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  • ItemEmbargo
    Towards unravelling the mechanism and function of exoelectrogenesis in cyanobacteria
    Wroe, Evan; Wroe, Evan [0009-0001-8766-9772]
    Photosynthetic microorganisms, including algae and cyanobacteria, are able to export electrons derived from the photosynthetic electPhotosynthetic microorganisms, including algae and cyanobacteria, are able to export electrons derived from the photosynthetic electron transport chain to the cell exterior, in a phenomenon known as exoelectrogenesis. This phenomenon has implications for an emerging field of biotechnology which seeks to employ photosynthetic biocatalysts to produce solar electricity, chemicals and fuels (bioelectrochemical systems). However, both the underlying mechanism by which exoelectrogenesis occurs and the function that exoelectrogenesis plays in photomicrobial communities are poorly understood. This gap in understanding represents a barrier to the intelligent design of biophotoelectrochemical systems, hindering their development into a practical technology. At a fundamental level this also represents a gap in the understanding of the biochemical mechanisms of photosynthesis, one of the most important and widespread biochemical pathways in life. The cyanobacteria *Synechocystis* sp. PCC 6803 is used throughout this work as a model organism for the study of photosynthesis and exoelectrogenesis. The research presented in this thesis aims to deepen understanding of the mechanism and function of exoelectrogenesis in cyanobacteria by generating and employing a workflow for identifying the endogenous redox mediator, developing a platform for *in operando* electrochemistry and microscopy, and using it to study the dynamic spatial variations in biofilm behaviour during exoelectrogenesis. First, the question of the mechanism of exoelectrogenesis is examined. Exoelectrogenesis is known to occur in cyanobacteria through the secretion of a small, soluble redox mediator, with the identity of this mediator having remained unknown for many years. Recently, a proposal for the identity of this mediator was put forward: this work begins by critically assessing the veracity of this proposal and testing whether it holds true under standard conditions. Finding that it does not, a workflow is developed for the identification of the mediator. Through this workflow, spectroscopic and electrochemical assays are applied to mediator-enriched samples, identifying unique features that may be attributable to mediator-like species. Chemical species composing these samples are separated out by high performance liquid chromatography, and putative features annotated by liquid chromatography-mass spectrometry with post-processing metabolomics analysis. The workflow identifies several plausible candidates in the cyanobacterial exometabolome which are increased in concentration under light exposure, providing solid movement towards elucidating the identity of the endogenous mediator of exoelectrogenesis. Next, insights are sought into the mechanism and function of exoelectrogenesis at a multicellular level. The current toolkit used in cyanobacterial exoelectrogenesis research predominantly employs bulk techniques to examine the process; what these bulk techniques gain in signal intensity, they sacrifice in dynamic, spatial information and understanding of diversity and heterogeneity within the multicellular community (the biofilm). Exoelectrogenesis is a single-cell behaviour that has a function within a multi-cellular community, so insights into both mechanism and function are to be gained by the study of single cells within the population. To this end, new tools are needed. A platform for *in operando* electrochemical microscopy is developed, to facilitate microscopic imaging during (photo)electrochemical measurements of the cyanobacterial biofilm. Similar techniques are increasingly being used to study other microbial community behaviours, however they have yet to be rigorously applied to the study of cyanobacterial exoelectrogenesis. This platform is optimised for both electrochemical and microscopic measurements, and further developed to apply spatially-patterned stimuli (voltage and light). A series of screening experiments are conducted to identify suitable fluorescence probes for biochemical parameters, as well as the most suitable chassis organism for microscopic studies. During this screening, the Nernstian membrane potential reporter Thioflavin T (ThT) is identified as a compatible and favourable probe for use in this system. In recent years, membrane potential reporters (including ThT) have been used to inform on many aspects of microbial community behaviour, including exoelectrogenesis in heterotrophic bacteria. The connection between membrane potential (Vm) and exoelectrogenesis in cyanobacteria has so far not been studied, but it is hypothesised that exoelectrogenesis would influence Vm and so the application of a probe for membrane potential could act as a useful proxy for exoelectrogenic activity. By conducting photoelectrochemistry of cyanobacterial biofilms on the microscope, with ThT as a Vm reporter, a robust, reproducible Vm response is observed during exoelectrogenesis. This response is tested under a series of relevant conditions, including inhibiting and increasing exoelectrogenesis. Methods for investigating the heterogeneity across the biofilm in three dimensions are developed, and a diversity of Vm responses demonstrated. Finally, by application of precision light stimulation, it is shown that Vm changes propagate from cells experiencing high light to those in darkness, presenting a route toward understanding why cyanobacteria export photo-excited electrons. Overall, this work presents movements towards identifying the mediator of exoelectrogenesis: a recent proposal is ruled out, several assays for the mediator identified and a system for isolating and identifying the mediator developed, which, with further work, can reveal its identity. A robust and flexible platform for *in operando* electrochemistry and microscopy is developed, optimised and successfully screened for use with appropriate fluorescence probes, and is shown to be well suited for further work, perhaps with other microorganisms or microscopes. Finally, membrane potential across the cyanobacterial biofilm is studied during exoelectrogenesis, showing that membrane potential changes occur and can be monitored during electron export, at the single- and multi-cellular level, and that changes in membrane potential are propagated across the biofilm in response to precise illumination. ron transport chain to the cell exterior, in a phenomenon known as exoelectrogenesis. This phenomenon has implications for an emerging field of biotechnology which seeks to employ photosynthetic biocatalysts to produce solar electricity, chemicals and fuels (bioelectrochemical systems). However, both the underlying mechanism by which exoelectrogenesis occurs and the function that exoelectrogenesis plays in