No description available.

This thesis describes the development and application of multi-dimensional fluorescence imaging to the study of membrane lipid microdomains. The work makes extensive use of spectrally- and temporally- resolved fluorescence microscopy and the novel, phase-sensitive, membrane-partitioning dye di-4-ANEPPDHQ. The methods are applied to live cell imaging including imaging of the immunological synapse between effector cells and their targets. It is demonstrated that the 60 nm spectral blue-shift in the fluorescence emission of di-4-ANEPPDHQ between the disordered and ordered phases that has been presented in the literature is accompanied by a 1900 ps lengthening in fluorescence lifetime. After optimising conditions, it is further demonstrated that the two contrast parameters are applicable to live cell microscopy and that they provide useful biological information about the order in the membrane. As the dye is excited in the blue spectral region and is relatively photostable, it can be used with single-photon excitation from commonly available sources and is compatible with widefield and TIRF microscopy. A rapid, optically-sectioning, line-scanning hyperspectral fluorescence lifetime imaging microscope is presented, characterised and demonstrated. As well as being able to extract intrinsic contrast from unstained biological tissue, this system is capable of imaging the spectral and lifetime changes of di-4ANEPPDHQ simultaneously in both artificial membrane constructs and live cells. From this, it is shown that it is likely that the dye is sensitive to the differential penetration of water into the lipid bilayer. The use of the probe is then demonstrated in a range of biologically-interesting examples. These include monitoring the change in lipid order during cholesterol depletion from the membrane using a high-speed, sectioning fluorescence lifetime imaging microscope, imaging diseased and healthy primary human T cells and imaging membrane nanotubes. Finally, membrane lipid order at the immune synapse between effector cells (T and NK cells) and their targets is studied. It is shown that order is patterned at the synapse in a distribution compatible with the so-called supramolecular activation clusters that have been observed in the protein distributions in these systems. These results show membrane lipid microdomains may play a key role in T cell activation and that multidimensional fluorescence microscopy and di-4-ANEPPDHQ are useful tools for imaging their spatial and temporal organisation.

Bacteria antibiotic resistance to diverse clinically used compounds is becoming a major health risk. One strategy of multidrug resistance is the active extrusion of toxic compounds out of the bacterial cell walls by efflux pumps. These pumps are often regulated at gene transcriptional level. Pseudomonas putida, one of. the most resistant bacteria, is used as a model system to study the gene regulations involved in multidrug resistance. P. putida exhibits resistance to organic molecules, antibiotics and plant antimicrobials by three efflux pumps. One of them contains three membrane proteins TtgABC coded in one operon. The regulation of the efflux pumps genes ttgABC is controlled by a DNA binding repressor protein TtgR that is coded adjacent to the efflux pump operon and transcribed divergently. TtgR forms a homo dimer and consists of a ligand binding domain and a helix tum helix DNA binding domain. TtgR senses and interacts with ligands, leading to its release from DNA and the transcription of the efflux pump genes. The crystal structures of TtgR in complex with chloramphenicol and tetracycline antibiotics, phloretin, quercetin and naringenin plant antimicrobials and the organic solvent butylparaben have .,been determined to high-resolutions. These structures allow us to identify ligand binding pockets of TtgR and explain its diverse ligand binding properties. The structural information are confinned and complement~ by biochemical studies including isothermal titration calorimetry (lTC). Pseudomonas aeruginosa is a main pathogenic invader of the lungs of cystic fibrosis patients and exhibits multidrug resistance by activation of $e efflux pumps. Mex.Z, a homology of TtgR, is shown to be one of the most frequently mutated genes in bacteria isolated from cystic fibrosis patients, highlighting its importance in bacterial adaptation and survival. We have determined the crystal structure of MexZ, allowing us to explain the structural consequences of some of the clinically important mutations.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.