Les carcinomes cortico-surrénaliens (CCS) sont des tumeurs malignes rares de mauvais pronostic et pour lesquelles les options thérapeutiques efficaces sont inexistantes. Il est donc indispensable de comprendre les mécanismes moléculaires impliqués dans le développement des CCS, afin d’améliorer leur prise en charge. Les deux altérations les plus fréquentes dans les CCS sont une surexpression du facteur de croissance IGF2 et l'activation constitutive de la voie Wnt/β-caténine. Le laboratoire a mis en évidence le rôle oncogénique de la β-caténine à l'aide d'un modèle murin (souris ΔCat) présentant une activation constitutive de la β-caténine dans la cortico-surrénale. Toutefois, la faible pénétrance du phénotype malin suggère la nécessité d'autres altérations pour la progression tumorale. L’objectif initial de ma thèse était de tester le pouvoir oncogénique de IGF2, seul ou en association avec l’activation constitutive de la β-caténine. Les modèles de surexpression de Igf2 dans la cortico-surrénale nous ont permis de montrer que Igf2 n'initie pas le développement de tumeurs cortico-surrénaliennes. Dans un contexte d'activation de la β-caténine, la surexpression de Igf2 favorise le développement tumoral à des stades tardifs. Toutefois la formation de tumeurs malignes reste un évènement rare. Ces résultats suggèrent donc que la surexpression de Igf2 et l'activation de la β-caténine ne sont pas suffisantes dans notre modèle pour induire le développement de CCS. Une analyse rétrospective des données de transcriptome nous a permis de mettre en évidence une surexpression de l’oncogène putatif et histone méthyl-transférase EZH2, qui est associée à un mauvais pronostic. Mes travaux in vitro suggèrent que EZH2 est surexprimé en réponse à une surexpression des facteurs E2F et qu’il pourrait être impliqué dans le contrôle de la prolifération, de l'apoptose et de certaines caractéristiques tumorales des cellules cortico-surrénaliennes humaines H295R. Des inhibiteurs pharmacologiques étant disponibles, EZH2 pourrait constituer une cible thérapeutique intéressante pour le traitement des CCS. En parallèle de ces travaux, nous avons cherché à identifier les mécanismes impliqués dans la zonation du cortex surrénalien. Au cours du renouvellement tissulaire, les cellules acquièrent d’abord une identité glomérulée puis fasciculée. L'identité de la zone glomérulée repose en partie sur l'activité de la voie Wnt/β-caténine. Cette voie de signalisation induit l'expression de gènes essentiels à l'identité de cette zone et inhibe l'identité fasciculée. La différenciation fasciculée des cellules doit donc reposer en partie, sur l'inhibition de cette voie de signalisation. Nous avons donc émis l'hypothèse que la signalisation PKA, activée dans la zone fasciculée par la liaison de l'ACTH à son récepteur MC2R, s'oppose à l'activité de la β-caténine pour permettre la différenciation fasciculée. A l'aide d'approches pharmacologiques et génétiques, nous avons pu mettre en évidence que l'activation de la PKA inhibe la voie Wnt/β-caténine dans le cortex surrénalien et que ceci est à l'origine d'une perte de la zone glomérulée au profit d'une expansion de la fasciculée. L'effet de la PKA sur la voie Wnt résulte au moins en partie de l'inhibition de l'expression de Wnt4 en réponse à l'activation de la PKA. En effet une diminution d'expression de Wnt4 est observée en réponse à l'activation de la PKA dans la cortico-surrénale et l'invalidation de Wnt4 spécifiquement dans le cortex induit un phénotype proche de celui observé lors de l'activation de la PKA. Au delà des mécanismes moléculaires de la zonation, nous avons également montré que l’effet inhibiteur de la PKA sur la signalisation Wnt était capable de s’opposer aux effets oncogéniques de la β- caténine dans la cortico-surrénale. Ces observations pourraient s’avérer pertinentes, la voie de signalisation ACTH/PKA étant inhibée dans les CCS. / Adrenocortical carcinoma (ACC) is a rare tumour associated with poor prognosis and for which, efficient therapeutic approaches are not available. It is therefore essential to understand the molecular mechanisms involved in CCS development in order to improve their clinical management. The two most frequent alterations in ACC are overexpression of IGF2 and constitutive activation of β-catenin. Our lab has previously demonstrated the oncogenic activity of β-catenin in the adrenal cortex by developing a mouse model of constitutive