Polypyrrolic (or aromatic) macrocycles have attracted considerable recent interest due to their potential utility in various applications, including as novel materials, anion receptors, and therapeutic leads. In order to explore further the range and utility of this generalized class of molecules, various sapphyrin and sapphyrin analogues were synthesized. Among the new compounds prepared is an inverted sapphyrin, a species that displayed weak, but noticeable aromaticity. Another new system, benzoxasapphyrin was found to display a reduced anion binding affinity compared to other analogous macrocycles. Finally, several derivatized sapphyrins were prepared as potential anticancer leads. Separate from the above, efforts were made to develop anion receptors based on the indole motif. This substructure plays a key role is stabilizing a range of non-covalent interactions in complex biological structures. However, prior to the present study, indole-type hydrogen bonding donors had not been widely used to prepare synthetic anion receptors. Accordingly, a diindolyl quinoxaline system was prepared; it served to demonstrate that small molecule indole receptors can effectively bind phosphate anions in organic media. Finally, a set of cyanide anion indicators were prepared; these were predicated on the use of a cyanide-specific reaction, namely the benzil rearrangement reaction. The benzil reaction-based indicators produced in this way were found to be quite selective for the cyanide anion in organic solvents. Further, their study helped provide insights into the mechanism of this as-yet not fully studied reaction. In the context of developing this specific anion indicator, a review of other reaction-based indicators was reviewed. This is provided in Chapter 3. / text

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published_or_final_version / abstract / toc / Botany / Master / Master of Philosophy

published_or_final_version / Molecular Biology / Master / Master of Philosophy

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We investigated the role of metabotropic glutamate receptors (mGluRs), and related intracellular second messengers, in the development of morphine tolerance and dependence. The mGluRs are divided into three groups: group I mGluRs are positively coupled to phosphatidylinositol (PI) hydrolysis, while group II and III mGluRs are negatively coupled to cyclic adensoine-3$ sp prime$,5$ sp prime$-monophosphate (cAMP) production. Opioid receptors are also coupled to these same systems, and have been shown to elicit changes in these messenger systems during chronic treatment. / We showed that chronic intracerebroventricular (i.c.v.) administration of selective group II and III mGluR antagonists concurrently with subcutaneous (s.c.) morphine significantly reduced the severity of precipitated withdrawal symptoms. Conversely, acute i.c.v. injection of a selective group II mGluR antagonist just prior to the precipitation of withdrawal significantly exacerbated the severity of abstinence symptoms. In addition, acute i.c.v. injection of a selective group II mGluR agonist just prior to the precipitation of withdrawal significantly reduced abstinence symptoms. From these results we hypothesized that chronic opioid treatment may induce a desensitization of group II mGluRs. / We also demonstrated that chronic i.c.v. infusion of a selective group I mGluR antagonist concurrently with s.c. morphine significantly attenuated the precipitated withdrawal syndrome. In addition, we showed that chronic i.c.v. antagonism of $ delta$-opioid receptors with a highly selective antagonist also decreased the development of morphine dependence, as well as tolerance. Since both group I mGluRs and $ delta$-opioid receptors are positively coupled to PI hydrolysis, further evidence for a role of products of PI hydrolysis in the development of morphine dependence was obtained when we showed that selective chronic inhibition of protein kinase C (PKC) activation, as well as selective chronic inhibition of intracellular Ca$ sp{2+}$ release, concurrently with morphine treatment significantly reduced the severity of abstinence symptoms. Thus, compensatory changes usually elicited by chronic opioid treatment may be counteracted by antagonizing receptors positively coupled to PI hydrolysis, as well as by inhibiting products of PI hydrolysis. / In the General Discussion, we propose a model based on the possible interaction of mGluRs and opioid receptors, via related intracellular second messengers, to explain the development of morphine dependence.

Standard local control theory, which describes Ca²⁺ release during excitation-contraction coupling (ECC), assumes that all Ryanodine Receptor (RyR) complexes are equivalent. Recent data from our laboratory has called this assumption into question. Specifically, we have shown that RyR complexes in ventricular myocytes differ depending on their location within the cell. This, and other data, has led us to hypothesize that similar differences occur within the rat atrial cell. To test this hypothesis, we have triple-labeled enzymatically-isolated, fixed myocytes to examine the distribution and colocalization of RyR, calsequestrin (CSQ), voltage-gated Ca²⁺ channels (Cav1.2), sodium-calcium exchangers (NCX), and caveolin-3 (cav-3). All images were acquired on a wide-field microscope, deconvolved, and subject to extensive analysis, including a novel method of measuring statistical significance of the recorded colocalization values. Overall, eight surface RyR populations were identified, depending on its binding partners. One of these groups, in which RyR, Cav1.2, and NCX colocalize, may provide the structural basis for ‘eager’ sites of Ca²⁺ release in atria, while other groups were defined based on their association with cav-3, and are therefore highly likely to be under the influence of other signaling molecules located within caveolae. Importantly, although a small portion of the surface RyR in atria do colocalize with NCX alone, the majority are tightly linked to Cav1.2 alone or Cav1.2 and NCX together. Therefore, it appears likely that Cav1.2-mediated calcium-induced calcium release (CICR) is the primary method of initiating Ca²⁺ release from the SR during EC coupling.