This dissertation is a composite of some of the research that I have conducted during the course of my PhD study. The larger goal of this dissertation is to renew the interests among the scientific community for an otherwise under-appreciated technique called Isothermal Titration Calorimetry. The resurgence of calorimetry in the biophysical community and the shift to investigations of more complex biological systems signal a real need for more sophisticated analysis techniques. This dissertation expounds on new ITC analysis methods that we have developed as well as results from the study of thermodynamic properties of higher order DNA structures. In 1978, Peter Privalov described the first use of microcalorimetry to obtain the thermodynamic properties for removing calcium from parvalbumin III protein. Fast forward 36 years: modern day electronics, highly efficient thermally conductive and chemically inert materials, in conjunction with sensitive thermal detectors, has transformed the original calorimeter into a device capable of measuring heat changes as small as 0.05 nanowatts, which is equivalent to capturing heat from an incandescent light bulb a kilometer away. However, analytical methods have not kept pace with this technology. Commercial ITC instruments are typically supplied with software that only includes a number of simple interaction models. As a result, the lack of analysis tools for more complex models has become a limiting factor for many researchers. We have recently developed new ITC fitting algorithms that we have incorporated into a userriendly program (CHASM©) for the analysis of complex ITC equilibria. In a little over a year, CHASM© has been downloaded by over 370 unique users. Several chapters in this dissertation demonstrate this software’s power and versatility in the thermodynamic investigations of two model systems in both aqueous and non-aqueous media. In chapter VI, we assembled a model NHE-III1 : a novel structure of Gquadruplex in a double stranded form and studied its structural complexity and binding interactions with a classical G-quaduplex interactive ligand known as TMPyP4. In chapter VII, we reported the thermodynamic properties of a novel PAH system in which weak dispersion forces are solely responsible for formation of the supramolecular complexes.

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Breast cancer is the second leading cause of death due to cancer in women. A number of effective therapeutic strategies have been implemented in clinics to cope with the disease yet recurrent disease and toxicity reduce their effectiveness. Hence, there is a need to identify and develop more effective therapies with reduced toxic side effects to improve overall survival rates. This dissertation investigates the mechanisms of action of two natural forms of vitamin E and a cholesterol lowering drug, simvastatin, as a therapeutic strategy in human breast cancer cells. Vitamin E in nature consists of eight distinct forms which are fat soluble small lipids. Until recently, vitamin E was known as a potent antioxidant but emerging work suggests they may be resourceful agents in managing a number of chronic diseases including cancer. Anticancer properties of vitamin E have been identified to be limited to the γ- and δ- forms of both tocopherols and tocotrienols. Gamma-tocopherol ([gamma]T) and gamma-tocotrienol ([gamma]T3) have both already been identified to induce death receptor 5 (DR5) mediated apoptosis in breast cancer cells. Studies here show that similar to [gamma]T3, [gamma]T induced DR5 activation is mediated by c-Jun N-terminal kinase/C/EBP homologous protein (JNK/CHOP) proapoptotic axis which in part contributed to [gamma]T mediated dowregulation of c-FLIP, Bcl-2 and Survivin. Also, both agents activate de novo ceramide synthesis pathway which induces JNK/CHOP/DR5 proapoptotic axis and downregulates antiapoptotic factors FLICE inhibitory protein (c-FLIP), B-cell lymphoma 2 (Bcl-2) and Survivin leading to apoptosis. Simvastatin (SVA) has been identified to display pleiotropic effects including anticancer effects but mechanisms responsible for these actions have yet to be fully understood. In this dissertation, it was observed that simvastatin induced apoptosis in human breast cancer cells via activation of JNK/CHOP/DR5 proapoptotic axis and down regulation of antiapoptotic factors c-FLIP and Survivin which are in part dependent on JNK/CHOP/DR5 axis. The anticancer effects mediated by simvastatin can be reversed by exogenously added mevalonate and geranylgeranyl pyrophosphate (GGPP), implicating the blockage of mevalonate as a key event. Furthermore, work has been done to understand the factors responsible for drug resistance and identify therapeutic strategies to counteract the same. It was observed that development of drug resistance was associated with an increase in the percentage of tumor initiating cells (TICs) in both tamoxifen and Adriamycin resistant cells compared to their parental counterparts which was accompanied by an increase in phosphorylated form of Signal transducer and activator of transcription 3 (Stat3) proteins as well as its downstream mediators c-Myc, cyclin D1, Bcl-xL and Survivin. Inhibition of Stat3 demonstrated that Stat3 and its downstream mediators play an important role in regulation of TICs in drug resistant breast cancer. Moreover, SVA, [gamma]T3 and combination of SVA+[gamma]T3 has been observed to target TICs in drug resistant human breast cancer cells and downregulate Stat3 as well as its downstream mediators making it an attractive agent to overcome drug resistance. From the data presented here, the mechanisms responsible for the anticancer actions of [gamma]T, [gamma]T3 and SVA have been better understood, providing the necessary rationale to test these agents by themselves or in combination in pre-clinical models. / text

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.