photomicrobial communities are poorly understood. This gap in understanding represents a barrier to the intelligent design of biophotoelectrochemical systems, hindering their development into a practical technology. At a fundamental level this also represents a gap in the understanding of the biochemical mechanisms of photosynthesis, one of the most important and widespread biochemical pathways in life. The cyanobacteria Synechocystis sp. PCC 6803 is used throughout this work as a model organism for the study of photosynthesis and exoelectrogenesis. The research presented in this thesis aims to deepen understanding of the mechanism and function of exoelectrogenesis in cyanobacteria by generating and employing a workflow for identifying the endogenous redox mediator, developing a platform for in operando electrochemistry and microscopy, and using it to study the dynamic spatial variations in biofilm behaviour during exoelectrogenesis. First, the question of the mechanism of exoelectrogenesis is examined. Exoelectrogenesis is known to occur in cyanobacteria through the secretion of a small, soluble redox mediator, with the identity of this mediator having remained unknown for many years. Recently, a proposal for the identity of this mediator was put forward: this work begins by critically assessing the veracity of this proposal and testing whether it holds true under standard conditions. Finding that it does not, a workflow is developed for the identification of the mediator. Through this workflow, spectroscopic and electrochemical assays are applied to mediator-enriched samples, identifying unique features that may be attributable to mediator-like species. Chemical species composing these samples are separated out by high performance liquid chromatography, and putative features annotated by liquid chromatography-mass spectrometry with post-processing metabolomics analysis. The workflow identifies several plausible candidates in the cyanobacterial exometabolome which are increased in concentration under light exposure, providing solid movement towards elucidating the identity of the endogenous mediator of exoelectrogenesis. Next, insights are sought into the mechanism and function of exoelectrogenesis at a multicellular level. The current toolkit used in cyanobacterial exoelectrogenesis research predominantly employs bulk techniques to examine the process; what these bulk techniques gain in signal intensity, they sacrifice in dynamic, spatial information and understanding of diversity and heterogeneity within the multicellular community (the biofilm). Exoelectrogenesis is a single-cell behaviour that has a function within a multi-cellular community, so insights into both mechanism and function are to be gained by the study of single cells within the population. To this end, new tools are needed. A platform for in operando electrochemical microscopy is developed, to facilitate microscopic imaging during (photo)electrochemical measurements of the cyanobacterial biofilm. Similar techniques are increasingly being used to study other microbial community behaviours, however they have yet to be rigorously applied to the study of cyanobacterial exoelectrogenesis. This platform is optimised for both electrochemical and microscopic measurements, and further developed to apply spatially-patterned stimuli (voltage and light). A series of screening experiments are conducted to identify suitable fluorescence probes for biochemical parameters, as well as the most suitable chassis organism for microscopic studies. During this screening, the Nernstian membrane potential reporter Thioflavin T (ThT) is identified as a compatible and favourable probe for use in this system. In recent years, membrane potential reporters (including ThT) have been used to inform on many aspects of microbial community behaviour, including exoelectrogenesis in heterotrophic bacteria. The connection between membrane potential (Vm) and exoelectrogenesis in cyanobacteria has so far not been studied, but it is hypothesised that exoelectrogenesis would influence Vm and so the application of a probe for membrane potential could act as a useful proxy for exoelectrogenic activity. By conducting photoelectrochemistry of cyanobacterial biofilms on the microscope, with ThT as a Vm reporter, a robust, reproducible Vm response is observed during exoelectrogenesis. This response is tested under a series of relevant conditions, including inhibiting and increasing exoelectrogenesis. Methods for investigating the heterogeneity across the biofilm in three dimensions are developed, and a diversity of Vm responses demonstrated. Finally, by application of precision light stimulation, it is shown that Vm changes propagate from cells experiencing high light to those in darkness, presenting a route toward understanding why cyanobacteria export photo-excited electrons. Overall, this work presents movements towards identifying the mediator of exoelectrogenesis: a recent proposal is ruled out, several assays for the mediator identified and a system for isolating and identifying the mediator developed, which, with further work, can reveal its identity. A robust and flexible platform for in operando electrochemistry and microscopy is developed, optimised and successfully screened for use with appropriate fluorescence probes, and is shown to be well suited for further work, perhaps with other microorganisms or microscopes. Finally, membrane potential across the cyanobacterial biofilm is studied during exoelectrogenesis, showing that membrane potential changes occur and can be monitored during electron export, at the single- and multi-cellular level, and that changes in membrane potential are propagated across the biofilm in response to precise illumination.
  • ItemOpen Access
    Diamond Energy - A systematic conformation searching method
    Wei, Mengman
    Fully understanding the properties of molecules requires a good knowledge of all low-energy conformations. A python program, Diamond Energy, was developed based on an assumption that the structures of local minima are similar to a diamond lattice. These conformers could be found by calculations with integer arithmetic and evaluated with simple energy equations. Both of these should speed up systematic searching. Test results on alkane showed that Diamond Energy was able to do systematic conformational searching for acyclic alkanes exhaustively, accurately and very fast. The scope of the program’s application was extended beyond acyclic alkanes to include six-membered rings, and then further extended to include saccharides. This extension was facilitated by the development of an automatic pipeline for searching suitable energy evaluation parameters when introducing new atom types. Finally, using the large quantity of data generated by the Diamond Energy test process, a machine learning pipeline was applied and fed with Diamond Energy data. This made it possible to explore the geometry distribution of diamond lattice framework and help to improve the conformational searching process.
