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  • ItemOpen Access
    Development of novel imaging technology to study cell signalling
    Li, Bing
    Currently it is difficult to study signalling on living cells at the single molecule level. One problem is that it is not possible to trigger signalling in a controlled manner and there is no effective method that can both introduce a precise amount of molecules onto or into a single cell at a specific position and then simultaneously image the cellular response using single molecule fluorescence. Here, we have developed local-delivery selective-plane illumination microscopy (ldSPIM) to address this issue. ldSPIM uses a nanopipette to accurately deliver individual proteins to a defined position. For single-molecule fluorescence detection, we implemented single-objective SPIM using a reflective atomic force microscope cantilever to create a 2 μm thick light sheet. Using this setup, we demonstrated that ldSPIM can deliver single fluorophore labelled proteins onto the plasma membrane of HEK293 cells or into the cytoplasm and characterise the interaction between cells and the delivered molecules in 3D at the same time. Then, we applied ldSPIM to characterize TLR4 activation and Myddosome signalling. The TLR4 agonist lipopolysaccharides (LPS) and aggregates of amyloid-β, which are supposed to be one of the key toxic species in Alzheimer’s disease, were delivered onto single macrophage stably expressing a MyD88-eGFP fusion construct. Whole-cell 3D light sheet imaging enabled the live detection of MyD88 accumulation and the formation of the Myddosome signalling complexes. Kinetics analysis of the trajectory of the assembly of individual Myddosomes suggested that amyloid-β triggered a significantly different Myddosome response compared with canonical LPS-triggered signalling. The nanopipette was also used to locally deliver interferon β onto mouse embryonic fibroblasts, to trigger the interaction between the interferon alpha and the beta receptor subunit 1 and the interferon alpha and beta receptor subunit 2. Lastly, in order to improve the light sheet imaging capability, we designed and assembled an epi-illumination SPIM (eSPIM), which is a next generation single-objective light sheet microscope with much faster scanning and is compatible with all kinds of sample dishes including multi-well plates. Overall, this thesis describes the building of new instrumentation to study cell signalling at the single molecule level, combining controlled delivery, via a nanopipette, and light sheet imaging, and the application of this instrumentation to study TLR4 signalling and the kinetics of Myddosome formation.
  • ItemEmbargo
    Towards a Fundamental Understanding of Aqueous Organic Redox Flow Batteries – A Study of Degradation, Aggregation, and Reactivity
    Hey, Dominic
    Renewable energy sources such as wind, solar, and hydropower are becoming more prominent in the global energy mix. To address intermittency and ensure a reliable energy supply, grid-scale energy storage systems are crucial. Redox flow batteries (RFBs) are a useful tool for grid-scale energy storage and are able to address the intermittent nature of renewable energy sources. RFBs are well-suited for grid-scale applications due to their scalability, long cycle life, flexibility, separation of power and energy, rapid response, and, most importantly, safety. The most widely used RFB is the all-vanadium system, which contains toxic and expensive metal ions. Thus, organic RFBs are a promising alternative to replace the all-vanadium system. To date, many different organic molecules, including quinones, viologens, phenazines, and alloxazines, have been investigated as potentially cheaper RFB active molecules. While a few molecules have shown good performance, most organic molecules considered for RFBs have lower energy density and generally experience degradation, reducing cell lifetime. Thus, fundamental insight at the molecular level is required to improve their performance. In this work, different analysis methods were employed to understand the inter- and intramolecular interactions of the organic molecules in the negative electrolyte. *In-situ* methods based on various spectroscopic techniques were utilised: nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR); ultraviolet-visible (UV-Vis); and infrared (IR) spectroscopy. In the first results chapter, a detailed understanding of degradation processes is developed for the negative electrolyte flavin mononucleotide (FMN), a redox-active biomolecule which is readily derived from vitamin B2. These findings were then used to improve performance dramatically. FMN hydrolysis products were identified via *in-situ* NMR and EPR, and it was shown that these products give rise to the additional plateau seen during charging of a FMN-cyanoferrate battery. The redox reactions of the hydrolysis product are not reversible. However, capacity retention was observed even after substantial hydrolysis, albeit with reduced Voltaic efficiency, with FMN acting as a redox mediator. Critically, degradation was mitigated, and battery efficiency was substantially improved by lowering the pH from 14 to 11. *Ex-* and *in-situ* NMR analyses were then used to study 2,6-dihydroxyanthraquinone (DHAQ), a well-studied organic molecule in RFBs, and discussed in the second research chapter. The electrochemical degradation processes during cycling to high voltages and the electrochemical recomposition of decomposed DHAQ at low voltages are discussed. NMR was used to understand the previously unassigned electrochemical mechanisms, as well as the nature of the decomposition products. At high voltages, DHAQ decomposes to 2,6 dihydroxyanthrone (DHA) and its tautomer, 2,6-dihydroxyanthranol (DHAL). Suppression of the water solvent signal enabled the complete assignment of the obtained NMR spectra. Comparisons between deuterated water (D2O) and deionised water (H2O) were made as more NMR signals can be observed when H2O is used to avoid proton-deuterium exchange. DHAQ can then be electrochemically regenerated from the decomposition products through an oxidation to the dimer (DHA)24− followed by another low voltage oxidation. This electrochemical regeneration is a novel process that could only be identified by using the combination of *ex-* and *in-situ* NMR methods. In the third chapter, intermolecular processes, such as aggregation, were studied via *in-situ* UV-Vis and related to the electrochemistry of DHAQ. The intermolecular interactions become stronger with higher concentration, and due to the drive towards using highly concentrated electrolytes in practical RFBs, an understanding of these interactions is crucial. The combination of non-negative matrix factorisation (NMF), a principal component analysis, and UV-spectroscopy enabled detailed characterisation of the inter- and intramolecular processes. A tendency for DHAQ to aggregate was identified via an *ex-* and *in-situ* UV-Vis concentration study coupled with *ex-situ* NMR experiments. This aggregate appears to directly influence the radical concentration during cycling and increases the reduction potential. In the fourth research chapter, a novel *operando* IR cell was designed. With the cell, an analysis of the reaction kinetics and diffusion on the example system DHAQ is possible. The combination of microscopy and IR spectroscopy allowed for the direct targeting of carbon electrode fibres. Thus, the interfacial reactions of DHAQ on the carbon electrodes were better understood. Additionally, in combination with the line scan and mapping tools of the set-up, an estimation of the kinetics of the system, especially the diffusion, preferred reaction sites, and the rate of reaction of comproportionation – which has been to date unclear in the literature – was enabled. In this doctoral thesis, new analysis tools were used to improve the fundamental understanding of chemical and electrochemical reactions in organic RFBs. By understanding degradation processes, aggregation, and interfacial processes, we are able to provide information for designing electrolytes more effectively, working with optimised parameters, such as pH and supporting electrolyte concentration, and provide valuable insight into the electrode structures and their reaction centres.
