L'objectif de cette thèse était de caractériser les éléments appartenant à la voie de signalisation énergétique AMP-activated protein kinase chez l'huître creuse Crassostrea gigas afin de comprendre son implication dans la gestion de l'énergie, en particulier en réponse à des conditions physiologiques qui sollicitent de l'énergie telles que la reproduction, ou à des stress environnementaux comme l'hypoxie ou le jeûne. Au niveau génomique, les trois sous-unités constitutives du trimère AMPK ainsi que plusieurs éléments impliqués dans cette voie de signalisation et dans les métabolismes glucidiques et lipidiques, potentiellement cibles de l'AMPK, ont été décrits. Au niveau protéique, plusieurs anticorps hétérologues ciblant les isoformes de la sous-unité α et la phosphorylation du résidu thréonine 172 de la sous-unité α, témoin indirect de l'activité AMPK, ont été utilisés. Deux sous-unités α tronquées dans le domaine kinase ont été caractérisées principalement dans les tissus musculaires suggérant leurs implications dans la fonction musculaire. Au cours d'un stress hypoxique, une augmentation significative des quantités de sous-unités α tronquées a été observée dans le muscle lisse. Ce résultat suggère que pendant une durée d'au moins 6 h, ces protéines tronquées sont nécessaires au maintien du métabolisme aérobie dans le muscle lisse, lui permettant ainsi de remplir son rôle de fermeture statique des valves. Nous avons suggéré une hypothèse indiquant que l'accumulation in vivo de ces sous-unitésα tronquées pourrait exercer un rôle de modulation ou de transdomination négative de l'activité de la sous-unité α entière. Dans la gonade, nous avons observé une activation de l'AMPK tout au long du processus de gamétogénèse afin de supporter les processus cataboliques de création de gamètes. Une diminution de cette activation a été observée lors du stade anabolique de mise en réserve des ovocytes. Enfin, lors d'un conditionnement en milieu contrôlé, une approche physiologique par privation de nourriture et une approche pharmacologique par injection d'AICAR ont été réalisées pour provoquer une modulation de l'AMPK. Les analyses ont montré que ni le jeûne ni l'AICAR n'ont induit une augmentation de la phosphorylation de la sous-unité α. Cependant, plusieurs changements liés à l'injection de l'AICAR ont été observés sur la physiologie de l'huître : la modification du rapport AMP:ATP chez les huîtres nourries en comparaison aux huîtres à jeun, et une mortalité dépendante de la dose injectée d'AICAR chez les huîtres mises à jeun. La caractérisation de l'AMPK chez C. gigas ouvre de nombreuses perspectives exigeant des études fonctionnelles poussées afin de démontrer le rôle pivot de cette kinase dans la gestion de l'énergie, comme démontré chez de nombreuses espèces de vertébrés, et ainsi décrypter le métabolisme énergétique de l'huître.

Acute lung injury (ALI) is a major cause of mortality after lung transplantation. Recent studies indicate protein kinase C delta (PKCδ) could be an effective target to treat ALI. We have developed a gold nanoparticle (GNP)-peptide hybrid that can inhibit PKCδ signaling. PKCδ inhibitor peptide (PKCi) and 95P2P4 stabilizing peptides were conjugated onto GNP. Physicochemical properties of the nanoformulations were examined. A lung transplant-simulated cell culture model was used to evaluate therapeutic efficacy in vitro. A pulmonary ischemia-reperfusion (IR) model was used to test therapeutic efficacy in vivo. GNP-Peptide hybrids showed good stability with high cellular uptake. GNP-PKCi formulations demonstrated anti-inflammatory and anti-apoptotic effects in vitro. When administered to rats under IR stress, GNP-PKCi formulation improved blood oxygenation, reduced pulmonary edema and histological lung injury. In conclusion, we have successfully formulated a clinically-applicable nanoparticle with therapeutic potential to ameliorate lung injury and inflammation. Our formulation strategy could be used to deliver other peptide-based drugs.

Acute lung injury (ALI) is a major cause of mortality after lung transplantation. Recent studies indicate protein kinase C delta (PKCδ) could be an effective target to treat ALI. We have developed a gold nanoparticle (GNP)-peptide hybrid that can inhibit PKCδ signaling. PKCδ inhibitor peptide (PKCi) and 95P2P4 stabilizing peptides were conjugated onto GNP. Physicochemical properties of the nanoformulations were examined. A lung transplant-simulated cell culture model was used to evaluate therapeutic efficacy in vitro. A pulmonary ischemia-reperfusion (IR) model was used to test therapeutic efficacy in vivo. GNP-Peptide hybrids showed good stability with high cellular uptake. GNP-PKCi formulations demonstrated anti-inflammatory and anti-apoptotic effects in vitro. When administered to rats under IR stress, GNP-PKCi formulation improved blood oxygenation, reduced pulmonary edema and histological lung injury. In conclusion, we have successfully formulated a clinically-applicable nanoparticle with therapeutic potential to ameliorate lung injury and inflammation. Our formulation strategy could be used to deliver other peptide-based drugs.