  • ItemOpen Access
    Towards Photoredox-Cleavable Prodrugs
    Mandrup Kandemir, Jiyan
    Photouncaging is a field of study in which light is used to release or activate molecules from inactive precursors, e.g. prodrugs. Photouncaging is most often achieved through a direct release approach employing a chromophore as the photocaging group, which absorbs light and followingly undergoes cleavage. While this approach operationally simple, it requires complex photocaging groups, which have the inherent drawback of being unstable towards light. Photouncaging can also be achieved by a less-explored sensitised approach in which the photocaging group itself is inert to light and uncaging is mediated by a photosensitiser, which engages the photocaging group in energy transfer or redox reactions. The sensitised photouncaging approach overcomes the challenges encountered in direct photouncaging by employing simple light-stable photocaging groups. With the increasing popularity of visible-light photoredox chemistry in organic synthesis, there is an increasing number of photosensitiser-mediated reactions being reported, which can also be applied to photouncaging. For this project it was envisaged that a photooxidative approach would be useful for release of prodrugs carrying electron-rich benzyl groups as a photocage, which would be oxidised by a photocatalyst with molecular oxygen as a terminal oxidant. It was found that only sufficiently electron-rich benzyl groups underwent efficient photooxidative cleavage, and in particular the 2,4-dimethoxybenzyl group was identified as a suitable photocaging group. With the identified 2,4-dimethoxybenzyl photocaging group it was found that 2,4-dimethoxybenzylcarbamates, ethers and sulfonamides all underwent photooxidative cleavage, and release could be achieved by use of transitionmetal and organic photocatalysts, including riboflavin. The rate of release was found to be dependent on concentration, and the cleavage proceeded in minutes under high dilution. Attempts at extending the photooxidative cleavage to a proof-of-concept for prodrugs met with mixed results. While rapid prodrug release could be observed when releasing at μM concentrations, an *in vitro* proof-of-concept for this methodology was not achieved. In some cases, drug stability was too low under the cleavage conditions, while in other cases prodrug stability was not sufficiently high. Overall, this study represents a useful addition of knowledge to the fields of photoredox-mediated photouncaging and photocleavable prodrugs, though further work will be necessary to prove its applicability in prodrugs.
  • ItemEmbargo
    Investigations of Catalysts Which Utilise Non-Covalent Interactions to Control Enantioselectivity in Rhodium-Catalysed Amination of C(sp3)–H Bonds
    Paterson, Kieran
    Investigations into enantioselective benzylic C(sp3)–H amination via bis-sulfonated rhodium(II) dimers ion-paired with N-quaternised Cinchona alkaloids are described herein. Design and initial synthesis of these novel catalysts was performed prior by Dr. Alex Fanourakis, and their application towards substrate-directed amination of aryl alcohols via a key alcohol-sulfonate hydrogen bond was already underway; the first section of this thesis describes the author’s contributions to this work, collaborating closely with Dr. Alex Fanourakis and Dr. Ben Williams. The second section of this thesis examines the corresponding amination of aryl tertiary amides, a deceptively divergent system given the loss of a substrate-based hydrogen bond-donor. This substrate class proved effective, however, and both yield and enantioselectivity here surpassed those for the analogous alcohol-directed methodology. Variation of the amide directing group, arene-based functionality, and methylene chain length were all viable and well tolerated, with collaborative work from Dr. Amit Dahiya in the latter. Additional contributions from Harry Palmer are also noted. Utilisation of these tertiary amide directing groups for site-selective unactivated C(sp3)–H amination was attempted, wherein the ion-paired dirhodium(II) catalyst displayed much higher reactivity relative to Du Bois’ Rh2(esp)2. Interesting effects on regioselectivity were also observed, but results here were ultimately uninspiring. The final section of this thesis sought to construct a possible binding model between the amide substrate and ion-paired catalyst, with control experiments and NMR titration performed to this effect. Both provided support for a central hydrogen bond between the substrate-based carbonyl and chiral cation-based hydroxyl, with ancillary binding also shown to be viable. Additionally, the key role of a hypervalent iodine additive was uncovered. Overall, this thesis seeks to expand the utility of ion-paired chiral cations for enantioselective C–H functionalisations, an under-developed strategy within asymmetric catalysis.
  • ItemOpen Access
    On Machine Learning Loss Landscapes
    Niroomand, Maximilian
    Loss functions are pivotal to the training of every machine learning model. When evaluating the loss function over a large range of parameters, a loss landscape is obtained. In this thesis, loss landscapes for various classes of machine learning models are explored. Geometric properties of the loss landscape can provide deep insights into machine learning models and their decision making process. Today, the most critical questions in machine learning are matters of model performance, robustness and interpretability. Here, tools from the energy landscapes field in theoretical chemistry are used to study these questions for neural networks and Gaussian processes. This cross-disciplinary approach is facilitated by a collection of analogues between both fields, such as the concept of heat capacity, monotonic sequence basins, or catastrophe theory, as developed in this work. The energy landscape perspective of loss landscape proves to be a helpful approach to examine critical questions in the machine learning field. Energy landscapes tools are applied to neural network ensemble generation and reveal that different minima of the loss landscape specialise on different sections of the input data, improving classification accuracy when combined. A second application is the evaluation and selection of loss functions, guiding hyperparameter choices. By comparing the appAUC loss function with conventional alternatives, I identify that the appAUC, while more accurate, is less robust and therefore inferior for real-life problems. Lastly, monotonic sequence basins can be used to group minima for which conserved weights can be identified to decode algorithmic decision making. Using this approach, I am able to identify and quantify input feature relevance for classification problems, a fundamental step towards trustworthy and accountable machine learning systems. For Gaussian processes, I present an analysis of the loss function hyperparameter space over different kernel choices, specifically over changes in the Matérn smoothness parameter $\nu$, which is equivalent to different kernel choices. I identify fold catastrophes in the landscape as $\nu$ changes around critical points and highlight the non-optimality of half-integer parameterisations of $\nu$ that are commonly employed in the field. Towards the end of this dissertation, a comparison of loss landscapes between machine learning models is provided, and suggestions for future work are highlighted.