  • ItemEmbargo
    Synthesis and Optical Characterisation of Diphenylhexatriene Derived Intramolecular Singlet Fission Materials
    Millington, Oliver; Millington, Oliver [0000-0001-6787-8553]
    There is strong motivation for the detailed exploration of physical processes that present the potential for enhancing the power conversion efficiencies of solar photovoltaic devices. One key assumption underlying the theoretical Shockley-Queisser limit of the efficiency of photovoltaic devices is that the absorption of one photon can only produce a single electron-hole pair; all excess energy above the bandgap of the semiconductor is lost to thermalisation. Singlet fission (SF) is a physical process in certain organic materials that enables the production of two excited states, each with triplet multiplicity, from a lone photoexcited singlet state. It has been widely recognised that integration of an appropriately energetic and efficient singlet fission material into a photovoltaic device may facilitate circumvention of the Shockley-Queisser efficiency limit. This requires that the energy of both SF-born triplets can be harvested independently and transferred to the semiconductor. In covalently connected assemblies of a singlet fission active chromophore, intramolecular singlet fission (iSF) can generate two triplet states upon a single molecule. The singlet fission properties of the molecule can be tailored by synthetic engineering of the interchromophore connectivity. Only a modest sub-set of the chromophores that have been identified to undergo SF in condensed phases have been investigated in molecular systems for iSF activity. 1,6-Diphenylhexa-1,3,5-triene (DPH) is a promising chromophore that is known to undergo singlet fission in the solid state. The high triplet energy of DPH (~ 1.5 eV) makes it a particularly attractive chromophore for further studies, given the identification of high triplet energy as a critical requirement for the viable application of SF in photovoltaic systems. In the work described in this thesis, the first studies of iSF activity in DPH materials were conducted. This thesis describes the design and chemical synthesis of the initial dimeric DPH derivatives for investigation as iSF candidates. The optical characterisation of these materials using transient absorption spectroscopy is reported and two of the initial five materials are found to form an ultrafast equilibrium between singlet and triplet-pair states. Following the initial studies, several subsequent generations of DPH dimers and oligomers are investigated. Facile iSF to generate a triplet-pair state is achieved for several materials but reducing triplet-pair annihilation, in order to achieve persistent pairs of triplets, proves elusive. Nevertheless, significant progress is achieved with the formation of spatially separated triplet-pairs being identified for contiguously connected oligomers, presented in the final chapter. Stabilisation of these spatially separated triplet populations remains the outstanding challenge for the generation of persistent pairs of triplets.
  • ItemEmbargo
    Utilising Non-Covalent Interactions in Developing Enantioselective Radical Transformations
    Bacos, Paul David
    This thesis details the development of catalytic, enantioselective strategies for radical reactions, utilising non-covalent interactions to control the asymmetry of these processes. The first research chapter describes the use of chiral phosphoric acids to control the enantioselectivity in the reactions of α-amino radicals. Firstly, a Giese reaction with α,β-unsaturated carbonyls is reported. This was found to be successful for a range of substrates, with the catalyst being able to exert control over the stereocentres originating from both the amine nucleophile and the acrylamide acceptor. Secondly, a reaction with polarity-mismatched acceptors, in the form of electron-rich enamides, is disclosed. While high enantioselectivities were achieved in some cases, the reaction was found to be effective only for a limited range of substrates. Thirdly, the Minisci-type addition of cyclic α-amino radicals into heteroarenes is detailed, including substrate design and limitations of the process. The second research chapter details preliminary studies into developing asymmetric nickel-catalysed functionalisations of α-amino radicals, using two different approaches. Initially, investigations were performed to determine whether chiral phosphoric acids, bound to the substrate via hydrogen bonding, could induce asymmetry. The subsequent approach relied on using an anionic ligand, ion-paired to a chiral cation derived from cinchona alkaloids, to create a chiral environment around the metal centre. The challenges encountered for both approaches are discussed. The third research chapter describes the incipient phase of a project based on the use of cinchona alkaloids-derived catalysts to perform enantioselective and desymmetrising hydrogen atom transfer from vicinal diols. The α-hydroxy radicals generated in the process were utilised in Giese additions and stereochemical isomerisation reactions, with high levels of enantioselectivity.
