Theses - Chemistry
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- ItemOpen AccessDevelopment of novel lysine targeting covalent inhibitors for Bruton’s tyrosine kinase and casein kinase 2 and synthesis of novel hybrid androgen receptor inhibitorsBizga Nicolescu, Radu CostinCancer is the lead cause of death worldwide, accounting for 10 million deaths in 2020, or 1 in 6 deaths. While current therapies have significantly advanced in the last decade, they suffer from severe limitations such as resistance, lack of selectivity and life-threatening side effects. Herein we present three novel strategies of targeting prostate cancer, breast cancer and leukaemia. 1) The development of a novel class of hybrid compounds designed through covalently linking enzalutamide and EPI-001 through triazole-PEG linkers is reported. The compounds are accessed in 6 synthetic steps performed in parallel for the 5 final target compounds. The compounds display a 50-fold improvement in the cell killing potency compared to the gold standards of therapy, enzalutamide and EPI-001 (LC50 EPI = 85 μM, LC50 Enza = 65 μM, LC50 hybrid = 1.6 μM). The best in line compound was proven to exhibit its toxicity exclusively through AR mediated pathways, yielding the first-in-class hybrid AR inhibitor. 2) We report the design, computational validation and synthesis of a proposed lysine targeting allosteric CK2 inhibitor. The challenging synthetic steps towards the peptidomimetic electrophile are presented, along with the biological characterisation of the final target molecule. Protein mass spectrometry and tandem mass spectrometry studies indicate that the electrophile does not target Lys158, as intended, but that instead it forms a transient, water unstable, covalent bond with the protein. 3) We developed a novel class of lysine targeting covalent inhibitors, targeting the PH domain of BTK, a previously unreported targeting strategy. 3 families of analogues were rapidly constructed using an efficient 4 step synthetic strategy, yielding 23 analogues and 14 co-crystal structures with the BTK PH domain. The high percentage of crystal structures represents a success rate of 61%, which validates the fragment elaboration strategy, whereby all fragments covalently label Lys12. The binding selectivity was validated by protein mass spectrometry and differential scanning fluorimetry, whereby all analogues induce expected responses with the WT BTK, but fail to induce a response with the loss of function mutant R28C, as expected. A 6-fold improvement in binding affinity from the parent compound was achieved through a hit-to-lead optimisation campaign (160 μM to 30 μM).
- ItemOpen AccessEnzyme and Directed Evolution Technologies For Nerve Agent NeutralisationBriseño-Roa, LuisOwing to the magnitude of the utilisation of organophosphorus (OPs) insecticides and the possibility of using OPs nerve agents (NA) against civilian populations, the research and development of enzymes involved in the biotransformation and detoxification of OPs has attracted considerable attention in recent years. A number of enzymes have been identified that can catalyse the hydrolysis of OPs, including nerve agents. Two of the best characterised are Pseudomonas diminuta phosphotriesterase (PTE) and PON1, a mammalian member of the Serum paraoxonase (PONs) family. These enzymes have excellent catalytic properties towards some OPs, but relatively poor activities against others. It has been possible to alter PTE substrate specificity by rational site-mutagenesis, but with little improvements on the wild-type rates. A more successful approach has been the application of directed-evolution strategies. The aim of the present work has been to create variants of PTE with an increased catalytic efficiency towards OPs nerve agents. To this end, a directed evolution platform was developed to enable screening for organophosphatase activity. This methodology relies on the screening of Escherichia coli colonies transformed with PTE-variant libraries. Twelve fluorogenic NA analogues, with a 3-chloro-7-hydroxy-4-methylcoumarin leaving group, were tested for suitability as substrates for PTEs and PON1. Included in this series were analogues of the pesticides Paraoxon and Parathion, and the chemical warfare agents DFP, Dimefox, Tabun, Sarin, Cyclosarin, Soman, VX, and Russian-VX . These chemical surrogates have a similar structure but do not share the same physico-chemical properties as the nerve agents themselves. The directed evolution platform developed and used consisted of two parts. First, partially lysed Escherichia coli colonies were screened using the fluorogenic nerve agents analogues as probes. Second, the selected (positive) clones were grown in microplates filled with liquid medium, and their organophosphatase activity was measured in vivo. Several gene libraries were synthesised in each of which four codons of the residues forming PTE’s substrate binding site were selectively randomised. The PTE variant S5a was used as template for the libraries, as it expresses at 20-fold higher level than the wild type, in bacterial hosts, while retaining its kinetic properties for the wild-type substrate, Paraoxon. These libraries were screened using analogues of Russian-VX and Parathion as probes; approximately 106 clones were screened in total. The twenty most active variants, as determined in vivo, were expressed, purified, and their kinetic parameters for Paraoxon and the NA analogues were determined. PTE-S5a itself hydrolysed 8/VX, 9/Sarin and 10/Russian-VX analogues between 2.5 and 3.5 times more readily than PTE-wt. In contrast, towards 11/Soman and 12/Cyclosarin analogues its activity, was only 70% of that of the wild type enzyme. Three of the selected clones, PTE -A (I106T), C (I106L), and H (I106T/F132V/S308A/Y309W), exhibited a higher kcat than PTE-S5a towards Paraoxon. The latter exhibited a 5-fold increased in its turnover rate (31,016 s-1); this rate is higher than that of the in vitro evolved PTE-H5 (26,294 s-1). PTE variants A (I106T), C (I106L), D (I106A/F132G), E (I106V/F132L), and F (I106L/F132lG) exhibited between 2 and 4-fold increases in their kcat/KM towards the Paraoxon analogue relative to PTE-S5a. Variants Q (G60V/I106L/ S308G), S (G60V/I106M/L303E/S308E), and T(G60V/I106S/L303P/S308G) showed between 2 and 14-fold improvements in their activities towards Russian-VX, Soman and Cyclosarin analogues. The selectivity for this latter group towards phosphonate NA analogues increased up to 107-fold, relative to the wild type PTE. Each PTE monomer binds two divalent transition metal ions via a cluster of four histidines (His-55, His-57, His-201 and His-230) and one aspartate (Asp-301). In addition, the two metal ions are linked together by a carbamate functional group, formed by the carboxylation of the e-amino group of Lys-169 and a water (or hydroxide ion) from the solvent. A case study is presented in which using both site-directed mutagenesis and directed evolution strategies, the possibility of replacing the carboxylated lysine (Lys-169) by any other residue was assessed.
