Thesis (Ph. D. in Pharmacology)--Vanderbilt University, May 2006. / Title from title screen. Includes bibliographical references.

PURPOSE: Uveal melanoma (UM) is the most common intraocular malignancy in adults with an incidence of six per one million individuals each year. Globe conserving treatments are currently the standard of care, but unfortunately, despite successful local control, a substantial mortality risk exists due to eventual emergence of distant metastasis, which is invariably lethal. There is therefore an unmet need for novel, effective, targeted therapies for metastatic UM. Somatic mutations in the G-protein α subunits, Gαq and Gα, are present in a mutually exclusive pattern in approximately 80% of UMs, and they abolish the GTPase activity, resulting in a constitutively active protein. We have previously demonstrated that GNAQ-mutant (GNAQ^mt) UMs are addicted to the oncogenic effect of the mutant GNAQ protein and dissected the GNAQ pathway in an attempt to identify druggable targets. Our findings that the mutant GNAQ protein activates the PKC/PKD axis, which activates beta-catenin (β-Catenin), prompted us to investigate the role of PKC and β-Catenin in GNAQ^mt UM. EXPERIMENTAL DESIGN: The GNAQ^mt UM cell lines Mel202 and OMM1.3 were treated with either the PKC inhibitor bisindolylmaleimide I (BIM) alone, the Wnt/β-Catenin inhibitors FH535 or cardamonin alone, the Wnt/β-Catenin activator Wnt-3a alone, or siRNAs for β-Catenin in combination with BIM, and their viability was assessed with the MTT assay. Levels of β-Catenin, phosphorylated AKT, ERK1/2, caspase 3 and LC3BII were assessed with western blotting. β-Catenin mRNA levels were assessed with microarray analysis and RT-PCR. RESULTS: GNAQ^mt UM cells are very sensitive to PKC inhibition and respond with a decrease in cell viability that involves autophagy and cleavage and translocation of LC3BII in autophagosomes, but not caspase activation. PKC inhibition results in the upregulation of β-Catenin protein, but not mRNA levels, through a post-translational mechanism that involved the phosphorylation and activation of AKT, but not ERK1/2. β-Catenin inhibition by either small molecule inhibitors or siRNA resulted in a dose-dependent increase of cell proliferation, whereas β-Catenin activation by Wnt-3a had the opposite effects, resulting in a decrease in cell viability. CONCLUTIONS: Our study demonstrates that PKC is a mediator of the oncogenic effect of mutant Gα protein in UM through the Wnt-3/β-Catenin signaling pathway. These results open exciting opportunities for the development of personalized targeted therapies for UM in a genotype-dependent fashion.

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Several compounds possessing diverse chemical structures have been shown to inhibit the action of protein kinase C (PKC). A general property of some of these compounds is that they inhibit bilayer to hexagonal phase interconversion in phosphatidylethanolamine bilayers. In addition, a large number of PKC inhibitors are positively charged. To clarify the relationship between charge and enzyme inhibition, the effect of the cationic amphiphile sphingosine was studied at different pH's. Inhibition by sphingosine was found to be pH dependent. Above pH 7.75, sphingosine has little or no inhibitory effect. In fact, at pH 8.5 sphingosine slightly enhances enzyme activity above that which occurs when the enzyme is stimulated by diacylglycerol and phosphatidylserine. After correcting for electrostatic repulsion, the intrinsic pK for sphingosine in Triton micelles is 8.5. Inhibition of PKC by sphingosine at physiological pH's therefore correlates with the presence of positive charge. In an attempt to optimize the structural features necessary for inhibition of PKC, a number of compounds which incorporate both positive charge as well as bilayer stabilizing ability were designed. These compounds possess a hydrophobic backbone which does not perturb hydrocarbon packing and have a tertiary or quaternary nitrogen functionality in the head group. All designed amphiphiles inhibit PKC activity; the potency of the amphiphile correlates with the presence of positive charge. Quaternary ammonium bilayer stabilizers are 10 fold more potent than their tertiary amine precursors, generally inhibiting in the 10 - 60 (mu)M range using the Triton mixed micelle assay. Aside from charge, factors such as the structure of the amine containing head group and its length from the hydrocarbon moiety did not markedly influence inhibitor potency. In contrast, the hydrocarbon backbone did influence potency. Cationic amphiphiles containing asteroid backbone were more potent than their straight chain analogues. These amphiphiles do not appear to alter the partitioning of PKC from the aqueous phase to the membrane surface. A number of bilayer stabilizing compounds possessing carboxylate and sulfate anions in addition to the quaternary nitrogen functionality were also designed. Although inhibitor potency correlated with the amount of charge present on the amphiphile, charge could not account for all the observed effects. Changes in the position of the charged functionalities and hydrocarbon length resulted in marked differences in amphiphile potency. Some inhibited in the submicromolar range. The results of these studies suggest that a combination of positive charge and a bilayer stabilizing structural characterisitic provides a basis for the rational design of PKC inhibitors. / Thesis / Master of Science (MS)

