Applied cancer research is heavily focused on the development of diagnostic tools with high sensitivity and specificity that are able to accurately detect the presence and anatomical location of neoplastic cells, as well as therapeutic strategies that are effective at curing or controlling the disease while being minimally invasive and having negligible side effects. Recently, much effort has been placed on the development of nanoparticles as diagnostic imaging and therapeutic agents, and several of these nanoplatforms have been successfully adopted in both the research and clinical arenas. This thesis describes the development of a nanoparticulate liposome system for use in a number of applications including multimodality imaging with computed tomography (CT) and magnetic resonance (MR), longitudinal vascular imaging, image-based biodistribution assessment, and CT detection of neoplastic and inflammatory lesions. Extensive in vitro and in vivo characterization was performed to determine the physico-chemical properties of the liposome agent, including its size, morphology, stability and agent loading, as well as its pharmacokinetics, biodistribution, tumor targeting and imaging performance. Emphasis was placed on the in vivo CT-based quantification of liposome accumulation and clearance from healthy and tumor tissues in a VX2 carcinoma rabbit model, gaining insight not only on the spatial but also the temporal biodistribution of the agent. The thesis concludes with a report that describes the performance of liposomes and CT imaging to detect and localize tumor and inflammatory lesions as compared to that of 18F-fluorodeoxyglucose (FDG) – positron emission tomography (PET). The outcome of the study suggests that liposome-CT could be employed as a competitive method for whole body image-based disease detection and localization. Overall, this work demonstrated that this liposome agent along with quantitative imaging systems and analysis tools, has the potential to positively impact cancer treatment outcome through improved diagnosis and staging, as well as enable personalization of treatment delivery via target delineation. However, in order to prove clinical benefit, steps must be taken to advance this agent through the regulatory stages and obtain approval for its use in humans. Ultimately, the clinical adoption of this multifunctional agent may offer improvements for disease detection, spatial delineation and therapy guidance.

G protein-coupled receptors (GPCRs) are known to exist as oligomers, but there is much uncertainty over the oligomeric size, the number of interacting G proteins and the stability of that interaction. The present approach to these questions has been threefold. Monomers of the M2 muscarinic receptor were purified from Spodoptera frugiperda (Sf9) cells and reconstituted in phospholipid vesicles, where they spontaneously formed tetramers. The size of the reconstituted complex was determined from its electrophoretic mobility after cross-linking and inferred from a quantitative, model-based assessment of cooperative effects in the binding of two muscarinic antagonists: N-methylscopolamine and quinuclidinylbenzilate. Binding of the agonist oxotremorine-M to receptor reconstituted with purified G proteins revealed at least three classes of sites that interconverted from higher to lower affinity upon the addition of guanylylimidotriphosphate (GMP-PNP). The binding properties resemble those of muscarinic receptors in myocardial preparations, thereby implying the existence of tetramers in native tissues. G proteins that copurify with the M2 receptor from cardiac membranes also were found to exist as oligomers, some of which contain both alpha(o) and alpha(i2), and the purified complexes contained receptor and G protein in near-equal amounts. A tetrameric receptor implies a tetramer of G proteins, a conclusion that is supported by the distribution of sites between different states identified in the binding of [35S]GTPgammaS to the purified complex. Covalent adducts of a GPCR fused to a Galpha-subunit provide a model system in which the relationship between receptor and G protein complex is defined with respect to stability and composition. Such a fusion of the M2 receptor and Galpha(i1) underwent a cleavage near the amino terminus of the alpha-subunit, however, flagging the likelihood of similar effects in other such adducts. Truncation of the amino terminus prior to fusion generated a stable product that revealed GMP-PNP-sensitive, biphasic binding of oxotremorine-M and noncompetitive interactions between N-methylscopolamine and quinuclidinylbenzilate. A covalent RG complex therefore exhibits the functional properties of M2 receptors in native systems. These observations are consistent with the notion that signaling through the M2 receptor occurs via cooperative interactions within a stable complex that comprises four receptors and four G proteins.

Advances in anti-cancer drug development have vastly improved cancer treatment strategies over the past few decades. Chemotherapeutic agents are now being replaced with targeted therapies that have much greater potency and far fewer unpleasant side effects. At the center of this, cell signaling pathways have been targeted as they moderate gene expression, control proliferation and are often dysregulated in cancer. The signal transducer and activator of transcription (STAT) proteins represent a family of cytoplasmic transcription factors that regulate a pleiotropic range of biological processes in response to extracellular signals. Of the seven mammalian members described to date, Stat3 has received particular attention, as it regulates the expression of genes involved in a variety of malignant processes including proliferation, survival, migration and drug resistance. Aberrant Stat3 activation has been observed in a number of human cancers, and its inhibition has shown promising anti-tumour activity in cancer cells with elevated Stat3 activity. Thus, Stat3 has emerged as a promising target for the development of cancer therapeutics. While Stat3 signaling can be inhibited by targeting upstream regulators of Stat3 activation (such as Janus kinase 2), direct inhibition of Stat3 protein may offer improved response, larger therapeutic windows for treatment and fewer side effects. The work presented within this thesis is focused on optimizing known Stat3 inhibitor S3I-201, a small molecule Stat3 SH2 domain binder that was discovered in 2007. We have performed an extensive structure activity relationship study that has produced some of the most potent Stat3 inhibitors in the scientific literature. These compounds showed high-affinity binding to Stat3’s SH2 domain, inhibited intracellular Stat3 phosphorylation and selectively induced apoptosis in a number of cancer cell lines. Lead agents further inhibited tumour growth in xenograft models of human malignancies and had favourable pharmacokinetic and toxicity profiles.

