associated with splice-switching approaches makes complementary therapeutic strategies necessary. Increasing the total pool of SMN2 transcripts and increasing the translational efficiency of these transcripts are strategies that can be used in combination with splice correction. I sought to determine whether the 5' untranslated region (5'UTR) of SMN2 contains an element that reduces its expression that can be targeted as a means of increasing SMN levels. I found that ASOs targeting the 5' end of SMN2 increase SMN mRNA and protein levels, and that this effect is due to ASO-mediated inhibition of SMN2 mRNA decay. Moreover, using the 5'UTR ASO in combination with a splice-switching oligonucleotide (SSO) increases SMN levels above those attained with the SSO alone. I show that the effect of this 5'UTR ASO is human-specific. These results add to the current understanding of SMN regulation and point toward a new therapeutic target for SMA.

Through analysis of asylum governance mechanisms, this thesis opens the 'black box' of asylum governance processes during the Italian 'refugee crisis'. Between 2014 and 2018 Italian asylum policies became increasingly restrictive, asylum-seekers' reception was highly inefficient, and asylum-seeking migration became more politicised. This thesis complements and challenges existing accounts of the 'crisis', which tend to focus on governance outputs and outcomes without providing satisfactory explanations about how these were produced in the first place, and largely neglecting actors' understandings and decision-making processes. To address this gap, I develop an actor-centred 'macro-micro-macro' approach applied across different governance levels, based on framing and sensemaking perspectives and network analysis. Drawing from 127 interviews and an extensive document analysis, the thesis shows that the understandings, actions and interactions of Italian asylum governance actors contributed to shape asylum-seeking migration as a social and political problem, constituting the 'refugee crisis' itself. I reach this conclusion by developing six interrelated empirical findings. First, at the regional level, actors' assessments of the situation around them were driven by perceptions of public attitudes to immigration and of its salience. Second, these perceptions were often disconnected from objective evidence and grounded on pre-existing narratives. Third, these perceptions decisively shaped political decision-makers' actions. Fourth, the interaction of decision-makers that exhibited different approaches to asylum-seekers' reception influenced the production of key outputs and outcomes: the efficiency of regional reception systems, levels of politicisation of asylum and the consequent emergence of migration-related mobilisations by the public. Fifth, these outputs, in turn, decisively influenced actors' perceptions of public opinion, mostly reinforcing pre-existing narratives. Sixth, these recursive regional governance processes had powerful effects on the national governance level, significantly influencing the understandings, decisions and actions of national policy actors. They ultimately drove and shaped the restrictive policy approach adopted by the Italian government since 2017.

Fusion inhibitors are a class of antiretroviral drugs used to prevent entry of HIV into host cells. Many of the fusion inhibitors being developed, including the drug enfuvirtide (ENF), are peptides designed to mimic, and thereby competitively inhibit, the viral fusion protein gp41. An exception to this is a class of cyclic, cationic, antimicrobial peptides known as θ-defensins, which are produced by many non-human primates and exhibit broad-spectrum antiviral and antibacterial activity. Currently, the θ-defensin analog RC-101 is being developed as a microbicide to prevent sexual transmission of HIV-1. Understanding potential RC-101 resistance, and how resistance to other fusion inhibitors affects RC-101 susceptibility, is critical for future development. Partial drug resistance due to genetic variability within HIV-1 presents a major hurdle in microbicide development. Drug-resistance mutations, whether naturally occurring or resulting from selection during treatment, often apply to many drugs in a particular class. Combining different drug classes into a single microbicide should provide greater protection against the growing variability observed in HIV. Our work has identified the beneficial effects of combining the fusion inhibitor RC-101 and the RT inhibitor CSIC to prevent transmission of clinically isolated and drug-resistant HIV-1. Several aspects of HIV-1 virulence and pathogenesis are mediated by the envelope protein gp41. Additionally, peptides derived from the gp41 ectodomain have been shown to induce chemotaxis in monocytes and neutrophils. While this chemotactic activity has been characterized, it is not known how these peptides could be produced under biological conditions. Our findings demonstrate that the epithelial serine protease matriptase efficiently cleaves the gp41 HR1 region at conserved residues into a chemotactic peptide. Here, we present evidence that advances our understanding of resistance to peptide entry inhibitors, reveals a potential benefit to combining specific drugs in an antiviral microbicide, and identifies a pathway by which HIV-1 may generate peptides to exploit host immunity. This work thereby facilitates improved methods in countering drug resistance and the development of new antiviral approaches to prevent HIV-1 transmission. Additionally, we have revealed basic mechanistic evidence that shed light on our current understanding of HIV-1 infection. Specifically, our focus on gp41 provides much needed insight into its role in membrane fusion, drug susceptibility, and modification of host responses.

