School of Chemistry - Theses
Permanent URI for this collection
Search Results
1 - 2 of 2
-
ItemExploring novel blue turn-on fluorescent probes for the direct detection of nitric oxide and free radicals in living cells( 2017)Bacterial biofilms are causing considerable damage to different areas in industry such as food industry, oil industry and dentistry. Traditional methods to control biofilm formation and to treat the surfaces affected by these microorganisms has mostly focused on biocidal and antibacterial strategies. The drawbacks of these approaches is related to the development of tolerances that decrease effectiveness of chemicals apply to eradicate these microorganisms. The growth of biofilms therefore is linked to a significant adaptation by bacteria cells to control changes in their environment. In this regard, the development of efficient methods to control biofilms formation as well as their irreversible eradication from affected surfaces is an important area of scientific research. Bacterial biofilms at times undergo regulated and coordinated dispersal events, where sessile biofilm cells convert to free-swimming, planktonic bacteria. Nitric oxide (NO) is an important biochemical signalling molecule that has been linked to the inhibition of biofilm formation and activation of dispersal through the generation of nitrosative and oxidative stress. Therefore, the availability of methods that enable sensing and visualizing NO is critical to reveal details of the biological functioning of this molecule. Knowledge of these will provide important guidelines for the development of strategies to combat biofilm formation. In this thesis two different approaches for detecting NO and oxidative stress were explored, that are based on fluorescence measurements using coumarin as fluorophore. The first strategy explores “turn-on” fluorescence for direct detection of endogenously produced NO. A family of five blue fluorescent probes CB1-5 were designed and synthesized and the photophysical properties studied in detail. These probes feature a substituted 7-hydroxy coumarin chromophore coupled to 2-methyl-8-aminoquinoline, which act as tridentate ligand for Cu(II) and active site for monitoring NO using the replacement strategy. The UV-vis absorption and fluorescence emission characteristics of the probes are significantly influenced by the substitution pattern on the coumarin ring, as well as by solvent polarity and pH. Time-dependent Density Functional Theory (TD-DFT) calculations for CB4 and CB5 showed that the absorptions are due to π ® π* transitions localised on the coumarin system, with a small charge transfer contribution from the quinoline system at higher pH where the 7-hydroxycoumarin moiety is deprotonated. Complexation of the probes with Cu(II) leads to fluorescence quenching, which switches back on upon reaction with NO. In vitro studies revealed that the probes detect NO with high selectivity in nM concentrations and do not respond to other oxidizing species. In vivo studies for CB4 and CB5 showed that these probes enable detection of NO in living bacterial cells in multi-dye imaging experiments. Furthermore, CB5 also enables to detect NO in macrophages, where it is an important effector molecule in host defence against bacterial pathogens. Using confocal microscopy, it was shown that the probe can be trapped by the cells and reacts directly and specifically with NO, rendering it a promising tool for imaging NO in response to pharmacological agents that modulate its level, for example during bacterial infections. The second strategy explored in this work was the development of a profluorescent nitroxide probe, which can be utilized for detecting the formation of reactive nitrogen and oxygen intermediates and associated changes in redox states within microcolonies. Attachment of a nitroxide to a fluorophore leads to fluorescence quenching, which upon free radical scavenging, metabolism or redox processes, returns the molecule to its native fluorescent state. A large variety of synthetic approaches and procedures were explored to construct such structure, but unfortunately none of them were successful.
-
ItemPhotophysical studies of 2-aminopurine in DNA( 2017)Deoxyribonucleic acid (DNA) forms the basis of all known living organisms. Despite the essential role played by DNA, its dynamic system and functional behaviour are still not completely understood. The work presented in this thesis aims to explore the structural dynamics of DNA systems, using fluorescence-based approaches, and to attempt to develop a technique for the measurement of fluorescence decays of biological molecules on the ultrafast (femtosecond) timescale. Absorption of UV radiation by DNA is known to lead to mutations and damage to DNA structure and functionality. For the majority of absorbed photons, the excitation energy dissipates harmlessly as heat, but in some instances this energy transfers to regions of DNA that are more susceptible to damage. 2-Aminopurine (2AP), a fluorescent analogue of the native DNA base adenine, can be incorporated into DNA with minimal perturbation to the DNA structure, and can be used to investigate inter-base electronic energy transfer. By selectively exciting the native DNA base in 2AP-containing dinucleotides and utilising 2AP fluorescence as an energy acceptor, the mechanism of electronic energy transfer has been investigated. Analysis of the resulting fluorescence lifetimes of 2AP has revealed that energy transfer preferentially excites conformations in which the bases are highly stacked, and the fluorescence of 2AP is highly quenched. This has led to a re-evaluation of energy transfer efficiencies between the natural bases and 2AP, and has shown that transfer efficiencies cannot be determined correctly from steady-state fluorescence measurements. To investigate the influence of base dynamics on the quenching of 2AP fluorescence in DNA, time-resolved fluorescence measurements were carried out on 2AP-containing systems in frozen solution at 77 K. These studies included dinucleotides, single–strand oligonucleotides and their corresponding duplexes. In all cases, comparison of the fluorescence decay parameters measured at room temperature with those measured at 77 K showed that elimination of base dynamics prevented rapid quenching, on the 10s of ps timescale or faster, although quenching on the 100s of ps timescale persisted for 2AP in single strands and duplexes. The multi-exponential fluorescence decay of 2AP in DNA and its high sensitivity to local environment is commonly exploited to investigate DNA-enzyme interactions. Transposases are enzymes involved in the movement of sections of DNA (transposons) within the genome. The Mos1 transposase catalyses the movement of a transposon via a cut-and-paste mechanism involving several intermediate complexes. Understanding the complex mechanism by which the transposase can remove and insert a section of DNA would allow these enzymes to be used as biomolecular tools. The structure of the intermediate Mos1 strand-transfer complex (STC) has been investigated by incorporating 2AP into several regions of the transposon and analysing the fluorescence decay. The involvement of a base-flipping-like mechanism has been identified in the mechanism of strand transfer for the Mos1 transposon. The time-resolved fluorescence measurements performed in this thesis are limited to time resolution of ~20 ps and longer using TSCPC. However, an abundance of photophysical events in DNA occur on the femtosecond timescale. Development of a methodology utilising fluorescence gating techniques (such as sum-frequency generation or diffraction from a transient grating) have been attempted, in order to construct an experimental system that enables the broadband detection of ultrafast fluorescence decays. Despite the lack of immediate success in recording the fluorescence decay from a sample, due to technical issues and time-constraints, initial characterisation of the set-up was performed and the prospect of broadband detection was demonstrated. Overall, this thesis gives insight into some of the dynamic processes taking place in DNA and presents work performed to develop a system that would allow the extension of these studies to processes occurring on the fs timescale.