  • ItemRestricted
    Adsorption to and Dissolution of Carbonate Minerals
    Farren, Lana; Farren, Lana [0000-0002-5199-4290]
    [Restricted]
  • ItemOpen Access
    Studying Models of the Amyloid Cascade Hypothesis Using Single-Molecule Imaging
    Fertan, Emre
    Observation of beta-amyloid (Aβ) plaques in the brains of Alzheimer’s disease (AD) patients led to the development of the amyloid cascade hypothesis (ACH), which postulates the accumulation of Aβ as the initiator of AD. Aβ can be found in multiple forms in the AD brain; while the insoluble plaques are the most studied species, the smaller, soluble aggregates may be more toxic, leading to neuroinflammation, oxidative stress, and tau phosphorylation. However, their small size and low abundance makes it challenging to study the soluble Aβ aggregates. Our group has been developing single-molecule and super- resolution microscopy techniques to characterise these aggregates. While these methods were previously applied to human samples, they have not been used to study disease models. Cellular and animal models have numerous advantages over human samples, primarily less intra-group variation and the ability to manipulate individual pathways. Thus, these models enable researchers to gain insight over disease mechanisms in a controlled system and test hypothesis such as ACH. Throughout my PhD, I have developed and applied single- molecule detection techniques to induced pluripotent stem cells and organoids, as well as transgenic murine models to test the predictions of the ACH. This thesis provides data on AD-like pathology caused by elevated Aβ aggregation, which can be accelerated by immune challenges and rescued by reducing Aβ levels, the role of glia in the pathogenesis of AD and their relationship to Aβ aggregation, peripheral Aβ aggregates in a mouse model, and the impact of tissue processing on the harvested aggregates. Moreover, novel methodologies to normalise conditioned media samples and study Aβ aggregates in the synapses have been described, and therapeutics against Aβ has been characterised. Overall, this thesis investigated the ACH in a range of cellular and murine models, providing supporting data for the hypothesis and revealing new details of the molecular processes that initiate AD.
  • ItemEmbargo
    Supramolecular effects in pyridinium compounds and their application towards practical redox flow battery systems
    Carrington, Mark
    Aqueous-organic redox flow batteries (AORFBs) have emerged as a promising technology class for stationary energy storage. To date, however, synthetic strategies for organic electrolytes have emphasised molecular complexity as a means of targeting desired performance characteristics. Consequently, materials typically exhibit high initial production costs and require large-scale manufacturing to re-establish cost competitiveness, undermining current efforts to achieve ultra-low-cost storage over the coming decade. In the present report, supramolecular complexity is leveraged as an alternative and low-cost design paradigm, that is developed toward augmented device functionality. Such capabilities are demonstrated for pyridinium-based systems and *operando* spectroscopic techniques are used to gain fundamental insights into structure and mechanisms. From such insights, methods are unlocked to both harness and suppress supramolecular effects towards regimes of performance inaccessible to conventional systems. In a first demonstration, both the host-guest chemistry and existing tonne-scale production capacity of both cucurbit[*n*]uril (CB[*n*]) macrocycles and methyl viologen are leveraged to advance an additive concept for AORFBs that enables stable and tuneable multi electron accessibility – without the need for synthetic modification. By encapsulating methyl viologen in all oxidation states, CB[7] macrocycles improve the solubility of reduced viologen species, suppress parasitic side reactions with trace contaminants, and provide resistance to electrochemical degradation at potentials more negative than any demonstrated for methyl viologen and its analogues to date. Host-guest binding is readily modulated by several independent parameters including temperature, concentration, supporting salt choice and supporting salt concentration. Electrolyte formulation additionally results in the production of industry-grade by-products such as CB[5], CB[6], and CB[8], which can be sold to access average installed electricity costs among the lowest projected to date. In a second demonstration, *operando* NMR and EPR spectroscopies, as well as *operando* mass spectrometry are used to understand and harness the inherent assembly processes of pyridinium radicals towards improved battery performance. Making use of a scalable and versatile reaction chemistry, a synthetic library of extended bispyridinium compounds featuring a diverse array of electronically tuneable aromatic cores is demonstrated. These compounds span the widest potential range demonstrated for pyridinium based redox flow batteries to date and are electronically analogous to classical diradical systems (e.g., Thiele’s and Chichibabin’s hydrocarbons), permitting prediction of electrochemical irreversibility and previously unidentified capacity fade mechanisms. Using coupled *operando* NMR and EPR spectroscopies, the redox behaviour of these electrolytes is elucidated under representative flow battery conditions and the presence of two distinct regimes (*narrow* and *wide* singlet triplet energy gaps) of electrochemical performance is identified. In both regimes, capacity fade is tied to the formation of free radical species, and further show that π-dimerisation plays a decisive role in suppressing reactivity between these radicals and trace impurities such as dissolved oxygen. This result stands in direct contrast to prevailing views surrounding the role of π-dimers in aqueous organic redox flow batteries and enables us to efficiently mitigate capacity fade from oxygen even upon prolonged (days) exposure to air. In a final demonstration, *operando* paramagnetic NMR spectroscopy is used to detect and understand the formation of triplet diradical species at high states of charge (SOC) – a completely new capacity fade mechanism within the field that occurs in high voltage systems. It is found that formation of triplet diradicals and their associated structures can be suppressed by careful management of operating temperature and state of charge. Through such operation, full cells made from previously unusable materials can be operated stably under practical conditions. Using such strategies, the materials rendered accessible substantially expand the available design space for future bispyridinium flow battery development towards higher voltage systems. Coupled with the known tendencies of such systems to resist side reactions with oxygen through controlled π-dimerisation, they enable the highest voltage air-stable AORFB demonstrated to date presenting new horizons for achievable energy densities in such systems. Collectively, these findings provide new insights into the degradation and stabilisation of pyridinium electrolytes during cycling and further unlock libraries of previously unusable materials enabling new material design strategies moving forward. They do so largely by mediating supramolecular assembly processes during cycling, and therefore, do not introduce synthetic modifications which ultimately bear out in system costs. As both π-mediated association and triplet diradical formation are exhibited by a wide range of organic electrolytes, it is anticipated that the insights described herein may enable new design paradigms not only for pyridinium systems, but broad classes of organic electrolyte relevant to redox flow batteries.