Advances in anti-cancer drug development have vastly improved cancer treatment strategies over the past few decades. Chemotherapeutic agents are now being replaced with targeted therapies that have much greater potency and far fewer unpleasant side effects. At the center of this, cell signaling pathways have been targeted as they moderate gene expression, control proliferation and are often dysregulated in cancer. The signal transducer and activator of transcription (STAT) proteins represent a family of cytoplasmic transcription factors that regulate a pleiotropic range of biological processes in response to extracellular signals. Of the seven mammalian members described to date, Stat3 has received particular attention, as it regulates the expression of genes involved in a variety of malignant processes including proliferation, survival, migration and drug resistance. Aberrant Stat3 activation has been observed in a number of human cancers, and its inhibition has shown promising anti-tumour activity in cancer cells with elevated Stat3 activity. Thus, Stat3 has emerged as a promising target for the development of cancer therapeutics. While Stat3 signaling can be inhibited by targeting upstream regulators of Stat3 activation (such as Janus kinase 2), direct inhibition of Stat3 protein may offer improved response, larger therapeutic windows for treatment and fewer side effects. The work presented within this thesis is focused on optimizing known Stat3 inhibitor S3I-201, a small molecule Stat3 SH2 domain binder that was discovered in 2007. We have performed an extensive structure activity relationship study that has produced some of the most potent Stat3 inhibitors in the scientific literature. These compounds showed high-affinity binding to Stat3’s SH2 domain, inhibited intracellular Stat3 phosphorylation and selectively induced apoptosis in a number of cancer cell lines. Lead agents further inhibited tumour growth in xenograft models of human malignancies and had favourable pharmacokinetic and toxicity profiles.

Many biosurfactants show antimicrobial activity and some are found to be superior for isolating membrane proteins. This study was aimed towards a general understanding of the interactions of biosurfactants with lipid membranes on a molecular level. To this end, a new, fluorescence lifetime-based membrane leakage assay has been established that does not only quantify membrane permeabilization more precisely but reveals also the leakage mechanism. This mechanism, referred to as graded or all-or-none leakage, is crucial for interpreting potential biological activities and modes of action. Lipopeptides of the surfactin, fengycin, and iturin families as produced by Bacillus subtilis were studied along with synthetic surfactants. Their membrane permeabilizing activity and selectivity mirrored, to some extent, the active concentrations and fungicidal selectivity of the compounds in vivo. Furthermore, the effects of co-surfactants and co-solvents (glycerol, urea, DMSO) have been investigated to better understand and predict means of improving the performance of fungicidal products as well as conditions for membrane protein solubilization.

Many biosurfactants show antimicrobial activity and some are found to be superior for isolating membrane proteins. This study was aimed towards a general understanding of the interactions of biosurfactants with lipid membranes on a molecular level. To this end, a new, fluorescence lifetime-based membrane leakage assay has been established that does not only quantify membrane permeabilization more precisely but reveals also the leakage mechanism. This mechanism, referred to as graded or all-or-none leakage, is crucial for interpreting potential biological activities and modes of action. Lipopeptides of the surfactin, fengycin, and iturin families as produced by Bacillus subtilis were studied along with synthetic surfactants. Their membrane permeabilizing activity and selectivity mirrored, to some extent, the active concentrations and fungicidal selectivity of the compounds in vivo. Furthermore, the effects of co-surfactants and co-solvents (glycerol, urea, DMSO) have been investigated to better understand and predict means of improving the performance of fungicidal products as well as conditions for membrane protein solubilization.

Objective: CSL360 is a chimeric IgG1 mAb recognizing CD123+/CD131- LSCs responsible for acute myeloid leukemia (AML). The in vitro targeting properties of 111In-labeled CSL360 modified with nuclear localization sequence (NLS) were evaluated in AML cells. Methods: 111In-NLS-CSL360 was constructed and its binding affinity, cellular uptake and nuclear importation were analyzed on CD123+ cells. Cytotoxicity was evaluated by clonogenic assays on AML cells (CD123+/CD131-). Results: 111In-NLS-CSL360 exhibited preserved binding to CD123. High cellular and nuclear uptake was observed at 266 nM after 24 hour of incubation. Nuclear uptake of 111In-NLS-CSL360 (266 nM) was 2.0-fold higher than 111In-CSL360 (266 nM) after 24 hour of incubation. Clonogenic survival (CS) of AML cells was reduced to 27.5 ± 4.1%. The nuclear uptake and cytotoxicity were reduced when pre-exposed to unlabeled CSL360, indicating 111In-NLS-CSL360 was CD123-specific. Conclusion: 111In-NLS-CSL360 could be a promising radioimmunotherapeutic agent specific for LSCs.