  • ItemEmbargo
    Carbon dots as sustainable photocatalysts for organic synthesis
    Lage, Ava
    As easily scalable, cost-effective and environmentally benign materials, carbon dots (CDs) have the potential to replace costly or harmful photocatalysts for a range of synthetically relevant transformations. This dissertation aims to establish CDs as versatile, sustainable photocatalysts for organic synthesis. Chapter 2 explores CDs as photocatalysts for net-oxidative and redox-neutral C-C bond formation. A variety of aromatic substrates and biological motifs were successfully trifluoromethylated under aerobic conditions. To utilise the full potential of this reaction, the net-oxidative trifluoromethylation was coupled to H2-evolution, thereby generating two products simultaneously: a trifluoromethylated aryl and feedstock for hydrogenation. Chapter 3 subsequently introduces CDs as photocatalysts for net-reductive reactions by example of dehalogenation of aryl-iodides, -bromides and -chlorides. The C-halogen bond was successfully cleaved for all three substrate groups despite the strong reduction potential required particularly for the bromides and chlorides. As the CD reduction potential, while appreciable, is not sufficient to drive some of these reactions, mechanistic studies were undertaken to illuminate possible reaction pathways. Chapter 4 expands the application of CDs to C-C bond formation reactions that are currently difficult to access without transition metal catalysts or under visible light irradiation. The examples in this chapter include cross-coupling of 1,4-Dicyanobenzene with aldehydes and ketones as well as pinacol coupling of aldehydes and ketones. Chapter 5 branches out to dual catalytic systems by using CDs in combination with a Ni-catalyst to perform cross-couplings to achieve C-O and C-N bond formation. Additionally, photoluminescence quenching and transient absorption studies were undertaken to further examine the interaction between the CDs and Ni-catalyst, as well as gain insight into the CD states involved in the reaction. The data suggests the possible involvement of an energy transfer pathway, which would be the first example of CDs as an energy transfer catalyst in organic photocatalysis.
  • ItemOpen Access
    Investigating the atmospheric composition and climate response to mitigation: a methane emissions-driven approach
    Staniaszek, Zofia; Staniaszek, Zofia [0000-0002-1789-4368]
    Methane is the second most important greenhouse gas after carbon dioxide, and also plays a central role in the chemistry of the atmosphere. The combination of its shorter lifetime and higher effectiveness as a greenhouse gas makes it an attractive option for near-term mitigation of climate change. Methane is also a key tropospheric ozone precursor: ozone is a greenhouse gas, and acts as an air pollutant in the troposphere. Therefore, mitigation of methane has both climate and air quality benefits. A new configuration of the UK Earth System Model, UKESM1-ems, has been developed with an updated methane treatment. Methane emissions are input directly, rather than prescribing a global surface concentration. This thesis focuses on UKESM1-ems and the new capabilities it provides: a more process-based treatment of methane; simulating feedbacks in the methane cycle, and the ability to directly perturb methane emissions. When compared to the previous, concentration-driven model, UKESM1-ems simulates the methane distribution with a better correlation compared to observations, including an improved latitudinal distribution, interhemispheric gradient and vertical gradient. The observed trend in methane over time is also reproduced, combining the methane emissions inputs, online wetland emissions and online chemistry and transport to simulate the methane mixing ratio. The modelled absolute methane mixing ratio is lower than observations: this is likely due to an underestimate in methane emissions, within the current large uncertainty range for emissions. Experiments following different emissions pathways are explored using UKESM1-ems. Firstly, an idealised scenario where all anthropogenic methane emissions are removed instantaneously, to attribute the role of future anthropogenic methane. Methane declines to below pre-industrial levels within 12 years and global surface ozone decreases to levels seen in the 1970s. By 2050, 690,000 premature deaths per year and 1 degree of warming can be attributed to anthropogenic methane. Secondly, the same low-methane scenario is used, with perturbed nitrogen oxide (NOx) and carbon monoxide (CO) emissions, to investigate their impact on the atmospheric oxidising capacity and test the hydroxyl (OH) relationship to NOx and CO. The effect of methane on NOx is also explored. Decreased methane emissions perturb both the NO/NO2 ratio and the partitioning between NOx and reservoir species, leading to increased NOx in low-methane scenarios. Finally, a Global Methane Pledge scenario is simulated. This pledge aims to reduce methane emissions by 30% globally by 2030, compared to 2020 values. The new ability of UKESM1-ems to mask emissions from different countries is used to implement this scenario and study regional impacts. The global mean methane mixing ratio decreases by 13% compared to 2020 levels. The expected temperature benefit (0.2°C) following this scenario is not seen in this experiment - this signal is too small and is within the noise and interannual variability of UKESM1-ems. There are global benefits for air quality, with ozone concentrations and population exposure to ozone decreasing in all countries. Global Methane Pledge member countries, where emissions reductions take place, see greater local air quality benefits than non-member countries.
  • ItemEmbargo
    Developments in White Pigment Production Using Biocompatible Cellulose-Based Materials
    Zhang, Yating
    White pigments are widely used in various everyday products, including paints, food, and cosmetics. The primary choice for achieving white colouration in industries has traditionally been high refractive index inorganic materials, notably titanium dioxide (TiO2), which recently has raised significant concerns among consumers. Consequently, there is a growing need to explore safer and more biocompatible alternatives. Over the last few years, cellulose-based materials have gained interest in different industrial sectors as well as in fundamental research, basically due to its abundance, biocompatibility, biodegradability, and environmental friendliness. This thesis presents the development of three distinct methods for producing white pigments using cellulose-based nanomaterials. Firstly, an inkjet printing method based on the evaporation-induced phase separation of ethyl cellulose (EC) was developed. The resulting porous films demonstrate enhanced reflectance compared to TiO2 formulations with equivalent solid content. The inherent biocompatibility of cellulose materials, in conjunction with the simplicity and versatility of inkjet printing, renders this approach highly promising for advanced white colouration production in the printing industry. Secondly, a scalable and straightforward spray-freeze-drying method using natural cellulose nanofibrils (CNFs) was introduced. This approach enables the production of ultra-light and highly porous cellulose aerogel microparticles, thereby opening up the possibility of using natural cellulose material for white pigment particle production. Lastly, a novel method termed as "organic solvent mixing" was developed using cellulose microparticles (CMPs) with optimised scattering dimensions. This proposed method combines the concepts of evaporation-induced phase separation and solvent exchange to address the issue of densification upon water drying. The method successfully yields films with optical properties comparable to those obtained through previously complex techniques. Moreover, this method offers notable advantages, including ease of operation, straightforward implementation, reduced toxicity, and lower cost.