Duchenne muscular dystrophy (DMD) is a classical monogenic disorder characterised by severe muscle weakness. Progressive muscle loss ultimately culminates in fatality due to failure of the heart and diaphragm muscles, typically in the 2nd-4th decade of life. The genetic cause of DMD is disruption of the DMD gene encoding for the dystrophin protein. Dystrophin is important for cytoskeletal structure and normal muscle function, and it plays a vital role in maintaining membrane stability and signalling functions. However, the downstream molecular events that follow dystrophin loss are incompletely understood. Moreover, pharmacodynamic biomarkers that can inform on disease progression are severely lacking. This thesis aimed to identify the effects of the dystrophin protein absence on the cardiac transcriptome, skeletal muscle proteome and extracellular microRNA (miRNA) secretome with pathological progression. To improve the understanding of the dystrophic cardiomyopathic advancement in the various anatomical regions of the heart, transcriptomic arrays were performed on RNA taken from the distinct chambers to identify novel pathways involved in pathology. Analysis identified early molecular changes in the right atrium and ventricle, mostly related to cell migration and cardiac remodelling. Furthermore, Sfrp2 (secreted Frizzled-related protein 2), a major regulator of the Wnt signalling pathway, was found to be highly differentially expressed between control and dystrophic hearts. It was observed that expression of the non-canonical c-Jun N-terminal kinase (JNK)-arm of the Wnt cascade was reduced. Interestingly, cellular fractionation demonstrated decreased levels in the cytoplasmic/extracellular region and increased expression surrounding the nucleus, which suggests a potential mislocalisation of the sFRP2 protein in dystrophic hearts. Protein expression in skeletal muscle was characterised by high-resolution mass spectrometry-based proteomics in two dystrophic mouse models over time. Analysis of the two models at different time points allowed for protein identification irrespective of mutation or age. The results highlighted the involvement of many proteins in DMD, such as Rho GTPases, SLC25A24, RICTOR, CAV3 and MVP. Expression of major vault protein (MVP) increased during differentiation of myoblasts in vitro and interestingly, Mvp knockdown was observed to potentially influence Dmd expression levels, suggesting a novel role for MVP in DMD pathology. The miRNA secretome was examined to investigate the relationship between skeletal muscle dystrophin expression and extracellular miRNA abundance. Interestingly, while muscle-related miRNAs (myomiRs, indicative of muscle turnover) were not restored to wild-type levels in mdx-XistΔhs mice, which express low levels of dystrophin from birth (~8-44%), other miRNAs such as miR-193b-3p, miR-370-3p and miR-483-3p did exhibit a decrease in extracellular abundance. In contrast, exon skipping treatment achieving similar dystrophin re-expression levels was able to completely restore serum miRNA levels. The disparity between the mdx-XistΔhs and mdx mice treated with exon skipping was attributed to uneven dystrophin distribution at the sarcolemma in the former. The observed patchy, within-fibre mosaicism was unable to restore extracellular myomiR abundance, and by extension muscle turnover. These results are directly relevant to the development of therapeutic strategies for dystrophin re-expression. In conclusion, this work has identified novel molecular pathways involved in cardiac and skeletal muscle dystrophic pathology. Furthermore, the results have demonstrated the importance of uniform dystrophin distribution at the sarcolemma for pathological correction. Together, these findings provide new insights into the molecular pathology of DMD with implications for therapy.