Objective: CSL360 is a chimeric IgG1 mAb recognizing CD123+/CD131- LSCs responsible for acute myeloid leukemia (AML). The in vitro targeting properties of 111In-labeled CSL360 modified with nuclear localization sequence (NLS) were evaluated in AML cells. Methods: 111In-NLS-CSL360 was constructed and its binding affinity, cellular uptake and nuclear importation were analyzed on CD123+ cells. Cytotoxicity was evaluated by clonogenic assays on AML cells (CD123+/CD131-). Results: 111In-NLS-CSL360 exhibited preserved binding to CD123. High cellular and nuclear uptake was observed at 266 nM after 24 hour of incubation. Nuclear uptake of 111In-NLS-CSL360 (266 nM) was 2.0-fold higher than 111In-CSL360 (266 nM) after 24 hour of incubation. Clonogenic survival (CS) of AML cells was reduced to 27.5 ± 4.1%. The nuclear uptake and cytotoxicity were reduced when pre-exposed to unlabeled CSL360, indicating 111In-NLS-CSL360 was CD123-specific. Conclusion: 111In-NLS-CSL360 could be a promising radioimmunotherapeutic agent specific for LSCs.

Carbonyl and oxidative stress augment the development of diabetic complications. We evaluated the cytoprotectiveness of walnut and hazelnut extracts and catechins for decreasing cytotoxicity, lipid peroxidation, reactive oxygen species (ROS) formation, and protein carbonylation in cell death models of carbonyl and oxidative stress. Polar extracts (methanol or water) showed better cytoprotection than the non-polar (ethyl acetate) nut extracts against hydroperoxide-induced hepatocyte cell death and oxidative stress markers. Catechin flavonoids found in plants, including walnuts and hazelnuts, prevented serum albumin carbonylation in a carbonyl stress model (using glyoxal or methylglyoxal). Hepatocyte protein carbonylation and cell death were prevented and UV spectra data suggested a catechin:methylglyoxal adduct was formed. We conclude that (a) bioactive nut constituents in polar extracts were more protective than non-polar extracts against oxidative stress, and (b) catechins were effective under physiological temperature and pH, at preventing dicarbonyl induced cytotoxicity likely by trapping dicarbonyls or reversing early stage carbonylation.

G protein-coupled receptors (GPCRs) are known to exist as oligomers, but there is much uncertainty over the oligomeric size, the number of interacting G proteins and the stability of that interaction. The present approach to these questions has been threefold. Monomers of the M2 muscarinic receptor were purified from Spodoptera frugiperda (Sf9) cells and reconstituted in phospholipid vesicles, where they spontaneously formed tetramers. The size of the reconstituted complex was determined from its electrophoretic mobility after cross-linking and inferred from a quantitative, model-based assessment of cooperative effects in the binding of two muscarinic antagonists: N-methylscopolamine and quinuclidinylbenzilate. Binding of the agonist oxotremorine-M to receptor reconstituted with purified G proteins revealed at least three classes of sites that interconverted from higher to lower affinity upon the addition of guanylylimidotriphosphate (GMP-PNP). The binding properties resemble those of muscarinic receptors in myocardial preparations, thereby implying the existence of tetramers in native tissues. G proteins that copurify with the M2 receptor from cardiac membranes also were found to exist as oligomers, some of which contain both alpha(o) and alpha(i2), and the purified complexes contained receptor and G protein in near-equal amounts. A tetrameric receptor implies a tetramer of G proteins, a conclusion that is supported by the distribution of sites between different states identified in the binding of [35S]GTPgammaS to the purified complex. Covalent adducts of a GPCR fused to a Galpha-subunit provide a model system in which the relationship between receptor and G protein complex is defined with respect to stability and composition. Such a fusion of the M2 receptor and Galpha(i1) underwent a cleavage near the amino terminus of the alpha-subunit, however, flagging the likelihood of similar effects in other such adducts. Truncation of the amino terminus prior to fusion generated a stable product that revealed GMP-PNP-sensitive, biphasic binding of oxotremorine-M and noncompetitive interactions between N-methylscopolamine and quinuclidinylbenzilate. A covalent RG complex therefore exhibits the functional properties of M2 receptors in native systems. These observations are consistent with the notion that signaling through the M2 receptor occurs via cooperative interactions within a stable complex that comprises four receptors and four G proteins.