β-catenin activation (ΔCat mice). However, the low malignant progression in ΔCat mice suggests that other alterations are necessary for acquisition of malignancy. The initial aim of my thesis was to test the oncogenic potential of IGF2 alone or associated with constitutive β-catenin activation. We showed that overexpression of Igf2 in the adrenal cortex does not trigger adrenal cortex tumourigenesis. In a context of constitutive β-catenin activation, overexpression of Igf2 promotes tumour development at later stages. However the formation of malignant tumours remains a rare event. These data suggest that the overexpression of Igf2 and constitutive activation of β-catenin are not sufficient to trigger malignant tumour progression. Retrospective analysis of available ACC transcriptome data highlighted overexpression of the putative oncogene and histone methyltransferase EZH2 in ACC, which was associated with poor prognosis. My in vitro studies suggest that EZH2 is overexpressed in response to overexpression of E2F transcription factors and that it could be involved in control of proliferation, apoptosis and oncogenic capacities of adrenocortical carcinoma cells H295R. Interestingly, the availability of pharmacologic inhibitors suggests that EZH2 could be a novel therapeutic target for the treatment of ACC. In parallel, we sought to identify the mechanisms involved in zonation of the adrenal cortex. During adrenal cortex renewal, cells first differentiate as glomerulosa before switching to fasciculata as they move within the cortex. Establishment of glomerulosa identity relies on the Wnt/β-catenin pathway, which induces expression of genes involved in glomerulosa differentiation and inhibits fasciculata identity. These data suggest that β-catenin has to be inhibited in order to allow the lineage conversion from glomerulosa to fasciculata. We thus postulated that PKA signalling pathway, which is triggered by ACTH binding to its receptor MC2R in zona fasciculata, played a role in repressing Wnt/β-catenin signalling to allow fasciculata differentiation. Using pharmacologic and genetic models, we have shown that PKA inhibits β-catenin signalling, which leads to loss of zona glomerulosa and expansion of zona fasciculata. The inhibitor effect of PKA on β-catenin pathway could be the result of decreased expression of Wnt4. Indee, a decrease of Wnt4 expression is observed in response to PKA activation and inactivation of Wnt4 in the adrenal cortex phenocopies PKA activation. We have also shown that PKA inhibits oncogenic effects of β-catenin in the adrenal cortex. The observation of decreased ACTH/PKA signalling in ACC suggests that this inhibition could be relevant to human adrenal tumour development.

Membranas e organelas constituem estruturas presentes nas células dos organismos. Estas estruturas representam interfaces entre eletrólitos. Uma tentativa de descrever, interpretar e compreender a distribuição iônica nas vizinhanças destas estruturas é feita neste trabalho com a análise de resultados de experimentos obtidos na investigação de alguns sistemas: 1) Sistemas constituídos por micelas de SDS. Medidas do pH nas vizinhanças de superfícies das micelas por sondas derivadas do ácido salicílico mostra variações do pH em termos da concentração de SDS e da concentração de sal adicionado. O objetivo dos experimentos é inferir o comportamento do pH nas vizinhanças de membranas biológicas que, por dissociação de alguns fosfolipídios, podem apresentar segmentos da membrana com carga na superfície. 2) A natureza \"zwitteriônica\" das membranas biológicas motivou o estudo da \"ligação iônica\" em micelas \"zwitteriônicas\", imersas em soluções com eletrólito, em concentrações variadas de sais de Cl- e Br- com os cátions monovalentes, Li+, Na+, K+, Rb+, Cs+ e tetrametil amônio (TMA+) e bivalentes Mg2+ e Ca2+. Os experimentos consistiram em determinar a concentração de haletos próximos a micela. A técnica de captura química mostra que há um grau de seletividade que não é determinado apenas pela carga iônica. 