Cells release nanosized membrane-enclosed vesicles termed extracellular vesicles. These naturally occurring vesicles have been established as important players in communication between cells, mainly through transfer of their content. This inherent ability makes extracellular vesicles outstanding candidates for development into drug delivery vehicles. Cells are considered to release a number of extracellular vesicle types with unique size, biogenesis, and cargo: exosomes, microvesicles, and apoptotic bodies. Exosomes are the most well studied vesicle type and are considered to have homogenous characteristics. However, fractionation of isolated exosomes with sucrose density gradient flotation allowed for identification of two distinct exosome subpopulations with unique size and composition. Subpopulations derived from melanoma cells were found to have differential effects on gene expression of recipient cells. Secondly, development of a size exclusion chromatography based approach allowed for more in-depth study on exosome heterogeneity. Four exosome subpopulations with different size and composition were derived from ovarian cancer cells. Preliminary findings point to a potential pathophysiological role of ovarian-cancer derived exosomes, with unique roles of subpopulations in ovarian cancer metastasis. In addition, differential properties of exosome subpopulations were explored in development of B cell targeted exosomes. Single chain variable fragments targeting B cell surface antigens CD19 and CD22 were successfully expressed on exosomes. Targeting of CD22 resulted in increased and rapid uptake as compared to targeting of CD19. Moreover, data suggested that specific exosome subpopulations are responsible for the latter observation. Findings demonstrate a promising approach for the delivery of biotherapeutics into malignant B cells. In conclusion, this works shows the heterogeneous nature of exosomes and the release of subpopulations with potential unique biological roles. Addressing heterogeneity is one of the key topics of research on extracellular vesicles and will allow for in depth study on their underlying biogenesis and in turn benefit development of extracellular vesicle based therapeutics.

Spinal muscular atrophy (SMA) a neuromuscular disorder caused by loss of the survival motor neuron (SMN) protein and characterized by motor neuron loss and muscle atrophy. Recent clinical trials of SMN restoration therapies changed the SMA therapeutic landscape and led to the first approval of a treatment. SMN restoration clearly has a positive impact on SMA pathology but is not a cure; patients will now live longer with a less severe form of SMA, leading to a growth of the patient population with additional therapeutic needs. Novel second-generation therapies are therefore to alleviate SMA related muscle pathology that can be used in combination with SMN restoration treatments. In the first part of the thesis, an unbiased approach was applied in which proteomic and transcriptomic analyses of Smn-depleted and SMN-restored SMA skeletal muscle were used to investigate dysregulated pathways, presenting the opportunity to identify novel muscle-specific treatments. Proteomic and transcriptomic analyses performed on skeletal muscle from pre-symptomatic and symptomatic Smn-/-;SMN2+/- and WT mice firstly show that SMN restoration completely normalizes protein and transcript expression in Smn-/-;SMN2+/- mice. Next, Connectivity Map (CMap), a drug repositioning strategy using publicly available databases, was employed to analyze proteomic and transcriptomic data and identify treatments able to restore aberrant pathways in SMA muscle. CMap analyses on 'omics studies provided an extensive list of potential treatments for SMA, including some already tested in clinical and preclinical studies and new options, such as harmine. Reference genes produced by the CMap analysis for harmine shows certain genes to be upregulated and others downregulated in SMA mouse tissues and harmine treatment can reverse these expressions in various cell lines and Smn-/-;SMN2+/- mice. Importantly, harmine treatment significantly increases lifespan in severe Smn-/-;SMN2+/- and moderate Smn2B/- mice, rescues myofiber size and prevents motor neuron death. In addition to validating CMap as a method for identifying new treatments for SMA, while using harmine as a proof-of-concept we also generated an extensive list of drugs that can similarly be evaluated. Next, a biased approach was applied in which the TWEAK/Fn14 pathway, critical to the regulation of denervation-induced muscle atrophy and muscle proliferation, differentiation and metabolism, was investigated in SMA, since SMA is typified by neurodegeneration, muscle atrophy and metabolic perturbations. We investigated the expression profile of Tweak and Fn14 together with downstream metabolic effectors in SMA muscle and found significantly decreased levels during disease progression in Smn-/-;SMN2+/- and Smn2B/- mice. Using C2C12 myoblasts and human myoblasts, we confirmed a regulated expression of the TWEAK/Fn14 pathway during differentiation, which is dysregulated in SMA fibroblasts. siRNA mediated knockdown in C2C12s suggests a role for SMN in the aberrant expression of the TWEAK/Fn14 pathway in SMA. Overlap in dysregulation of myopathy, myogenesis and glucose metabolism pathways in SMA mice and TWEAK and Fn14 KO mice reinforced this finding. Through pharmacological interventions that target TWEAK/Fn14 pathway signaling, SMA disease phenotypes were improved in two distinct SMA mouse models. Taken together, these findings show that both biased and unbiased approaches can be used in order to identify modulators of muscle pathology in SMA as well as demonstrates that new investigative approaches, such as CMap, can augment the progress made by SMN restoration therapies.