- ItemOpen AccessHeteroatom-Doped Graphitic Materials for Energy StorageClark, CassiusThe transition to a zero-carbon society will require a new generation of energy storage technology with high energy density. Central to this are novel electrode materials that possess high specific capacities (mAh g−1) and long cycling lifetimes. Modern lithium-ion batteries utilise graphite as an anode, being a cheap, safe and stable material. Sodium-ion batteries, a promising alternative to their lithium counterpart, utilise hard-carbon as an anode, a disordered matrix of graphene nano-sheets, due to the inability of sodium to bind effectively with pure graphite. However, the theoretical specific capacity of carbonaceous materials (372 mAh g−1 and 300 mAh g−1 for Li in graphite and Na in hard carbon, respectively) is low compared to other materials such as phosphorus (2596 mAh g−1 ) or silicon (3579 mAh g−1). Materials such as these come with their own caveats. High volume expansions, chemical instability, cost, or conductivity problems are a few potential issues encountered with Si or P anodes. Alternatively, chemical enhancement of graphite is considered an effective way of modifying the electrochemical ion-storage properties whilst retaining a high level of stability. Heteroatom substitution of carbon in a graphite lattice has been shown to produce high-capacity anode materials suitable for Li- and Na-ion batteries. This thesis develops an understanding of the structure and function of element-doped graphite within Li- and Na-ion batteries. Turbostratic doped graphitic materials were produced through pyrolysis of organic material. In Chapter 2, investigations into nitrogen-doping are made. Alterations to the synthetic procedure in combination with thorough structural and compositional analysis helps in understanding what factors make nitrogen-doped graphite effective as an anode. Notably, it was found that pyrolysis of organic precursors produced dense, solid spheres, previously thought to be hollow. Use of X-ray photoelectron spectroscopy, combined with ion-etching, revealed how the nitrogen dopant environment varied with increasing depth, and across annealing parameters. The effect of these environments on electrochemical performance could then be assessed. Chapter 3 focuses on boron as a dopant. The electron deficiency of boron compared to carbon is predicted to aid electron transfer, and subsequently facilitate intercalation of Li+ or Na+. Literature on boron-doped graphite largely considers systematic changes to the boron quantity present or focuses solely on applications. However, there is some debate about whether different precursors affect the final structure and performance of the graphite. In this chapter, a particular focus is on investigating the ability of different precursors to produce substitutionally-incorporated B-doped graphite. 11B solid-state nuclear magnetic resonance spectroscopy (SSNMR) combined with structural analysis are used to identify the phases boron is present in and how this is related to performance in Li- and Na-ion batteries. Relating the boron environments present to the voltage at which battery capacity is observed allows for the interpretation of how boron doping affects and facilitates the intercalation of Li+ and Na+ ions. In Chapter 4, an in-depth study of the function of phosphorus-doped graphite is presented. Ex-situ 31P SSNMR spectroscopic studies of doped-graphites, partially cycled to different voltages in Li- and Na-ion cells, led to proposed mechanisms of lithiation or sodiation. Furthermore, alternations to the synthetic procedure allowed reliable encapsulation of white phosphorus between graphene layers, enabling effective, reversible cycling of red phosphorus without degradation.
- ItemOpen AccessMolecular dynamics investigation of the long wavelength dielectric response of classical dipolar liquids using Bloch function methodsLloyd, HaydnPeriodic Boundary Conditions (PBCs) are commonly utilised in many simulations of great importance in computational science, both as methods to accurately simulate perfect crystals, as well as to approximate macroscopic systems. When considering the latter case, the use of these boundary conditions restricts the wavevectors for which the Fourier components of quantities calculated from these systems can be non-zero. In particular, the minimum non-zero wavevector that can be examined, is inversely proportional to the size of the simulation cell. This means the examination of small, non-zero wavevectors can require the use of large systems, that may become prohibitively expensive to simulate. This can potentially inhibit the study of long-range contributions to the properties of the system. In this work, Bloch boundary conditions are introduced for systems that include charges. In these, the particle positions and momenta are periodic, as in PBCs, but the charge multipoles in image cells are changed in phase controlled, by a wavevector within the first Brillouin Zone, q, that is characteristic of the boundary conditions. In such cases, the accessible wavevectors are then all shifted by q, and so the minimum accessible wavevector is now q. A demonstration of how the Ewald summation is modified for energy and force calculations necessary for the simulation of these systems is given, explicitly for that of charges and dipoles, and a scheme to derive this for higher multipoles is given. This framework is then applied to the longitudinal and transverse dielectric constants of dipolar fluids subject to static electric fields, both Stockmayer and polarisable dipole fluids. This is used to verify the ratio of the transverse and longitudinal susceptibility being the dielectric constant, in the limit q goes to zero. A brief analysis of how this may be applied to systems with dynamic electric fields is also given.
