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  • (2019) Du, Haojin
    Thesis
    This work provides a systematic study on engineering nickel hydroxide-based nanomaterials with enhanced electrochemical properties for the applications of supercapacitor and overall water splitting catalyst. Silver nanowires (Ag NWs) and Ni(OH)2 nanosheets based hybrid materials were prepared, which can simultaneously realize bifunctions of flexible transparent electrodes and all-solid supercapacitors. Ag NWs provide high conductivity while ensuring flexibility of the electrodes. Ni(OH)2 also has a variety of functions, one of which can reduce the contact resistance by compacting Ag NWs, and the second one is to act as a collector as a pseudo-capacitor. Through the combination of two multifunctional materials, all-solid-state supercapacitor with high cycle stability and flexibility has been demonstrated, which may have potential applications for flexible supercapacitor applications. Bifunctional overall water splitting catalysts based on Ni(OH)2 nanomaterials have been fabricated by the corporation of the hydrothermal method with calcination, which can enhance both oxygen evolution reaction and hydrogen evolution reaction capability. The morphology and crystal structure of hexagonal Ni(OH)2 nanosheets at different temperatures were investigated. It has proved that the content of nickel is an important factor for the improvement of electrochemical activity. Graphene contains a large number of defects which provided sufficient electrochemical active sites and improve the adhesion of the Ni(OH)2 nucleation process to the graphene substrate. It solves the poor catalytic stability attributed to the weak adhesion between catalysts and the conductive substrate.

  • (2019) Kuppusamy, Rajesh
    Thesis
    Antimicrobial resistance is a major worldwide threat to public health and there is an urgent need for the development of novel antibacterial agents. This research project focused on the development of short cationic peptidomimetics that employ 3'-amino-[1, 1 '-biphenyl)-3-carboxylic acid and anthranilic acid backbones segregated by hydrophobic and cationic groups. The biphenyl peptidomimetic compounds showed that simple diaminoethanes and their respective guanidine cationic groups were sufficient to mimic lysine and arginine amino acids of natural antimicrobial peptides. The biphenyl backbone was important for antibacterial activity and tryptophan was important for bacterial cell membrane permeability. The most active compound showed good minimum inhibitory concentrations (MIC) against S. aureus (15.6 μM) and E.coli (7.8 μM) but was inactive against P. aeruginosa strain PA01. Based on these results, anthranilamide derivatives with tryptophan and simple amine cationic groups were developed. The anthranilamide peptidomimetic compounds showed that the guanidine group was important for good antibacterial activity against S. aureus (3.9 μM), E.coli (15.6 μM), and these compounds had low cytotoxicity (>100 μM). Active compounds disrupted 75% of established S. aureus biofilms. Biphenyl could be used as an alternative to naphthoyl groups to give hydrophobic groups to the mimetics. Increasing the net charge by adding lysine decreased antibacterial activity compared to compounds containing simple amine groups but improved the compound's cytotoxicity.Various alkyl-substituted guanidine compounds were investigated. Increasing the lipophilicity (adding alkyl groups) at the guanidine residues decreased antibacterial activity. Increasing the cationicity increased antibacterial activity against P. aeruginosa. The most active compound showed broad-spectrum antibacterial activity of against S. aureus (2.0 μM), E.coli (7.8 μM), and P. aeruginosa (32.0 μM). The active compounds at 4.0-8.0 μM showed significant disruption (55-77%) of preformed S. aureus biofilms and one compound at 15.6 μM disrupted 45% of E.coli biofilms. Peptidomimetics are promising future antibiotics. These compounds can potentially circumvent current antimicrobial resistance that is generated when bacteria produce biofilms.

