Thesis (Ph.D.)--University of Tennessee Health Science Center, 2008. / Title from title page screen (viewed on November 5, 2008). Research advisor: Jie Zheng, Ph.D. Document formatted into pages (xiii, 140 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 105-116).

Thesis (M.Sc.). / Written for the Dept. of Biochemistry. Title from title page of PDF (viewed 2009/06/23). Includes bibliographical references.

Thesis (Ph.D. in Pharmacology) -- University of Colorado Denver, 2008. / Typescript. Includes bibliographical references (leaves 148-159). Free to UCD Anschutz Medical Campus. Online version available via ProQuest Digital Dissertations;

Thesis (Ph.D.)--University of Alabama at Birmingham, 2010. / Title from PDF title page (viewed on June 28, 2010). Includes bibliographical references.

Thesis (Ph. D.)--University of Virginia, 2006. / Includes bibliographical references (leaves 181-233). Also available online through Digital Dissertations.

Antibiotic resistance has emerged as one of the World's leading public health challenges. The inexorable emergence of drug resistant pathogens, combined with a steep decline in antibacterial drug discovery, has led to a major crisis. One of the most common drug resistance mechanisms involves bacteria adapting to reduce intracellular drug accumulation. To understand these resistance mechanisms, one needs quantitative information about the membrane permeability of drugs. In this Thesis, we develop a novel optofluidic permeability assay that allows us to quantify the permeability coefficient of drugs crossing lipid membranes. Lipid vesicles are used as model systems and drug molecules are tracked directly using their autofluorescence in the ultraviolet. The permeability coefficient of the drug is inferred by studying the increase in drug autofluorescence intensity within vesicles as they traverse a microfluidic network while exposed to the drug for well defined times. This provides a novel platform from which we can develop membrane models for understanding drug permeability. We incorporate the Escherichia coli outer membrane protein OmpF in vesicles and quantify its role in the transport of fluoroquinolone antibiotics. We provide direct visualisation of OmpF mediated fluoroquinolone transport. We study the pH dependence of antibiotic transport both through pure phospholipid membranes and through OmpF, and present a physical mechanism to explain the pH dependence of E. coli fluoroquinolone susceptibility. We also show the importance of lipid composition on drug permeability - changing the lipid composition of the membrane is shown to change antibiotic permeability by over an order of magnitude. Finally, we report on the discovery of a novel signalling mechanism in E. coli that relies on the transport of small drug-like molecules, and discuss the role it plays in stress response in the microbial community.

Le sujet principal de ces travaux de thèse concerne la synthèse et la caractérisation de nouveaux matériaux membranaires bioinspirés dont la fonction première est le transport dirigé d'espèces chimiques. La réalisation de ces systèmes est basée sur l'auto-assemblage de briques élémentaires (moléculaires, polymériques) dont les propriétés vont induire la création de chemins de transport directionnels au sein des nouveaux matériaux. Tout d'abord, la synthèse de nouveaux composés bola-amphiphiles a tout d'abord été présentés. Ces derniers ont ensuite été étudiées par spectroscopie de fluorescence et par diffusion dynamique de la lumière ont permis de mettre en évidence leur capacité à former des canaux ioniques, des canaux protoniques et des canaux d'eau au sein d'une bicouche lipidique. Une seconde étude a consisté en l'élaboration de nanomatériaux membranaires mésoporeux. Ces derniers ont été fonctionnalisés par des groupements hydrophobes afin de permettre le confinement de composés au sein des mésopores via des interactions de van der Waals. Enfin ,une dernière étude a porté sur la l'élaboration de matériaux membranaires à base d'Aquaporines artificielles dans le but d'obtenir un matériau permettant un transport dirigé d'eau au travers de la membrane. / The main topic of this thesis concerns the synthesis and characterization of novel bioinspired membrane materials whose primary function is the transport of chemical species directed . The realization of these systems is based on the self-assembly of building blocks ( molecular , polymeric ) whose properties will induce directional transport in new materials.First, the synthesis of new bola - amphiphilic compounds was shown . They were then examined by fluorescence spectroscopy and dynamic light scattering allowed to highlight their ability to form ion channels, proton channels and water channels in a lipid bilayer.A second study involved the development of mesoporous nanomaterials membrane. These have been functionalized with hydrophobic groups to allow the confinement of the compounds in the mesopores via van der Waals interactions.Finally, a recent study has focused on the development of materials membrane based on artificial Aquaporins in order to obtain a material to transport water directed through the membrane.

