Acinetobacter baumannii is an opportunistic pathogen which has been implicated in a variety of nosocomial infections among immunocompromised patients worldwide. Recently, Multi-drug resistant (MDR) isolates of A. baumannii have been isolated from military personnel returning from service in Iraq and Afghanistan. Antibiotic resistance of A. baumannii has limited the number of active antibacterial, making very difficult to treat these types of infections. This work investigated the role of Resistance-Nodulation-cell Division (RND) efflux pumps in the antibiotic resistance mechanism of A. baumannii. Expression of six different RND pumps was analyzed in clinical isolates of A. baumannii. A novel RND family pump, AdeMNO, was found to be present in a majority of isolates. The adeMNO operon was cloned, sequenced, and characterized using the single copy gene expression system in an efflux sensitized surrogate Pseudomonas aeruginosa strain. Antibiotics, trimethoprim, chloramphenicol, and clindamicin were identified as the substrates of this pump. In order to understand the mechanisms of regulation of adeMNO operon, a putative regulator belonging to the lysR-family was identified, cloned, and sequenced from the upstream region of the operon. Promoter regions of the adeMNO operon were also sequenced from various clinical isolates and sequence polymorphisms identified that could be implicated in the regulation of adeMNO expression. / UOIT

During pregnancy, 64-96% of women take at least one prescription drug. The placenta is the primary barrier between substrates in maternal and fetal circulation. The blood-brain barrier (BBB) acts as an additional barrier for the fetal brain, which is particularly susceptible to the effects of xenobiotics. Multidrug resistance phosphoglycoprotein (P-gp; encoded by Abcb1 mRNA) and breast cancer resistance protein (Bcrp1; encoded by Abcg2 mRNA) are efflux transporters localized on placental syncytiotrophoblast and capillary endothelial cells of the BBB. Placental Abcb1/P-gp and Abcg2/Bcrp1 limit maternal-fetal transfer of endogenous and exogenous substrates. Similarly, the neuroprotective roles of Abcb1/P-gp and Abcg2/Bcrp1 in the adult BBB have been demonstrated. However, developmental changes in expression and function and regulation of Abcb1/P-gp and Abcg2/Bcrp1 in these tissues are poorly understood. This thesis investigates gestational changes in expression and function of Abcb1/P-gp and Abcg2/Bcrp1 in the placenta and fetal brain, in addition to regulation by steroids, progesterone and glucocorticoids. The effects of glucocorticoids on Abcb1/P-gp and Abcg2/Bcrp1 in the placenta and fetal brain are of importance given that 10% of pregnant women are treated with synthetic glucocorticoids during the management of threatened preterm labour. These studies demonstrate that the decrease in placental Abcb1/P-gp mediated fetal protection near term is compensated by an increase in Abcb1/P-gp and Abcg2/Bcrp1 mediated neuroprotection in the fetal brain; likely in preparation for life ex-utero. The lack of effects of progesterone and the dose-, age- and sex- dependent regulatory effects of synthetic glucocorticoid have highlighted the complexity associated with regulation of these transporters. Further, these studies are the first to report sexually dimorphic glucocorticoid effects on Abcb1/P-gp and Abcg2/Bcrp1 expression and function, with the female fetus being particularly susceptible to glucocorticoid these effects. In this regard, Abcb1/P-gp and Abcg2/Bcrp1 transport capacity may be altered when synthetic glucocorticoid is administered as a co-therapy, and as such, recipient sex should be considered during pharmacotherapy. Understanding the regulation of Abcb1/P-gp and Abcg2/Bcrp1 expression and function in the placenta and fetal brain during normal development and under pathological conditions is critical for fetal health and development, particularly when therapeutic strategies are utilized in pregnancy.