- ItemEmbargoSingle-molecule techniques for mapping protein aggregatesLam, YuiProteins in our body are usually folded to carry out their physiological functions. However, they can become misfolded and aggregated throughout the lifetime of a cell. These proteins can be degraded or cytotoxic, the latter of which leads to diseases such as neurodegeneration and cancers. Unfortunately, the compositions of native protein aggregates are still not fully known. Mapping them could help us understand the diseases they cause, and provide the basis for early diagnosis and monitoring therapy. Since protein aggregates are heterogeneous in sizes and structures, single-molecule techniques are required to unravel their compositions. Super-resolution microscopy is particularly useful, as these aggregates are smaller than the diffraction limit of light. Compared to conventional light microscopy, the fluorescence of the nearby molecules in super-resolution microscopy fluoresces at different times. With this, even though the fluorescence of the single fluorophore is still blurred by the diffraction limit (~200 nm), the fluorophore is inferred as in the centre of the blurred spot. Thus, the resolution in super-resolution microscopy can reach down to sub 20 nm. This allows us to super-resolve the protein aggregates and may therefore help us decipher the mysteries of disordered and aggregated proteins. However, applying super-resolution microscopy to these complexes is a challenge. Although there are already a range of developed methods, there are still important limitations. In this thesis, I present a series of advances in single-molecule techniques for mapping protein aggregates. Firstly, an advanced illumination module for super-resolution microscopy to facilitate accurate measurements was implemented and characterised. Functionalisation of antibodies, which are commonly used to map native protein aggregates in super-resolution microscopy, was then optimised. Next, single-molecule pull-down assays were investigated with the aim of capturing and imaging protein aggregates with higher selectivity, sensitivity, and speed on glass coverslips for super-resolution microscopy. Meanwhile, two classes of DNA-small molecule conjugates, with PET-ligand analogues and aggregate-targeting peptides, were synthesised and tested. These novel probes can potentially selectively target protein aggregates in super-resolution microscopy. Finally, a newly developed, state-of-the-art microscope for super-resolution microscopy, NanoPro, is presented. The theme that unites the presented work is improving single-molecule techniques for mapping protein aggregates.
- ItemOpen AccessSupramolecular Approaches to Viscoelastic Biomaterials and Their ApplicationsPark, JuneViscoelasticity encompasses the characteristics of both the shape-conserving elasticity and the shock-absorbing viscosity. The extracellular matrices (ECM), or the protein-polymer scaffolds surrounding the cells in our body, possess unique tissue-dependent viscoelasticity to protect and provide mechanical queues to the nearby cells. Viscoelasticity in polymeric hydrogels can be achieved in several ways, one of which is by incorporating the host-guest supramolecular components. The following works explore the application of cucurbituril (CB)-based host-guest chemistry to achieve tuneable viscoelasticity in hydrogels. First, an ECM-mimetic CB-based biomaterial tuned to the viscoelasticity of the lung is developed for stem cell engraftment into the solid organs. The crosslinker designed with a MMP-cleavable sequence enabled the enzymatic and cell-mediated changes in the gel mechanical properties, supported the organoid culture, and facilitated the functional engraftment and differentiation of the lung stem cells in the mouse lungs, highlighting the importance of tuning the viscoelasticity and cell-responsiveness of stem cell-carrying biomaterials for engraftment into solid organs. The second work reports of biofabricating the flexible electronics through a parylene surfacemodification chemistry and a novel hybrid network consisting of CB and 1-benzyl-3vinylimidazolium (BVI) host-guest crosslinkers and gelatin-methacrylate (GelMA). The gel was designed to match the stress relaxation profile of the brain, facilitating a more brain ECM-mimetic biomaterial that is resistant to the high strain of the flexible neural devices. Combined with the ability to photo-pattern and combine with the different types of cells, this work presented the potential ways of multiplexing different types of biomaterials and cells to augment the bioelectronics functionalities, opening a new era of regenerative bioelectronics. The final work describes a new coumarin-based monomer that can be incorporated into the various free-radical-polymerised and-crosslinked materials to achieve reversibly phototuneable viscoelasticity. Harnessing coumarin’s UV wavelength-dependent cyclo-additionvi and-reversion, an extraordinary range ( 0.01 seconds to 1 million seconds) of stress relaxation half time (τ1/2) and storage modulus (100-10,000 Pa) were achieved with UV irradiation alone without changing the molecular composition. Unlike the previous approaches of directly functionalising the coumarin onto the polymer backbones, generating a CB-coumarin monomer provided a powerful platform to augment the existing materials to possess reversibly tuneable viscoelasticity.
