Philosophiae Doctor - PhD / Background: Extended-spectrum beta-lactamase-producing (ESBL) and carbapenemaseproducing Gram-negative bacilli showing resistance to cephalosporins and carbapenems respectively, have been reported from several countries globally and recently among Libyan combatants who have been transferred to European countries for advanced medical care. However, there is a lack of data about their presence in Misrata and in Libya in general. This is the first documented study aimed at investigating the prevalence and resistance mechanisms of ESBL and carbapenemase-producing K. pneumoniae isolates from Misrata. Materials and Methods: Two hundred Gram-negative bacillus isolates were collected and identified from hospitals and pathology laboratories in Misrata. Following antimicrobial susceptibility screening, those showing resistance to cephalosporin and carbapenem were tested for ESBL activity using the Modified double disc synergy test, Sensititer ESBL confirmatory MIC plates and MAST AmpC detection sets D52C and D68C. Carbapenemase activity was detected using RAPIDEC CARBA NP test, Modified Hodge test (MHT), carbapenem inactivation methods (CIM), carbapenem combined test (CCT), and by MAST carba puls set. ESBL and carbapenemases genes were detected using multiplex PCR. Results: K. pneumoniae was the predominant species (85/200) of the 14 species identified, with 56 (65.8%) showing carbapenem resistance, 16 (18.8%) were cephalosporin-resistant carbapenem-susceptible and 13 (15.2%) were susceptible to all antibiotics except ampicillin. OXA-48 was the only carbapenemase detected, with SHV, TEM and CTX-M group 1 found in almost all carbapenem and cephalosporin resistant K. pneumoniae. Rep-PCR analysis revealed multiple clones and some K. pneumoniae strains were genetically related or indistinguishable despite differences in ESBL genes or carbapenemase activity. Conclusion: The findings of this study show that carbapenemase- and ESBL-producing K. pneumoniae are prevalent in Misrata and emphasize the urgent need for optimized infection control and antibiotic stewardship programmes in the Libyan hospitals to prevent further spread of these organisms.

Chemotherapy is used in the treatment of cancer. Unfortunately, drugs often fail due to multidrug resistance (MDR) caused by P-glycoprotein (P-gp1or ABCB1) and the multidrug resistance-associated protein (MRP1 or ABCC1). These proteins bind and transport drugs out of cancer cells, thereby conferring MDR. / The second chapter of this thesis addresses an unexplained phenomenon that accompanies P-gp1 expression, collaterally sensitive to verapamil. The collective results of this work demonstrated that treatment of cells that over-express P-gp1 with verapamil induces apoptosis. Furthermore, the findings show that the ATPase activity of P-gp1 was activated by verapamil. The degree of ATPase activation was proportional to the level of apoptosis and the increased demand for ATP resulted in the production of reactive oxygen species (ROS). Finally, the production of ROS led to cell death mediated by apoptosis in that experimental model system. / Chapters three and four are devoted to understanding the binding characteristics of MRP1 with two of its physiological substrates, glutathione (GSH) and leucotriene C4(LTC4). Photoreactive derivatives of these substrates were synthesised to address this objective, IAAGSH and IAALTC4. Photolabelling and transport studies showed that these derivatives have similar binding characteristics as the native compounds. In addition, photolabelling of MRP1 occurred with a high specificity with both compounds. IAAGSH and IAALTC 4 were also used to determine the locations of GSH and LTC4 binding sites. This was accomplished using MRP1-variants containing hemagglutinin (HA) epitopes at specific locations in the amino acid sequence. Through photoaffinity labelling, immunoprecipitation, and trypsin digestion, a map of binding sites for IAAGSH or IAALTC4 was obtained. Both LTC4 and GSH bound to transmembrane (TM) regions 10-11 and 16-17 which have been previously implicated in drug binding. Furthermore, novel binding sites for both substrates were discovered. IAALTC4 photolabelled a novel site within the first five TMs (TMD0) of MRP1, whereas IAAGSH labelled two cytoplasmic regions (L1 and L0). These may represent specific binding sites for LTC4 and GSH. / The work within this thesis explores some of the biochemical characteristics of Pgp1 and MRP1 that are not directly related to drug resistance and may lead to new strategies in cancer treatment.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2006. / Vita. Bibliography: p. 92-107.

Thesis (honors)--Georgia State University, 2007. / Title from file title page. Under the direction of Dabney White Dixon. Electronic text (88 p. : col. ill.) : digital, PDF file. Description based on contents viewed Sept. 30, 2008. Includes bibliographical references (p. 46-47).