3) Resultados preliminares do grau de dissociação no interior de vesiculas de, cloreto de dimetildioctadecil amônio, DODAC, indicam que 8% dos monômeros estão dissociados em vesículas com o diâmetro médio em torno de 150 e 300 nm. As leis fundamentais usadas para compreender os resultados estão aliadas a hipótese de que os sistemas estudados estão em equilibrio termodinâmico, que a interação eletrostática é predominante e ao potencial eletrostático é conferido o papel do potencial da força média que atua nos íons. A simplificação adicional consistindo em admitir que os íons se comportam como cargas puntiformes no que se refere à interação eletrostática conduz o modelo teórico à equação de Poisson-Boltzmann que, linearizada, resulta na equação de Debye-Hückel. Hipóteses adicionais se fazem necessárias para formular o modelo como um problema matemático com condições de contorno. Para cada situação as hipóteses adicionais são discutidas. Sistemas hipotéticos são analizados com o intuito de comparar os resultados provenientes da equação de Poisson-Boltzmann e da equação de Debye-Hückel. A análise teórica dos sistemas conduz a resultados em acordo com os valores medidos. Entre as conclusões obtidas, neste trabalho, são mencionados: 1- As sondas derivadas do ácido salicílico mantêm os grupos dissociáveis próximos a superfície, a distância é da ordem de 0,1 nm, mesmo para sondas que apresentam cadeia longa entre o grupo nitrogênio e o grupo dissociável. 2- A especificidade iônica é bem descrita utilizando, além da carga elétrica, a massa do íon. Os íons na superfície de uma micela zwitteriônica têm liberdade translacional e portanto superfícies zwitteriônicas em solução de eletrólito apresentam condutividade elétrica na superfície. 3- As concentrações iônicas no interior de vesículas são uniformes em praticamente toda a região interna, apresentando variações apenas nas vizinhanças da superfície interna carregada eletricamente. / Membranes and organelles are structures present in biological systems. Such structures are interfaces between electrolytes. Addressing to the description, interpretation and comprehension of the ionic distribution around the structures an attempt is done is this work analyzing experimental results from the investigation of the following systems: 1) SDS micelles. \"pH\" measurements in the micellar surface neighborhood using salicylic acid probes show the pH values dependence with the SDS and added salt concentrations. The experiments aimed to infer the pH behavior in biological membranes where same phospholipids may dissociate and portions of the surface can acquire electrical charges. 2) The zwitterionic nature of biological membranes leads to the investigation of ion binding in zwitterionic micelles in electrolyte solutions with varied concentrations of Cl- or Br<SUP- salts with the cations Li+, Na+, K+, Rb+, Cs+ e tetramethylammonium (TMA+) and the bivalent cations Mg2+ e Ca2+. Halide concentration in the micellar vicinity was measured. Chemical trapping method shows there is a selectivity degree that does not depend only on the ionic charge. 3) Preliminary results in the determination of the inner dissociation degree of dioctadecyldimethylammonium chloride vesicles, DODAC, show that about 8% of the vesicle constituent monomers are dissociated in vesicles with 150 and 300 nm mean diameter. The theoretical description is based upon the thermodynamics equilibrium hypothesis about the systems and that the electrostatic interaction is the stronger interaction and also it is attributed to the mean electrostatic potential the role of the mean force potential acting on the ionic species. A further simplification in considering the ions as point charges with respect to the electrostatic interactions leads the model to the Poisson-Boltzmann equation and under linearization results in the Debye-Hückel alternative description. Additional hypothesis are necessary in order to have the model as a mathematical problem with boundary conditions and are discussed for each system. Hypothetical systems are analysed aiming the comparison between Poisson-Boltzmann and Debye- Hückel descriptions. Some conclusions derived in the analysis are: 1- The salicylic acid probes have the dissociable groups always near (~0,1nm) the micellar surface, even to the probes that have a long chain between the nitrogen group and the dissociable group. 2- In a zwitterionic micelle the ions on the surface have translational freedom and this is way the zwitterionic membranes in electrolyte solutions are conducting surfaces. 3- The ionic concentrations in the vesicle interior have uniformly value almost everywhere showing variations only in the vicinity of the electrically charged interior surface. The theoretical study of the three systems considered gives results in accord with experimental data.

