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Migraine, the third most prevalent disease in the world (global prevalence of 12- 14.7%), is a complex and multifaceted neurovascular disorder, characterised by severe attacks of headache and often associated with nausea and sensory hypersensitivity. Numerous contraindications and adverse events are associated with traditional medicines and, more importantly, 40% of patients receive little-to- no benefit from them. Calcitonin gene-related peptide (CGRP) and its receptor are now recognised to play a key role in migraine pathophysiology and evidence suggests that the CGRP receptor could be antagonised with synthetic peptides obtained by truncation of the native CGRP. Peptide-based therapeutics have been an active area of research for decades and have found clinical applications in a number of disease areas, e.g., metabolic disorders, cancers, cardiovascular diseases, and infection diseases, reaching the milestone of 100 approved peptide drugs in 2020. In this project, information on regulatory approved peptide therapeutics and diagnostics was collated in an open- source database, PepTherDia (http://peptherdia.herokuapp.com), and a detailed structural analysis was performed to uncover key structural trends to help with future peptide drug design. The use of peptides as medicines is still limited due to poor bioavailability after oral administration and, more generally, poor stability due to their susceptibility to proteolytic degradation. Various strategies, including fluorine-editing, peptoid- editing, or lipidation, could be explored to prepare proteolytically stable peptide or peptidomimetic analogues. Towards this goal, a library of peptide and peptidomimetic analogues of the C-terminal portion of CGRP were designed and synthesised, starting from the structure of a potent CGRP receptor antagonist (P006) previously developed in collaboration with PharmNovo AB. These compounds were tested for antagonism at the CGRP receptor, lipophilicity and toxicity. Methods to assess peptide stability in blood and plasma protein binding were also optimised, validated, and applied to study the pharmacokinetic profile of the library of compounds. This led to the discovery of potent CGRP receptor antagonists with vii extremely favourable pharmacokinetic profiles. Among them, the most promising compounds of the series were three N-terminal benzoylated analogues (i.e., LJMU027, LJMU017, and LJMU018) presenting significantly increased estimated half- lives (> 3 hours) compared to P006 (estimated half-life of ~13 min), while maintaining comparable activity against the CGRP receptor. Due to first pass metabolism and poor oral absorption, peptides are often administered parenterally, thus usually resulting in low patient acceptability and compliance. Alternatively, peptides could be delivered via the intranasal route, offering the advantages of rapid direct delivery to the brain, greater acceptability and compliance, and lower adverse event profiles due to inferior systemic effects. Nano- sized structures derived from peptide self-assembly in water were obtained (diameter size between 14 and 30 nm) and peptide-loaded dry powder microparticles prepared from low molecular weight chitosan suitable for nasal delivery were manufactured (average diameter size of 9.55 ± 0.74 μm). In conclusion, this investigation showed that benzoyl modifications performed at the N-terminus of P006 did not disrupt antagonistic activity but yielded stable analogues that were up to 90% intact after 60 min incubation in pooled human serum, with a tendency to bind to plasma proteins. The ability of these peptides to self-assemble was exploited to prepare nano-sized structures, which could be directly administered parenterally or loaded into mucoadhesive carriers for nasal delivery.

Abstract: Radical cation catalyzed methodology was adapted for the synthesis of disubstituted quinolines, to address the requirements for continuous oxygen sparge and high reaction temperatures. Using previously reported aldehydes and 5,6,7,8-tetrahydronaphthylamine, the reaction required optimization of radical cation salt and reaction conditions. A series of radical cation salts were synthesized by the Buchwald-Hartwig amination of para-substituted iodobenzene derivatives screened using similar para-substituted anilines. The triarylamines were oxidized to the radical cations using triphenylammonium hexachloroantimonate (TPASbCl6), tris(p-methylphenyl)ammonium hexachloroantimonate (TTASbCl6), tris(4-biphenyl)ammonium hexachloroantimonate (TBPhASbCl6), and triphenylammonium hexafluorophosphate (TPAPF6), to compare with commercial tris(4-bromophenyl)ammonium hexachloroantimonate (TBPASbCl6). After optimizing other reaction variables, the best results were obtained using TPASbCl6 as the catalyst. This method of radical catalysis was compared to our previously optimized acid/iodide methodology with a series of aniline substrates. Radical cation catalysis was comparable to the HI method, with some substrates providing increased yields. Bidirectional MCR reactions were evaluated using 1,5-diaminonaphthalene to target the formation of tetrasubstituted 4,10-diazachrysene compounds under the radical cation conditions. These reactions were successful, but resulted in isolation of the dihydroquinoline as product, which does not undergo aerobic oxidation to the fully aromatic ring system. Preliminary exploration of an electrochemical method for generating the aniline radical cation was pursued. The electrochemical cell allows for oxidation of a soluble redox mediator, leading to the same radical cation mediated MCR quinoline synthesis. Initial results were poor, but lithium bromide as electrolyte provided improved but still modest yields. The dual role of lithium bromide as the electrolyte and the amine redox mediator requires more investigation to better understand its purpose in the reaction process.

Mouse embryonic stem cells (mESCs), an in vitro model for naive pluripotent differentiation into primed pluripotent epiblast-like cells (EpiLCs), have been used to reveal transcriptional and epigenetic control of early embryonic development. However, the control and significance of morphological changes during lineage specification remain less defined. We show with quantitative time-lapse imaging marked changes in morphology and actin architectures during differentiation that depend on activity of the Arp2/3 complex, an actin filament nucleator. Inhibiting Arp2/3 complex activity pharmacologically or genetically does not block exit from naive pluripotency but attenuates increases in EpiLC markers compared with controls. We found that loss of Arp2/3 complex activity delays entry into the intermediate formative pluripotent state, resulting in globally defective lineage specification as indicated by RNA-sequencing with marked effects on TBX3-depedendent transcriptional programs. Further, we identified two previously unreported indicators of pluripotent status; MRTF and FHL2, which have inverse actin-dependent nuclear translocation as competing SRF co-factors that is dependent on Arp2/3 complex but not formin activity. These data reveal a previously unrecognized role for Arp2/3 complex-dependent actin remodeling in mESC differentiation, timing of formative pluripotency, and TBX3-dependent lineage specification, as well as newly identified MRTF and FHL2 nuclear shuttling. Moreover, our current findings on a role for Arp2/3 complex activity in mESC differentiation compared with the established role for formin activity in epithelial to mesenchymal transition indicate that distinct actin nucleators regulate distinct modes of epithelial plasticity.

This practice-led research project examines how the 3D video game image is constructed and understood within photographic frameworks. Through artistic practice and scholarly research, this project unpacks the ways that computer-generated and photographic images are interrelated, how their processes generate meaning and affect, and how artists can deconstruct and reimagine the potential of these images. The thesis specifically examines theories of photography and new media to analyse artworks by Jon Raman, Pierre Huyghe and Philippe Parenno. These artists, as well as my own practice, appropriate and redeploy 3D video game images to investigate how their emotive, symbolic and narrative qualities reinforce dominant modes of representation. Through my own practice, I combine analogue photographic processes and digital, 3D imaging technologies to redirect the expected affective outcomes of the video game image. Together, the thesis and artistic practice interrogate the ways that the technical structure of computer-generated 3D video game images draw directly from photographic technologies. This project argues that the link to photography is crucial in understanding not only how 3D video game images reproduce the affective, symbolic and narrative possibilities of photography, but also how artists can intervene and reorient these patterns of meaning and affect.