  • (2019) Du, Eric
    Thesis
    Biomimetic scaffolds have been an area of great interest in recent years for cell culture materials. Self-assembled peptide hydrogels are a type of biomimetic material that has excellent potential in this field owing to its ease of modification and unique physical characteristics. As many of nature’s peptides contain various biological motifs, it is easy to incorporate these motifs into a peptide gelator sequence. In addition, self-assembled peptide hydrogels are chemically well-defined, and the resulting fibrous networks are reversible due to the noncovalent interactions that mediate the self-assembly process. The encompassing aim of this Thesis is to monitor and control cell growth in and on multicomponent self-assembled peptide hydrogels. This was achieved by synthesising a small library of peptides containing biological motifs and using them in multicomponent systems to mimic the extracellular matrix which is a combination of many fibre types. The gels that were formed were characterised for the physical characteristics which revealed co-assembly and destructive interactions between different combinations of gels. A concentration study of the gelators also elucidated interesting concentration dependent traits. The hydrogels were also used in a series of in vitro experiments whereby factorial combinations (that is, all possible combinations of factors) were used to culture fibroblasts and human embryonic stem cells in 2D and 3D which revealed that cells not only survived, but on some surfaces were able to proliferate and attach. A novel chemiluminescent hydrogel was also synthesised to monitor oxidative stress in cells as an alternative to current assays to monitor cell behaviour. The gelator was shown to not only be able to respond to oxidative burst in T cells, but also be able to respond to the classic glucose oxidase and horse radish peroxidase reaction. This gel, therefore, holds good potential in multiple biomedical applications.

  • (2019) Lizarme Salas, Yuvixza
    Thesis
    Control molecular conformation via fluorination is a novel strategy for optimising the potency and selectivity of biological target binding. This project investigated selective fluorination strategies to control the conformations of diverse but structurally-related drug candidates. Two are quorum sensing (QS) inhibitor candidates, and the other two are Alzheimer’s disease (AD) drug candidates. QS controls several functions including antibiotic resistance, hence disrupting QS systems through inhibitors is a potential strategy for new antimicrobial therapies. In this project, six different fluorinated analogues of each QS inhibitor candidates were synthesised. The fluorination patterns were predicted to control the shape of the analogues to pinpoint the optimal conformation for QS inhibitory (QSI) activity. The conformation of these analogues was investigated, followed by docking studies to predict the best conformation for QSI activity, and finally, QSI activities were tested in bacteria and reveals that the activities of all analogues were improved compared to their respective lead compounds. AD is associated with low levels of the neurotransmitter acetylcholine (ACh). Therefore, drugs that mimic ACh and bind to its receptors are desired to treat AD. An example is xanomeline, which has been shown to improve cognitive function and reduce neurodegeneration, however, it has poor selectivity. It was hypothesised that fluorination may deliver a molecule with improved receptor selectivity through conformational control. Therefore, six fluorinated analogues were synthesised. It reveals that these analogues have different conformation. However, the binding affinities of all analogues to all receptor tested were very similar to that of xanomeline. Inhibition of acetylcholinesterase is another strategy to combat ACh deficit. Piperine has AChE inhibitory activity and possesses a rigid shape due to the presence of double bonds. However, the double bonds can undergo photoisomerisation. It was hypothesised that replacing the double bonds with fluorine atoms may avoid the photoisomerisation problem whilst maintaining inhibitory activity. Therefore, a difluorinated analogue that mimics the rigid shape of piperine was synthesised. It was revealed that this analogue is photostable and inhibitory activity is improved. Overall, this research has contributed to the field of medicinal chemistry by developing a novel strategy for controlling the conformations of four drug candidates.

  • (2019) Barnett, Christopher
    Thesis
    Three main subsets of the imidazol-2-ylidene scaffold have been synthesised and their electronic properties have been investigated. The respective [PdBr2(iPrBenz)(L)] complexes, where L = the imidazol-2-ylidene, were prepared in order to use the 13C NMR chemical shift of the benzimidazol-2-ylidene fragment NCN as a measure of σ-electron donating ability of the imidazol-2-ylidene. The respective selenone adducts were also prepared, with the 77Se NMR chemical shift used as a measure of the π-electron accepting ability of the imidazol-2-ylidenes. The subsets of NHCs consisted of imidazol-2-ylidene derivatives with systematically altered substituents. The first subset, discussed in Chapter Two, contained consistent N-methyl substituents with various electron withdrawing and donating groups at the 4,5-positions. This chapter describes the synthesis of the NHCs and NHC precursors, as well as the preparation of the palladium and selenium probes, and discusses the findings. The second group, discussed in Chapter Three contained consistent 4,5-diprotio groups and systematically altered N-alkyl groups chosen to vary in steric bulk (methyl, ethyl, iso-propyl, tert-butyl, and adamantyl). This chapter briefly discusses the preparation of the precursors, then the preparation of the palladium and selenium probe species and discusses the results. The third group, discussed in Chapter Four, contained consistent N-mesityl substituents with various electron donating and withdrawing groups at the 4,5-positions. This chapter discusses the preparation of the NHCs and NHC precursors, as well as the palladium and selenium probes, and the results obtained from the probes. With derivatives in these three sets, the previously reported extremes of both σ-electron donating and π-electron withdrawing abilities for the imidazol-2-ylidene class were extended. A fourth minor subset, discussed in Chapter Five, was investigated designed to compare sterically similar scaffolds with distinct electronic properties. 1,3-Dimesityl-4-methylimidazol-2-ylidene was compared to 1,4-dimesityl-3-methyl-1,2,3-triazol-5-ylidene, using the same [PdBr2(iPrBenz)(L)] complexes and selenium adducts, as well as complexes of [IrCl(CO)2(L)], and [AlH3(L)], where L = the ligand. The information obtained using the electronic character probes was used in conjunction with established electronic parameters of the substituents to examine correlations to allow prediction of the electronic character of related species. The performance of a selection of the analysed imidazol-2-ylidenes and the 1,2,3-triazol-5-ylidene was investigated in two organocatalytic reactions: the formation of a γ–butyrolactone from trans-cinnamaldehyde and para-bromobenzaldehyde, and the formation of a spiro-γ-butyrolactone from trans-cinnamaldehyde and 1,2-cyclohexanedione, and these results were rationalised through consideration of the quantified electronic characteristics.