Dans la majorité des cas, l’hypersécrétion de cortisol résulte d’un adénome hypophysaire sécrétant de l’ACTH (maladie de Cushing). Plus rarement, le syndrome de Cushing est la conséquence d’un adénome corticosurrénalien unilatéral ou d’une hyperplasie bilatérale des surrénales (HMBS) sécrétant du cortisol. Ces deux pathologies appartiennent à la catégorie des hypercortisolismes dits ACTH-indépendants en raison des taux plasmatiques effondrés d’adrénocorticotrophine (ACTH). Les mécanismes moléculaires à l’origine de la sécrétion accrue de cortisol par ces lésions sont longtemps restés méconnus. Au cours des dernières années, des avancées considérables ont été réalisées dans la compréhension des mécanismes moléculaires impliqués dans la physiopathologie du syndrome de Cushing. Deux grands types d’anomalies paraissent impliqués dans la pathogénie de l’hypercortisolisme : des mutations germinales et somatiques activant des voies de signalisation intracellulaires et l’expression illégitime de récepteurs membranaires. Dans la surrénale humaine normale, la sérotonine (5-HT), synthétisée et libérée par les mastocytes sous-capsulaires, stimule la sécrétion des corticostéroïdes via son récepteur 5-HT4. Ce dernier est principalement localisé à la surface des cellules de la zone glomérulée mais faiblement exprimé au niveau des cellules de la zone fasciculée, expliquant l’action stimulante prédominante de la 5-HT sur la sécrétion d’aldostérone. Dans la dysplasie micronodulaire pigmentée des surrénales, l’activation de la voie AMPc/PKA par une mutation du gène PRKAR1A est responsable d’une surexpression de la tryptophane hydroxylase (TPH) de type 2, enzyme limitante de la synthèse de 5-HT, et des récepteurs sérotoninergiques 5-HT4, 5-HT6 et 5-HT7 couplés positivement à la voie AMPc/PKA dans les cellules cortisolosécrétrices. Chez l’Homme, la sécrétion de cortisol est physiologiquement stimulée par l’ACTH également via la voie AMPc/PKA. Les patients souffrant de maladie de Cushing, d’un syndrome de Cushing paranéoplasique (paraCS), d’un déficit en 21-hydroxylase ou d’HMBS présentent des taux d’ACTH plasmatique ou intrasurrénalienne élevés. Chez ces patients, nous montrons que la stimulation chronique de la voie AMPc/PKA par l’ACTH provoque une surexpression de la TPH de type 1, du récepteur eutopique 5-HT4 et des récepteurs ectopiques 5-HT6 et 5-HT7 dans les cellules stéroïdogènes. Pour l’un des patients avec paraCS, nous avons pu montrer que les cellules corticosurrénaliennes en culture sécrètent du cortisol en réponse à la 5-HT ou à des agonistes des récepteurs 5-HT4 ou 5-HT7. Par ailleurs, le rôle de la voie Wnt/-caténine dans l’apparition des récepteurs illégitimes reste controversé. Nous avons donc évalué l’expression des récepteurs 5-HT4, 5-HT6, 5-HT7, du LH-R et du GIP-R dans une tumeur corticosurrénalienne avec mutation germinale du gène APC et deux modèles d’activation constitutive de la voie Wnt/-caténine dans le cortex surrénalien, incluant des souris génétiquement modifiées et des cellules corticosurrénaliennes humaines en culture primaire. Nos résultats indiquent que l’activation de la voie Wnt/-caténine favorise une surexpression significative du LH-R dans les 3 modèles étudiés. Globalement, les données issues de notre travail montrent que l’activation de voies de signalisation intracellulaire, comme la voie AMPc/PKA par l’ACTH ou la voie Wnt/-caténine par des mutations génétiques, favorise l’émergence de systèmes de régulation surrénaliens aberrants. Ils indiquent en outre que la 5-HT intrasurrénalienne est impliquée dans l’hypersécrétion de corticostéroïdes associée à différentes pathologies incluant la maladie de Cushing et le syndrome de Cushing paranéoplasique, le bloc en 21-hydroxylase et l’HMBS. Le recours à des inhibiteurs sélectifs de la tryptophane hydroxylase pourrait donc permettre de réduire l’excès de stéroïdes chez les patients atteints de ces affections. / In most cases, cortisol hypersecretion (Cushing’s syndrome; CS) results from ACTH-producing pituitary adenoma (Cushing’s disease). Occasionally, CS is the consequence of a unilateral adrenal adenoma or a bilateral macronodular adrenal hyperplasia (BMAH) producing cortisol. In these conditions, hypercortisolism is referred to as “ACTH-independent” owing to suppressed plasma ACTH levels. The molecular mechanisms underlying the maintenance of cortisol hypersecretion by adrenocortical adenomas and BMAHs in the absence of circulating ACTH has long remained unknown. However, major advances have been made during the past recent years in the comprehension of the pathophysiology of primary adrenal CS. Two main types of molecular defects have been shown to favor cortisol hypersecretion