Septic shock is a major cause of deaths resulting from uncontrolled inflammation and circulatory failure. Recent studies suggest that the vascular isoform of ATP-sensitive K+ (KATP) channels is an important contributor to septic susceptibility. To understand the molecular mechanisms for channel regulation during sepsis, we performed studies in isolated endothelium-denuded mesenteric rings. Lipopolysaccharides (LPS) induced vascular relaxation and hyporeactivity to phenylephrine. The LPS-treated aortic smooth muscle cells displayed hyperpolarization and augmentation of KATP channel activity. Both were due to an up-regulation of Kir6.1 and SUR2B surface expression. The up-regulation relied on transcriptional and translational mechanisms, in which nuclear factor-¦ÊB (NF-¦ÊB) and Protein kinase A (PKA) played a critical role. Oxidative stress occurs during sepsis and may act as another regulatory mechanism affecting KATP channel activity and vascular contractility. We found that micromolar concentrations of H2O2 impaired the pinacidil-induced vasodilation. The effect attributed to the suppression of KATP channel activity, which can be fully produced by reactivity oxidants. Unlike the Kir6.1/SUR2B channel, the Kir6.2/SUR2B channel was insensitive to 1mM H2O2, indicating that the modulation sites are located in Kir6.1. Site-directed mutational analysis showed that three cysteine residues located in N-terminus and the core region of Kir6.1 were likely to mediate the redox-dependent channel modulation. Arginine vasopressin (AVP) is a vasoconstrictor that is successfully applied to manage sepsis. However, the downstream target of AVP is uncertain. Our studies show that AVP-induced vasoconstriction depended on V1a receptor, Protein kinase C (PKC) and KATP channel. Additionally, AVP decreased Kir6.1/SUR2B channel activity through V1a receptor. The inhibitory effect was caused by a suppression of the channel open state probability. The channel inhibition was mediated by phosphorylation of the channel protein by PKC. The widespread involvement of the vascular KATP channel in vascular responses to endotoxemia strongly suggests that the temporospatial control of channel activity may constitute an important intervention to vascular tone, blood pressure and organ-tissue perfusion in septic shock. Such a control appears feasible by targeting several modulatory mechanisms of intracellular signaling, Kir6.1/SUR2B expression, redox state and channel protein phosphorylation as demonstrated in this dissertation.

Cyclic-AMP dependent protein kinase A (PKA) regulates traffic of multiple proteins at different stages along the constitutive secretory pathway. PKA effects are regulated by protein phosphatases, which reverse the actions of PKA by dephosphorylation of PKA-substrates. Localization of specific PKA effects is mediated by the binding of A-kinase anchoring proteins (AKAPs). Apolipoprotein E (apoE) is an important regulator of lipid metabolism and atherosclerosis, and represents a large proportion of total protein constitutively secreted from macrophages. The signalling and trafficking pathways regulating secretion of apoE are unknown. Catalase is a peroxisomal enzyme which contributes to defence against hydrogen peroxide (H2O2). The primary hypothesis of this thesis is PKA and related protein phosphatase pathways are involved in the regulation of apoE secretion. The secondary hypothesis is that these pathways also regulate cellular clearance of H2O2. In Chapter Three, I have investigated the role of PKA in apoE secretion from primary human macrophages. Structurally distinct inhibitors of PKA (H89, KT5720, inhibitory peptide PKI14-22) all decreased basal secretion of apoE by between 50-80% whereas apoE mRNA or cellular protein are unaffected. Disruption of PKA-AKAP anchoring also significantly inhibited apoE secretion from human macrophages. Secretion of apoE was not immediately stimulated by PKA activity, suggesting that although PKA activity may be permissive for apoE secretion, it is in itself insufficient to stimulate apoE secretion above basal levels. Data from confocal microscopy and live cell imaging revealed PKA inhibition paralysed apoE vesicular movement from and to the plasma membrane. In Chapter Four, I investigated the effects of protein phosphatase 2B (PP2B) inhibition on apoE secretion by cyclosporin A (CsA). This was found to dose- and time-dependently inhibit secretion of apoE from primary human macrophages and increased cellular accumulation of apoE without affecting apoE mRNA levels. The role of PP2B in regulating apoE secretion was confirmed by using additional peptide and chemical inhibitors of PP2B. This effect was independent of the known inhibition of ABCA1 by CsA. Live cell imaging and confocal microscopy all demonstrated that inhibition of PP2B did not affect the apparent cellular distribution of apoE. Biochemical and microscopy studies indicated distinct mechanisms for PKA and PP2B regulation of apoE secretion. Chapter Five identified PKA-anchoring AKAPs in human macrophages, and investigated AKAP220 expression and its role in PKA-dependent processes relevant to atherosclerosis. AKAP220 protein was absent in human monocytes but was detectable after their differentiation into macrophages, with stable expression during late stages of maturation. It was also present in Chinese Hamster Ovary cells (CHO) cells. AKAP220 silencing had no effects on lipoprotein cholesteryl ester accumulation, total cellular apoE levels, apoE secretion or cholesterol efflux from human macrophages. Confocal microscopy in CHO cells revealed peroxisomal localisation of AKAP220. Catalase activity was confirmed to be PKA-regulated process, and AKAP220 was found to be a negative regulator of catalase activity, such that cell lysate catalase activity increased during AKAP220 silencing. AKAP220 silencing also decreased basal secretion of H2O2, detected using a sensitive and specific Amplex?? Red assay kit from intact CHO monolayers. In conclusion, this thesis has provided evidence that apoE secretion occurs via PKA- and PP2B-dependent pathways in human macrophages, and has identified the A-kinase anchoring protein AKAP220 as a regulator of cellular H2O2 clearance. These results will provide a basis for future investigations into the roles of PKA-related pathways in apoE secretion and catalase activity.