During pregnancy, 64-96% of women take at least one prescription drug. The placenta is the primary barrier between substrates in maternal and fetal circulation. The blood-brain barrier (BBB) acts as an additional barrier for the fetal brain, which is particularly susceptible to the effects of xenobiotics. Multidrug resistance phosphoglycoprotein (P-gp; encoded by Abcb1 mRNA) and breast cancer resistance protein (Bcrp1; encoded by Abcg2 mRNA) are efflux transporters localized on placental syncytiotrophoblast and capillary endothelial cells of the BBB. Placental Abcb1/P-gp and Abcg2/Bcrp1 limit maternal-fetal transfer of endogenous and exogenous substrates. Similarly, the neuroprotective roles of Abcb1/P-gp and Abcg2/Bcrp1 in the adult BBB have been demonstrated. However, developmental changes in expression and function and regulation of Abcb1/P-gp and Abcg2/Bcrp1 in these tissues are poorly understood. This thesis investigates gestational changes in expression and function of Abcb1/P-gp and Abcg2/Bcrp1 in the placenta and fetal brain, in addition to regulation by steroids, progesterone and glucocorticoids. The effects of glucocorticoids on Abcb1/P-gp and Abcg2/Bcrp1 in the placenta and fetal brain are of importance given that 10% of pregnant women are treated with synthetic glucocorticoids during the management of threatened preterm labour. These studies demonstrate that the decrease in placental Abcb1/P-gp mediated fetal protection near term is compensated by an increase in Abcb1/P-gp and Abcg2/Bcrp1 mediated neuroprotection in the fetal brain; likely in preparation for life ex-utero. The lack of effects of progesterone and the dose-, age- and sex- dependent regulatory effects of synthetic glucocorticoid have highlighted the complexity associated with regulation of these transporters. Further, these studies are the first to report sexually dimorphic glucocorticoid effects on Abcb1/P-gp and Abcg2/Bcrp1 expression and function, with the female fetus being particularly susceptible to glucocorticoid these effects. In this regard, Abcb1/P-gp and Abcg2/Bcrp1 transport capacity may be altered when synthetic glucocorticoid is administered as a co-therapy, and as such, recipient sex should be considered during pharmacotherapy. Understanding the regulation of Abcb1/P-gp and Abcg2/Bcrp1 expression and function in the placenta and fetal brain during normal development and under pathological conditions is critical for fetal health and development, particularly when therapeutic strategies are utilized in pregnancy.

Multidrug resistance phenotype of tumor cells describes resistance to wide range of structurally unrelated anticancer agents and is a serious limitation to effective chemotherapy. It is a multifactor yet not fully elucidated phenomenon by the involvement of diverse cellular pathways. Aim of this study was to investigate the resistance mechanisms developed against docetaxel and doxorubicin that are widely used in the treatment of breast cancer in model cell line MCF-7. Resistant sublines were developed by application of drugs in dose increments and effect of docetaxel and doxorubicin on drug applied cells were investigated by cell viability assays. Expression analysis of P-gp, MRP1, BCRP, Bcl-2, Bax and &amp / #946 / -tubulin isotypes were performed by RT-PCR, qPCR, Western blot and immunocytochemistry. Genome-wide expression analysis was also performed by cDNA microarray. According to cell viability assays, drug applied cells developed varying degree of resistance to docetaxel and doxorubicin. Gene expression analysis demonstrated that de novo expression of P-gp contributed significantly to drug resistance. Expression levels of class II, III and V &amp / #946 / -tubulin isotypes increased in docetaxel resistant sublines. According to microarray analysis, a variety of genes showed significantly altered expression levels particularly drug metabolizing and detoxification enzymes (i.e. increased GPX1 and GSTP1 with decreased POR), survival proteins (e.g. decreased TRAIL together with increased decoy receptors and CD40), extracellular matrix components (e.g. increased integrin signaling), growth factors and cytokines (e.g. EGFR1, FGFR1, CTGF, IL6, IL8 and IL18 overexpression), epithelial-mesenchymal transition proteins (i.e. increased vimentin and N-cadherin with decreased E-cadherin and occludin) and microtubule dynamics related proteins (e.g. increased MAP1B and decreased MAP7). Development of cross-resistance and combined drug effects on resistant sublines were also studied. Results demonstrated that docetaxel and doxorubicin resistant cells developed cross-resistance to paclitaxel, vincristine, ATRA, tamoxifen and irradiation. Finally, modulatory effects of verapamil and promethazine in combined drug applications were investigated and verapamil and promethazine were shown to decrease MDR1 expression level thus reverse the MDR. They also showed synergic and additive effects in combined docetaxel and doxorubicin applications. Identification of resistance mechanisms may personalize chemotherapy potentially increasing efficacy of chemotherapy and life quality of patients.