DNA and RNA structures are involved in major biological processes, such as regulating gene expression, and can play an important role in cancer and neurodegenerative diseases. Huntington's disease (HD) is the most common monogenic neurological disorder caused by the expanded and unstable CAG trinucleotide repeat in the first exon of the huntingtin gene (HTT). The pathology of HD has been traditionally associated with the mutant HTT protein, which confers toxic gain of function leading to dysfunction and death of neurons. However, recent evidence indicates that at least part of the gain of function may operate at the RNA level. In the first part of this thesis, the transcriptional and epigenetic regulation of the HTT gene in HD is investigated. It is shown that expanded CAG repeats of endogenous HTT transcript adopt double-stranded RNA structures that lead to RNA Polymerase II transcriptional repression and heterochromatin formation of the HTT gene in cis, providing a molecular link between dsRNA and the pathology of expansion diseases. Double-stranded RNA is a key player in numerous Biological activities in cells, however little is known about the proteins that bind to dsRNA. Therefore, a proteomics approach was established, employing the dsRNA pull-down to identify dsRNA-binding proteins in control and HD fibroblast cells. Analysis of the dsRNA interactome indicated that interaction with the top candidate ENO1 is enriched in HD and promotes dsRNA-mediated transcriptional repression of the HTT gene. This thesis work also elucidates on the importance of RNA/DNA hybrids in cancer as their accumulation is a major cause for genomic instability. The research demonstrated that cancer cells overexpressing oncogenes such as HRASV12, lead to increased RNA synthesis and RNA/DNA hybrid accumulation that can be correlated with replication stress. These events lead to DNA damage, revealing a strong link between R-loop-mediated genome instability and human disease. Overall, nucleic acid structures and their interactors could determine the ability of a single gene product to perform multiple functions, as potential disease modifiers and future targets to modulate diseases.

Clinical ototoxicity - permanent hearing loss caused by medications - is estimated to affect millions of patients annually. Two classes of drug are largely to blame: platinum-based chemotherapeutics, primarily cisplatin, and aminoglycoside antibiotics. Development of methods to prevent ototoxicity depends upon an understanding of its mechanisms and may benefit from an understanding of native protective pathways of the inner ear. As the mechanisms behind cisplatin ototoxicity remain unclear, I first sought, and herein report, a refined mouse model of cisplatin ototoxicity which will allow for further in vivo investigation of cisplatin ototoxicity and potential methods for its prevention. This low-dose, multi-cycle model was found to accurately reproduce cisplatin ototoxicity as it has been described clinically and histopathologically. I then used this mouse model of cisplatin ototoxicity to investigate cisplatin pharmacokinetics in the cochlea and their role in driving cisplatin ototoxicity. Cisplatin was found to be retained within the cochlea for months following its administration. This initial finding in mice was extended to cochlear tissue samples from deceased human patients. Analysis of intra-cochlear cisplatin distribution in murine and human tissue identified the stria vascularis region of the cochlea as a promising target for intervention. With the nature of aminoglycoside ototoxicity better understood, I investigated a native inner ear protective pathway which could be leveraged to promote sensory hair cell survival. The improved hair cell survival that has previously been demonstrated as a result of heat stress was found to be mediated by cell-cell communication via extracellular vesicles. Further, hair cell protection against aminoglycosides could be reproduced through the application of exogenous, non-inner ear-derived extracellular vesicles. In sum, these data provide new insight into mechanisms of ototoxicity and details of cellular pathways which can help protect against it.