Cyclic-AMP dependent protein kinase A (PKA) regulates traffic of multiple proteins at different stages along the constitutive secretory pathway. PKA effects are regulated by protein phosphatases, which reverse the actions of PKA by dephosphorylation of PKA-substrates. Localization of specific PKA effects is mediated by the binding of A-kinase anchoring proteins (AKAPs). Apolipoprotein E (apoE) is an important regulator of lipid metabolism and atherosclerosis, and represents a large proportion of total protein constitutively secreted from macrophages. The signalling and trafficking pathways regulating secretion of apoE are unknown. Catalase is a peroxisomal enzyme which contributes to defence against hydrogen peroxide (H2O2). The primary hypothesis of this thesis is PKA and related protein phosphatase pathways are involved in the regulation of apoE secretion. The secondary hypothesis is that these pathways also regulate cellular clearance of H2O2. In Chapter Three, I have investigated the role of PKA in apoE secretion from primary human macrophages. Structurally distinct inhibitors of PKA (H89, KT5720, inhibitory peptide PKI14-22) all decreased basal secretion of apoE by between 50-80% whereas apoE mRNA or cellular protein are unaffected. Disruption of PKA-AKAP anchoring also significantly inhibited apoE secretion from human macrophages. Secretion of apoE was not immediately stimulated by PKA activity, suggesting that although PKA activity may be permissive for apoE secretion, it is in itself insufficient to stimulate apoE secretion above basal levels. Data from confocal microscopy and live cell imaging revealed PKA inhibition paralysed apoE vesicular movement from and to the plasma membrane. In Chapter Four, I investigated the effects of protein phosphatase 2B (PP2B) inhibition on apoE secretion by cyclosporin A (CsA). This was found to dose- and time-dependently inhibit secretion of apoE from primary human macrophages and increased cellular accumulation of apoE without affecting apoE mRNA levels. The role of PP2B in regulating apoE secretion was confirmed by using additional peptide and chemical inhibitors of PP2B. This effect was independent of the known inhibition of ABCA1 by CsA. Live cell imaging and confocal microscopy all demonstrated that inhibition of PP2B did not affect the apparent cellular distribution of apoE. Biochemical and microscopy studies indicated distinct mechanisms for PKA and PP2B regulation of apoE secretion. Chapter Five identified PKA-anchoring AKAPs in human macrophages, and investigated AKAP220 expression and its role in PKA-dependent processes relevant to atherosclerosis. AKAP220 protein was absent in human monocytes but was detectable after their differentiation into macrophages, with stable expression during late stages of maturation. It was also present in Chinese Hamster Ovary cells (CHO) cells. AKAP220 silencing had no effects on lipoprotein cholesteryl ester accumulation, total cellular apoE levels, apoE secretion or cholesterol efflux from human macrophages. Confocal microscopy in CHO cells revealed peroxisomal localisation of AKAP220. Catalase activity was confirmed to be PKA-regulated process, and AKAP220 was found to be a negative regulator of catalase activity, such that cell lysate catalase activity increased during AKAP220 silencing. AKAP220 silencing also decreased basal secretion of H2O2, detected using a sensitive and specific Amplex?? Red assay kit from intact CHO monolayers. In conclusion, this thesis has provided evidence that apoE secretion occurs via PKA- and PP2B-dependent pathways in human macrophages, and has identified the A-kinase anchoring protein AKAP220 as a regulator of cellular H2O2 clearance. These results will provide a basis for future investigations into the roles of PKA-related pathways in apoE secretion and catalase activity.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 95-113).

Diss. (sammanfattning) Stockholm : Karol. inst., 2006. / Härtill 4 uppsatser.

Dissertation (Ph.D.)--University of South Florida, 2007. / Includes vita. Includes bibliographical references. Also available online.