  • ItemEmbargo
    Understanding the Limits of Lithium-Air Batteries – NMR and Thermodynamic Studies
    Ellison, James; Ellison, James [0000-0002-4578-5804]
    Lithium-air batteries promise to deliver exceptionally high energy density while only using common materials, such as carbon, in their cathode structure. To do this, they oxidise a metallic lithium anode to release Li+ ions, which combine with O22- ions, produced from the reduction of atmospheric oxygen. However, such batteries are yet to be commercialised due to problems in cell operation, such as their high overpotentials, poor rate capabilities and, most critically, poor cell lifetimes. This work sets out to quantify the realistic expectations that should be had of a lithium-air battery should they be realised and the cell geometry and support systems such a battery would likely need. It goes on to discuss the theory of the chemical structural motifs that promising new solvents would likely have. To aid in studying the breakdown products formed in the lithium air batteries, which limit their lifetime, operando 17O nuclear magnetic resonance was developed. This technique can non-destructively and in real time track and quantify the formation and removal of all common breakdown products, this information challenging to access by any other technique. Operando diffraction can in principle access it, however it typically requires a synchrotron and if often limits to crystalline products. Operando Raman is typically surface sensitive and chemical tests are destructive. Here 17O NMR is used to investigate the relative contributions of singlet oxygen, chemical and electrochemical breakdown to the observed decomposition products in the cell. To support this work Gaussian Process regression was utilized. It was found that Gaussian processes can also be used to denoise NMR data, matching or outperforming current denoising methods in many cases. Finally, a potential additive to the electrolyte, lithium iodide, is discussed. Lithium iodide had previously been proposed to reduce the charge overpotential and switch the discharge product in the battery to LiOH, thereby avoiding many of the corrosive species formed in the cell. Here, the thermodynamics and kinetics associated with this reaction are explored, and the range of conditions where this reaction is possible is discussed.
  • ItemOpen Access
    Characterisation of neurodegenerative diseases derived protein aggregates using improved single-molecule pull-down
    Zhang, Yu
    The development of therapies and diagnostic methods for major neurodegenerative diseases (ND), including Alzheimer’s (AD) and Parkinson’s disease (PD), remains challenging due to our limited understanding of their pathology. Since more and more reports have revealed the pathogenic significance of protein aggregates in ND, this PhD thesis aims to answer 2 key questions in the field: How to characterise these protein species in the complex human samples and How to find the key species associated with these diseases. To address these questions, this thesis begins by establishing an improved single-molecule pull-down (SiMPull) assay, which was used to characterise soluble α-Synuclein (αS) and Amyloid-β (Aβ) aggregates in serum from patients with PD and controls. Our findings reveal that the ratio of detected aggregates (αS/(Aβ+αS)) and morphological information of αS aggregates can be differentiated between PD samples and controls. We further improved the assay's surface coating using Rain-X, a common household chemical, for simpler and more specific detections. With this improved assay, we explored the diagnostic potential of an inflammasome-related protein, apoptosis-associated speck-like protein containing a CARD (ASC specks) protein, characterised αS and Aβ aggregates in saliva, assessed the binding affinity of rationally designed anti-Islet amyloid polypeptide (IAPP) antibodies, and tested ultra-sensitive single-molecule detection of protein aggregates in human serum. Additionally, for the first time, we demonstrated that small soluble αS can co-aggregate with Aβ and tau in human serum, highlighting that protein aggregation can occur between different protein species in a soluble state, while previous reports mainly focus on larger insoluble aggregates. In summary, we established a single-molecule approach to characterise protein aggregates in human samples and identified several key protein aggregates in samples from patients with PD. This research platform can be extended to other systems and samples, enabling the acquisition of more detailed single-molecule information on target species. As such, it serves as a fundamental research tool in the study of neurodegenerative diseases.
  • ItemOpen Access
    On Critical Care Data and Machine Learning Loss Function Landscapes
    Cafolla, Conor Thomas; Cafolla, Conor [0000-0003-2021-974X]
    Intensive Care Units (ICUs) are constantly under strain, with many vulnerable people needing urgent critical care. Often doctors do not have the capacity to accommodate everyone, and so it is important that patients are discharged when it is safe to do so. However, discharging a patient at the wrong time may result in the patient being readmitted or simply not surviving; both of these cases are highly undesirable. Therefore, it is useful for there to be an understanding of what factors contribute most to the mortality rate of patients in intensive care units. In the present work, machine learning models are trained to predict the mortality of a given patient with certain measurements. Neural networks with one hidden layer and two outcomes (alive or deceased) are used in these models, and local minima of the neural network loss functions are found using basin-hopping methods implemented with the open source software GMIN. In this way, the landscape of the loss function defined by the neural network model can be explored. Area Under the Curve (AUC) values were used to evaluate these models. Two databases, MIMIC III and Amsterdam UMC db, are compared. There are many possible calculations to perform, and initially time-series for single variables and pairs of variables are used as inputs to the neural network. From MIMIC III, Glasgow Coma Scale (GCS) and Blood Urea Nitrogen (BUN) perform well, with AUCs just below 0.8 on their own, and an AUC above 0.8 together. From Amsterdam UMC db, Blood Pressure (BP) measurements perform well, with AUCs around 0.8. Generally the data from Amsterdam UMC db appears to outperform MIMIC III. The effect of using a model trained on one time window and evaluated on different time windows is also investigated, and we find that the AUC value decreases but not substantially for most clinical variables, suggesting the most recent data is the most useful for mortality prediction. There is a notable exception in Respiration Rate, where it is found that data from earlier times may actually provide more prognostic value than the most recent measurements. A permutational shuffling analysis is performed, which reveals patterns in the ways the data is organised, and sheds light on some innate properties of the data. The data from the two ICU databases are then applied to another model, where inputs to the neural network are the worst values of a set of pre-chosen clinical variables, inspired by a score used elsewhere in the medical prognosis picture (APACHE II). The AUCs obtained in this way are generally better than for the time-series data above, with AUCs reaching just under 0.8 for MIMIC III and 0.85 for Amsterdam UMC db. Synthetic spiral data is created to test some new machine learning methods, including an ensemble-like method where minima from the loss function landscape are combined in a process called Machine Learning Superposition (MLSUP). Minima are selected to maximise the diversity between them; pairs of minima are identified as suitable candidates for MLSUP by their misclassification index and their contributions to heat capacity peaks. We find that MLSUP outperforms a single neural network model for a larger neural network architectures, but is not as useful for a smaller ones. MLSUP is also applied to the ICU databases described above, however we find that there is little improvement in the AUCs obtained by the single neural networks. The synthetic data is further used to explore two new landscapes: the landscape defined by a loss function designed to closely resemble the AUC function, and a landscape where narrower minima are penalised in energy (Sharpness Aware Minimisation, SAM). In both cases, AUCs from synthetic data and the real data are comparable to the more conventional ``cross entropy'' loss function, but do not offer much improvement. Coupled with the fact that these new loss functions have higher order complexity, and hence take longer to evaluate, it is concluded that these landscapes are not practically useful. However they still offer great insight into the nature of machine learning models and their landscapes. This thesis concludes with an overview of the work completed, and some closing thoughts on the use of artificial intelligence (AI) in the healthcare setting, discussing how it should be used while adhering to some dangers it could present.
