The phenomenon of multidrug resistance (MDR) in cancer cells is generally associated with P-glycoprotein (P-gp) expression and presents an obstacle to successful chemotherapy. Attempts to overcome P-gp-associated MDR using P-gp modulators, such as verapamil, have been hindered by their intrinsic in vivo toxicity. In 1991, however, Scala et al. demonstrated the alteration of P-gp function by interferon-alpha (IFN-α) in vitro at non-toxic in vivo concentrations, suggesting a basis for the use of IFN-α clinically in patients exhibiting P-gp-associated MDR. Drug resistance in B-CLL has been linked to the phenomenon of MDR, however, publications regarding this have been conflicting. The contrasting results prompted further investigation of the role of P-gp-associated anthracycline resistance and, using isolated β-lymphocytes from B-CLL patients, this investigation examined P-gp expression, function and IFN-α modulation in vitro. Optimum conditions for in vitro analysis of P-gp-associated anthracycline resistance were determined by examining the stability of the anthracycline, daunorubicin, in varying cell culture conditions. The resulting system balanced conditions affecting drug stability with those affecting cell survival. While other investigations have neglected the issue of drug stability, this study demonstrates that the instability of daunorubicin may be a critical variable determining the outcome of drug sensitivity studies. In RPMI + 2mM L-glutamine and 10% (v/v) FBS, loss of drug concentration is due to both adsorption and degradation and these experiments show that the presumed availability of drug may be over-estimated in in vitro studies. Furthermore, the degradation products might interfere with P-gp function and modulation. MDRl gene mRNA was detected in the B-cells of forty-three out of fifty B-CLL patients analysed, whereas P-gp expression, as measured by flow cytometry, resulted in only sixteen patients out of fifty-five being classed as positive (> 10% increase in staining as compared to the control). P-gp functionality and modulation studies on the B-cells of eleven patients confirmed the existence of an efflux mechanism with identical characteristics to P-gp using verapamil, the dye rhodamine 123 (rho123) and daunorubicin. Four patients were classed as functional low expressers (functional P-gp with low P-gp expression (7-10% increase in staining)), six were classed as functional high expressors (functional P-gp with high P-gp expression (20-57% increase in staining)) and one as a non-functional high expressor (non-functional P-gp with high P-gp expression (13.4% increase in staining)). Verapamil modulated rho123 efflux in all ten patients classed as P-gp functional expressors, and daunorubicin efflux in eight of these patients. However, IFN-α modulated rho123 and daunorubicin efflux in only two and one patients, respectively, even at concentrations higher than 500I.U./ml. In contrast to Scala et al. (1991), this finding suggests that at a well tolerated concentration IFN-α may not be suitable for use as a P-gp modulating agent in vivo in B-CLL, although conclusive evidence would require a larger study.

abstract: In sub-Saharan Africa, an invasive form of nontyphoidal Salmonella (iNTS) belonging to sequence type (ST)313 has emerged as a major public health concern causing widespread bacteremia and mortality in children with malaria and adults with HIV. Clinically, ST313 pathovars are characterized by the absence of gastroenteritis, which is commonly found in “classical” nontyphoidal Salmonella (NTS), along with multidrug resistance, pseudogene formation, and chromosome degradation. There is an urgent need to understand the biological and physical factors that regulate the disease causing properties of ST313 strains. Previous studies from our lab using dynamic Rotating Wall Vessel (RWV) bioreactor technology and “classical” NTS strain χ3339 showed that physiological fluid shear regulates gene expression, stress responses and virulence in unexpected ways that are not observed using conventional shake and static flask conditions, and in a very different manner as compared to ST313 strain D23580. Leveraging from these findings, the current study was the first to report the effect of fluid shear on the pathogenesis-related stress responses of S. Typhimurium ST313 strain A130, which evolved earlier than D23580 within the ST313 clade. A130 displayed enhanced resistance to acid, oxidative and bile stresses when cultured in the high fluid shear (HFS) control condition relative to the low fluid shear (LFS) condition in stationary phase using Lennox Broth (LB) as the culture medium. The greatest magnitude of the survival benefit conferred by high fluid shear was observed in response to oxidative and acid stresses. No differences were observed for thermal and osmotic stresses. Based on previous findings from our laboratory, we also assessed how the addition of phosphate or magnesium ions to the culture medium altered the acid or oxidative stress responses of A130 grown in the RWV. Addition of either phosphate or magnesium to the culture medium abrogated the fluid shear-related differences observed for A130 in LB medium for the acid or oxidative stress responses, respectively. Collectively, these findings indicate that like other Salmonella strains assessed thus far by our team, A130 responds to differences in physiological fluid shear, and that ion concentrations can modulate those responses. / Dissertation/Thesis / Masters Thesis Microbiology 2017

The Tuberculosis infection rate has been generally escalating due to poor health conditions in the Gweru district of Zimbabwe. The study therefore seeks to identify the risk factors that affect TB treatment in the Gweru district. A cross sectional study was carried out in which a questionnaire was employed for data collection on 113 respondents. A binary logistic regression model was employed for data analysis. A total of 98 TB patients were interviewed: [50 respondents (44.0%) had Multi-drug resistant Tuberculosis and 63 respondents (56.0%) had general Tuberculosis). Before being enrolled into the study, an informed consent form was given to each of the participants. The data was then put into excel and later transferred to SPSS for analysis. Out of the 14 potential risk factors of TB treatment, only 6 variables (side effects, gender, alcohol use, HIV status, smoking during the treatment period and having been pre-exposed to TB drugs) were statistically significant in their association with treatment failure.