Thesis (Ph.D.)--University of Tennessee Health Science Center, 2008. / Title from title page screen (viewed on January 7, 2009). Research advisor: John D. Schuetz, Ph.D. Document formatted into pages (xi, 113 p. : ill.). Vita. Abstract. Includes bibliographical references (p. 92-113).

Thesis (M.S.) -- University of Texas Southwestern Medical Center at Dallas, 2005. / Embargoed. Vita. Bibliography: 29-34.

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No description available.

ATP-binding cassette (ABC) transporters use the power of ATP binding and hydrolysis to deliver a wide variety of molecules across membrane bilayers. Crystal structures in the past two decades have provided snapshots of these transporters under various conditions, which revealed conformational changes of transporters upon substrate and ATP binding along the transport pathway. However, understanding of kinetics of substate translocation and the knowledge of transient intermediates along the transport pathway remain primitive, especially for Type II ABC importers. In this thesis work, I employed fluorescence resonance energy transfer (FRET) to study the transport mechanism of BtuCD, a model type II ABC importer that transports vitamin B12. I also exploited the photophysical property of the transport substrate, B12, that quenches the fluorescence of certain fluorophores nearby through FRET, which enables the monitoring of B12 binding and release. Our ensemble FRET data showed the binding of B12 and nucleotide both weaken the interaction between BtuCD and BtuF. Our single-molecule FRET (smFRET) experiments not only revealed stepwise movement of substrate molecule through the transporter in real-time and at the single-molecule level, but also yielded the rates of transition between individual conformational states, which had not been previously characterized in any other transporters. The results showed that ATP hydrolysis, instead of ATP binding, drives the power-stroke for the transport cycle. They also showed two sequential ATP hydrolysis events are required to complete a transport cycle, with the first ATP hydrolysis event delivering B12 into the cavity of BtuCD and the second resetting the BtuCD-F complex for a new cycle of transport.

ABC transporters consist of two transmembrane domains (TMDs) that form the transport channel and two cytosolic nucleotide-binding domains (NBDs) that energize transport via ATP binding and hydrolysis. Using site-directed mutagenesis, the role of highly conserved residues in the NBDs of Abcb1a was investigated. / In both NBDs of Abcb1a the A-loop aromatic residue is a tyrosine: Y397 in NBD1 and Y1040 in NBD2. Another tyrosine (618 in NBD1 and 1263 in NBD2) also appears to lie close to the ATP molecule. These four tyrosine residues were mutated to tryptophan and the effect of these substitutions on transport properties, ATP binding, and ATP hydrolysis was analyzed. Y618W and Y1263W enzymes had catalytic characteristics similar to wild-type (WT) Abcb1a. On the other hand, Y397W and Y1040W showed impaired transport and greatly reduced ATPase activity, including an ∼10-fold increase in KM(ATP). Thus, Y397 and Y1040 play an important role in Abcb1a catalysis. / Since it was speculated that ABC transporters utilize a catalytic base to hydrolyse the beta-gamma phosphodiester bond of ATP, a search for that residue was undertaken. Six pairs of highly conserved acidic residues in the NBDs of Abcb1a were investigated. Removal of the charge in D558N and D1203N as well as in E552Q and E1197Q produced enzymes with severely impaired transport. These mutants were purified and characterized with respect to ATPase activity. Mutants D558N and D1203N retained some drug-stimulated ATPase activity and vanadate (Vi) trapping of 8-azido-[alpha32P]nucleotide confirmed slower basal and drug-stimulated hydrolysis. The E552Q and E1197Q mutants showed absence of ATPase activity but Vi trapping of 8-azido-[alpha 32P]nucleotide was observed, at a level similar to that of WT Abcb1a. Photolabelling by 8-azido-[alpha32P]nucleotide, in the presence or absence of drug, was also detected in the absence of Vi. The ATPase activity, binding affinity, and trapping properties of these glutamate residues were further analyzed. In addition to the E→Q mutants, the glutamates were individually mutated to D, N, and A. The double mutants E552Q/E1197Q, E552Q/K1072R, and K429R/E552Q were also analyzed. The results obtained suggest that 1) the length of the side-chain is important for the catalytic activity, whereas the charge is critical for full turnover to occur, 2) formation of the catalytic transition state does occur in the mutant site in the single-site mutants, suggesting that E552 and E1197 are not classical catalytic carboxylates, 3) steps after formation of the transition state are severely impaired in these mutant enzymes, 4) NBD1 and NBD2 are functionally asymmetric, and 5) the glutamates are involved both in NBD-NBD communication and transition-state formation through orientation of the linchpin residue.