  • ItemEmbargo
    Optically Active Metamaterials Made of Plasmonic Nanoparticles and Chitin Nanocrystals
    Lu, Zihao
    Engineering materials from the nanoscale to the macroscopic scale in order to achieve strong optical activity is important for many scientific disciplines from chemistry to material science and physics. This thesis work aimed to develop by self-assembly optically active metamaterials composed of plasmonic nanoparticles embedded in a helicoidal architecture made of chitin nanocrystals, and to understand how the material interact with light to generate macroscopic circular dichroism. The material fabrication was based on a unique wet-chemistry synthetic pathway to incorporate achiral plasmonic nanoparticles, of silver and gold, into an optically inactive chitin-nanocrystal template. Spectroscopic characterisation of the composite materials, wherein plasmonic nanoparticles appear as a disordered ensemble, showed intense and well-distinguished bisignate circular dichroism spectroscopic line shapes in the vicinity of plasmonic resonance. Tuneable spectral features of such chiroptical properties were also realized, through the control of particle number density, size, and type. Possibilities of macroscopic optical activity arising from a seemingly completely disordered ensemble were assessed theoretically, with supportive evidence of numerical modelling and calculations based on plasmonic ensembles extracted from experimental chiral composites. These findings not only expanded the experimental accessibility of optically active plasmonic metamaterials, but also theoretically demonstrated that macroscopic optical activity could arise from apparently disordered systems, thereby promoting the understanding of chiral light-matter interactions.
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    NMR Studies of Aqueous-Electrolyte Ion Sorption in Energy Storage Materials
    Lyu, Dongxun
    The ion adsorption at solid–water interfaces is the key underlying process for electrochemical energy storage, electrochemical separations, and electrocatalytic applications involving aqueous electrolytes. However, the impact of charged species including hydronium (H3O+) and hydroxide (OH-) on the ion adsorption and surface charge distribution are poorly understood, hindering the design and optimisation of devices with enhanced performance. It is challenging to achieve a microscopic level characterisation of mixed ion adsorption at the electrode-electrolyte interface due to the complex ion dynamics and the disordered structures of the porous carbon electrodes. In this work, nuclear magnetic resonance (NMR) spectroscopy has been applied for the study of ion dynamics and charge storage mechanisms of aqueous supercapacitors. The unique power of NMR spectroscopy for the characterisation of electrode-electrolyte interfaces is that it can differentiate ions adsorbed inside the pores apart from the ions in the bulk counterpart. The element selective feature of NMR spectroscopy allows the separate observation of different chemical species in the electrolyte. Specifically, the cation and anion adsorption of LiTFSI (lithium bis(trifluoromethylsulphonyl)imide) aqueous electrolyte in activated carbon, a common electrode material for supercapacitors, are quantified by 7Li and 19F NMR. Apart from quantifying the electrolyte ions, the H3O+ uptake by activated carbon is also measured through pH measurements, gathering a microscopic understanding of the mixed-ion adsorption at the carbon-electrolyte interfaces. Remarkably, the data suggests that H3O+ plays a key role in maintaining the local charge neutrality within carbon nanopores due to the surface basicity of the activated carbon. To explore this further, the carbon surface was modified to be close to neutral through chemical oxidation. By comparison with the pristine activated carbon, the functionalized carbon exhibit changes in the ion adsorption, leading to enhanced electrochemical capacitance of the modified carbon materials. To gain insights into the enhanced capacitance and the role of H3O+ in the charge storage mechanisms, operando NMR measurements were performed as a function of the electrolyte pH. These findings highlight the significance of H3O+ in electrochemical systems involving aqueous electrolytes, guiding the design and development in the fields of energy storage, colloidal systems, multiphase catalysis, and beyond.
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    The photoredox-catalysed generation and reaction of alkyl α-amino radicals for α-tertiary amine synthesis
    Harris, Georgia
    This thesis comprises three projects focused on the synthesis of α-trialkyl-α-tertiary amines, exploiting photo-reductively generated alkyl α-amino radicals as intermediates. The first project describes the development of a multicomponent method for the photoredox-mediated synthesis of α-trialkyl α-tertiary amines (ATAs). This highly hindered scaffold is generated from primary amine, alkyl ketone and alkene feedstocks, via the single electron reduction of an in-situ formed imine, followed by 1,2-radical addition. The transformation was found to be broad-scoping with respect to all three components and its utility demonstrated in the single step synthesis of the blockbuster drug fingolimod. Mechanistic studies enabled a catalytic cycle to be proposed and highlighted the importance of a key 1,5-HAT process in driving the forward reaction. Subsequently, it was discovered that by employing a chiral amine as a starting material, stereochemical information could be transferred to the newly formed ATA. Further investigation highlighted phenylglycinol methyl ether as the optimal chiral amine transfer (CAT) reagent, which enabled the generation of a variety of enantioenriched ATAs. Computational studies supported a stereochemical model in which an intramolecular hydrogen-bond rigidifies the transition state of the enantiodetermining step, allowing an efficient relay of chiral information to the fully substituted centre. The final project describes efforts toward the total synthesis of the α-trialkyl-α-tertiary amine containing marine alkaloid, (±)-cylindricine C. Whilst attempts to incorporate the previously outlined methodology proved challenging, the synthesis of unnatural isomer, (±) 2,13-di-epi-cylindricine C, was achieved in an overall yield of 5.5% over 12 linear steps.