No description available.

Multiple drug resistance (MDR) has been a severe issue in treatment and recovery from infection.Gram-negative bacteria intrinsically exhibit higher drug tolerance than Gram-positive microbes. In this thesis, two proteins involved in Gram-negative bacterial MDR were studied, AcrB and SurA. Resistance-nodulation-cell division pump AcrAB-TolC is the major MDR efflux system in Gram-negative bacteria and efficiently extrudes a broad range of substances from the cells. To study subtle conformational changes of AcrB in vivo, a reporter platform was designed. Cysteine pairs were introduced into different regions in the periplasmic domain of the protein, and the extents of disulfide bond formation were examined. Using this platform, an inactive mutant, AcrB∆loop, was created that existed as a well-folded monomer in vivo. Next, random mutageneses were performed on a functionally compromised mutant, AcrBP223G, to identify residues that restored the function loss. The mechanism of function restoration was examined. SurA is a periplasmic molecular chaperone for outer membrane biogenesis. Deletion of SurA decreased outer membrane density and bacterial drug resistance. The dependence of SurA function on structural flexibility and stability was examined. In addition, the effect of molecular crowding on SurA interaction with its outer membrane protein substrates was examined.

Chemotherapeutic treatment of Pseudomonas aeruginosa, a Gram-negative opportunistic pathogen, is substantially challenged by several membrane-spanning, multidrug-efflux pumps of the three-component RND family. Of these pumps, MexXY-OprM contributes to the intrinsic resistance of this organism by exporting clinically relevant antibiotics, most notably the ribosome-targeting aminoglycosides. Overproduction of MexXY-OprM is the most common mechanism providing pan-aminoglycoside resistance to P. aeruginosa cystic fibrosis clinical isolates. The mexXY genes are located in an operon, the expression of which is induced by ribosome-targeting antimicrobials. The mexXY operon is negatively regulated by MexZ, a repressor protein encoded by the divergently-transcribed gene mexZ. A second gene, PA5471, is also induced by ribosome-targeting antibiotics and is required for antibiotic induction of mexXY expression. One possibility is that PA5471 interacts with MexZ to alleviate repression of mexXY, thereby providing a mechanism for PA5471-dependent drug inducibility of mexXY. PA5471 interaction with MexZ was confirmed using a bacterial two-hybrid assay. To identify residues/regions of PA5471 important for interaction with MexZ, random chemical mutagenesis of the mexZ and PA5471 genes was carried out and the effects of these mutations on interaction of their protein products was assessed using the bacterial two-hybrid assay. Mutations of PA5471 that compromised interaction with MexZ included P68S, G76C, R216C, R221W, R221Q, G231D, and G252S, which occur within or in close proximity to a predicted surface-exposed α-helix of a PA5471 structural model that may contribute to the MexZ-interaction domain. Representative mutations P68S, G76C, R216C and R221W were placed into the chromosome of P. aeruginosa to assess their impact on drug-inducible mexXY expression. All of these mutations significantly reduced mexX upregulation in the presence of spectinomycin, where mutations R216C and R221W resulted in the near complete ablation of this antibiotic induction. These data suggest that PA5471 acts as a direct antirepressor of MexZ and that this interaction is key to mexXY upregulation in response to ribosome-targeting induction signals. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2013-02-26 13:32:39.307