  • ItemOpen Access
    Advancing Fluorescence Microscopy Techniques for Volumetric Whole-cell Imaging
    Zhang, Ziwei
    One revolution in the last decade has been the application of new imaging methods to modern biology, revealing new insights into cellular structure and dynamics. However, imaging methods capable of three-dimensional (3D) imaging at high spatial and temporal resolution are still lacking. This thesis describes two projects aimed at advancing volumetric whole-cell imaging based on fluorescence microscopy. The first project establishes a robust single-molecule localisation microscopy (SMLM) pipeline for volumetric imaging in the whole cell using the double-helix point spread function (DH-PSF) approach. The practical challenges associated with this method, such as aberration- and drift-induced inaccuracies, were comprehensively evaluated, and methods were developed to address all these problems. With a novel spontaneous blinking PAINT (Point Accumulation for Imaging in Nanoscale Topography) dye, HMSiR-Hoechst, super resolved SMLM images of DNA throughout the entire mammalian cell nucleus were obtained. The second project involved the design and construction of an epi-illumination selective plane illumination microscopy (eSPIM) system capable of rapid volumetric multi colour live fluorescence imaging of cellular samples. This system was one of the first of its kind in Europe, demonstrating excellent performance by yielding high resolution in line with established standards. A comprehensive comparison of eSPIM with three alternative volumetric imaging modalities was conducted, focusing on the dynamics of DNA and Heterochromatin Protein 1 beta (HP1β) within live mouse embryonic stem cells (mESCs), demonstrating the superior performance of the eSPIM. Collectively, this work makes a significant contribution to volumetric fluorescence microscopy, extending our capability to dissect and visualise cellular structure and interaction in whole-cell in 3D.
  • ItemOpen Access
    Improving de novo molecule generation for structure-based drug design
    Thomas, Morgan; Thomas, Morgan [0000-0002-1610-3499]
    *De novo* molecule generation for drug design has seen a resurgence in recent years, mostly due to the rapid advances in machine learning (ML) algorithms that utilise deep neural networks, resulting in a plethora of ML-based generative models. However, there is often a large disparity in published evaluations and applications of such approaches compared to the practical needs of real drug design projects (for example, optimizing QED *versus* optimizing binding affinity commonly approximated by structure-based approaches). Moreover, the density of approaches and often lack of relevant, standardized objectives makes it difficult to truly discern “state-of-the-art”. The work in this thesis aims to address some of these issues and improve the applicability and evaluation of *de novo* molecule generation for practical drug design. The first research chapter will outline the design and use of an open-source python-based software named MolScore. This configurable suite of scoring functions (including an interface to 5 docking algorithms and ~2,300 trained bioactivity models) can be used to design difficult yet relevant drug design objectives for standardized comparison, or practical usage with generative models. In addition, MolScore includes a graphical user interface to improve usability and a suite of common evaluation metrics to evaluate *de novo* generated molecules. Next, MolScore was implemented to compare the use of docking as a more difficult objective function for REINVENT (a generative model for goal-directed *de novo* molecule generation), as opposed to more commonly used predictive models of molecule bioactivity. This resulted in increased diversity of *de novo* molecules and improved coverage of known bioactive chemical space. However, the added computational expense required for generative model optimization is a practical disadvantage of docking as a scoring function. To address the computational expense of optimizing docking scores, a hybrid reinforcement learning algorithm (Augmented Hill-Climb) is proposed to improve the learning efficiency of language-based generative models. This significantly reduced the computational runtime while maintaining the chemical desirability of *de novo* molecules. Augmented Hill-Climb displayed superior efficiency against four other commonly used reinforcement learning algorithms, also displayed in an alternative model architecture. It was then benchmarked against 22 various generative models showing the best sample efficiency when additionally constraining for chemical desirability. Overall, the work outlined in this thesis contributes to the field of computational drug design by providing software, algorithmic developments, and benchmark results for different *de novo* molecule generation approaches.