by adrenocortical neoplasms: somatic mutations responsible for activation of intracellular signaling pathways and abnormally expressed (or illegitimate) membrane receptors by tumor cells. In the human adrenal gland, serotonin (5-HT), released by subcapsular mast cells stimulates corticosteroid secretion through activation of its type 4 receptor (5-HT4R). The 5-HT4R is principally expressed in zona glomerulosa cells but weakly expressed in zona fasciculata cells explaining why 5-HT strongly stimulates aldosterone production. Interestingly, in primary pigmented nodular adrenocortical disease (PPNAD) cells, activation of the cAMP/PKA pathway by PRKAR1A mutations triggers upregulation of the 5-HT synthesizing enzyme tryptophan hydroxylase (TPH) type 2 together with the 5-HT4, 5-HT6 and 5-HT7 receptors, positively coupled to cAMP/PKA signaling pathway. 5-HT strongly stimulates cortisol production and inhibition of TPH reduced corticosteroidogenesis in cultured PPNAD cells. In human, cortisol secretion is normally stimulated by ACTH also through activation of the cAMP/PKA signaling pathway. Patients suffering from Cushing’s disease, paraneoplastic Cushing’s syndrome (paraCS), 21-hydroxylase deficiency or BMAH display high plasma or intraadrenal ACTH levels. In these patients, we show that chronic stimulation of cAMP/PKA pathway by ACTH induces TPH type 1 and 5-HT4/6/7 receptors overexpression in steroidogenic cells. In primary cultured adrenocortical cells originating from a patient with paraCS, 5-HT and 5-HT4/7 receptors agonists were able to activate cortisol secretion. On the other hand, the role of Wnt/-catenin signaling pathway in the emergence of illegitimate receptors is still debated. We therefore evaluated 5-HT4, 5-HT6, 5-HT7, LH/hCG and GIP receptors expression in an adrenocortical tumor with APC germline mutation and two experimental models of constitutive activation of β-catenin in adrenocortical cells, namely genetically modified mice and human transfected adrenocortical cells. Our results indicate that Wnt/-catenin pathway activation promotes significant overexpression of LH/hCG receptor in the 3 models investigated. Globally, our data show that activation of intracellular signaling pathways such as the cAMP/PKA pathway by ACTH or Wnt/-catenin by genetic mutations favors the emergence of abnormal regulatory systems in the adrenal cortex. Our results also demonstrate that intraadrenal 5-HT is involved in corticosteroids hypersecretion related to different diseases including Cushing’s disease, paraneoplastic Cushing’s syndrome, 21-hydroxylase deficiency and BMAH. TPH inhibitors may thus represent a new therapeutic approach of corticosteroid excess in patients suffering from these disorders.

The soluble form of ADCYs, ADCY10, is ubiquitously expressed in the cytoplasm and distinct organelles including cell nucleus. In contrast to its membrane-associated isoforms (ADCY1-9) which are stimulated by G-protein-coupled receptors, ADCY10 is activated by bicarbonate (HCO3-) and can form cAMP in nearly all cell compartments. ADCY10 is involved in a variety of physiological as well as pathological processes including cell cycle control in tumor cells. However, the underlying mechanism is still unclear. Here the role of ADCY10 in cell cycle control and cell proliferation is studied in endothelial cells from human umbilical veins (HUVECs). The current study reveals that ADCY10 and α-Tubulin translocate and colocalize during mitosis suggesting a role of ADCY10 in cell division. In addition, FACS analysis demonstrated that ADCY10 plays a role in cell proliferation by modulating cell cycle control. Inhibition of ADCY10 by 0 mM HCO3- or 10 μM KH7 (specific ADCY10 inhibitor) induced cell accumulation in G2 phase rather than M phase determined by decreased mitotic indicator cyclin B1 level. Thus, ADCY10 inhibition leads to decreased cell proliferation. The known cAMP effectors, Epac and PKA, were assessed as possible downstream targets of ADCY10 in cell proliferation. It was shown that ADCY10 and Epac induce cell proliferation via ERK1/2-MAPK pathway. Inhibition of Epac was associated with suppressed cell proliferation. However, an arrest of cell cycle after Epac inhibition was observed in G0/G1 phases rather than S or G2/M phases. Thus, Epac inhibition causes a different arrest of cell cycle compared to ADCY10 inhibition. Regarding PKA, it was demonstrated that deficiency of PKA might play a role in either activation or inhibition of cell proliferation. However, direct inhibition of PKA by PKI and H-89 did not lead to cell accumulation in G2. This effect might be associated with broadened roles of PKA in different pathways. In contrast, direct