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    Artificial Transmembrane Signalling and Cooperativity in H-Bonded Networks
    Trevisan, Lucia; Trevisan, Lucia [0000-0002-9745-1275]
    Supramolecular chemistry is the study of non-covalent interactions and applications in the assembly of complex systems where multiple components interact with each other. This thesis addresses these two areas, with a first project on artificial transmembrane signalling, and a second on cooperativity in hydrogen bonded networks. Cells rely on complex signal cascades where proteins are responsible for the transmission and amplification of chemical signals across membranes. While numerous examples of biomimetic synthetic transport systems are reported in the literature, this first project focused on the development of membrane-anchored molecules capable of communication across the lipid bilayer of vesicles by exploiting an artificial transduction mechanism. The signalling relies on molecular motion across the bilayer which is controlled by switching the polarity of two different head-groups. A new external redox input signal based on ascorbic acid was successfully employed to trigger membrane translocation and signal amplification via activation of an internal catalytic process. Hydrogen bonds play a major role in determining structure and function in biology, chemistry and materials science. A few examples in the literature report cooperativity in hydrogen bonding networks involving hydroxyl groups. However, the magnitude of this effect is difficult to quantify experimentally. The aim of the second project was to develop a systematic structure-activity approach to the quantification of cooperativity. A series of phenol oligomers containing intramolecular hydrogen bonds was designed to make quantitative measurements of the effects of the intramolecular interactions on the strengths of intermolecular hydrogen bonding interactions between the terminal phenol hydrogen bond donor and hydrogen bond acceptors. Intramolecular hydrogen bonds strengthened the intermolecular hydrogen bond by up to 14 kJ mol⁻¹. The experimental results were corroborated by computational studies and a simple model for cooperativity in hydrogen bonded networks was proposed. The formation of each intramolecular hydrogen bond in the chain increases the polarity of the next hydrogen bond donor by 33%. A series of bisphenols variously substituted in the *para* position allowed to quantify the substituent effect. Addition of a *para*-nitro substituent to the phenol that acts as the intramolecular hydrogen bond donor strengthened the intermolecular hydrogen bond with the other phenol by 16 kJ mol⁻¹, and the effects of different substituents correlate with the corresponding Hammett parameters. Finally, a study of the hydrogen bonding donor properties of a series of benzyl alcohols intramolecularly hydrogen bonded to a phenol showed that changing both the size of the ring involved in the intramolecular hydrogen bond and the nature of the hydroxyl donor on the end of the chain had no significant impact on the magnitude of the cooperative effect.
  • ItemEmbargo
    Non-Covalent Interactions in Water Using Synthetic and Biological Receptors
    Tobajas Curiel, Gloria
    This thesis deals with two different topics, and they will be introduced and discussed separately. The first part of this thesis discusses the factors that govern the nature of aromatic interactions in water. The rational design and interpretation of the supramolecular behaviour of aromatic interactions in aqueous solution represents a major challenge. First, aromatic interaction energies are strongly affected by the geometry of the interaction. Second, molecular recognition in water involves contributions due to polar functional group interactions, partial desolvation of polar and non-polar surfaces and changes in conformational flexibility. Here, conformationally well-defined supramolecular complexes formed between four different calix[4]pyrrole receptors and thirty different pyridine N-oxide guests provide a platform for disentangling the factors that govern aromatic interactions in water and in chloroform. The three-dimensional structures of the complexes are fixed by four H-bonding interactions between the pyrrole donors at the bottom of the receptor and the N-oxide acceptor on the guest, locking the geometrical arrangement of interacting functional groups in the binding pocket at the other end of the receptor, so that an aromatic ring on the guest makes two edge-to-face and two stacking interactions with the four aromatic side-walls of the receptor. The thermodynamic contribution of these aromatic interactions to the overall stability of the complex has been quantified by chemical double mutant cycles using isothermal titration calorimetry and 1H NMR competition experiments. In chloroform, the free energy measurements of the aromatic interactions show that electron-withdrawing substituents increase the strength of the interactions by a factor of up to 20, highlighting the role of electrostatics in stabilising both the edge-to-face and stacking interactions. The effect of introducing a heteroatom depends on where it sits with respect to the aromatic side-walls of the cavity. In water, aromatic interactions are all significantly more favourable than in chloroform, and the free energy contributions measured in water have been rationalised by comparison with the magnitude of the corresponding measurements in chloroform. The enhanced interaction energies observed in water are due to entropic contributions associated with the desolvation of hydrophobic surfaces on the substituents. There are flexible alkyl chains that line the open end of the binding site and assist the desolvation of the non-polar π-surfaces of the guests, but at the same time allow water to interact with polar H-bond acceptor sites. Moreover, water can access cracks in the walls of receptor binding site without significantly affecting the geometry of the aromatic interactions. The results highlight the complexity of the solvation processes that govern molecular recognition in water. The second part of this thesis discusses attempts to use protein-templated dynamic combinatorial chemistry to speed up the structural optimisation of multivalent molecular probes that bind amyloid fibrillar aggregates. Building blocks consisting of amyloid-binding probes and linkers have been synthesised and attempts at equilibrating a dynamic library of exchanging building blocks between monovalent and divalent constructs under suitable conditions for templating have been made. However, the poor solubility of the library members prohibited the continuation of the study.