- ItemEmbargoThe Exploration of a Strategy for Regioselective Arene Amination Utilising Non-Covalent InteractionsGillespie, JamesThe formation of arene C-N bonds for the preparation of anilines is one of the most used reactions in industry and academia. Numerous methods which utilise reactive N-centred radicals for the synthesis of anilines from arenes have recently been developed. However, regiochemical control is a major challenge associated with these methods, with mixtures of regioisomers commonly obtained in most protocols. Non-covalent interactions have been used in a limited number of examples to control regioselectivity in radical reactions. We therefore decided to investigate whether non covalent interactions between an arene substrate bearing a suitable directing group and an incoming N-centred radical could be a viable strategy to selectively target the arene ortho position. Initial investigations focussed on using hydrogen bonding interactions between substrate and radical to direct radical addition to the arene ortho position. Whilst promising reactivity was seen in many cases, the regiochemical outcome was poor. Later studies focussed on the use of anionic substrates and investigating whether ion pairing interactions with a cationic N-centred radical could direct ortho-selective radical addition. It was found that reacting anionic phenylsulfamate substrates with reagents which generate aminium radical cations resulted in an ortho-selective radical amination, allowing access to ortho-phenylenediamine products. This work was subsequently expanded upon to include arenesulfonates and arenecarboxylates as substrates for ion pair-directed ortho-selective amination. In these cases, the reaction proceeds via rearrangement of readily accessible O- (arenesulfonyl)hydroxylamines and O-benzoylhydroxylamines, respectively, and constitutes a remarkably facile way to access the ortho-aminated products. This work provides a blueprint for further development of regioselective amination reactions and more generally showcases the potential for non-covalent interactions to control regioselectivity in radical chemistry.
- ItemEmbargoRemodelling Surface Site Interaction PointsStorer, Maria ChiaraThe Surface Site Interaction Point (SSIP) model describes the non-covalent interaction properties of molecules by abstracting a discrete number of points on the van der Waals surface of molecules. Each point is assigned a value based on empirical interaction scales and the calculated Molecular Electrostatic Potential (MEP). SSIPs have been used to provide predictions of partition coefficients, solvent effects on association constants for formation of intermolecular complexes, and the probability of cocrystal formation. In this thesis, secondary electrostatic interactions are shown to be highly overestimated at the van der Waals surface and to be more accurately described on electron density isosurfaces that lie closer to the nuclei. Interaction parameters calculated with these isosurfaces successfully account for the properties of arrays of multiple H-bond donor and acceptor groups in different configurations. Three MEP isosurfaces are required to describe soft H-bond acceptors, hard H-bond acceptors, and H-bond donors. The Atomic Surface Site Interaction Point (AIP) model has been developed to obtain interaction points on these three surfaces using empirical rules for different atom types. This new approach to obtain interaction sites ensures a correct description of secondary electrostatic interactions, an accurate placement of lone pairs, a consistent description of π-systems and a representation of short H-bond contacts with hard acceptors but longer contacts with soft acceptors. Partition data between n-hexadecane and water was used to fine-tune the AIP representation of non-polar functional groups, for which it is difficult to obtain accurate empirical interaction parameters. The phase transfer data is also used to analyse the effect of H-bond cooperativity on the AIPs of functional groups that can make more than one H-bond, like alcohols, ethers and carbonyls. Finally, two methods for fast calculation of AIPs are discussed. The first method is a fragment-based approach, which assigns AIPs of large compounds from the AIP representation of small molecules with matching substructures. The second approach relies on a neural network method developed by Astex to quickly calculate the MEP surface and obtain the AIPs. These methods extend the scope of the AIP model to describing large molecules or large libraries of compounds for applications such as virtual screening and modelling host-guest systems.
- ItemOpen AccessDissipative Matsubara DynamicsPrada, Adam; Prada, Adam [0000-0003-3601-1265]The two most widely used path-integral chemical dynamics methods are Ring-polymer molecular dynamics (RPMD) and Centroid molecular dynamics (CMD). Both of these were originally proposed as heuristic approximations, which were justified by analytic limits and empirical evidence.In 2015, the Matsubara-dynamics theory was proposed as a controlled minimal approximation combining quantum statistics with classical dynamics, while conserving the quantum Boltzmann distribution. It was then shown that both RPMD and CMD are approximations to Matsubara dynamics, thus providing a firm theoretical ground for these methods. Unfortunately, a naive implementation of Matsubara dynamics is too computationally expensive to be a useful method because of a severe sign problem, which limited the number of Matsubara modes in a calculation to around 10 or less. Therefore, Matsubara dynamics has never been directly compared to exact quantum results with the exception of the harmonic oscillator and a linear correlation function cropped with a window function. In this work, we present results for up to 200 Matsubara modes and fully converged non-linear correlation functions. This was achieved by developing dissipative Matsubara dynamics — a way of implicitly including a bath of harmonic oscillators in a Matsubara dynamics simulation.We first show that the fact that the bath is harmonic allows its inclusion in the simulation without exacerbating the sign problem. Then we proceed to show that the presence of the bath even allows simulations of analytically continued Matsubara dynamics, which does not suffer from the sign problem but was previously impossible due to the presence of unstable trajectories. These simulations allow the inclusion of almost an order of magnitude more Matsubara modes than previously feasible. To further improve the stability, we introduce the “real-noise” approximation, which allows the simulation of up to ≈ 200 Matsubara modes. However, even this number is insufficient to converge non-linear operators. Therefore, we developed a harmonic correction for the tail of the Matsubara distribution, with which we were able to obtain converged non-linear correlation functions, which nearly perfectly match the exact quantum results. This is the first time such a comparison has been done and it provides a strong validation of the Matsubara-dynamics theory.