  • ItemEmbargo
    Generating artificial metalloenzymes containing new-to-nature organometallic cofactors via controlled ligand exchange and microfluidic screening technologies
    Klein, Oskar James; Klein, Oskar James [0000-0003-2213-8003]
    Artificial Metalloenzymes (ArMs) containing non-natural organometallic cofactors have commanded increasing attention for their ability to catalyse transition-metal dependent transformations with the selectivity and mild conditions associated with enzymes. This work investigates novel ArMs where an organometallic cofactor has been introduced *via* ligand exchange reactions, thus ensuring an intimate link between the first and second coordination-sphere, with the latter being dictated by the protein fold. Further, the development of a high-throughput microfluidic screening platform is presented, with the aim to facilitate efficient directed evolution. The first chapter gives a theoretical description of the matter covered by this thesis and reviews the relevant fields, including considerations for the formation of ArMs, *in vitro* directed evolution and a brief discussion of transition metal catalysis, focusing on ruthenium. The second chapter covers the practical aspects of the work conducted, including detailed descriptions of synthetic processes as well as biochemical and microfluidic techniques. The third chapter details the design, synthetic challenges and characterisation of a range of substrate molecules used for the establishment of fluorescence assays for different ArM activities. Particular focus is placed on the synthesis and use of charged substrates that do not leak from the aqueous phase when used in water-in-oil droplet microfluidics. The fourth chapter focuses on the formation of ArMs from organometallic complexes. A systematic study is presented, describing how the steric and electronic properties of a ruthenium piano-stool complex, carrying a key bipyridine ligand, led to various speciation of cytochrome *b*562. Applying findings from this study, the scope of organometallic fragments that could be introduced was further expanded to include new metal centres and ligands. The resulting conjugates were characterised and investigated for catalytic activity. Finally, a method for evolving cofactor recognition using pre-fluorescent complexes is presented. The fifth chapter details the development of a microfluidic workflow compatible with the ArMs used in this thesis, aiming to rapidly encapsulate, express and sort large genetic libraries of ArMs and thus enable directed evolution. Coupling of the genotype and phenotype is achieved by covalent attachment to hydrogel beads, mitigating the possibility of catalytic poisoning when using an *in vivo* approach. The concluding chapter summarises the key results of this work and gives perspective on potential future developments.
  • ItemRestricted
    Genetic engineering of structural colour in the model organism, Flavobacterium IR1
    Caton Alcubierre, Laura; Caton Alcubierre, Laura [0000-0003-2905-3045]
    [Restricted]
  • ItemOpen Access
    The Design, Synthesis and in vitro Evaluation of Proteolysis Targeting Chimeras (PROTACs) for the Degradation of Protein Arginine Methyltransferase 1
    Martin, Poppy
    Protein arginine methyltransferase 1 (PRMT1) is a protein responsible for the asymmetric dimethylation of arginine. PRMT1 is upregulated in a wide range of cancers and the reduction of PRMT1 activity reduces cell proliferation and tumour growth in cell and animal models of cancer. A reduction in PRMT1 can also sensitise cancer cells to other treatments. Therefore, targeting PRMT1 is a promising therapeutic strategy in cancer treatment. Published inhibitors for PRMT1 show poor selectivity and dose-limiting toxicities that have precluded their translation to the clinic. The degradation of PRMT1 using a PROTAC may be superior to inhibition as PROTACs can act catalytically at a low dose. PROTACs can also exhibit high selectivity and cause a more pronounced functional outcome compared to inhibition. In this thesis, PRMT1 is explored as a target protein for PROTAC-induced degradation. The endogenous properties of PRMT1 were evaluated and PRMT1 was determined to be amenable to degradation by a PROTAC. PROTACs were designed that comprised a PRMT1 ligand, a linker and an E3-ligase ligand. Ten PROTACs that recruit the VHL E3-ligase and six PROTACs that recruit the CRBN E3-ligase were synthesised. The degradation of PRMT1 was assessed by Western blot and degradation was not observed for the PROTACs synthesised. Suitable pharmacokinetic properties and target engagement have been shown for selected candidates by the detection of the downstream effects of PRMT1 inhibition and by a NanoBRET assay for E3-ligase binding. Regioselectivity challenges in the synthesis of the CRBN-recruiting PROTACs led to the isolation of a heterobifunctional molecule with the linker attached to the binding pharmacophore of the CRBN ligand. This molecule was found to degrade PRMT1 and is proposed to be a monomeric degrader that destabilises the structure of PRMT1 upon binding. This thesis details a novel approach to degrade PRMT1 using a PROTAC and provides insights that may assist the rational design of PROTACs that target PRMT1 in the future.
