School of Chemistry - Theses
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ItemImaging the interactions of antimicrobial peptides with model and living cellular membrane systems( 2016)Since the discovery of penicillin over 80 years, the use of antimicrobial compounds has undoubtedly been one of the greatest contributions to modern science and medicine. Due to their use, misuse, and abuse over this time, numerous bacterial species have developed countermeasures to resist the effects of these antimicrobials. This rise in antimicrobial resistance has continued unabated since their first use, and now has the potential for devastating costs to humanity on a global scale; notably, mortality rates well into the millions over the coming decades. In turn, this has sparked interest in new therapeutic options not based on currently existing compounds. Antimicrobial Peptides (AMPs) are one such class of compounds. They can be found in virtually all forms of life, and act as the first-line- of-defence against a broad spectrum of pathogens at very low concentrations with limited resistance capacity. This makes AMPs extremely lucrative for future therapeutic design. Despite extensive investigations into their interactions, detailed mechanistic information about their behaviour with membranes remains elusive. Numerous models have been proposed to account for the observed behaviour of cell death, including pore-formation, detergent action, or some combination of the two. Mechanistic information regarding the progression of these models remains limited, however. In this work, we investigate the mechanistic behaviour of a model AMP with artificial and living bacterial membranes, primarily through the means of time- resolved fluorescence microscopy techniques. The antimicrobial peptide under study is an analogue of melittin; a well-characterised lytic peptide that displays significant activity to both eukaryotic and prokaryotic membranes. The analogue also bears a proline-to-lysine substitute around the centre of the peptide sequence, to which the fluorescent probe Alexa Fluor 430 is grafted. Using known photophysical information of the fluorescent peptide, information regarding the mechanistic progression of its interaction with membranes can be observed and deconvolved. This research progresses from developing a simple mechanistic understanding using model membrane systems, to increasingly complex living systems. In the liposome giant vesicle model, a slow two-step pore-formation mechanism was found to fit to the observed data, with major significance on the roles of aggregates. Leakage studies using the same liposome system found that the same pore-forming mechanism occurred in both unilamellar and multilamellar membranes, but with dramatically reduced pore stability in the latter. Similar experiments on E. coli bacteria revealed a more complex interaction that could not be adequately explained using either model systems. This finding has important ramifications for correlating information gained from model studies to living membranes. Finally, peptide interactions with Pseudomonas aeruginosa L-Forms were investigated in detail for the first time using super-resolution and fluorescence lifetime microscopies. The use of a biologically relevant environment was found to drastically alter the observed outcome of the peptide-membrane interaction, although the importance of peptide aggregates remains. Furthermore, little change in the fluorescence lifetime is observed over time, despite a clear time-resolved killing mechanism. The reasons for this, and the potential importance of the L-Form state, are explored in detail.
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ItemInvestigating the structure and dynamics of DNA with fluorescence and computational techniques( 2014)Nucleic acids, such as DNA, play an essential role in all known forms of life; however, despite their fundamental importance, there is still a significant lack of understanding surrounding their functional behaviour. This thesis explores the structure and dynamics of DNA by employing methods based on fluorescence and through the use of computational calculations. Time-resolved fluorescence experiments have been performed on dinucleotides containing 2-aminopurine (2AP) in various alcohol-water mixtures. 2AP, a fluorescent analogue of the nucleobase adenine, has been used extensively to investigate nucleic acids because of its ability to be incorporated into their structures with minimal perturbation and its high sensitivity to its local environment. Direct solvent effects on 2AP were established through measurements on the free fluorophore. Analysis of the complex fluorescence decays associated with the dinucleotides was challenging but has provided insight into their conformational dynamics. Solvent polarity was found to play a significant role in determining both photophysical and conformational properties in these systems. The complicated fluorescence decay of 2AP in nucleic acids highlights the need for accurate and unbiased analysis methods. Various time-resolved fluorescence analysis methods, including iterative reconvolution and the exponential series method, have been investigated with real and simulated data to obtain an overview of their benefits and limitations. The main outcome of the evaluation is that no single method is preferred in all situations and there is likely to be value in using a combination when there is ambiguity in the interpretation of the results. Regardless of the analysis technique used, the parameterised description of the observed fluorescence decay is meaningless if the underlying physical model is unrealistic. The advance of computational methods has provided a new means to rigorously test the viability of proposed models. Calculations have been performed at the M06-2X/6-31+G(d) level of theory to investigate the stability of 2AP-containing dinucleotides in conformations similar to those observed in the double-helical structure of DNA. The results help to explain the similarity of the time-resolved fluorescence behaviour of 2AP in dinucleotide and DNA systems but also bring to light subtle differences that could perhaps account for experimental discrepancies. The recent emergence of advanced optical microscopy techniques has offered the prospect of being able to directly visualise nucleic acid structure at the nanoscale but, unfortunately, limitations of existing labelling methods have hindered delivery of this potential. To address this issue, a novel strategy has been used to introduce reversible fluorescence photoswitching into DNA at high label density. Photophysical studies have implicated aggregation and energy-transfer as possible quenching mechanisms in this system, which could be detrimental to its future application. The reliability of fluorescence photoswitching was investigated at ensemble and single-molecule level and by performing optical lock-in detection imaging. These developments lay the foundations for improved and sequence-specific super-resolution microscopy of DNA, which could offer new insights into the 3D nanoscale structure of this remarkable biopolymer. In summary, the work presented in this thesis outlines important observations and developments that have been made in the study of the structure and dynamics of nucleic acids.