- ItemOpen AccessA Solid State NMR Investigation of Poly (ADP-ribose) and Its Involvement in Tissue CalcificationMurgoci, AdrianThe mechanisms of bone and vasculature biomineralisation are not fully understood. It is unclear how the calcium and phosphate ions are concentrated at the calcification site, and how the composition and structure of the mineral phase changes during maturation. This work aims to use solid state NMR spectroscopy to investigate the evolution of the mineral phase, and the interface between the apatite nanocrystals and the organic matrix at different time points on the mineralisation timescales in two in vitro models of biomineralisation, i.e. physiological bone mineralisation by MC3T3-E1 osteoblast-like cells (chapters 7 and 8) and pathological medial arterial calcification by bovine vascular smooth muscle cells (chapter 9). The advantage of solid state NMR to study biomineral in tissues is that it can be used on samples that are more or less in native state, without extensively dehydrating the tissue or removing the organic layers. However, in order to gain a holistic picture of the mineral at nanoscopic level, the calcified matrix samples studied by ssNMR have also been investigated by SEM/EDS and occasionally TEM, to explain the changes observed in the NMR spectra, and rationalise how the progression of mineralisation could occur in vivo. Another benefit of NMR spectroscopy is that no prior assumptions are needed about the composition of a sample to observe its components. The Duer group discovered that poly (ADP-ribose) (PAR), a biological polymer produced in response to DNA damage, is deposited in the calcified matrix of bone and pathologically mineralised arteries and could play an important role in mineralisation. PAR has affinity for calcium, forming “beads” that bind preferentially to the hole zones of collagen fibrils where mineralization is initiated. PAR mediates the biomimetic calcification of collagen fibrils in vitro in a periodic arrangement ofvi mineral density. The structure of PAR has been previously characterised by mass spectrometry following its isolation from different organs (but not calcified tissues like bones) via laborious and potentially damaging procedures. Chapter 6 aims to fully characterise the structure of poly (ADP-ribose) in 13C-enriched in vitro grown samples, without the requirement to extract it from its native environments, by determining the chemical shifts corresponding to the signals from the linear parts of the polymer, and potentially identify the chemical shifts of branching points and chain ends. Moreover, careful isotopic enrichment of cells and matrix with sugars or key amino acid residues help us define novel hypotheses about the interactions between collagen fibrils and PAR at a molecular level, and the involvement of the latter in the mineralisation of tissues (Chapter 8).
- ItemOpen AccessQuantitative analysis of protein liquid-liquid phase separationQi, Runzhang; Qi, Runzhang [0000-0002-7805-6501]Numerous biological proteins tend to demix into a protein-rich liquid condensate phase and a protein-poor dilution phase. This process of liquid–liquid phase separation (LLPS) has emerged as a crucial mechanism for describing the production of biological condensates in live cells, which have been found to be critical to a number of biological functions and activities. In this thesis, I have focused on the quantitative studies of protein LLPS. First, a microfluidic platform was developed for high-throughput characterisation of protein condensate phase diagrams. It was implemented by combining the technology of droplet microfluidics, fluorescent imaging, and quantitative computational analysis. This new approach, replacing manual step-wise experiments, enables to generate phase diagrams with minimal sample and time consumption as well as high resolution. It may also facilitate the research of protein condensates such as the effect of small molecules and further quantitative physical studies. Next, a convenient protein condensate model system was explored, with tunable sizes and dispersity using microfluidics. It was achieved by transferring the condensate formation process into a droplet-microfluidic-based solid microgel formation step, separating liquefaction to condensate formation in a crowding agent. Furthermore, the protein LLPS mechanism was investigated from the perspective of colloidal interactions. I developed an inverse Monte-Carlo based method to interpret smallangle X-ray scattering data for the purpose of fitting interaction potentials using Derjaguin- Landau-Verwey-Overbeek (DLVO) theory. It was achieved by constructing a Monte-Carlo model that can produce simulated scattering curves from interaction potential parameters, with a surrogate machine learning model that may produce comparable results within shorter time periods, and finding the best fit in the given parameter space. For outlook, it may be enhanced with an extended DLVO theory to include hydrophobic interactions as a key step towards elucidating protein colloidal interactions.