The emergence of drug-resistance of tumor cells is a major complication for succesful chemotherapy. In this study, the molecular mechanisms of resistance to prednisone, vincristine and melphalan in multiple myeloma cell lines, RPMI-8226 and U-266 were investigated. Drug resistance was induced by application of the drugs by stepwise dose increments and confirmed by XTT cytotoxicity assay. Gene expression analysis demostrated that MDR1 gene is one of the most important factor causing the multidrug resistance phenotype in prednisone, vincristine and melphalan resistant multiple myeloma cell lines. According to microarray analysis alterations in laminin, integrin and collagen genes were detected. Additionally, upregulation of some oncogenes and growth factors (Rho family of GTPases, YES1, ACT2, TGFBR, EPS15, PDGF) was shown to have a role in MDR in multiple myeloma. Significant downregulation of suppressors of cytokine signalling gene expressions and upregulation of different types of interleukine and interferon gene expressions (IL3 and interferon-gamma receptor) which are related to JAK-STAT signalling pathay was shown. Alterations in expression levels of genes related to ceramide metabolism were shown especially for melphalan resistance in multiple myeloma. The data from vincristine/prednisone and vincristine/melphalan drug combination studies were shown that the usage of vincristine on prednisone and melphalan resistant multiple myeloma cell lines increase the efficacy of the chemotherapy. On the other hand the cross-resistance development of prednisone and melphalan resistant sublines to irradiation was detected. These results may help to understand the molecular mechanisms of prednisone, vincristine and melphalan resistance in multiple myeloma model cell lines RPMI-8226 and U-266.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

Drug efflux represents an important protection mechanism against antibiotics and environmental toxic compounds in bacteria. Efflux genes constitute from 6% to 18% of all transporters in bacterial genomes, yet their regulation, natural substrates, and physiological functions are poorly understood. Among the 20 chromosomally encoded efflux genes in Escherichia coli K-12, only the AcrAB-TolC efflux system is constitutively expressed under the ordinary laboratory growth of E. coli. To explore conditions and circumstances that trigger the expression of additional efflux genes as well as their physiological functions, I examined the expression of all 20 efflux genes under a physiologically relevant circumstance for E. coli, which is anaerobic condition in this study. I found that expression of an RND type efflux pump MdtEF is up-regulated more than 20 fold when E. coli is cultured under anaerobic conditions. Mutagenesis studies revealed that the anaerobically induced expression of mdtEF is subject to the regulation of the anaerobic global transcription factor ArcA. Direct drug efflux and tolerance assay showed that anaerobically grown E. coli cells display an increased efflux activity and enhanced drug tolerance in an MdtEF dependent manner, confirming the functional up-regulation of the efflux pump MdtEF in the anaerobic physiology of E. coli. Since the up-regulation of mdtEF by anaerobic growth occurs in the absence of antibiotics and drugs, I speculate that MdtEF has physiological functions under the anaerobic growth of E. coli. To explore this, I first compared the viability of ΔmdtEF and WT MG1655 strains and found that ΔmdtEF caused a decreased cell survival during prolonged anaerobic growth of E. coli. Interestingly, this defect became more pronounced when cells grow in the presence of 10 mM nitrate, but no defect was observed in ΔmdtEF strain when cells grow in the presence of 40 mM fumarate under the same anaerobic conditions, suggesting that MdtEF has physiological roles relevant to the anaerobic respiration of nitrate. I further found that E. coli cells harboring the deletion of mdtEF are susceptible to indole nitrosative derivatives, a class of toxic by-products formed and accumulated within E. coli when the bacterium respires nitrate under anaerobic conditions, and deletion of the genes responsible for the biosynthesis of indole, tnaAB, restores the growth defect of the ΔmdtEF strain during anaerobic respiration of nitrate. Taken together, I conclude that the multidrug efflux pump MdtEF expels the nitrosated indole derivatives out of E. coli cells under anaerobic conditions. Since the production and accumulation of nitrosyl indole derivatives is ascribed to the reactive nitrogen species elicited when E. coli consumes nitrate, I propose that the up-regulated multidrug efflux pump MdtEF functions to protect E. coli from nitrosative damage in its anaerobic ecological niches. / published_or_final_version / Biological Sciences / Master / Master of Philosophy