Pseudomonas aeruginosa é um patógeno multirresistente capaz de produzir um biofilme protetor contra antibacterianos (ATB). O presente estudo avaliou estratégias terapêuticas contra biofilmes de cepas multirresistentes de P. aeruginosa representativas de clones e/ou genótipos de resistência endêmicos no Brasil. Os biofilmes foram formados in vitro utilizando um modelo adaptado do MBEC Assay e as estratégias terapêuticas utilizaram bacteriófagos líticos, combinação de ATB e/ou uso de força iônica alta (meio FIA). A aplicação de bacteriófagos líticos (φSPM-1) e a combinação de Aztreonam (ATM) e Piperacilina/Tazobactam (PPT), não foram capazes de eliminar o biofilme. Biofilme formado em meio FIA possui CIM similar ao modelo planctônico, tanto para ATM (4 mg/mL) quanto para PPT (16 mg/mL). Ambos os ATB apresentaram CIM reduzida (inferior a 2 mg/mL) quando aplicados em conjunto com meio FIA. Dependendo da concentração de NaCl, a aplicação de meio FIA possui efeito bactericida sobre bactérias planctônicas e efeito bacteriostático sobre biofilmes já formados. / Multidrug-resistant Pseudomonas aeruginosa is a pathogen capable of producing a protective biofilm against antibiotics (ATB). The present study evaluated therapeutic strategies against biofilms of multidrug-resistant strains of P. aeruginosa representative of clones and/or exhibiting resistance genotypes endemic in Brazil. Biofilms were formed in vitro using an adapted model of MBEC Assay and the therapeutic strategies used lytic bacteriophages, combination of ATB and/or use of high ionic strength (HIS medium). The application of lytic bacteriophages (φSPM-1) and the combination of Aztreonam (ATM) and Piperacillin / Tazobactam (PPT) were unable to remove the biofilm. The application of HIS during biofilm formation restored the bacteriostatic effect of both ATM (4 mg/mL) and PPT (16 mg/ml). Both ATB showed reduced MIC values (less than 2 mg/mL) when applied in conjunction with HIS medium. It was shown that HIS has a bacteriostatic or bactericidal effect on planktonic growth, which depend on the NaCl concentration, and bacteriostatic activity against mature biofilm.

Chai, Stella. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 108-126). / Abstracts in English and Chinese. / ABSTRACT --- p.i / 論文摘要 --- p.iii / ACKNOWLEDGEMENTS --- p.v / PATENT AND PUBLICAION --- p.vii / CONFERENCE ABSTRACTS AND PRESENTATIONS --- p.viii / AWARDS --- p.ix / ABBREVIATIONS --- p.x / LIST OF TABLES --- p.xii / LIST OF FIGURES --- p.xiv / TABLE OF CONTENT --- p.xviii / Chapter CHAPTER 1. --- INTRODUCTION --- p.1 / Chapter 1.1 --- Multidrug resistance (MDR) --- p.1 / Chapter 1.1.1 --- Cancer --- p.1 / Chapter 1.1.2 --- Mechanisms of MDR in cancer --- p.1 / Chapter 1.1.2.1 --- Drug entry --- p.3 / Chapter 1.1.2.2 --- Drug metabolism --- p.3 / Chapter 1.1.2.3 --- Drug sequestration --- p.4 / Chapter 1.1.2.4 --- Mechanisms activated after nuclear entry --- p.5 / Chapter 1.1.2.5 --- Evasion of drug-induced apoptosis --- p.5 / Chapter 1.1.3 --- Approaches in treating MDR --- p.5 / Chapter 1.1.3.1 --- Overcoming MDR by inhibiting transporters --- p.6 / Chapter 1.1.3.2 --- Overcoming MDR by altering signaling pathway --- p.6 / Chapter 1.1.4 --- ATP Binding Cassette (ABC) Transporters --- p.7 / Chapter 1.1.4.1 --- P-glycoprotein (P-gp) --- p.7 / Chapter 1.1.4.2 --- Multidrug resistance-associated protein 1 (MRP 1) --- p.9 / Chapter 1.1.4.3 --- Breast cancer resistant protein (BCRP) --- p.10 / Chapter 1.1.4.4 --- ABC drug transporters and drug absorption --- p.11 / Chapter 1.2 --- The Use of Traditional Chinese Medicine (TCM) in circumventing P-gp-mediated MDR --- p.12 / Chapter 1.2.1 --- Active