stimulation of PKA under ADCY10 inhibition revealed that PKA is a downstream molecule of ADCY10 as a regulator of cell cycle transition from G2 to mitotic phase. However, the underlying pathway remains to be investigated. The cell cycle transition of G2/M phase is regulated by an auto-amplification loop of cyclin B1/CDK1, which is controlled by the kinase WEE1 and the phosphatase PP2A. WEE1 content was regulated via ADCY10 but was independent of PKA or Epac. Direct inhibition of PP2A showed a suppression of cell proliferation and induced cell cycle arrest in G2. These results were in accordance with those observed after the ADCY10. Furthermore, inhibition of ADCY10 had no effect on PP2A expression level but rather affected PP2A activity and was independent of Epac and PKA. Therefore, this data provides evidence that ADCY10 controls cell proliferation and cell cycle regulation via PP2A. Taken together, ADCY10 coordinates the cell cycle progression in a complex framework. Downstream of ADCY10, Epac promotes G1/S transition, whereas PKA mediates cell cycle transition of G2/M.

Despite distinct dissimilarities, diverse cancers express several common pro-tumorigenic traits. We present here evidence that the pro-apoptotic protein Par-4 utilizes one such common tumorigenic trait to become selectively activated and induce apoptosis in cancer cells. Elevated PKA activity noted in cancer cells activated the apoptotic function of ectopic Par-4 or its SAC domain, which induces apoptosis selectively in cancer cells and not in normal or immortalized cells. PKA preferentially phosphorylated Par-4 at the T155 residue within the SAC domain in cancer cells. Moreover, pharmacological-, peptide- or siRNA-mediated inhibition of PKA activity in cancer cells resulted in abrogation of both T155 phosphorylation and apoptosis by Par-4. The mechanism of activation of endogenous Par-4 was similar to that of ectopic Par-4, and in response to exogenous stimuli, endogenous Par-4 induced apoptosis by a PKA and phospho-T155 dependent mechanism. Enforced elevation of PKA activity in normal cells resulted in apoptosis by the SAC domain of Par-4 in a T155-dependent manner. Together, these observations suggest that selective apoptosis of cancer cells by the SAC domain of Par-4 involves phosphorylation of T155 by PKA. These findings uncover a novel mechanism engaging PKA, a pro-cancerous activity commonly elevated in most tumor cells, to activate the cancer selective apoptotic action of Par-4.

No description available.

ENZYMATIC REGULATION OF OPIOID ANTINOCICEPTION AND TOLERANCE By Lynn C. Hull, Ph.D. A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University. Virginia Commonwealth University, 2009 Director: William L. Dewey, Ph.D. Department of Pharmacology and Toxicology The involvement of kinases in opioid actions has long been established. The acute actions of opioids, through the Gi/Go G-proteins, cause the inhibition of adenylyl cyclase and therefore a decrease in protein kinase A (PKA) activation. Additionally, acute opioid administration may cause the G-protein to activate the phospholipase C (PLC)-mediated cascade leading to the activation of protein kinase C (PKC). The phosphorylation of the MOR which can lead to both desensitization by uncoupling of the G-protein coupled receptors (GPCRs) from the G-proteins and to internalization by recruitment of β-arrestins has long been identified as a key process in tolerance. Phosphorylation by PKA and PKC leads primarily to uncoupling of the receptor from the G-proteins. Phosphorylation of the receptor by G-protein coupled receptor kinase (GRK) leads to the recruitment of β-arrestins and internalization of the receptor. Many in vitro studies have come to the conclusion that GRK induced internalization plays a more central role in the tolerance to high efficacy opioids and a lesser role in low- and moderate-efficacy opioid tolerance. In fact it has been hypothesized that morphine, a moderate-efficacy opioid, causes no internalization at all, while the desensitization of the receptor via phosphorylation by PKA and PKC plays a more central role in low- and moderate-efficacy opioid tolerance. We sought to test these in vitro findings in an in vivo model of opioid tolerance. Animals were made tolerant to one of a number of opioids of varying efficacy (low-efficacy meperidine, moderate-efficacy morphine and fentanyl, and high-efficacy [D-Ala2, N-MePhe4, Gly-ol]-enkephalin (DAMGO)) over an 8 hour period and then were administered one of the kinases’ inhibitors. Tolerance reversal was determined by challenging these mice with the same opioids to which they were tolerant. Calcium is known to play an important role in the