Clathrin coated vesicles were the first type of coated vesicle to be characterised. The coat consists of two components, clathrin and adaptor (or AP) complexes, the AP-1 complex is associated with the clathrin coated vesicles that bud from the TGN and the AP-2 complex is associated with the clathrin coated vesicles that bud from the plasma membrane. A new type of adaptor-like complex was discovered in our laboratory and was published in 1996. The complex has been shown to consist of two known proteins, beta3B and mu3B, and two unknown proteins of 160kD and 22kD. Unlike the conventional adaptor complexes this complex is not associated with clathrin. The aim of this thesis was to complete the characterisation of the adaptor-like complex and to establish its function. My studies have shown that, the adaptor-like complex consist of an alpha/gamma like subunit, delta, a beta subunit (beta3A/B), a mu subunit (mu3A/B) and a sigma subunit (sigma3A/B). We named the adaptor-like complexAP-3, by analogy with the AP-1 and AP-2 complexes. The AP-3 complex is localised to perinuclear and more peripheral membranes in non-neuronal cells, with little overlap with endocytic markers. The beta subunit of the AP-3 complex is the major target for phosphorylation. Analysis of mice with mutations in the beta3A subunit, and in the delta subunit of the AP-3 complex, have revealed that the beta subunit is required for the stability of the mu subunit and that the delta subunit is essential for the stability of the whole complex. Further analysis of the mutant mice indicated that the mice lack significant levels of functional AP-3 complex. Studies on fibroblasts generated from these mice revealed that the AP-3 complex plays a role in the trafficking of LAMPI to lysosomes.

2-ω-carboxyethylpyrrole (CEP) is a product of Docosahexaenoic acid (DHA) oxidation, which forms covalent adducts with different proteins. CEP-modified proteins can interact with macrophage receptor, integrin αDβ2. This study aims to compare αDβ2 binding to its physiological ligand, fibrinogen, and CEP-modified fibrinogen, which is formed during inflammation. We hypothesize that modification of fibrinogen changes its ligand-binding properties to integrin αDβ2 which can affect macrophage migration and retention. Recombinant αD I-domain and αDβ2-transfected HEK293 cells were used for the experiments. In biolayer interferometry (BLI) assays, fibrinogen was immobilized at pH 5.0 and fibrinogen-CEP at pH 3.5. Our results showed that the affinity of αD I-domain binding to fibrinogen-CEP was higher than fibrinogen, and the binding was inhibited by the anti-CEP antibody. The optimal expression of αD I-domain was found at 25°C. In cell adhesion assays, optimal concentrations were used for the inhibition assay; fibrinogen at 2µg/ml and fibrinogen-CEP at 8µg/ml. αDβ2-transfected cells demonstrated stronger adhesion to fibrinogen-CEP, and this adhesion was significantly inhibited by polyglutamic acid, which mimics CEP-mediated binding. These findings suggest that αDβ2's interaction with DHA-modified extracellular matrix (ECM) proteins significantly increases macrophage adhesion and may serve for macrophage retention during chronic inflammation. Developing small molecules that can inhibit αDβ2-CEP interaction could be a breakthrough in treating inflammatory diseases. The main conclusions drawn from the study are that CEP-modified fibrinogen has a higher affinity for αD I-domain binding than native fibrinogen, and this interaction can be inhibited by targeting CEP. The study provides insights into the potential therapeutic applications of inhibiting αDβ2-CEP interactions in inflammatory diseases.

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