- ItemEmbargoThree-Dimensional Single-Molecule Localisation Microscopy and Visualisation TechniquesHanda, AnoushkaQuantitative imaging in complex biological samples requires techniques such as, super-resolution (SR) imaging to better understand the morphology and stoichiometry of proteins below the diffraction limit. 3 Dimensional (3D) SR tools such as the 3D double helix-point spread function (DH-PSF) and 3D single-molecule light field microscopy (SMLFM) are techniques which have an increased depth of field (4 µm and 8 µm respectively) and are capable of isotropic resolutions of 25 nm (DHPSF). This makes 3D SR highly compatible to investigate biological processes such as sub-synaptic diversity in a physiologically relevant environment. This thesis demonstrates the development of visualisation software for 3D SR and the development of 3D SR techniques to push the boundaries of thick biological sample imaging. Through developing virtual reality (VR) visualisation software for single-molecule localisation microscopy (SMLM), we were able to explore, segment, analyse and export data through an intuitive medium. This thesis presents the first DH-PSF brain tissue imaging to understand the organisation of a scaffolding protein known as postsynaptic density 95 (PSD95). PSD95 is known to form nanoclusters which make up the basic structural unit of an excitatory synapse, however, through the sensitivity of 3D DH-PSF imaging, we were able to observe the presence of diffuse PSD95 protein. We have developed a series of quantitative imaging analysis algorithms to compare the role of inter and intra synaptic diversity in the hippocampus. This will further contribute to understanding how the organisation of PSD95 can lead to schizophrenia, learning disabilities, and autism. Finally, 3D SMLFM is able to improve acquisition time, labelling density, optical sectioning and signal-to-noise ratio in comparison to 3D DH-PSF for biological imaging. In summary, this thesis shows the development of visualisation software (vLUME), improved depth of field imaging (SMLFM) and utilised DH-PSF imaging to understand the role of PSD95 nanocluster and diffuse protein organisation in the brain.
- ItemMetadata onlyCombining Epidemiology and Biophysics in Drug Repurposing for DementiaGeiser, Ryan[Restricted]
- ItemEmbargoNew Catalysts for Asymmetric Nitrene Transfer Based on Ion-Pairing Between Sulfonated Rhodium(II,II) Tetracarboxylates and Chiral Cations: Design, Synthesis and Applications to Enantioselective Carbon-Nitrogen Bond FormationFanourakis, AlexanderNew Catalysts for Asymmetric Nitrene Transfer Based on Ion-Pairing Between Sulfonated Rhodium(II,II) Tetracarboxylates and Chiral Cations: Design, Synthesis and Applications to Enantioselective Carbon-Nitrogen Bond Formation – Alexander Fanourakis The work presented in this thesis investigates a new strategy for the catalytic, asymmetric synthesis of C(sp3)–N bonds and is divided into three parts. First, the design and synthesis of a novel class of chiral rhodium(II,II) tetracarboxylate dimer is presented. Inspired by a design strategy previously developed within the Phipps research group for bipyridine ligands, di-sulfonated variants of the Rh2(esp)2 dimer are ion-paired with two chiral cations based on quaternised cinchona alkaloids. The modular catalyst design is used to construct a large library with the aim of exerting catalyst-control over selectivity in the ensuing nitrene-transfer reactions. In the second part, proof of concept using these dimers is achieved in a challenging intermolecular enantioselective benzylic C(sp3)–H amination which was developed working with Dr Benjamin D. Williams and Kieran J. Paterson. A range of aryl alcohols are efficiently aminated with up to 93% ee and this study highlights the importance of substrate-direction by the terminal alcohol group and lays the foundation for the next stage. The final part of this thesis describes a more general reaction mediated by these catalysts: an asymmetric aziridination of alkenyl alcohols. This substrate-directed transformation was developed with Nicholas J. Hodson and Arthur R. Lit and is broad across alkene substitutions with an empirical mnemonic developed to predict substrate performance. It is hoped that this second reaction will constitute a small step in advancing asymmetric aziridination to the same level of utility as the current asymmetric epoxidation protocols.
- ItemEmbargoEnhancing solar fuel synthesis via complementary approaches: round-the-clock operation and heat utilisationPornrungroj, Chanon; Pornrungroj, Chanon [0000-0001-9886-8489]The synthesis of fuels from sunlight offers a promising sustainable solution for chemical energy storage. However, the inefficient utilisation of the solar spectrum limits its commercial viability. Requiring high energy photons to drive the reaction, a large portion of the solar spectrum, particularly visible (Vis) and infrared (IR) energy, could not be converted for most solar fuel synthesis, and thus lost in the form of wasted heat. Apart from fundamental improvements to the (photo)catalyst materials and device architectures, solar fuel production systems can also be designed to improve their solar energy utilisation and their overall functionality by integrating complementary technologies. In this PhD thesis, three distinct complementary approaches were explored to tackle two main challenges in the solar fuel research community; (i) the intermittent nature of renewable energy sources which prevent a sustained and predictable fuel production output and (ii) thermal-related energy losses due to inefficient utilisation of the solar spectrum. The first approach describes an integrated device that in addition to possessing photoelectrochemical (PEC) water splitting functionality, also operates as an electrolyser in the nocturnal period to achieve a steady round-the-clock fuel production. The device is based on a perovskite-BiVO4 tandem PEC architecture. The light-absorbers are interfaced with a H2 evolution catalyst and a Co-based water oxidation catalyst, respectively, which can also be directly driven by electricity. The system provides a solar-to-hydrogen (STH) efficiency of 1.3% under simulated solar irradiation and an onset voltage for water electrolysis at 1.8 V. The second approach integrates a thermoelectric (TE) with a PEC device, where TE converts wasted heat into additional electricity. A range of integrated TE-PEC systems, which benefit from the reactor heating to bolster water splitting under concentrated light irradiation, were demonstrated. Unassisted water splitting can already be achieved under 2 sun irradiation by wiring a TE element to a BiVO4 photoanode, whereas the photocurrent is further increased by introducing a perovskite solar cell. The third approach describes a hybrid solar vapour generator (SVG) and a photocatalytic (PC) device, where the full solar spectrum is harvested via a light management strategy. By floating the device on aqueous feedstock, it absorbs UV light via the PC top layer, and the SVG synergistically utilises the remaining Vis-IR light. The SVG evaporates aqueous feedstock generating distilled water vapour, which is absorbed by the PC layer to drive the overall solar-driven water splitting with STH of 0.11-0.14%. Because the PC layer does not make direct contact with the aqueous feedstock which may contain contaminants, the device confers over 150 hours stability for operation in seawater and other waste streams while providing 0.95 Kg m−2 h−1 of purified water as a secondary product. This work provides an example of how two solar driven processes could be coupled to provide a more versatile approach toward practical solar fuel synthesis, especially in an area where clean water is scarce. These strategies can improve the efficiency, production rates, and practicality of existing solar fuel systems, and therefore the overall economics of solar fuel production. More broadly, the approaches highlight the necessary collaboration between materials science and engineering to help drive the adoption of a sustainable energy economy using existing technologies.