  • (2019) Wang, Huixin
    Thesis
    Owing to the development of electrospray ionization (ESI), various ion dissociation techniques and software algorithms, mass spectrometry has become an indispensable tool for protein analysis. However, new approaches are needed to overcome some of the challenges in protein sequencing, including in the identification of post-translational modifications (PTMs), and in the analysis of protein complexes and protein-protein interaction networks. For PTM identification, commonly used database search algorithms for bottom-up proteomics often fail to identify peptides with unexpected PTMs due to their relatively low abundances and the absence of such modifications in the PTM databases. However, such unexpected PTMs can have important roles in biological functions. Here, we report a novel mass spectrometry method to identify the proteins targets of organophosphate (OP) insecticides in a non-targeted fashion. This method integrates a high-resolution twin-ion metabolite extraction program with Mascot database searching. Using this method, transmethylation was identified as a new reaction pathway for OP insecticides, in addition to the well-known phosphorylation modification that causes acute toxicity. Our results show that this method can be used for the reliable identification of unknown PTMs in complicated biomatrices which may ultimately benefit the discovery of protein biomarkers for a variety of conditions. For protein sequence identification, collision induced dissociation (CID) is the most widely used dissociation technique. In the CID of intact proteins, the fragmentation patterns of the protein ion depend strongly on the protein charge state. An optimal charge state can generate selective fragmentation resulting in a limited number of sequence ions in high abundances which can be useful for protein identification with high sensitivity. However, an accurate model to predict the fragmentation patterns of particular charge states is needed. Here, we report an approach to predict the specific cleavage sites of intact protein ions upon CID by use of an improved electrostatic model for calculating the proton configurations of protein ions at different charge states. The origin of highly selective cleavage sites in the CID of highly charged proteins ions is investigated using an improved electrostatic model, molecular dynamics and hybrid ONIOM simulations. The ONIOM results indicate that the protons located at low-basicity amino acid residues can dramatically reduce the reaction barrier to the cleavages at such amide bonds. The results from our electrostatic model suggest that unlike peptide ions at relatively high charge states, protons at low-basicity amino acid residue sites are electrostatically confined within a relatively narrow range of amino acid residues. Such confined protons can ‘trigger’ the fragmentation at the specific peptide bonds. Our model can potentially be used to predict the specific charge states that yield either specific sequence ions in high abundances, or fragment extensively for optimal protein sequence coverage. Recently, higher energy collision induced dissociation (HCD) has become available for the LTQ Orbitrap. There are some reports in the literature that demonstrate that HCD benefits the de novo sequencing of proteins and the identification of PTM sites. However, studies investigating the potential use of HCD for intact protein sequencing are relatively rare. Here, we systemically compared the performance of HCD and CID for intact protein analysis. Our results indicate that HCD yields significant performance gains compared to CID for obtaining high sequence coverage at relatively low charge states owing to higher ion trapping efficiencies and higher ion collision energies. The origin of the highly specific cleavages in the HCD of highly charged protein ions was investigated. We found that for HCD, highly specific fragmentation sites occur near the first sites that low-basicity amino acids are predicted to be protonated with increasing charge states, which is consistent with the mechanism for the formation of highly specific fragmentation of protein ions in CID. This result provides additional evidence that the fragmentation patterns of highly charged protein ions can be predicted to improve protein identification. Solution-phase labelling experiments have been increasingly used in combination with top-down proteomics to rapidly obtain structural information. However, the low charge state of protein ions formed from native solutions usually result in low sequence coverage which limits spatial resolution. Here, we demonstrate that theta-capillary nanoelectrospray ionization can be used to form protein ions with the highest known charge densities to date from native-like solutions by use of alkyl cyclic carbonate “supercharging” additives. It is anticipated this approach will be particularly promising for top-down hydrogen-deuterium exchange mass spectrometry to obtain significantly more protein structural information in native solutions than by use of more conventional approaches.