ingredients in TCM - Alkaloid --- p.12 / Chapter 1.2.2 --- Active ingredients in TCM - Saponin --- p.14 / Chapter 1.2.3 --- Active ingredients in TCM - Flavonoid --- p.15 / Chapter 1.2.4 --- Active ingredients in TCM - Others --- p.17 / Chapter 1.3 --- Polyoxypregnane compounds (POPs) --- p.17 / Chapter 1.3.1 --- Characterization --- p.17 / Chapter 1.3.2 --- POPs isolated from M. tenacissima --- p.18 / Chapter 1.4 --- Objectives of Current Study --- p.22 / Chapter CHAPTER 2. --- EFFECTS OF POLYOXYPREGNANE COMPOUNDS ON VIABILITY AND PROLIFERATION OF HUMAN RESISTANT CANCER CELLS --- p.24 / Chapter 2.1 --- Materials and Methods --- p.25 / Chapter 2.1.1 --- "Chemicals, Materials and Reagents" --- p.25 / Chapter 2.1.2 --- Methods --- p.26 / Chapter 2.1.2.1 --- Cell Lines and Cell Culture --- p.26 / Chapter 2.1.2.2 --- Preparation of POPs --- p.27 / Chapter 2.1.2.3 --- Sulforhodamine B assay --- p.27 / Chapter 2.1.2.4 --- Statistical analysis --- p.29 / Chapter 2.2 --- Results --- p.29 / Chapter 2.2.1 --- Effects of POPs on the viability of parental SW620 and P-gp-overexpressing resistant SW620/Ad300 cells --- p.29 / Chapter 2.2.2 --- Effects of POPs on the viability of parental MCF-7 and MRP1-overexpressing resistant MCF-7/VP cells --- p.33 / Chapter 2.2.3 --- Effects of POPs on the viability of parental MCF-7 and ABCG2-overexpressing resistant MCF-7/FLV1000 cells --- p.37 / Chapter 2.3 --- Discussion --- p.41 / Chapter 2.3.1 --- Structure activity relationship (SAR) --- p.43 / Chapter 2.3.2 --- Nine compounds relating to P-gp-mediated MDR --- p.46 / Chapter CHAPTER 3. --- MECHANISM OF NINE SELECTED POPS IN MODULATING P-GP-MEDIATED MDR --- p.49 / Chapter 3.1 --- Materials and Methods --- p.49 / Chapter 3.1.1 --- "Chemicals, Materials and Reagents" --- p.49 / Chapter 3.1.2 --- Methods --- p.53 / Chapter 3.1.2.1 --- Cell Lines and Cell Culture --- p.53 / Chapter 3.1.2.2 --- Extraction of nine POPs from M. tenacissima --- p.54 / Chapter 3.1.2.3 --- Sulforhodamine B (SRB) assay --- p.55 / Chapter 3.1.2.4 --- Flow cytometry assay --- p.55 / Chapter 3.1.2.5 --- P-gp ATPase assay --- p.56 / Chapter 3.1.2.6 --- Immuno-blot/Western blot analysis --- p.58 / Chapter 3.1.2.7 --- Reverse transcription and quantitative real-time PCR --- p.59 / Chapter 3.1.2.8 --- Statistical analysis --- p.60 / Chapter 3.2 --- Results --- p.60 / Chapter 3.2.1 --- Effects of nine selected POPs on the viability of sensitive human breast cancer MCF-7 cells --- p.60 / Chapter 3.2.2 --- Effects of nine selected POPs on the viability of MDR 1 -transfected HEK1 MDR1 cell line and its control vector transfected cell line HEK293 pcDNA3 --- p.61 / Chapter 3.2.3 --- Effects of nine selected POPs in inhibiting efflux of P-gp substrate --- p.64 / Chapter 3.2.4 --- Effects of nine selected POPs in modulating P-gp ATPase activity --- p.68 / Chapter 3.2.5 --- Effects of nine selected POPs in regulating P-gp protein expression --- p.69 / Chapter 3.2.6 --- MDR1 mRNA expression in various cell lines --- p.72 / Chapter 3.3 --- Discussion --- p.72 / Chapter 3.3.1 --- Effective POPs are targeting specifically P-gp overexpression --- p.73 / Chapter 3.3.2 --- Mechanistic understanding the circumvention of MDR by the effective POPs --- p.74 / Chapter 3.3.2.1 --- Relative potency for the reversal of P-gp-mediated MDR --- p.75 / Chapter 3.3.2.2 --- Inhibition of P-gp-mediated drug efflux across cell membrane by the effective POPs --- p.75 / Chapter 3.3.2.3 --- Stimulation of ATPase by the effective POPs --- p.76 / Chapter 3.3.2.4 --- No effect of POPs on the alteration of P-gp expression --- p.77 / Chapter 3.3.2.5 --- An overall summary of the mechanism of MDR reversal by the effective POPs --- p.78 / Chapter 3.3.3 --- Implication in drug disposition and drug-drug interactions --- p.79 / Chapter 3.3.4 --- Additional information for the structure activity relationship (SAR) --- p.80 / Chapter CHAPTER 4. --- EFFECTS OF CRUDE EXTRACT AND THREE MAJOR POLYOXYPREGNANES (POPS) OF MARS DEN I A TENACISSIMA --- p.81 / Chapter 4.1 --- Materials and Methods --- p.82 / Chapter 4.1.1 --- "Chemicals, Materials and Reagents" --- p.82 / Chapter 4.1.2 --- Methods --- p.82 / Chapter 4.1.2.1 --- "Preparation of M. tenacissima extract, artificial mixture and three fractions" --- p.82 / Chapter 4.1.2.2 --- Sulforhodamine B assay --- p.85 / Chapter 4.1.2.3 --- "Biotransformation study of POP68, POP69 and POP70" --- p.85 / Chapter 4.1.2.4 --- HPLC-MS analysis --- p.86 / Chapter 4.1.2.5 --- Animal care and housing conditions --- p.87 / Chapter 4.1.2.6 --- Toxicity studies of fraction 2 in mice --- p.88 / Chapter 4.1.2.7 --- Statistical analysis --- p.89 / Chapter 4.2 --- Results --- p.89 / Chapter 4.2.1 --- "Effects of crude extract, artificial mixture on the viability of sensitive human breast cancer MCF-7 cells" --- p.89 / Chapter 4.2.2 --- "Effects of crude extract, artificial mixture on the viability of sensitive SW620 and P-gp-overexpressing resistant SW620/Ad300 cells" --- p.90 / Chapter 4.2.3 --- "Metabolites of POP68, POP69 and POP70 after incubation with human intestinal microbiota" --- p.91 / Chapter 4.2.4 --- Toxicity of fraction 2 in mice --- p.94 / Chapter 4.3 --- Discussion --- p.98 / Chapter CHAPTER 5. --- FINAL DISCUSSION AND CONCLUSIONS --- p.105 / REFERENCES --- p.108

Ziel der vorliegenden Arbeit waren pharmakologische Untersuchungen zum antiproliferativen Effekt der beiden Laktatdehydrogenase (LDH)-Inhibitoren Natriumoxamat und Galloflavin sowie des MRP1-Inhibitors Reversan einzeln und in Kombination bei verschiedenen Sauerstoffkonzentrationen in vitro zu untersuchen. Zusätzlich wurde der antiproliferative Effekt der drei Inhibitoren mit dem antiproliferativen Effekt von 5-FU verglichen. Das Konzept zu dieser Arbeit basiert auf Gemeinsamkeiten zwischen LDH und MRP1 in malignen Zellen. Eine ist, dass beide Moleküle von zahlreichen Tumoren überexprimiert werden. Weiter sind beide an der Ausbildung von Chemoresistenz beteiligt und beide werden auch in Hypoxie exprimiert. Zudem wird das für die Funktion von MRP1 notwendige ATP in malignen Zellen hauptsächlich mit der hyperaktiven Glykoloyse gebildet, deren Stoffumsatz auch von der LDH-Aktivität abhängig ist. Eine kombinierte Inhibition beider Zielstrukturen scheint somit geeignet zu sein, um die Proliferation maligner Zellen gezielt zu hemmen. Da in großen Teilen solider Tumoren hypoxische bzw. anoxische Bedingungen vorherrschen, wurde die Wirksamkeit der drei Inhibitoren auch bei 5 % und 1 % Sauerstoff, die als tumorphysiologisch gelten, untersucht. Die wichtigsten Ergebnisse aus dieser Arbeit sind, dass die beiden LDH-Inhibitoren Natriumoxamat und Galloflavin und der MRP1-Inhibitor Reversan einen antiproliferativen Effekt bei kolorektalen Karzinomzellen auslösen, der auch für tumorphysiologische Sauerstoffkonzentrationen nachzuweisen war. So verringerte sich durch Natriumoxamat bzw. Galloflavin der Anteil vitaler Zellen um bis zu 45 % und durch Reversan um bis zu 60 % bei 5 % und 1 % Sauerstoff im Vergleich zur unbehandelten Kontrolle. Auch unterschiedliche Kombination aus Natriumoxamat, Galloflavin und Reversan führten zu einer Steigerung des antiproliferativen Effektes, der auch immer bei tumorphysiologischen Konzentrationen nachzuweisen war. Den stärksten antiproli-ferativen Effekt wies die Dreifachkombination aus Galloflavin, Natriumoxamat und Reversan auf. So verringerte sich der Anteil