Extracellular microRNAs (ex-miRNAs) and serum proteins are promising biomarkers for a variety of pathological conditions, including muscular dystrophies and, Duchenne Muscular Dystrophy (DMD) in particular. miR-1, miR-133 and miR-206 are muscle-specific miRNAs (myomiRs) that regulate myoblast proliferation and differentiation. These myomiRs are highly abundant in serum of DMD patients and dystrophic mdx mice and have therefore been investigated as biomarkers of dystrophic disease. The biological significance of miRNAs present in the extracellular space is currently not well understood. The work presented here demonstrates that ex-myomiRs are selectively released during periods of myogenic differentiation in cell culture and in vivo. Consequently, their release can be independent of dystrophic pathology, thus indicating that the presence of myomiRs in the serum may serve a physiological function in some contexts. In summary, serum myomiR abundance appears to be a function of the regenerative/degenerative status of the muscle, overall muscle mass, tissue expression levels and myomiR stability in serum. These findings have implications both for miRNA biology in normal muscle physiology as well as for the use of ex-myomiRs as biomarkers for DMD. Secondly, size-exclusion chromatography was identified as a useful methodology to fractionate biofluids and thereby separate extracellular vesicles (EV) from protein complexes and lipoproteins. Using this tool, it was demonstrated that ex-myomiR carriers in cell culture supernatant and murine serum are predominantly non-vesicular, and furthermore, that their release is independent of ceramide-mediated vesicle secretion. In addition, it was found that EVs isolated from dystrophic mice are smaller and more numerous compared to EVs derived from wild-type mice, thereby indicating a role for dystrophic pathology in the regulation of vesicle biogenesis and/or secretion. There is currently an urgent need for minimally invasive, therapy-monitoring DMD biomarkers for use in clinical trials. Serum protein biomarkers are desirable since they can typically be quantified by clinical biochemistry assays, which are technically facile and allow for rapid screening of large numbers of patients. Previously, identification of such biomarkers by mass spectrometry has been limited due to analytical challenges resulting from the massive complexity of the serum proteome. Here, an aptamer-based proteomics approach was utilised to profile 1,129 proteins in the serum of wild-type, mdx mice and, mdx mice treated with exon skipping therapy to restore dystrophin expression. A number of novel, therapy-responsive biomarkers were identified, and the leading candidate (ADAMTS5) was also found to be significantly elevated in DMD patient serum. In conclusion, this work demonstrates that myomiR release accompanies myogenic differentiation and shows that serum myomiR levels are influenced by a number of physiological and pathological factors. Furthermore, multiple novel, therapy-responsive protein biomarkers were identified in the serum of the mdx mouse with potential utility in clinical trials for DMD.

Huntington's disease (HD) can be regarded as a model neurodegenerative disorder to screen potential genetic based therapeutics and their carriers. It is an autosomal dominant disorder caused by a mutation in a single gene that leads to progressive neurodegeneration caused in part by protein misfolding. The mutation codes for an expanded polyglutamine tract within the Huntingtin gene (HTT) which leads to neuronal loss through a pathological cascade of events. Current treatment strategies include symptom management but no disease-modifying therapies exist. Research has shown that nucleic acid based therapeutics aimed at decreasing HTT expression can prevent, or reverse, the phenotype. Translating such therapies to the clinic is hindered by the blood brain barrier (BBB) and the lack of an easily administrable, non-toxic, immunologically inert delivery vehicle capable of bypassing the BBB. This study examines a range of nucleic acid based therapeutics for their potency and toxicity, and evaluates extracellular vesicles (EVs) as a delivery vehicle through investigation of the biodistribution of brain targeted EVs and an analysis of EV loading. A small interfering RNA (siRNA) targeting a region upstream of the repeat induced potent non-toxic silencing of HTT. In examining EVs as a carrier for therapeutics for neurological diseases including HD, it was found that targeting can increase brain accumulation of EVs but that the physiological characteristics of EVs which make them susceptible to clearance by the reticuloendothelial system (RES) must be further evaluated to bioengineer modified EVs to avoid fast clearance. Lastly, loading by electroporation was found to induce siRNA aggregation which can lead to overestimation of loading by increasing siRNA content in an ultracentrifugation pellet, but cholesterol-conjugated siRNA mixed with EVs was capable of generating silencing in vitro. This thesis examined EV based treatment for HD through the selection of a nucleic acid cargo to silence HTT, and examination of EVs as a delivery vehicle via biodistribution and loading studies. If the loading can be optimised and fast clearance avoided, there is promise in the use of EVs as a carrier of siRNA for HD.