  • (2019) Ng, Anuja
    Thesis
    This thesis investigated the relationship between future-directed intentions and emotions. Predictions were made in line with both conditioning and expectancy-based accounts of emotion. The first question was whether the intention to prevent an aversive outcome down-regulates anxiety, even prior to performance of the prevention behaviour. In a series of experimental tasks (Chapters 3 and 4), participants learned that certain stimuli predicted either shock or no-shock, and a response was available to prevent shock for one stimulus. Participants formed an intention to prevent the shock on some trials but not others. The results indicated that anxiety, as indexed by skin conductance levels (SCL), and shock expectancy, was higher when participants intended to prevent the shock, compared to when they intended not to prevent the shock. Further, anxiety, indexed by SCL and self-report, was lower when participants had high versus low self-efficacy for the prevention behaviour. The second question explored was whether the intention to abstain from a desired outcome down-regulates desire. In an experimental task (Chapter 5), participants were permitted to use their mobile phones recreationally at certain times. On some trials, participants intended to use their phone while on others, they intended not to. Participants rated their desire for mobile phone use at the start of each trial and immediately after they had formed intentions with respect to use. The results indicated that desire decreased following an intention to abstain from phone use and increased following an intention to use. Results in the final experiments (Chapter 6) indicated that even when participants were instructed to abstain from phone use, desire was higher with greater phone accessibility, and this appeared to be a cue-elicited effect. The experiments in this project demonstrate that intentions can elicit emotional changes even prior to performance of the intended behaviour and that factors such as self-efficacy and accessibility can moderate these changes. These findings have theoretical and practical implications for emotion regulation and highlight how intentions can themselves be effective strategies for down-regulating emotions.

  • (2019) Zhao, Manchen
    Thesis
    Understanding protein-protein interactions is important in the fields of biochemistry, molecular biology, clinical diagnostics and drug discovery. However, conventional study assays require many analytes on the biointerface to give a single averaged output. As a single signal constitutes the measurement, the main challenge is non-specific adsorption of other species, leading to the sophisticated design of the biointerface to repel non-specific species adsorption with molecular level control. Hence, such knowledge, like how protein-protein interactions behave on the biointerface and how well the biointerface works at a single-molecule level, is valuable to exploit. The goal of this thesis is to establish a novel biosensing platform to realize surface characterisation and quantitative monitoring of molecular interactions simultaneously. By employing super-resolution techniques, like single molecule localization microscopy (SMLM), individual molecules can be localized with a spatial resolution of 20 nm, allowing a greater density of single molecules being detected simultaneously. The strategy to achieve this goal is described here. Firstly, a low background surface for SMLM, poly-L-lysine grafted polyethylene glycol surface, was developed that limited non-specific protein adsorption even after many modification steps, satisfying the criteria to study bioaffinity reactions with SMLM. Secondly, with BSA and anti-BSA as the study model, dual-colour SMLM with a further physical analysis enabled to quantitatively monitor many antigen/antibody interactions and distinguish specific binding events from non-specific adsorption. Furthermore, the low proportion of specific binding events on the surfaces led to a study and comparison that revealed some factors that possibly affected the performance of biointerfaces with different interfacial designs or imaging parameters. Finally, as an important aspect of protein-protein interactions, protein stoichiometry could be also obtained from SMLM imaging, showing hemagglutinin (HA) tagged mEos2 was monomer and HA-mEos2-Folden was assembled into trimer when they were pulled down from cell lysates. These studies illustrated SMLM, an imaging tool as well as a quantitative analytical platform, to characterise surfaces and study molecular interactions simultaneously, to provide guidance for the design of new biointerfaces and even study protein stoichiometry, by determining one cluster of detected events acquired in SMLM from one single molecule or several closely spaced molecules.