The bovine polymorphonuclear leukocyte (neutrophil) is a central component of the acute inflammatory response, and is capable of reacting to a myriad of pro-inflammatory chemical signals that have been characterized in the context of bovine respiratory disease (BRD). Human neutrophils and bovine macrophages are known to react to pro-inflammatory signals as well; however, they are also capable of responding to anti-inflammatory signals from the autonomic nervous system. In particular, activation of the beta2-adrenergic receptor on these cells decreases several aspects of inflammatory activity, including reactive oxygen species production, chemotaxis, degranulation, and inflammatory mediator production. Dysfunction of beta-adrenergic receptors is known to contribute to the pathophysiology of numerous diseases in both people and animals. For example, congestive heart failure, asthma, cystic fibrosis, atopic dermatitis, pheochromocytoma, myasthenia gravis, hypertension, and sepsis have all been linked to decreased beta1- / beta2-adrenergic receptor density (depending on the cell type) and / or uncoupling of the respective receptor from its effector enzyme, adenylyl cyclase. Dysfunction of the beta2-adrenergic receptor mechanism has also been described in pulmonary airway and vascular smooth muscle tissue from cattle, sheep, and rats exposed to Manheimia haemolytica, which provides insight into the pathophysiology of BRD. Despite the prominent role of the bovine neutrophil in the acute inflammatory stage of BRD, and despite the potential for dysfunction following excessive exposure to inflammatory stimuli, there are no reports that describe the presence of the beta2-adrenergic receptor on bovine neutrophils, nor function of the components responsible for its signal transduction cascade. Without complimentary work with bovine neutrophils, using data from human neutrophils to examine treatment options for the acute inflammatory stage of BRD is unrealistic. For this reason, the present dissertation proposed that 1) bovine neutrophils possess the beta2-adrenergic receptor mechanism, 2) components of the beta2-adrenergic receptor mechanism work in concert to increase bovine neutrophil adenosine 3,5-cyclic monophosphate (cAMP) levels and suppress superoxide anion production, and 3) the beta2-adrenergic receptor mechanism is dysfunctional following exposure to inflammatory stimuli. Using the nonselective beta1- / beta2-adrenergic receptor antagonist [3H]CGP-12177 we observed a maximum specific binding density (Bmax) value of 0.19 fmol per 100,000 bovine neutrophils. Although this value is approximately equal to what we observed with dairy cow neutrophils, human neutrophil Bmax values with this radioligand are anywhere from five to ten-fold greater, which suggests a significant species difference. We further defined the adrenergic receptor population on bovine neutrophils to be dominated by the beta2-subtype. Next, we characterized the function of beta2-adrenergic receptors by stimulating cAMP production with the beta2-adrenergic receptor agonist, terbutaline. The role of the beta2-subtype was confirmed when the terbutaline-mediated effect was negated by ICI-118,551, a beta2-adrenergic receptor antagonist. Also, the role of the phosphodiesterase enzyme in cAMP recycling in bovine neutrophils was illustrated, as the terbutaline-mediated rise in cAMP concentration was dependent upon phosphodiesterase inhibition by 3-isobutyl-1-methylxanthine (IBMX). This study confirms the anti-inflammatory nature of the beta2-adrenergic receptor on bovine neutrophils by demonstrating the ability of terbutaline and IBMX to decrease superoxide anion production in a dose-dependent manner. The synthetic cAMP analog, 8-bromo-cAMP also decreased superoxide anion production, but the effect was time-dependent because of its need to diffuse across the cell membrane. Moreover, IBMX exaggerated the terbutaline-mediated effect on superoxide anion production, while cAMP exaggerated the IBMX-mediated effect on superoxide anion, demonstrating that the beta2-adrenergic receptor acts in concert with adenylyl cyclase, while the phosphodiesterase enzyme functions to decrease their signal. By increasing the dose of the inflammatory stimulant opsonized zymosan eight-fold, we were able to eliminate the ability of various concentrations of terbutaline and IBMX to reduce superoxide anion production. We sought to provide a more specific demonstration of this phenomenon by activating protein kinase C (PKC) via phorbol 12-myristate 13-acetate (PMA) administration. However, preincubation with PMA actually increased terbutaline-mediated cAMP production, in a dose and time-dependent manner. At this time, we cannot explain why increasing the dose of opsonized zymosan and PMA had opposite effects on beta2-adrenergic receptor mechanism function. The answer may reside in the many reported functions of PKC isoforms. Additional studies that identify the PKC isoform repertoire in bovine neutrophils may illustrate the potential for selective inhibition, and may lead to more specific identification and treatment of beta2-adrenergic receptor mechanism dysfunction. Also, it remains to be seen how the various components of the bovine neutrophil beta2-adrenergic receptor mechanism function in-vivo during the acute inflammatory stage of BRD. / Ph. D.