Extracellular vesicles (EVs) are nano-sized particles constitutively released from cells into biological fluids such as blood, plasma, saliva and urine. Interestingly, these vesicles contain genetic cargoes including proteins, RNA and bioactive lipids that can be functionally delivered and affect recipient cells. Hence, EVs are postulated to be an alternative source of cell-cell communication in both normal physiological systems and pathological situations. Recently, EVs from mesenchymal stem cells have been shown to promote regeneration of injured cells. Induced pluripotent stem cells (iPSCs) are a relative newer type of stem cells that is emerging as useful tools for re-modeling diseases. However, knowledge about EVs from iPSCs is relatively sparse. In initial experiments, we successfully purified EVs from mouse iPSCs using the differential ultracentrifugation (UC) method. However, we noticed a discrepancy between total particle counts and expression of EV markers, across different EV batches. One crucial prerequisite for EV research is the availability of a standardized workflow for collection and purification of EVs from biological sources. Increasing evidence from recent studies has suggested that the original UC method is limited by several shortcomings such as low EV yield, purity and altered biophysical properties. Hence, this has led to a new wave in the development of alternative EV purification strategies. In this thesis, we start with a systematic comparison study between UC and an alternative purification protocol, size-exclusion liquid chromatography (LC) of EVs from serum-free conditions. We found that LC is better than UC in terms of overall EV yields, purity and vesicle integrity. Subsequently, we demonstrate that LC allowed for the derivation of pure EVs from complex media sources used for growing stem cells like iPSCs, which was previously impossible with UC. Lastly, we describe novel data on the characterization of EVs from pluripotent stem cell sources and discuss the possible roles of these EVs in stem cell biology.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterised pathologically by the accumulation of amyloid-β plaques and neurofibrillary tangles within the brain. Currently, there are no disease-modifying therapeutic interventions available. This thesis describes the development and delivery of novel RNA-based therapeutics for AD. Recently, significant advances have occurred in the field of RNA interference (RNAi)-based therapeutics, allowing targeted silencing of disease-relevant genes through the use of short interfering RNAs (siRNAs) or microRNAs (miRNAs). However, development has been impeded by poor penetrance of the blood-brain-barrier (BBB) in vivo. Exosomes, as secreted extracellular vesicles (EVs), may overcome this problem as they naturally deliver RNA between cells and cross the BBB. Furthermore, recent evidence suggests that they may be loaded with exogenous RNA and preferentially targeted to neuronal tissue. Thus, this study investigated EV-mediated delivery of RNAi-based therapeutics shown to regulate AD-relevant genes. A comparison of EV-loading methods revealed that electroporation was confounded by the formation of large siRNA aggregates and indicated that endogenous loading strategies may be preferable. Endogenous loading of RNAi molecules was shown to be enhanced by co-expression with RNA-binding proteins, particularly following inclusion of an acylation domain. These findings highlight the potential for EV-mediated delivery of RNAi-based therapeutics. A combinatorial therapeutic approach is increasingly recognised to hold the most promise for the future of AD. Thus, this study also focused upon the development of novel multi-gene targeting locked nucleic acid-modified antisense oligonucleotides (LNA-ASOs) as a combinatorial approach towards AD therapy. Two multi-gene LNA-ASOs each produced potent silencing of two separate target genes simultaneously, resulting in a significant reduction in amyloid-β production in vitro. A murine-specific multi-gene LNA-ASO was also identified, paving the way for future in vivo studies. These findings suggest that multi-gene targeting LNA-ASOs may represent a promising novel therapeutic strategy for AD. In summary, this thesis has investigated novel strategies for both RNA delivery and multi-gene targeting, demonstrating the potential of RNA-based therapeutics for the treatment of AD.