  • (2019) Tan, Rui Ping Amanda
    Thesis
    NEGR1 is a cell adhesion molecule present within synapses at the pre- and postsynaptic terminals, making it likely to play a role in synaptogenesis and synaptic neurotransmission. NEGR1 has been associated with obesity as well as various neurodevelopmental disorders. In this thesis, we study the effect of NEGR1-overexpression as well as NEGR1 silencing in cortical and hypothalamic neurons. Our results indicate that NEGR1-overexpression reduces synapse formation in cortical and hypothalamic neurons. Numbers of spines and filopodia along the dendrites of NEGR1-overexpressing cortical neurons are reduced. Repetitive stimulation results in reduced synaptic exocytosis with subsequent stimulation in axons of NEGR1-overexpressing cortical neurons. Concurrently, NEGR1-overexpressing neurons present with increasing rates of endocytosis across subsequent stimulations. Neurons overexpressing NEGR1 show lower levels of synaptophysin in synaptic boutons suggesting a reduction in the pool of synaptic vesicles available for exocytosis. Levels of CSP, a synaptic chaperone protein involved in chaperoning SNARE proteins, are reduced in synapses of NEGR1-overexpressing neurons, suggesting that NEGR1-overexpression potentiates the degradation of CSP. NEGR1 silencing with NEGR1 siRNA-transfection in cortical neurons also results in reduced synapse formation. Actin polymerization and dendritic spine and filopodium formation along dendrites of NEGR1-deficient neurons are reduced. The pool of synaptic vesicles available for exocytosis in axons of NEGR1 siRNA-transfected neurons is reduced and repetitive stimulation results in reduced exocytosis and endocytosis of synaptic vesicles during subsequent stimulations. Synaptic levels of CSP and its substrate SNAP25 are reduced in NEGR1 silenced neurons. Furthermore, NEGR1 knock down causes the synaptic activity-dependent reduction in the synaptic pool of CSP and synaptophysin. Hence, we propose that NEGR1 regulates targeting of CSP to synaptic vesicle, most likely, by regulating CSP palmitoylation, which plays a key role in targeting of CSP to synaptic vesicles. Our results show that NEGR1 has an important role in the regulation of synaptogenesis and synaptic vesicle recycling with both processes being important in neuronal development and continual synaptic maintenance. Future research will investigate the role that NEGR1 plays in regulating palmitoylation of CSP and other synaptic substrates and changes in this regulation in neurodevelopmental and neurodegenerative disorders.

  • (2019) Curry-Hyde, Ashton
    Thesis
    Current comprehension of circRNAs is relatively limited as compared to the extensive body of research available on other well-known coding and non-coding RNAs. CircRNAs have been implicated in gene regulatory functions and have been reported to display endogenous translational capacities. Current research demonstrates the conservation of circRNAs in mammalian brains, with humans having the most complex circular transcriptome currently known. In addition, expanding circular transcriptomes in ageing brains demonstrates the increase in circular network complexity alongside increasing complexity in maturing brain neural networks. Combined, the lack of knowledge of circRNAs could prove to be an understudied, and potentially fundamental, basis for the comprehension of brain evolution through time and amongst species. This research identified four conserved circRNAs between human and mouse cortex where 760 and 60 circRNAs had been discovered respectively; a fact that is indicative not only of some level of circular conservation between the mammalian brains, but also the potential impact of the independent circular transcriptomes on the evolution of the human brain as it is known today. The common circular transcriptome determined in human oligodendrocytes and neurons indicates a moderately interconnected basis of brain function and development; however, the distinct differences in cirexons being predominantly already annotated linear mRNA exons in the human cortex, compared to the high proportion of intronic/intergenic circRNA fragments comprising cirexons in human cell lines, could provide a novel distinct comprehension of human brain function and evolution through time. Current research of circRNA functions have not yielded results that provide a substantive understanding of their function. To the extent that this understanding remains elusive, it provides an incomplete foundation upon which to base interpretations of the reults from this research. Nevertheless, what is distinct is the increase in circRNAs in human brains and the postulated role of circRNAs being gene regulators, implicating circRNAs as definitive in human brain evolution with regard to functional capacity and complexity as compared to other mammals.