Juvenile hormone (JH) is an important insect hormone that controls diverse biological processes in postembryonic development and adult reproduction. JH exerts its effects through the nuclear receptor Methoprene-tolerant (MET). MET is a transcription factor of the basic helix-loop-helix (bHLH)/Per-Arnt-Sim (PAS) family. In the presence of JH, MET forms a heterodimer with its DNA-binding partner Taiman (TAI). The MET-TAI complex directly binds to the regulatory regions of some JH target genes and regulates their transcription. However many questions remain unanswered regarding the JH-regulated gene expression. The work in this report aims to determine the role of protein kinase C (PKC) in JH signaling in adult mosquitoes and to find the direct target genes of Krüppel homolog 1 (Kr-h1), a zinc finger transcription factor encoded by a JH early response gene. We discovered that PKC is an essential component of a membrane-initiated JH signaling pathway. PKC was activated by JH in a phospholipase C (PLC)-dependent manner. Inhibition of PKC activity dramatically decreased the JH-induced gene expression. RNAi experiment indicated that several PKC isoforms were involved in the JH action in adult female mosquitoes. We showed that PKC modulated the transactivation activity of MET by enhancing the binding of MET and TAI to the promoters of JH target genes. Kr-h1 is rapidly upregulated by JH in newly emerged mosquitoes. RNAi-mediated depletion of AaKr-h1 caused a substantial decrease in oviposited eggs, indicating that this protein plays an essential role in mosquito reproduction. We combined chromatin immunoprecipitation (ChIP) with cloning of the generated DNA and have identified chromatin binding sites of AaKr-h1 in Aedes aegypti. After adult emergence, binding of AaKr-h1 to its in vivo targets increased with the JH-induced increase in AaKr-h1. Interestingly, depletion of AaKr-h1 in newly emerged mosquitoes led to considerable upregulation of some AaKr-h1 target genes but downregulation of other target genes. The results suggest that AaKr-h1 acts downstream of AaMET to regulate gene expression in response to JH and that AaKr-h1 can activate or repress the expression of individual target gene. / Ph. D.

Thesis (M.S.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains v, 105 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

Thesis (Ph. D.)--West Virginia University, 2004 / Title from document title page. Document formatted into pages; contains vii, 350 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 322-346).

Thesis (Ph. D.)--West Virginia University, 2003. / Title from document title page. Document formatted into pages; contains v, 149 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.