- ItemEmbargoSelf-Assembly of Tetra-Anilines into Metal-Organic ArchitecturesDavies, JackThe internal cavities of supramolecular capsules can be used to bind and sense guests, stabilize reactive molecules and catalyse chemical transformations. To improve the selectivity and scope of substrates for the aforementioned applications, design rules for capsules that extend beyond those of high-symmetry and moderate size are required. The work presented in this thesis aims to explore design strategies for the formation of new or under-explored structure types of metal-organic architectures. The structure types targeted are expected to provide interior spaces distinct from those provided by the current generation of metal-organic capsules with well-understood design principles. Previously under-explored rigid rectangular ligand panels provide the cornerstone for this work. In particular, the subcomponent self-assembly of tetra-aniline subcomponents with 2-formylpyridine and Zn(II) cations. First, it will be demonstrated that rectangular tetra-anilines, even those with relatively high aspect ratios (i.e. they show significant deviation from a square), can assemble into Zn₈L₆ pseudo-cubes. There are myriad possible diastereomeric configurations that could be adopted; however, remarkable diastereoselectivity is observed for each of the six tetra-anilines investigated. A thorough analysis of the solid state structures allowed the elucidation of guiding principles for the formation of Zn₈L₆ pseudo-cubes. The observed stereochemical configuration for each structure was rationalised by considering subcomponent aspect ratio and conformational flexibility. The second project presents the impact of incorporating a twist into the tetra-aniline subcomponent upon the product of self-assembly. A Zn₈L₆ pseudo-cube is a kinetic self-assembly product, while a much larger Zn₁₆L₁₂ tetrahedral capsule is the thermodynamically favoured product. The third project places the geometric design rules elucidated in project one in the context of heteroleptic (i.e. mixed-ligand) self-assembly. This work demonstrates that by selecting combinations of two-dimensional subcomponents with well-matched side lengths, mixed-ligand assemblies can be selectively prepared. Design rules underpinning the formation of heteroleptic prismatic architectures are explored. A Zn₆L₃L’₂ triangular prismatic structure type assembles from the combination of tetra- and tri-topic subcomponents, while mixing two different tetratopic subcomponents constructs a Zn₈L₂L’₄ tetragonal prism. Taken together, the presented work provides an insight into the potential of using geometry-based design strategies for the construction of new structure types of metal-organic architectures.
- ItemOpen AccessOptimisation of Gaussian process regressions of molecular potential energy surfacesAlbertani, FabioMachine learning methods applied to multi-dimensional surface learning pose some fundamental questions on the importance of the mathematical expression of the feature dimensions of the input space. Moreover, for Gaussian processes which are particularly popular in regression problems, the choice of an appropriate kernel function to construct the desired model is not straightforward. The feature spaces, onto which one projects molecular geometries, have many degrees of freedom and do not necessarily consider the physics of the problem. One then has to consider the consequence of the properties of the feature spaces onto the latent functions of the corresponding Gaussian processes. Moreover, for our particular interest of potential energy surface learning, one usually deals with sparsity in the training data given the high computational cost of ab initio electronic structure calculations. This sparsity creates instability in the models that a Bayesian approach to Gaussian processes creates and the expression of the training data on the feature space is thus important. This thesis covers three aspects of the learning process. Firstly, Gaussian processes that project the input molecular geometries onto spaces that have different properties. Secondly, the optimisation of a Morse projected feature space, which is controlled through its projection parameters. The latter can be selected either with a Bayesian optimisation approach or a separate optimisation. Finally, a different noise kernel that treats to move away from the usual homoscedastic noise treatment. The latter emerges from learning surface from stochastic electronic structure methods which provide, as well as energy data, error estimations of the calculations.