vitaler Zellen bei 1 % Sauerstoff durch diese Kombination auf bis zu 28 % bei vier der fünf kolorektalen Karzinomzelllinien. Die Dreifachkombination wies einen gleichstarken bzw. stärkeren antiproliferativen Effekt auf als das Chemotherapeutikum 5-FU und zwar ebenfalls bei 5 % und 1 % Sauerstoff. Die Ergebnisse der vorliegenden Arbeit zum antiproliferativen Effekt von Natriumoxamat, Galloflavin (beides LDH-Inhibitoren) und Reversan (MRP1-Inhibitor) in vitro lassen den Schluss zu, dass das Konzept der Arbeit, einen antiproliferativen Effekt auch bei tumorphysiologischen Sauerstoffkonzentrationen zu induzieren, grundsätzlich bestätigt wurde. Auch löste die gemeinsame Hemmung von LDH und MRP1 einen teilweise stärkeren antiproliferativen Effekt aus als 5-FU. Weitere Untersuchungen sind aber ohne Frage nötig, um die molekularen Interaktion zwischen LDH und MRP1 sowie ihrer Inhibition im Detail zu verstehen. / The aim of the present study was to investigate the antiproliferative effect of the two lactate dehydrogenase (LDH) inhibitors sodium oxamate and galloflavin and the MRP1 inhibitor reversan at different oxygen concentrations in vitro. The inhibitors were used individually and in combination. In addition, the antiproliferative effect of the three inhibitors was compared with the antiproliferative effect of 5-FU. The concept of this study is based on similarities between LDH and MRP1 in malignant cells: their overexpression by numerous tumors; their contribution to chemoresistance and their expression in hypoxia. In addition, the ATP necessary for the function of MRP1 is mainly formed in malignant cells by an increased turnover of the hyperactive glycolysis, which also depends on the LDH activity. Thus, a combined inhibition of both targets appears to inhibit tumor cell proliferation effectively. Since hypoxic or anoxic conditions prevail in large parts of solid tumors, the efficacy of the three inhibitors was also investigated at 5% and 1% oxygen, which are considered to be physiological for solid tumors. The most important results of the study are that both sodium oxamate and galloflavin, as well as reversan trigger an antiproliferative effect in colorectal carcinoma cells, even in the presence of tumor physiological oxygen concentrations. For example, the pro-portion of viable cells decreased up to 45% with sodium oxamate or galloflavin and up to 60% with reversan, even at 5% and 1% oxygen compared to untreated control cells. Different combinations of sodium oxamate, galloflavin and reversan resulted in en-hanced antiproliferative effects, which were also demonstrated at tumor physiological oxygen concentrations. The strongest antiproliferative effects were observed with the triple combination of galloflavin, sodium oxamate and reversan. In this combination, the proportion of viable cells decreased to 28% at 1% oxygenation in four of the five colorectal carcinoma cell lines. The triple combination caused an antiproliferative effect that was equal to or even more potent than the antiproliferative effect of the chemotherapeutic agent 5-FU also at 5% and 1% oxygen. The results of this study on the antiproliferative effect of sodium oxamate, galloflavin (both LDH inhibitors) and reversan (MRP1 inhibitor) in vitro seems to confirm the aim of the study, which was to induce an antiproliferative effect even in tumor physiologi-cal oxygen concentrations. In part, the combined inhibition of LDH and MRP1 caused a stronger antiproliferative effect than 5-FU. However, further investigations are neces-sary to comprehend the molecular interaction between LDH and MRP1 as well as its inhibition in detail.

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.