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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.

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.

Living organisms rely on many different mechanisms to adapt to changes within their environment. Protein phosphorylation and dephosphorylation events are one such way cells can communicate to generate a response to environmental changes. In the Kennelly laboratory we hope to gain insight on phosphorylation events in the domain Archaea through the study of the acidothermophilic organism Sulfolobus solfataricus. Such findings may provide answers into evolutionary relationships and facilitate an understanding of phosphate transfer via proteins in more elaborate systems where pathway disturbances can lead to disease processes. A λ-phage expression library was generated from S. solfataricus genomic DNA. The immobilized expression products were probed with a purified protein kinase, SsoPK4, and radiolabeled ATP to identify potential native substrates. A protein fragment of the ORF sso0563, the catalytic A-type ATPase subunit A (AtpA), was phosphorylated by SsoPK4. Full length and truncated forms of AtpA were overexpressed in E. coli. Additional subunits of the ATPase were also overexpressed and ATPase activity reconstituted in vitro. Phosphoamino acid analysis and MS identified the phosphorylation sites on AtpA. Several variants of AtpA were derived via site-directed mutagenesis and assayed for ATPase activity. Chemical cross-linking was employed to determine possible ATPase subunit interactions; tryptic digests of AtpA and its mutant variants were performed to examine protein folding. The phosphorylated-mimic variant of AtpA, T98D, resulted in an inactive ATPase complex as determined by ATPase activity assays and native-PAGE indicating potential phosphoregulation by SsoPK4 on enzyme activity. Ultimately, any findings would need verification with in vivo studies. / 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.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [133]-143).

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.