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(2018)ThesisElectrospray ionisation (ESI) mass spectrometry (MS) has emerged as a central technique for protein sequence analysis. Protein analysis by mass spectrometry typically proceeds by either the (i) top-down method, where an intact protein is ionised and fragmented inside the mass spectrometer; or (ii) the bottom-up method, where a protein is chemically digested to form peptides, which are subsequently measured by liquid chromatography (LC)-MS. However, protein and peptide ions are usually formed in low charge states and are often not ionized efficiently, which reduces the performance of MS based techniques for protein identification. Using small chemical additives (e.g. 1,2-butylene carbonate), proteins and peptides can be ‘supercharged’ to significantly improve the performance of ESI mass spectrometry for protein analysis. For top-down protein analysis, the use of 1,2-butylene carbonate and ESI can result in the formation of protein ions in sufficiently high charge states that they can protonate atmospheric gases (e.g., N2, O2, and Ar) in room-temperature ion-molecule reactions. These results suggest that protein ion charging is limited by proton transfer reactions between multiply charged protein ions and atmospheric gases. By performing ESI at reduced pressure, protein ion charge states can be increased by over 40% compared to the use of conventional chemical supercharging. The most highly charged protein ions can transfer a proton to helium, which indicates that such ions are the most acidic entities isolated to date. For bottom-up protein analysis, a T-junction was used to introduced 1,2-hexylene carbonate into the LC eluent in LC-MS measurements to significantly increase peptide ion abundances. For a whole protein HeLa digest, the average ion abundances improved by ~5.5 times from 2.2 x106 (no additive) to 1.2 x 107 (1,2-hexylene carbonate). This results in a 100% and 50% increase in the respective number of peptide and protein identifications. Using 1,2-hexylene carbonate, peptides can be identified in sub-nanogram protein loadings. These data suggesting that 1,2-hexylene carbonate will be beneficial for improving bottom-up protein analysis by LC-MS.
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(2019)ThesisThis 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.
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(2019)ThesisAntimicrobial 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.
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(2018)ThesisTiO2 coatings were fabricated by anodizing unpolished or polished Ti6Al4V in 1 M H3PO4 or 1-3 M H2SO4 at room temperature at 120 V for 10-20 min and selected coatings were annealed at 300-500C for 8 h. Analyses included mineralogy (GAXRD, Raman), morphology (FESEM, AFM), topography (3D confocal microscopy), microstructure (FIB), chemistry (XPS), optical (UV-Vis), thermodynamic, human osteoblast-like cellular adhesion and proliferation, UV- or X-Ray-activated photocatalytic performance (MB degradation), UV- or X-Ray-activated antibacterial performance (Staphylococcus aureus). Anatase was the principal polymorph in the anodized TiO2 coatings although rutile was observed as a minor phase that resulted from dielectric breakdown and associated heating. The surface preparation of substrates determined not only the grain and pore sizes but also the morphology and topography of the coatings. Consequently, the presence of the amorphous TiO2 passivating layer, which is inherent to the unpolished substrates, resulted in thicker and more irregular anodized coatings compared to those of the anodized metal surfaces exposed by polishing. Large-scale delamination steps were observed in coatings on unpolished substrates while localized delaminations occurred on polished substrates with longer anodization times. 3D confocal microscopy allowed distinction between the fine-scale morphology (large open pores) and the coarse-scale topography (delaminations). These data showed that increasing time and acid concentration resulted in similar trends of increasing roughness (morphology) and unevenness (topography). The influence of the oxidation strength of the acid was pervasive in that it impacted on the crystallinity, microstructural homogeneity, coating thickness, Ti3+ concentration, gas generation during arcing to form pores, and resultant pore size and distribution density. The pores formed a subsurface network of variable continuity, which has a significant impact on the surface area and associated density of photocatalytically active sites, access by liquids and gases to the coating interior, penetration depth of incident radiation, gas condensation, and residual liquid trapping. These data and related thermodynamic analyses of the acids, anodization processes, and oxidation processes revealed that surface, bulk, and microstructural effects governed the photocatalytic performance. Nanostructural analysis allowed differentiation between fine-scale (small open pores from arcing) and coarse-scale (large open pores from arcing and oxidation eruptions) features. While the anodized coatings were biocompatible, cellular adhesion was dominated by the large pores and proliferation depended largely on the small pores. The anatase crystallinity was enhanced by increasing H2SO4 concentration, which increased the S-based species content, but most by annealing, which decreased it. Photocatalytic performance revealed the opposing effects of anatase crystallinity, which enhanced the performance, and blockage of active sites, which reduced the performance. The antibacterial performances against Staphylococcus aureus followed the same trends for unannealed coatings but the annealed coatings showed inferior performance owing to S depletion and associated reduction of availability for bacterial consumption. The performances of the X-irradiated coating were inferior to those of the UV-irradiated coatings because the absorption of the characteristic X-rays of the W source was very low.