Genetic neuromuscular diseases remain essentially untreatable. Duchenne muscular dystrophy (DMD) is one such example and another is C9ORF72-related frontotemporal dementia/amyotrophic lateral sclerosis (c9FTD/ALS). Both these conditions may, however, be amenable to treatment with antisense oligonucleotide (ASO) compounds. Therapeutic ASO development for DMD is already in the clinical trial phase, while the applicability of ASO therapies in c9FTD/ALS remains to be clarified. No reliable method has yet been devised in order to target these compounds to the central nervous system following systemic administration. This thesis has investigated the ability to deliver therapeutic ASOs to the brain following systemic administration in the mdx mouse model of DMD. It has also investigated the molecular genetics of c9FTD/ALS to see whether ASO treatment may be a beneficial approach. The integrity of the blood-brain barrier (BBB) was assessed in mdx mice. The mdx mouse BBB was found to be structurally intact by light and electron microscopy. Brain gene expression of BBB components was similar between mdx and wild-type mice. The barrier function of the mdx BBB was found to be largely equivalent to that of wild-type mice using systemically administered fluorescent tracers. Peptide-conjugated phosphorodiamidate morpholino oligonucleotides (PPMOs) were tested by intravenous injection in mdx mice. Pip6a-PMO was found to be the most active compound for inducing exon skipping in the brain. Fluorescent labelling of Pip6a-PMO showed body-wide distribution after systemic delivery, including increased accumulation in brain compared to unconjugated PMO, particularly in choroid plexus. c9FTD/ALS patient fibroblasts were analysed for C9ORF72 promoter methylation, expansion size and gene expression. No clear relationship was found between C9ORF72 expansion size and levels of promoter methylation or gene expression. An unexpected positive correlation was found between the degree of promoter methylation and gene expression. LNA-gapmer knockdown of a putative C9ORF72 natural antisense transcript resulted in upregulation of gene expression. These experiments suggest that the BBB is not severely defective in DMD. Furthermore, peptide conjugation of ASOs can be used to induce detectable exon skipping in brain tissue following systemic injection, suggesting that certain PPMOs may cross the BBB. Finally, ASOs may also provide a useful therapeutic approach in c9FTD/ALS, although further work will be needed in future to confirm this and to better characterise the disease.

The ability to reprogram adult somatic cells into induced pluripotent stem cells (iPSCs) and subsequent development of protocols for their differentiation into disease-relevant cell types have enabled in-depth molecular analyses of multiple disease states as hitherto impossible. Parkinson's disease (PD) is one such example, in which the dopaminergic neurons that manifest pathology are embedded in an inaccessible region of the midbrain, the substantia nigra pars compacta, making it unfeasible to obtain samples of the affected region from living patients. Overabundance of alpha-synuclein, encoded by the SNCA gene, has long been implicated in the pathogenesis of PD. However, the precise mechanism by which overexpression of alpha-synuclein leads to the demise of dopaminergic neurons remains elusive. Neurons differentiated from PD patient-specific iPSCs carrying multiplications of the SNCA gene may provide a means to recapitulate molecular phenotypes of the disease in vitro. The application of CRISPR/Cas9 to mammalian systems is likewise revolutionizing the utilization of genome editing in the study of molecular contributors to the pathogenesis of numerous diseases, including PD. In this body of work, I have utilized the double-nicking CRISPR/Cas9 system to mediate site-specific mutagenesis of SNCA in PD patient-specific iPSCs harboring a triplication of the SNCA gene locus, resulting in isogenic cells which can be used to phenocopy normal alpha-synuclein expression from two alleles. I have further demonstrated the utility of nuclease null or "dead" Cas9, for transcriptional silencing of alpha-synuclein expression. Finally, I have used these systems to interrogate phenotypic outcomes of SNCA triplication, demonstrating that alpha-synuclein induces endoplasmic reticulum stress and aberrant activation of a highly conserved arm of the unfolded protein response. The tools generated in these studies can be applied in future efforts to dissect the contributions of alpha-synuclein overexpression to PD pathogenesis and as a platform for the discovery of disease-modifying therapeutic approaches for PD.