  • ItemEmbargo
    To the Frontier of RNA Glycosylation: Novel Insights from Metabolic Labelling
    Hazemi, Madoka Eurika; Hazemi, Madoka [0000-0003-4690-1114]
    The recent discovery of glycosylated RNA (glycoRNA) has redefined our comprehension of the glyco-conjugate universe, uncovering an unprecedented link between glycans and RNA. The pioneering exploration of this novel biopolymer was driven by the metabolic carbohydrate reporter, per-O-acetylated N-azidomannosamine (Ac4ManNAz), that emphasises the unknown potential of the field of glycobiology and RNA modification. However, the nascent field raises numerous questions concerning the molecule's authenticity, structure, and intricate biological roles, warranting further investigation. This thesis systematically expands upon this initial finding by harnessing additional metabolic probes, including per-O-acetylated N-azidoacetylglucosamine (Ac4GlcNAz), N-azidoacetylgalactosamine (Ac4GalNAz), and 6-azidofucose (Ac4FucAz). Our successful integration of Ac4GlcNAz and Ac4GalNaz into intracellular RNA has added another dimension to our knowledge of RNA glycosylation. We further developed an effective pulldown and release method, which ensures a high-confidence detection and analysis of our azido sugar-RNA. We uncover new dimensions of RNA glycosylation, including a peculiar lack of overlap between RNA and sugar probe signal, as well an unusual resilience of sugar-RNA bonds to enzymatic cleavage. This led us to unravel a novel non-enzymatic sugar-RNA linkages, adding a new layer of intrigue to the glycoRNA narrative. Through meticulous sequencing and a novel annotation workflow, we have used high-confidence transcript profiling to identify distinct sets corresponding to each sugar probe, thereby highlighting the diversity of these sugar-RNA species. Collectively, this thesis provides a robust and critical understanding of the largely unexplored field of glycoRNA. We present findings that reinforce the concept of RNA glycosylation, while also revealing major uncertainties surrounding the nature and authenticity of glycoRNA. This work thus serves as a foundation for future research in this nascent field, underscoring the importance of decisively unravelling the linkage structure of glycoRNA and maintaining rigorous scrutiny in the pursuit of early-stage scientific investigations of this field.
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    Automated Synthesis of Duplex-Forming Recognition-Encoded Oligomers
    Dhiman, Mohit
    The functional properties of biological polymers are encoded by the sequence of monomer building blocks. Synthetic polymers made from sequences of different monomer building blocks would allow exploration of chemical space for new structures with functional properties in non- aqueous media. Previously in the Hunter group, recognition-encoded melamine oligomers (REMOs) composed of alternating piperazine and triazine units, equipped with phenol and phosphine oxide hydrogen-bonding groups, have been shown to form sequence- and length- selective duplexes and are a promising candidate for such a system. An automated solid-phase synthesis (SPS) route was optimised to yield high-purity REMOs with precise sequence control. Loaded Wang resin was reacted alternatingly with piperazine and dichlorotriazine monomers, appended with phenol donor (D) or phosphine-oxide acceptor (A) recognition units, under microwave conditions. Major side products were identified using liquid chromatography-mass spectrometry (LCMS) and the undesired reactions were supressed by the choice of resin, solvent and coupling conditions. A 42-mer REMO was synthesised and purified by high-performance liquid chromatography, displaying the scope of the SPS approach. The tetramer sequence DADA, appended with terminal azide and alkyne groups, was synthesised via SPS. Duplex formation in dichloromethane was investigated by using the copper-catalysed azide-alkyne cycloaddition (CuAAC) reaction to trap the species present. LCMS was used to identify the macrocyclic self-complementary duplex as a major product. This trapping approach, as well as 31P NMR denaturation and Förster resonance energy transfer, were used to investigate duplex formation by complementary homo-oligomers A6•D6 and A12•D12. CuAAC trapping was also used to demonstrate sequence selective duplex formation in mixed sequence libraries of 6-mers prepared by SPS. In the Hunter group, an oligotriazole trimer equipped with benzoic acid and phenol recognition units was previously replicated via covalent template-directed synthesis, but folding of the backbone led to significant side-reactions. Here, attempts were made towards a new, more rigid oligotriazole replicator. The backbone was assembled via CuAAC reactions between dialkyne bearing phenol or benzoic acid monomers and an aryl diazide linker. Attempts to replicate a benzoic acid trimer via covalent template-directed synthesis yielded none of the desired trimer copy, and base-pair formation by esterification of carboxylic acid units on the template proved more challenging on this backbone, which limited the ultimate utility of this approach.
  • ItemOpen Access
    A photochemical method for the detection of cytosine modifications in DNA
    Mortishire-Smith, Benjamin; Mortishire-Smith, Benjamin [0000-0002-0733-1676]
    Covalent modifications to DNA bases represent an important form of epigenetic information which contributes to the regulation of gene expression in many cellular processes throughout development and differentiation. Aberrant modification patterns can also signal or drive the emergence of genetic disease states. Techniques that enable non-canonical bases to be detected and mapped throughout the genome are therefore of academic and diagnostic interest. To this end, a toolbox of chemical and enzymatic methods has been developed to selectively manipulate the unique functionalities of covalent DNA modifications; however, bisulfite sequencing, the benchmark technique for the detection of 5-methylcytosine and its oxidised derivatives, is highly destructive and greatly reduces the sequence complexity of DNA samples. These limitations are being addressed through the development of bisulfite-free sequencing methods that seek to detect modified DNA bases under non-degrading conditions. This thesis describes the discovery and development of a photochemical treatment that alters the hydrogen bonding pattern of 5-carboxycytosine to a uracil analogue. The photochemical conversion is selective for the unique carboxylate functionality of 5-carboxycytosine over other canonical and modified bases. The reaction was first identified in experiments on nucleoside monomers, before being optimised in model oligonucleotides and genomic DNA fragments. The transformation was investigated using experimental, physical and computational techniques and a plausible mechanistic pathway was proposed. The conversion successfully enables the detection of 5-carboxycytosine within a modified oligonucleotide and, in conjunction with enzymatic oxidation by TET, the detection of 5-methylcytosine in a model modified genome via next-generation sequencing. The mild irradiation with visible light in aqueous conditions is well-suited to the manipulation of complex biological macromolecules such as nucleic acids, and this reaction represents the first example of a photochemical method for profiling cytosine modifications at single-base resolution.