  • ItemEmbargo
    Design and Frabication of Optical Li+ Sensors for Application in Li-ion Batteries
    Francis, Haydn
    Li-ion batteries have redefined expectations for consumer electronics and are the key technology in enabling the electrification of road transport. Their contribution to decarbonisation only stands to increase in the coming decades as demand for Li-ion batteries continues to soar. However, as production continues to accelerate, maximising device-level level sustainability over the lifetime of cells, modules, and battery packs is of increasing importance. Currently, there is a broad suite of techniques available in laboratory settings for the analysis of Li-ion battery performance and degradation during operation. Studying batteries using these techniques has led to the design of safer, longer-lasting batteries over the past few decades. However, most of these techniques use large and/or expensive instrumentation or require the design of bespoke cells and are therefore not applicable as methods for monitoring commercial battery systems in the field. Currently, only a small collection of non-disruptive diagnostic techniques are available for these applications. In addition, these techniques generally measure broad metrics, which are extrapolated using statistical methods to estimate and predict cell performance, often resulting in systematic underutilisation of battery systems. Optical techniques offer a potential solution to this problem owing to their low-cost, facile integration as part of commercial battery systems and their potential to monitor a range of mechanical, thermal, and chemical parameters through the application of optical fibres. By providing specific and detailed data in real-time, optical techniques coupled with a smart battery management system have the potential to improve battery lifetimes, reduced safety risks, and facilitate more advanced characterisation ahead of end-of-life protocols (e.g., re-use, recycling, etc). In this thesis, we discuss the development of a novel optical sensing platform for monitoring [Li+] in battery electrolytes. The first chapter introduces key concepts around the basic operation of Li-ion batteries and the degradation processes that lead to loss of performance during their operation. Following this, the range of *in situ* and *in operando* techniques that have facilitated our current understanding of processes occurring inside Li-ion batteries are discussed as well as the state-of-the-art in the application of optical techniques. This chapter ends with a summary of the thesis and its objectives and serves as an introduction to the context in which this work was originally formulated. The second chapter begins with an introduction to the field of optically active organic molecules, ionophores, and their convergence to form highly selective and sensitive optical chemosensors. Subsequently, the design, synthesis, and characterisation of two novel Li+-selective fluorescent chemosensors based on a naphthalene diimide core are described. Although both displayed poor solubility in salt-solvent systems reflective of the conditions in a Li-ion battery electrolyte, they were deemed to be a promising starting point for the development of a solid-supported chemosensor-based optical sensing platform for our applications. Chapter III starts with a summary of the fabrication and operation principles of planar and nanoparticle optodes as well as their application as part of optical fibre sensors. Following this is a discussion of potential strategies for the immobilisation of a chemosensor onto a solid support for applications as an optode sensor. Subsequently, the chapter describes the optimisation of experimental parameters towards the development of a fabrication procedure for planar optodes functionalised with one of the novel chemosensors synthesised in Chapter II. The final planar optodes used a silica-based substrate loaded with a mesoporous surface film, which is functionalised with a modified derivative of the chemosensor. Characterisation of the planar optodes demonstrated their promising properties for monitoring [Li+] selectively in concentration ranges and solution conditions relevant to Li-ion battery electrolytes. This chapter also summarises attempts to apply the same fabrication procedure for the functionalisation of optical fibres to form ‘dip probes.’ However, these were largely unsuccessful. To further characterise the properties of the optode sensor in emission mode, the planar optodes were integrated as part of microfluidic devices. This provided precise control over liquid samples on the optode surface and enabled full characterisation of their Li+-sensing capabilities in emission mode. Chapter IV provides a summary of this data, which showed the planar optodes to possess the necessary sensitivity and selectivity for application as an emission mode concentration sensor in battery electrolytes. In addition, the platform was shown to demonstrate strong cyclability, a rapid time response, and a level of spatial resolution that suggested the optodes could be applied for dynamic chemical imaging using fluorescence microscopy. Chapter V is the final chapter and describes the application of the optode-based microfluidic devices for two proof-of-concept experiments. Firstly, ex-situ [Li+] measurements were taken using the devices on a pristine electrolyte and electrolyte extracted from a Li-ion pouch cell after cycling. These results provided a quantitative indication of bulk Li+ depletion in the electrolyte during cycling with a comparable level of accuracy to ICP-OES measurements run in parallel. Secondly, simple experimental conditions were designed to establish the ability of the optodes to track the evolution of Li+ concentration gradients in solution with time. These experiments yielded the first recorded example of spatial Li+ tracking using a solid-supported optical sensor to our knowledge. Modelling of the diffusion data showed that the experimental results could provide a comparative estimation of the Li+ self-diffusion coefficients for different solvent systems.
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    Molecular Encapsulation of Conjugated Polymers for Organic Electronics
    Moiseanu, Teodora; Moiseanu, Teodora [0000-0003-2400-1772]
    Conjugated polymers have been the subject of extensive research, particularly for their application in the field of organic electronics. Attributes such as their semiconducting nature, solution processability, degree of flexibility, and infinite tunability have led to their successful implementation as light-harvesting or light-emitting materials. In the first chapter, we introduce and describe several key concepts within the area of organic electronics. These concepts include the understanding of how conjugated polymers function and their relevance within the field. We explore the various applications with detailed device characteristics. Additionally, we examine various strategies for modifying monomers to adapt and control the molecular bandgap of the polymers to suit specific applications. Furthermore, we consider the influence of conjugation length and aggregation on the electronic properties and device performance. Lastly, we discuss different synthetic procedures employed to achieve high molecular weight conjugated polymers. The second chapter begins with the introduction of different techniques to lower the formation of aggregates in conjugated polymers. It describes both non-covalent and covalent encapsulations that have been previously employed, offering examples of their efficacy in reducing nonradiative decay processes. Past research on molecularly encapsulated conjugated polymers has extensively focused on molecular backbones such as diketopyrrolopyrrole (DPP), naphthalene diimide (NDI) and perylene diimide (PDI). In the context of this work, the research is centered around narrow bandgap encapsulated conjugated polymers based on benzodithiophene (BDT). Consequently, this chapter delves into BDT-based conjugated copolymers and their naked (unencapsulated) counterparts. This study highlights that molecular encapsulation serves to suppress intermolecular interactions, resulting