Duchenne muscular dystrophy (DMD) is a fatal, muscle-wasting disorder due to mutations/deletions in the dystrophin gene. Whilst improvements in palliative care have increased the life expectancy of patients, cardiomyopathy and respiratory complications are still the leading causes of death. A potential therapy for the treatment of DMD is antisense oligonucleotides (AOs), which modulate dystrophin pre-mRNA splicing to restore the dystrophin reading frame and generate a truncated functional protein. Conjugation of AOs to cell penetrating peptides (CPP), such as Pip5e-, significantly improves delivery to skeletal muscles and to the heart, which is imperative given the impact of cardiomyopathy to mortality. However, it should be noted that the contribution of skeletal muscles, such as the core respiratory muscle, the diaphragm, in dystrophic cardiopulmonary function is poorly understood. The specific aims of the work in this thesis were to (i) understand the effect of the diaphragm on cardiac function using magnetic resonance imaging (MRI), (ii) screen a number of derivatives of Pip5e (Pip6) in an effort to discover further promising peptides and define the properties integral to heart penetrating capacity, and (iii) assess whether Pip6-PMOs restore cardiac function (MRI) following a repeat, low dose regimen. In short, the specific restoration of dystrophin in the diaphragm of the dystrophic mouse model, the mdx mouse, did not improve cardiac function, highlighting the importance of a body-wide therapy. The screening of multiple Pip5e-PMO derivatives revealed 3 promising peptides with improved cardiac splicing capacity; however, serial deletions of amino acids from the central core resulted in the diminution of dystrophin restoration, possibly due to a reduction in hydrophobicity. Finally, the Pip6-PMO treatment regimen substantially restored dystrophin protein (28% in heart) and stabilised cardiac function, even with an increased work load. In conclusion, this study illustrates the importance of a body-wide treatment, such as the CPP strategy (Pip-PMO). These Pip-PMO conjugates demonstrate high dystrophin restoration in a number of muscles, including cardiac muscle, and have a beneficial effect on cardiac function.

For many dominant diseases, conventional treatment options are limited. This makes them attractive candidates for gene therapy, which may be directed to specifically silence a disease-causing allele. However, many mutations are not easily amenable to this technique, including nucleotide repeat expansions, which cause numerous neurodegenerative diseases such as Huntington’s disease and several Spinocerebellar Ataxias. Combined gene knockdown and replacement (K&R) may present a more practical approach for such conditions, whereby the gene of interest is subject to mutation-independent non-allele-specific silencing and concurrently replaced with a functional copy. Artificial mimics of naturally occurring intronic microRNAs are theoretically ideal for this purpose, since they can be nested within the replacement gene. This thesis investigates the development of mimics of two different intronic miRNA systems (mirtron miR-1224 and the miR-106b cluster) to silence Ataxin-7 and to be incorporated into novel single K&R constructs for testing in vitro. Artificial mirtrons and intronic miRNAs were successfully developed and shown to silence Ataxin-7 mRNA in numerous cell lines. An RNAi-resistant gene was developed and mirtrons could be successfully incorporated as introns. Patient-derived fibroblasts and iPSC-derived neuronal cells were investigated as models for testing of gene silencing therapies. This work suggests that mirtron-based K&R is achievable, and warrants further investigation.

Duchenne muscular dystrophy (DMD) is a progressive muscle wasting disorder caused by absence of functional dystrophin protein. This thesis describes investigations into the role of small non-coding RNAs in both DMD pathology, and as potential therapeutic molecules. MicroRNAs (miRNAs) are a class of small RNAs that regulate gene expression and are implicated in wide-ranging cellular processes and pathological conditions. This study has compared differential miRNA expression in proximal and distal limb muscles, diaphragm, heart and serum in the mdx dystrophic mouse model relative to wild-type controls. Global transcriptome analysis revealed muscle-specific patterns of differential miRNA expression as well as commonalities between tissues, including previously identified dystromirs. miR-1, miR-133a and miR-206 were found to be highly abundant in mdx serum, suggesting that these miRNAs are promising disease biomarkers. Indeed, the relative serum levels of these miRNAs were normalised in response to peptide-PMO mediated dystrophin restoration therapy. This study has revealed further complexity in the miRNA transcriptome of the mdx mouse, an understanding of which will be valuable for the development of novel DMD therapeutics and for monitoring their efficacy. Myostatin is a secreted growth factor that negatively regulates muscle mass and is therefore a potential pharmacological target for the treatment of muscle wasting disorders such as DMD. This study describes a novel myostatin inhibition approach in which small interfering RNAs (siRNAs) complementary to a promoter-associated transcript induce transcriptional gene silencing (TGS) in cultured myotubes. Silencing was sensitive to treatment with the histone deacetylase inhibitor Trichostatin A, and the silent state chromatin mark H3K9me2 was enriched at the myostatin promoter following siRNA transfection, suggesting epigenetic remodelling underlies the silencing effect. These observations suggest that long-term epigenetic silencing may be feasible for myostatin and that TGS is a promising novel therapeutic strategy for the treatment of muscle wasting disorders. The work in this thesis therefore demonstrates the potential of small RNAs as therapeutic agents and as disease biomarkers in the context of DMD.