Although some progress has been made in the treatment of cancer over the last sixty years, the majority of chemotherapeutics has fallen short. Because general chemotherapies that target DNA replication have only a limited efficacy and significant non-target side-effects, a new paradigm for cancer drug development has been adopted. Using a molecular targeted approach, new gene and protein targets have been identified and the development of chemotherapies that are specific to these targets has already begun. In this study, compounds that interact with two key cancer targets, the G-quadruplex of the c-Myc promoter and p-glycoprotein, have been investigated. By developing such compounds, improvements in treatment efficacy is anticipated with an aspiration for decreased mortality attributable to cancer.Formation of DNA secondary structures, such as the G-quadruplex, in the NHE III1 region of the c-Myc promoter has been shown to repress c-Myc transcription. Because c-Myc is an oncogene that is overexpressed in a variety of cancers, stabilization of the G-quadruplex by small molecules would be advantageous in cancer treatment. Using Fluorescence Resonance Energy Transfer, with Taq Polymerase Stop assays for confirmation, a group of compounds were identified that stabilize the c-Myc G-quadruplex structure. Using a colon cancer model, two compounds were shown to decrease c-Myc gene and protein expression. Also, exposure to the compounds for 48 hours results in an induction of caspase-3, indicative of apoptosis. Furthermore, surface plasmon resonance suggests that compound-induced stabilization of the c-Myc G-quadruplex can prevent sustained binding of the regulatory protein NM23-H2 by increasing its dissociation from the G-quadruplex. This may subsequently prevent unraveling of the G-quadruplex.Because resistance to chemotherapy reduces its effectiveness, development of multidrug resistance (MDR) modulators was also studied. Psorospermin is a topoisomerase II-directed DNA alkylating agent active against MDR cell lines. In a study examining the mechanism of psorospermin's P-glycoprotein modulation, Flow Cytometry demonstrated that doxorubicin-resistant multiple myeloma cells pre-treated with psorospermin enhanced intracellular retention of doxorubicin. Because neither transcription of mdr1 nor translation of P-glycoprotein was downregulated by psorospermin, resistance reversal is most likely due to a direct interaction between the side chain of psorospermin and P-glycoprotein, inhibiting drug efflux.

No description available.

Resistance to the avermectin/milbemycin class of anthelmintics in nematodes has become a serious problem worldwide due to their unrestricted usage. Resistance to these compounds is attributed to the over-expression of the transport protein, P-glycoprotein (P-gp). P-gp acts by pumping drug molecules out from the cell or organism, P-gp efflux activity can be blocked using multidrug resistance (MDR) modulators associated with chemotherapy to enhance their therapeutic effect. A series of experiments was undertaken to determine if the association of the anthelmintics, ivermectin (IVM) and moxidectin (MOX), and MDR modulators would increase the anthelmintics' efficacy against resistant parasites. Using an in vitro migration assay, IVM and MOX in the presence or absence of verapamil (VRP), CL347,099 and cyclosporin A (CyA) were used against IVM- and MOX-selected strains of H. contortus. The modulators alone had no effect on reducing the number of migrating larvae, IVM and MOX had a significant increase in efficacy of 52.7 and 58,3% respectively, when used in association with VRP, above that obtained with the anthelmintics alone. CL347,099 was also able to significantly increase the IVM and MOX efficacy by 24.2 and 38.9%, respectively. The effect of IVM and MOX in combination with VRP and CL347,099 was determined in jirds infected with selected strains of H. contortus. The combinations of VRP with either IVM or MOX significantly reduced worm counts of the selected strains compared with the untreated controls, whereas IVM or MOX alone did not. CL347,099 plus MOX combination was significantly more efficacious than moxidectin alone against the selected strains. To evaluate the effect of VRP on the pharmacokinetic behaviour of the anthelmintics IVM and MOX, the drug combination was given to sheep. The IVM plus VRP treatment resulted in an increase of the pharmacokinetic parameters of IVM. The peak concentration (83%) and area under the curve (54%) were significantly differen