in polymers exhibiting a lower degree of energetic disorder and increased spacing between polymer chains. However, it is noteworthy that the photoluminescence quantum yield (PLQY) in the solid state is unexpectedly lower (1%) compared to previous studies. Nevertheless, when assessing the performance of devices in comparison to the record breaker PM6:Y6 system, our materials demonstrate remarkably high efficiencies. These outcomes prompt a deeper investigation into the reasons underlying the reduced emissivity with encapsulation, and whether this phenomenon is linked to the BDT core or the acceptor comonomer component. The third chapter focuses on increasing the order within the polymer backbone by having a higher density of encapsulation throughout the entire polymer chain. To achieve this objective, this chapter explores the synthesis of BDT-based encapsulated conjugated homopolymers, and their reference counterparts. The control of interpolymer distance is achieved through various ways including: the nature of the solubilising chain, the nature of the encapsulating chain, and the length of the encapsulated chain. Through ultraviolet-visible (UV-Vis) and photoluminescence (PL) measurements, our studies reveal that encapsulation mitigates aggregation to a certain extent. Nevertheless, even with increasing the distance between polymer chains, the emission remains suppressed. Notably, as excimer formation can be discarded due to the reduced cross-communication, our findings point towards nonradiative loss being attributed to an intramolecular process. Through thorough transient absorption (TA) measurements, we identify this loss as being linked to polaron pair formation. Collectively, these results lead us to the conclusion that BDT-based polymers might not be as suitable for solar cell applications as was previously believed, with the root cause lying in intricate intramolecular processes that prove challenging to control effectively. The final chapter (chapter IV) explores a distinct property offered by molecular encapsulation. It firstly describes chirality within conjugated polymers, providing examples of materials holding higher dissymmetry factors of absorption and emission, albeit at the cost of the conjugation length of these polymers. Therefore, the research focus was shifted towards the synthesis of main-chain planar chiral conjugated polymers, aiming to achieve a high degree of chiroptical properties. Specifically, the study centeres on [n]paracyclophane-based ([n]PC) conjugated polymers, designed to investigate various effects. These include altering the cyclophane chiral centre by changing the ansa unit, modifying the polymer morphology by changing the side chains, and varying the choice of comonomers used for polymerisation to encompass a broader spectal range. It was demonstrated that the chiral response can be predominantly influenced either by the size of the encapsulating chain, resulting in more twisted conjugated polymers, or by the morphology of the sample, with higher responses observed for less crystalline materials. Furthermore, the synthesis of the paracyclophane comonomers is enantioselective, rendering it more suitable for industrial applications and thus next-generation circularly polarised organic light-emitting diodes (CP-OLEDs) materials.
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    Characterisation and Detection of P53 Aggregates
    Wu, Yunzhao
    P53 is a tumour-suppressing protein whose primary function is to protect the integrity of the genome, specifically by regulating DNA repair, cell cycle arrest, cell proliferation, and apoptosis. Wild-type (WT) p53 has multiple aggregation-prone regions and can form amyloid aggregates. Some mutants of the p53 protein, such as the R248Q mutant, exhibit higher aggregation propensity than the WT. P53 aggregation is associated with loss-of-function, gain-of-function, and dominant-negative effects of the protein and thus plays a crucial role in cancer. Understanding the properties of p53 aggregates and their pathological implications provides new insights into cancer biology and may lead to novel diagnostic and therapeutic strategies. Previous studies on p53 aggregates mainly focused on the properties of bacteria-derived p53 core-domain fragments, which have different post-translational modifications from human p53 and may behave distinctly from full-length p53 proteins. The p53 concentrations used in these studies were also higher than the physiological concentrations. Meanwhile, most kinetic studies on p53 aggregation employed microplate reader-based assays, which are limited in sensitivity and cannot provide morphological information about the aggregates. Hence, the biophysical properties of full-length p53 aggregates at physiological concentrations are not well characterised. Also, the detection of p53 aggregates in biopsies has not been demonstrated. In this thesis, a series of advances in single-molecule assays are presented to characterise full-length p53 aggregates and detect p53 aggregates in human plasma. Firstly, a single-molecule fluorescence microscope with flat-field illumination was constructed and characterised. Secondly, a Python-based computational suite for automated fluorescence image analysis was implemented. Based on these two techniques, the aggregation kinetics, membrane disruptive ability, and morphological features of insect cell-derived full-length WT and R248Q p53 aggregates were characterised. Specifically, p53 aggregation at physiologically relevant concentrations was found to be dominated by a nucleation-elongation process. Meanwhile, both WT and R248Q p53 aggregates exhibit membrane-disruptive abilities. Furthermore, a single-molecule array assay was developed to detect p53 aggregates in plasma samples, demonstrating p53 aggregates as a promising diagnostic biomarker for glioblastoma. The presence of p53 aggregates in the plasma samples of glioblastoma patients was validated using the single-molecule pull-down assay. The work presented in this thesis extends the understanding of full-length p53 aggregates and highlights the implications of p53 aggregates in cancer diagnosis.
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    Chemical Softness as a Predictor for Reactivity at Metal Surfaces
    Gunton, Amy
    Heterogeneous catalysis is an important global industry, but there are many gaps in our understanding of catalytic selectivity. Reactivity indices are helpful for predicting selectivity, and it would be useful to have a reactivity index which can be applied to metal surfaces and adsorbates. The local softness is a reactivity index based on Pearson’s theory of hard and soft acids and bases. It is the derivative of the local electron density with respect to the chemical potential, at constant external electric potential. It can be calculated simply for molecules or nanoparticles which have a band gap. However, the calculation for conductors is less straightforward. In this work, a method was developed to calculate the local softness of metal surfaces using density functional theory. This required a solution to the problem of increasing the chemical potential while keeping the external electric potential constant, which is difficult to do in charged cells with periodic boundary conditions. This problem was solved by correcting for a shift in energy reference with charge and by extrapolating to an infinitely sized unit cell. The local softness was visualised using isosurfaces and colourplots and was used to compare predicted reactivity between different sites on various metal surfaces. In order to get a measure of the softness of individual atoms on a surface, Bader’s theory of atoms in molecules was used to integrate the local softness over the regions of atomic volume. The resulting reactivity index, the atomic softness, was used to predict the adsorption energy of carbon monoxide on eighteen different metal surfaces. The local and atomic reactivity indices were also used to study directing effects for aromatic adsorbates on the Pt\{111\} surface. The local and atomic softness were found to be useful for predicting reactivity trends between different sites on metal surfaces and for adsorbates.