  • ItemOpen Access
    Investigating the Structure and Physical Stability of Glp1-Like Peptides Using Mass Spectrometry
    Gibson, Katherine
    Working with fibrillation prone peptides is an adventure, to say the least, but harnessing their therapeutic potential is a challenge worth facing. On comparison to small molecules, peptides provide improved efficacy coupled with reduced toxicity; the difficulty lies in their pharmaceutical developability, and ensuring their fidelity throughout a defined shelf-life. In addition to chemical degradation, physical aggregation of peptides into oligomers and fibrils can compromise the safety, efficacy and bioavailability of peptide therapeutics. It is imperative that we understand these processes, not only to mitigate for them, but so we can develop a set of measurable criteria which we use to confidently predict fibrillation propensity. The research conducted here focuses on Glucagon-like Peptide 1 (GLP-1-like) scaffolds. Translation of GLP-1 itself as a therapeutic was burdened with poor bioavailability and metabolic instability. Thus, literature precedent describes the search for alternatives. These peptide agonists are particularly attractive as treatments for Type II Diabetes Mellitus (TIIDM) as they offer glucose dependent signalling with subsequent insulin production and glucose regulation. The majority of GLP-1-like peptides are currently still dosed via liquid injection but explaining the origin of batch-to-batch variation in physical stability of these drug substances is difficult and not yet fully understood In this work, two model peptides were studied as each showed batch-to-batch variation in their physical stability. The first system looked at two batches of a lipidated GLP-1-like peptide (RS19 and RS57), that had shown batch dependent fibrillation propensity throughout early stage development despite being manufactured with no process changes. The second system used four batches of C-terminally amidated GLP-1(7-37) analogue (GLP-1-Am) that had been synthesised in-house where each batch was a result of a different purification pathway. Each of these batches fibrillated differently to each other, as well as to a commercial batch of comparable purity. Mass spectrometry (MS) is an increasingly important tool in biopharmaceutical characterisation, with great potential for understanding the conformational bias of inherently dynamic systems like fibrillating peptides. Two complementary structure based MS techniques are travelling wave ion mobility MS (TWIMS) and hydrogen-deuterium exchange MS (HDX-MS), and the two peptide systems in this work provided different case studies with which structural mass spectrometry could be used to investigate the origins of batch variation in physical stability. TWIMS was used to investigate the five GLP-1-Am batches, and the two batches of lipidated GLP-1-like peptide to see if process related changes manifested as differences in conformation and oligomer distribution. Experiments were completed on samples that were freshly prepared or stressed to see if there was a correlation to physical stability. HDX-MS was used to investigate GLP-1-Am batches only. In both systems, differences were observed between batches however issues with reproducibility meant that determining any unambiguous links with fibrillation propensity was not possible. Two current state-of-the art instruments, cyclic ion mobility spectrometry (cIMS) and millisecond HDX-MS were also used in this work to improve the resolution with which we study therapeutically relevant peptides. cIMS was successfully applied to the lipidated GLP-1-like peptide system to detect and identify the location of isoAsp residues, based on conformational differences when Asp or isoAsp were present. As an example of a dynamic system where fast amide proton exchange was expected, millisecond HDX-MS was used in this work, and showed differences in the deuteration rate of the five GLP-1-Am batches for experiments acquired in the same dataset. However, the reproducibility of results between days remained a significant challenge. Despite this, the analysis presented here highlights the potential of developing these techniques.
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    A Nuclear Magnetic Resonance Approach Towards Understanding Surface Modification and its Influence on Ni-rich Layered Oxides in Li-ion Batteries
    Chen, Richard Lin Bin
    Rechargeable lithium-ion batteries (LIBs) are crucial for transitioning towards a carbon neutral society. Nickel-rich layered oxides are the state-of-the-art material of choice, offering high practical capacities. However, accessing this high capacity induces structural degradation and parasitic electrolyte reactions at the surface. One strategy to mitigate this problem is surface modification with coating materials like Al2O3 – cycling life is extended but the fundamental reasons behind the protection are not well understood. Therefore, the aim of the thesis is to identify the characteristics of coatings that contribute to this protection with polycrystalline LiNi0.8Mn0.1Co0.1O2 (NMC811) coated with Al2O3 by atomic layer deposition (ALD) being studied. Varying the number of ALD cycles allows investigation of the influence of coating thickness (sub-nm differences) on surface degradation. Coupled with electrochemical cycling, it is revealed that an optimal coating thickness exists, balancing initial capacity with retentions. X-ray spectroscopy and diffraction confirm surface degradation is reduced with coating on the NMC811. Meanwhile, 27Al solid state (ss)NMR indicates that the chemical evolution of the coating is not correlated with coating thicknesses. Detailed structural and chemical evolution of the Al2O3 coating at different stages of the electrochemical cycling process is tracked using a multinuclear NMR approach. Probing dipolar interactions between 27Al and 1H/19F/7Li shows the coating is susceptible to reactions involving acidic and protic species from the electrolyte, but no lithiated alumina phase is formed. The changes are concomitant with stabilisation of the surface oxygen electronic structure by the coating, supported by less electrolyte degradation. Finally, the heat treatment and performance of Al2O3 coated NMC811 is presented to provide insights into the effect of a range of coating structures. 27Al ssNMR shows that heating induces Al doping into the bulk NMC structure in parallel to crystallisation of the initial amorphous phase. However, the electrochemical cycling suggests a conformal coating performs better than these modifications. From these studies, it is clear that Al2O3 surface modification have a chemical role in passivating the NMC811 surface, with strong implications for material engineering of long-lasting and high energy density LIBs.