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My research deals with the interplay between metabolism and transport of resveratrol and its metabolites. It takes into account the role of uptake and efflux transporters and enterohepatic circulation in the disposition of resveratrol and conjugated metabolites of resveratrol. The issue of enzyme- and transporter-mediated drug-drug interaction (DDI) is also addressed. Chapter 1 presents an introduction to resveratrol, its biological activities as well as its interactions with enzymes and transporters. It provides a background for enzyme inhibition. It also explains the hypotheses and describes in short, the studies performed. Chapter 2 is based on P450 enzyme inhibition. In the first part of this chapter, we explored the ability of sandwich cultured cryopreserved human hepatocytes to predict inhibition parameters and drug-drug interaction (DDI) values. Two lots of cryopreserved human hepatocytes were used to predict inhibition parameters. The predicted DDI values were compared with those reported in literature and clinical studies and were found to be within 1.5 fold of those reported in clinical studies. The second part of this chapter focuses on the potential of resveratrol or resveratrol-3-glucuronide (R3G) to inhibit CYP2C8. CYP2C8 has been found to be inhibited by glucuronide metabolite such as gemfibrozil-O-β-glucuronide and clopidogrel-β-glucuronide. Hence, we examined the potential of resveratrol, R3G and resveratrol-3-sulfate (R3S) to inhibit CYP2C8. We found that resveratrol, R3G and R3S inhibited CYP2C8 in a reversible manner. Chapter 3 details studies performed in human cancer cell lines (HT-29 and Caco-2) to study the role of uptake transporters in the disposition of resveratrol and R3G. Western blotting was initially performed to examine the expression of OATP1B transporters in cancer cell lines. Uptake studies were performed in HT-29 and Caco-2 cell lines with atorvastatin as a positive control. Both, western blots and uptake studies were inconclusive in detecting the presence of OATP1B transporters. Our studies showed that resveratrol undergoes passive diffusion and sulfation in Caco-2 cell line. The uptake of R3G in Caco-2 cell line was not detectable. In chapter 4, we evaluated the impact of inhibition of efflux transporters on the disposition of resveratrol, R3G and R3S. Mrp2 and bcrp inhibition studies were performed in mice and resveratrol, R3G and R3S were monitored using LC-MS/MS. Non-compartmental analysis was performed to obtain pharmacokinetic parameters. We observed that the inhibition of efflux transporters had a greater impact on area under the curve (AUC) of R3S as compared to R3G and resveratrol. Resveratrol and R3G have been shown to undergo enterohepatic circulation (EHC). This occurs due to the action of gut bacterial β-glucuronidase. This enzyme converts the glucuronide metabolite into parent, which is reabsorbed into enterocytes. The impact of inhibition of gut bacterial β-glucuronidase due to antibiotics was studied in chapter 5. Elimination of gut microbiome was attempted by using a combination of neomycin and bacitracin. Non-compartmental analysis was performed on the observed data. There was no observable difference in the AUCs of resveratrol, R3G and R3S. Chapter 6 deals with simulations performed using an existing pharmacokinetic model to explain the data obtained upon transporter and EHC inhibition. The simulations showed that the inhibition of transporters seemed to decrease the elimination rate constant of R3G and R3S. In summary, we investigated the impact of transporters on pharmacokinetics of resveratrol and its major metabolites. We also investigated P450 inhibition in sandwich cultured human hepatocytes and the potential of resveratrol, R3G and R3S to inhibit rCYP2C8. We were able to show that inhibition of transporters does impact pharmacokinetics of R3S and R3G. / Pharmaceutical Sciences

In this work, a hydrogel made from hyaluronic acid is synthesized and utilized to study neural stem cell behavior within a custom tailored three dimensional microenvironment. The physical properties of the hydrogel have been optimized to create an environment conducive for neural stem cell differentiation by mimicking the native brain extracellular matrix (ECM) environment. The physical properties characterized include degree of methacrylation, swelling ratios, enzymatic degradation rates, and viscoelastic moduli. One dimensional proton nuclear magnetic resonance (1HNMR) confirms modification of the hyaluronic acid polymers, and is used to quantify the degree of methacrylation. Rheological measurements are made to quantify the viscoelastic moduli. Further post-processing by lyophilization leads to generation of large voids to facilitate re-swelling and cell infiltration. ReNcell VM (RVM), and adult human neural stem cell line derived from the ventral mesencephalon, are cultured and differentiated inside the hydrogel for up to 2 weeks. Differentiation is characterized by immunocytochemistry (ICC) and real time quantitative polymerase chain reaction (qRT-PCR). / Bioengineering

There is a large amount of interest and research currently going into studying the effects of low dose radiation on humans, and bridging the gap with the data from the effects of high dose radiation. Much work is to be done to understand low dose exposures such as from medical treatments and those who work with or around radiation. Two popular and widely known examples of low-dose phenomena are the radiation induced bystander effects and the radioadaptive response (RAR). This research involves the study of the impact of a low dose of radiation that is administered several hours after a high – even fatal – dose is given, which contrasts the traditional RAR where a low priming dose is given before a high dose and can lead to increased cell survival. Many different parameters were checked to see if cell survival can be enhanced or diminished depending on the stage of the cell cycle, cell growth conditions, and cell profiling differences in protein function (namely the TP53 gene). Additionally, the post-conditioning response was contrasted to see if it was possible to see any effects from the newly emerging area of bystander signalling, UV BioPhotons, would be present in cell lines that either did or did not exhibit a post-conditioning effect. It was shown that post-conditioning has a protective effect on survival of the cells in certain dose ranges and certain cell lines. The post-conditioning effect also appears to be stronger in magnitude than the classic RAR. No relationship between gamma-induced biophoton signalling and post-conditioning was observed, nor is it certain whether an acute gamma-field can induce significant UV biophoton damage. This thesis is aimed to explore the various parameters by which post-conditioning effects occur on various Human cancers. / Thesis / Master of Science (MSc) / This research looks at the effects of radiation on cells. More specifically, how do low doses of radiation affect cells after they have already been treated with higher doses of radiation. Moreover, can cells communicate through non-physical methods, such as through invisible light? The research focuses primarily on cancer cells and their responses to varying doses of radiation treatments.