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ItemAssessment of perylene-based profluorescent nitroxides for monitoring polyester degradation upon weathering( 2013)A profluorescent nitroxide possessing an isoindoline nitroxide moiety linked to a perylene fluorophore was developed to monitor radical mediated degradation of melamine-formaldehyde crosslinked polyester coil coatings in an industry standard accelerated weathering tester. Trapping of polyester derived radicals (most likely C-radicals) that are generated during polymer degradation leads to fluorescent closed-shell alkoxy amines. Time-dependent degradation profiles were generated to assess the relative stability of different polyesters towards weathering. The nitroxide probe couples excellent thermal stability and satisfactory photostability with high sensitivity and enables detection of free radical damage in polyesters under conditions that mimic exposure to the environment on a time scale of hours rather than months or years required by other testing methods. There are indications that the profluorescent nitroxide undergoes partial photodegradation in the absence of polymer derived radicals. It was also found that UV-induced fragmentation of the NO-C bond in closed-shell alkoxy amines leads to regeneration of the profluorescent nitroxide and the respective C-radical. The maximum fluorescence intensity that could be achieved with a given probe concentration is therefore not only determined by the amount of polyester radicals formed during accelerated weathering, but also by the light-driven side reactions of the profluorescent nitroxide and the corresponding alkoxy amine radical trapping products. Studies to determine the optimum probe concentration in the polymer matrix revealed that aggregation and reabsorption effects lowered the fluorescence intensity at higher concentrations of the profluorescent nitroxide, but lower probe concentrations, where these effects would be avoided, were not sufficient to trap the amount of polyester radicals formed upon weathering. The optimized experimental conditions were used to assess the impact of temperature and UV irradiance on polymer degradation during accelerated weathering.
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ItemSynthesis and application of molecular probes for biologically important metal ions( 2012)The synthesis and characterization of new molecular probes aimed towards studies with metal ions in biology is described. One synthetic platform involves 1,2-phenylenediamine-based ligands which are divided into two classes. The first consists of ligands with carboxylic acid functional groups. These resemble the classic hexadentate ligand H4Edta. Previous studies demonstrated that functional groups substituted at the aromatic backbone affect the binding affinities for metal ions. This proves a way of preparing a family of ligands with similar coordination properties but different binding affinities. Interesting cobalt complexes were characterised by single crystal X-ray diffraction studies. Two cobalt atoms, each complexed to one ligand in a single crystal of one of the complexes, had different coordination geometries. A fluorescent group Bodipy was successfully attached to the aromatic backbone to make a new water soluble probe which exhibited a 2-fold fluorescence enhancement in presence of Zn(II). A second class of 1,2-phenylenediamine-based ligands was established by preparation of aminopyridine ligands, which were designed to optimize selectivity for Cu(II) and Cu(I) ions. A particular focus was the length of the linker to the pyridine arms for flexible coordination around the metal centre, with a particular Cu(II) complex demonstrating the point. Interesting redox behaviours were observed in the preparation of copper complexes. One ligand H2L binds Cu(I) to form salt [CuI(H2L)]BF4 which undergoes aerial oxidation in solution. A diamagnetic salt [CuIIL•-]BF4 was isolated where π-radical anion L•- is the (1e-, 2H+) oxidation product of H2L. This analysis was confirmed by a number of techniques including monitoring of the oxidation process by electrochemistry and electronic and NMR spectroscopies. The second synthetic platform focused on bis(thiosemicarbazone) compounds. New water soluble ligands were prepared by selective transamination reactions using sulfanilic acid as the transaminating group. Studies with the new ligands revealed that good water solubility, 1:1 binding stoichiometry and the ability of ligands to form chromophoric species with Zn(II) with buffered solutions make them potential molecular probes for studies with this ion. An affinity of KD = 6.0 x 10-9 M-1 for Zn(II) was determined for one ligand Na[H2L7] via competition assays with H2Egta2-. The new ligand was applied in estimating the binding affinities of two plant proteins.
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ItemSynthesis and study of organic light emitting materials( 2011)Since the discovery of electroluminesence from organic materials, there has been a considerable amount of research, time and efforts devoted to developing conjugated materials as the active units in light emitting devices for use in display applications. This thesis describes the synthesis and study of organic materials to achieve high efficiency in organic light emitting devices (OLEDs). A number of blue fluorescent and green to blue phosphorescent host polymers based on dibenzophosphole oxides have been synthesized and studied. Effect of the structure of polymer backbone and different substituents on optical properties has been studied and described. In addition, the synthesis and the study of polymers based on phosphine oxides have been described which are potential charge-transporting high triplet energy materials. All these polymers are thermally stable, blue fluorescent, potential hosts for green and blue phosphorescent guests and expected to facilitate charge transport in OLEDs.