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(2018)ThesisCampylobacter concisus is a Gram-negative bacterium that is associated with inflammatory bowel disease (IBD), due to its significantly higher prevalence in the intestinal tract of patients with IBD. IBD is a chronic inflammatory condition of the gastrointestinal tract with Crohn s disease (CD) and ulcerative colitis (UC) being the two major clinical forms. This thesis examined mobile genetic elements in C. concisus strains and their clinical relevance in IBD. The zonula occludens toxin (Zot) is a prophage encoded virulence factor in C. concisus. The detailed motifs of Campylobacter Zot proteins were found. Identification and comparison of zot-containing prophages from different Campylobacter species showed that they were individually acquired. The genomes of 63 oral C. concisus strains isolated from patients with IBD and healthy controls were examined, of which 38 genomes were sequenced in this thesis. A novel secreted enterotoxin B homologue, Csep1 was identified. The csep1 gene was presented in the pICON plasmid or the chromosome. A six-nucleotide insertion at the position 654-659 bp in csep1 (csep1-6bpi) was found, which was associated with active CD. The effects of immunosuppressive drugs azathioprine (AZA) and mercaptopurine (MP), and anti-inflammatory drug 5-aminosalicylic acid (5-ASA) on the growth of C. concisus and other enteric microbes were investigated. AZA and MP significantly inhibited the growth of all 11 C. concisus strains. Interestingly, 5-ASA showed inhibitory effect on some C. concisus strains, while it promoted the growth of other C. concisus strains. In summary, this thesis reports the identification of detailed motifs of Zot proteins and zot-containing prophages in different Campylobacter species and a novel molecular marker, the csep1-6bpi gene, which is associated with active CD. Furthermore, immunosuppressive drugs used for IBD treatment were shown to inhibit the growth of C. concisus strains, suggesting an additional therapeutic mechanism of these medications in treatment of IBD, which also explains why csep1-6bpi positive C. concisus strains were absent in CD patients in remission.
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(2018)ThesisRecently, resistive switching devices have emerged as promising candidates for next generation non-volatile memory and neuromorphic computing applications. In general, resistive switching device consists of a two-terminal metal-insulator-metal structure, in which metal oxide is widely employed as the insulator. Among a variety of metal oxides, SrTiO3 has attracted extensive attention owing to its superior physical and chemical properties. In this dissertation, novel SrTiO3 resistive switching devices through solution processed approaches have been developed and their electrical properties are tuned through engineering the defects and interfaces. The thesis includes the following parts: (1) Specific cation doping is utilized to modulate the electrical properties of SrTiO3 nanoparticles film. In Cr-doped SrTiO3 device, oxygen vacancies are induced since the Ti4+ is partly replaced by Cr3+, leading to reversible hysteresis loops upon application of voltage, while negligible resistance change is observed in the undoped device. (2) By insertion of a reduced graphene oxide layer between SrTiO3 and the bottom electrode, a transition from digital switching to analog switching is demonstrated. Typical potentiation and depression behaviours are implemented towards neuromorphic computing applications. (3) To explore the potential applications of silver nanowires as electrodes and artificial filaments, controlled fragmentation of silver nanowires has been realized through ultraviolet (UV)/ozone irradiation and a low-temperature annealing process. Based on the fragmented silver nanowire network, the device exhibits a reliable threshold switching effect with a selectivity of 5 × 105. (4) A facile sol-precipitation method is introduced to prepare SrTiO3 nanocubes and thin films. Using silver top electrode deposited by ink-jet printing, the device shows a typical bipolar switching behaviour. After deposition of silver nanowires onto the bottom electrode, unipolar switching with a high on/off ratio (~105) is demonstrated. This work provides facile and cost-effective solution-based methods to fabricate SrTiO3 devices for potential resistive switching applications. Meanwhile, the systematic study on modification of switching behaviour of SrTiO3 devices may give a better understanding of switching mechanism and offer ways to improve the device performance.
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(2019)ThesisBiomimetic 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.
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(2019)ThesisControl 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.
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(2019)ThesisThree 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.
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Fundamentals and Applications of Tandem Mass Spectrometry in Top-down and Bottom-up Protein Analysis(2019)ThesisOwing 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.