published_or_final_version / Medicine / Master / Master of Philosophy

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The structural elucidations of nine new natural products from Thevetia ahouia, Eremocarpus setigerus, Trichilia hispida, Chrysothamus paniculatus, Uvaria acuminata, Wikstroemia monticola, and Uvaria zelanica are discussed in detail. Also included are some known compounds that were found in these plants. Of the new compounds discussed, two are diterpenes (eremone, chrysothame), two are relatively simple triterpenes (hispidols A, B), two are highly oxidized triterpenes (hispidins A, B), one is a fatty acid derivative (uvaricin), one is a shikimate-derived metabolite (1-epizeylenol), and one has both steroidal and carbohydrate features (3'-OMe-evomonoside). ¹H and ¹³C NMR spectral data are given for all the new compounds. Assignments were made by analogy with model compounds, computer simulation, and ¹H-¹H and ¹H-¹³C decoupling where indicated. High resolution mass spectral fragmentations are given for most of the new compounds, and individual structural assignments made for intense peaks. An X-ray analysis (eremone) was done with all pertinent information included. Most of the compounds have been tested for anti-tumor activity, and several exhibit strong cytotoxicity (hispidins A and B, 3'-OMe-evomonoside, huratoxin, uvaricin).

A number of mathematical models of cancer growth and treatment are presented. The most significant model presented is of the interactions between a growing tumor and the immune system. The equations and parameters of the model are based on experimental and clinical results from published studies. The model includes the primary cell populations involved in effector-T-cell-mediated tumor killing: regulatory T cells, helper T cells, and dendritic cells. A key feature is the inclusion of multiple mechanisms of immunosuppression through the main cytokines and growth factors mediating the interactions between the cell populations. Decreased access of effector cells to the tumor interior with increasing tumor size is accounted for.The model is applied to tumors of different growth rates and antigenicities to gauge the relative importance of the various immunosuppressive mechanisms in a tumor. The results suggest that there is an optimum antigenicity for maximal immune system effect. The immunosuppressive effects of further increases in antigenicity outweigh the increase in tumor cell control due to larger populations of tumor-killing effector T cells. The model is applied to situations involving cytoreductive treatment, specifically chemotherapy and a number of immunotherapies. The results how that for some types of tumors, the immune system is able to remove any tumor cells remaining after the therapy is finished. In other cases, the immune system acts to prolong remission periods. A number of immunotherapies are found to be ineffective at removing a tumor burden alone, but offer significant improvement on therapeutic outcome when used in combination with chemotherapy.Two simplified classes of cancer models are also presented. A model of cellular metabolism is formulated. The goal of the model is to understand the differences between normal cell and tumor cell metabolism. Several theories explaining the Crabtree Effect, hereby tumor cells reduce their aerobic respiration in the presence of glucose, have been put forth in the literature; the models test some of these theories, and examine their plausibility.A model of elastic tissue mechanics for a cylindrical tumor growing within a ductal membrane is used to determine the buildup of residual stress due to growth. These results can have possible implications for tumor growth rates and morphology.

There are currently no ideal treatments or acceptable vaccines for cutaneous leishmaniasis, a worldwide health problem caused by infection with a number of species of the dimorphic protozoa Leishmania. Therefore, there is an urgent need to search for simple, safe, effective, and affordable treatments. Imiquimod is an immune-response modifying agent. Recently, 5% imiquimod cream (Aldara(TM)) received approval by the Food and Drug Administration in the United States and is currently available for the treatment of external genital and perianal warts caused by human papillomavirus infection. The antiviral activity of this drug is mediated through stimulation of cytokine release from many cell types including macrophages resulting in a local immune response at the site of application. Moreover, imiquimod has been shown to enhance cell-mediated immune responses (CMIR). Since imiquimod activates macrophages, the exclusive host cells of Leishmania, and stimulates CMIR which are required for host defence against Leishmania, we have investigated the potential of using imiquimod and its related compound, S-28463, as agents for treating leishmaniasis. It is demonstrated within that imiquimod and S-28463 effectively stimulated leishmanicidal activity both in vitro in macrophages and in vivo in a mouse model. These compounds also stimulated signal transduction associated with the induction of nitric oxide synthesis in macrophages. Imiquimod and S-28463 induced leishmanicidal activity in macrophages in the absence of any other cell types. We have demonstrated that S-28463 generated macrophage leishmanicidal activity by inducing genes involved in macrophage activation and inflammatory responses. Finally, we have also performed an analysis on the influence of L. donovani on macrophage gene expression using a cDNA array analysis, a similar methodology to study the effect of S-28463 on macrophage gene expression. Intramacrophage infection with L. donovani was shown to cause general

The development of multidrug resistance (MDR) in tumour cells to a wide range of anticancer drugs has become a major obstacle in the chemotherapeutic treatment of cancer. Molecular characterization of MDR tumour cells has led to the identification of several cell-based genetic alterations including the overexpression of a membrane protein, P-glycoprotein (P-gp). P-gp is a ATP dependent drug efflux pump and P-gp ATPase activity has been demonstrated to be essential in drug transport. In an effort to understand how P-gp ATPase activity is coupled to drug binding and transport, we examined the effects of N-ethylmaleimide (NEM), a potent inhibitor of P-gp ATPase, on P-gp drug binding and transport. Our results show that short term treatment of MDR cells with NEM led to a concentration-dependent increase in P-gp drug binding and phosphorylation. In addition, NEM increases [3H]-vinblastine accumulation in drug resistant cells but not in sensitive cells. Our study suggests that inhibition of P-gp ATPase activity, and not increased phosphorylation of P-gp by NEM, is responsible for the observed increase in P-gp-drug binding. / Selection of tumour cell lines in vitro has led to multiple cellular changes that may mediate drug resistance to anticancer drugs. The role of other mechanisms, in addition to P-gp and multidrug resistance protein (MRP) in drug resistance, is supported by evidence from studies with tumour cell lines and clinical tumours. In an effort to identify other cellular changes that may be important in tumour drug resistance to anticancer drugs, we have used a differential immunodot blot method to isolate monoclonal antibodies that bind to proteins in drug resistant but not in drug sensitive cells. By using the immunodot blot method, we have isolated a monoclonal antibody (IPM96) which recognized a 40 kDa protein (P-40) in several MDR cell lines. The expression of P-40 is concurrent with the level of drug resistance. Biochemical characterization showed P-40 to be associated with the cell membrane and in the soluble fraction. Molecular cloning of P40 cDNA revealed that P-40 is identical to annexin I, a substrate for the epidermal growth factor receptor tyrosine kinase. The observed increase in P-40 (or annexin I) protein levels in drug resistant cells is due to the elevation of P-40 transcripts. The pharmacological characterization of P-40 cDNA transfectants (P-40-MCF-7) has demonstrated that overexpression of P-40 in drug sensitive cells is capable of conferring drug resistance to adriamycin, actinomycin D, Taxol and cisplatin. Taken together, our study provides convincing evidence that annexin I is important in the development of drug resistance in cancer cells. In addition, it suggests a novel mechanism of drug resistance that is different from the ATP-dependent drug efflux pumps that mediate P-gp- and MRP-associated MDR