- ItemRestrictedInvestigating the aggregation of TDP-43 in models of Amyotrophic Lateral SclerosisMatharu, NaunehalTrans-Activation Response DNA-binding Protein 43 (TDP-43) is a major constituent of proteinaceous inclusions characteristic of most forms of amyotrophic lateral sclerosis (ALS) and ubiquitin-positive frontotemporal lobar degeneration (FTLD). Normally a nuclear protein, TDP-43 translocates to the cytoplasm and forms pathogenic inclusions in the disease-state, where the protein is often phosphorylated and cleaved and co-localises with stress granules. This thesis explores the features of TDP-43 pathology in established cell models using microscopy techniques to investigate the relationship of pathological aggregate species and cellular dysfunction. We have characterized stable cell models expressing EGFP-TDP-43 to identify key pathological hallmarks of disease at the molecular level, using confocal microscopy imaging. Using various chemical stress inducers to recapitulate disease pathology, we were able to induce TDP-43 aggregation and to characterise features of the self-assemblies within the cells. We further investigated the properties of these putative pathogenic aggregate species using FLIM (Fluorescence lifetime imaging microscopy) and demonstrate decreased fluorescence lifetimes of EGFP-tagged TDP-43 following MG132 stress induction and we correlated these data with an in vitro model system of liquid droplet formation. Using structured illumination microscopy (SIM) we detail the interactions of TDP-43 aggregate species with subcellular structures to begin investigating the relationship between the aggregate species and the cellular milleu. We demonstrate the use of SIM to enhance the use of mitochondria and lysosomes as markers to screen for TDP-43-induced cellular dysfunction. Finally, we describe the use of a novel cell model utilising a biarsenical dye system as a promising alternative to commonly used fluorescent proteins for studying TDP-43 dynamics. We demonstrate improved scope for this model by incorporating it into a more physiologically relevant cell line and introducing the A315T and M337V disease-related mutational variants. This model holds promise for studying the mechanisms underlining the cell-to-cell spread of pathology and to identify therapeutic targets.
- ItemEmbargoSoft matter engineering for cellular bioelectronic interfacesDenduluri, Akhila JyothyIn nature, interactions between living systems and charged entities occur across multiple scales (cells, tissues, organ systems). These interfaces between electrical and living systems, known as bioelectronics, can be leveraged to monitor and modulate biological functions. In partic- ular, investigation of electronic and ionic charge interactions at the individual cell level exposes singular behaviours otherwise obscured by an ensemble average. However, a key limitation is the availability of effective approaches that enable such investigations in a micro-environment mechanically and biologically conducive to individual cells. In this thesis, micron-scale soft-matter (droplets and microgels) were leveraged to develop two such cellular-bioelectronic interfaces. One such interface to leverage cellular charge generating behaviour was developed by in- tegrating commercial flow-cytometric cell-sorting with single-cell double-emulsions(DEs). This platform, known as photosynthetic-organism directed DE-FACS (podDE-FACS), enables rapid in- vestigation of cyanobacteria, microbes with electrogenic capabilities widely used as the biological source of anodic electrons in the development of renewable power-generation devices, known as biophotovoltaics (BPV). However, the efficiency of such devices is too low to translate into practical applications. With an aim to enhance BPV photocurrents through increased electron availability, podDE-FACS was developed to systematically identify rare, highly electrogenic cyanobacteria using Resazurin, a redox-sensitive fluorescent-marker. The platform was used for the isolation, recovery and growth of a previously-unknown highly-reducing cyanobacterial strain. Shifting the focus from charge generation to charge provision, a second soft-matter interface was developed to create a 3D cell-hosting electro-active platform. The fundamental challenge in interfacing cells with electrical set ups, such as electrodes, is the mechanical and dimensional mismatch between biotic-abiotic components. Recent advances in charge providing conducting biomaterials has seen chemical and structural innovation in reducing mechanical mismatch, however, the spatial and dimensional mismatch is yet to be addressed. A novel, bottom-up fabrication approach which enables co-assembly of cells with conducting hydrogel microparti- cles (HMPs) an organic conjugated polymer, PEDOT:PSS (poly(3,4-ethylenedioxythiophene) polystyrene-sulfonate). This new class of truly three-dimensional, electrically active cell-laden gels are not only micro-porous and injectable, but provide the basis for a 3D conducting cell-gel electrode that closely mimics native tissue micro-architecture.
- ItemEmbargoProtein and Peptide Solubility - In Silico and In Vitro ApproachesOeller, Marc; Oeller, Marc [0000-0003-0597-5950]Solubility is a crucial property to optimise in the development of peptides and proteins as therapeutic compounds. Biologics, which are drugs based on biological compounds such as proteins, inherently possess many beneficial properties such as low toxicity and high specificity. Often, however, they exhibit only limited solubility, which makes them unsuitable to be developed into drugs. Screening out molecules of insufficient solubility is a standard step used in drug development. Usually, this step is done in vitro, at a late stage in the process as it is expensive. In silico methods, at least in principle, offer the advantage of reducing the cost of this screening, while simultaneously eliminating unsuitable candidates much earlier in the process. One such method is CamSol , a sequence-based solubility predictor developed in the Centre for Misfolding Diseases, to assess and improve the solubility of proteins. The goal of this PhD was to develop a new method, built upon the foundations of CamSol, that predicts the solubility of proteins and peptides containing non-natural amino acids and to incorporate the effects of formulation pH. A particularly exciting aspect of this project concerning the translation into industrial applications, is that it was carried out in collaboration with AstraZeneca. The effects of pH on the solubility of proteins are well understood at least in outline, but there are no sequence-based methods that can reliably predict the solubility of biologics at varying pH values. Similarly, no prediction method is currently capable of assessing the effects of non-natural amino acids on the solubility of proteins. On the experimental side, a high-throughput, low material requirement in vitro method was developed to measure the solubility of proteins and peptides to verify the predictions. Our results show that the correlation between the predicted and measured solubilities is high, confirming that these new capabilities of CamSol are valid tools that can be employed to facilitate drug development.