acute antinociceptive actions of opioids as well as in opioid tolerance. Therefore it is important to determine how opioids are affecting the regulation of intracellular calcium. Our laboratory has previously shown that Calcium Induced Calcium Release (CICR), the ryanodine receptor and intracellular microsomal Ca2+ pools all play a role in opioids’ actions. It is also well known that mammalian ADP-ribosyl cyclase, CD38’s, product cADPR acts on the ryanodine receptor to cause Ca2+ release into the intracellular space. We chemically and genetically altered CD38 and then tested the acute effect of morphine as well as what effect these treatments had on morphine tolerance to determine what role if any, that CD38 may play in the acute actions of morphine antinociception as well as in morphine tolerance. Together, studies focusing on the role of an ADP-ribosyl cyclase, CD38, and 3 separate kinases, PKA, PKC and GRK, in opioids’ actions were performed in order to better understand the roles of these enzymes’ pathways in the actions of opioid-induced antinociception and subsequent development of tolerance. It is hoped that the results herein add useful knowledge to the general understanding of this drug class, and will one day be of use in the development of future analgesics and in the clinical treatment of pain and reduction in tolerance.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

"The accurate, fast and low cost computational tools are indispensable for studying the structure and dynamics of biological macromolecules in aqueous solution. The goal of this thesis is development and validation of continuum Fuzzy-Border (FB) solvation model to work with the Polarizable Simulations Second-order Interaction Model (POSSIM) force field for proteins developed by Professor G A Kaminski. The implicit FB model has advantages over the popularly used Poisson Boltzmann (PB) solvation model. The FB continuum model attenuates the noise and convergence issues commonly present in numerical treatments of the PB model by employing fixed position cubic grid to compute interactions. It also uses either second or first-order approximation for the solvent polarization which is similar to the second-order explicit polarization applied in POSSIM force field. The FB model was first developed and parameterized with nonpolarizable OPLS-AA force field for small molecules which are not only important in themselves but also building blocks of proteins and peptide side chains. The hydration parameters are fitted to reproduce the experimental or quantum mechanical hydration energies of the molecules with the overall average unsigned error of ca. 0.076kcal/mol. It was further validated by computing the absolute pKa values of 11 substituted phenols with the average unsigned error of 0.41pH units in comparison with the quantum mechanical error of 0.38pH units for this set of molecules. There was a good transferability of hydration parameters and the results were produced only with fitting of the specific atoms to the hydration energy and pKa targets. This clearly demonstrates the numerical and physical basis of the model is good enough and with proper fitting can reproduce the acidity constants for other systems as well. After the successful development of FB model with the fixed charges OPLS-AA force field, it was expanded to permit simulations with Polarizable Simulations Second-order Interaction Model (POSSIM) force field. The hydration parameters of the small molecules representing analogues of protein side chains were fitted to their solvation energies at 298.15K with an average error of ca.0.136kcal/mol. Second, the resulting parameters were used to reproduce the pKa values of the reference systems and the carboxylic (Asp7, Glu10, Glu19, Asp27 and Glu43) and basic residues (Lys13, Lys29, Lys34, His52 and Lys55) of the turkey ovomucoid third domain (OMTKY3) protein. The overall average unsigned error in the pKa values of the acid residues was found to be 0.37pH units and the basic residues was 0.38 pH units compared to 0.58pH units and 0.72 pH units calculated previously using polarizable force field (PFF) and Poisson Boltzmann formalism (PBF) continuum solvation model. These results are produced with fitting of specific atoms of the reference systems and carboxylic and basic residues of the OMTKY3 protein. Since FB model has produced improved pKa shifts of carboxylic residues and basic protein residues in OMTKY3 protein compared to PBF/PFF, it suggests the methodology of first-order FB continuum solvation model works well in such calculations. In this study the importance of explicit treatment of the electrostatic polarization in calculating pKa of both acid and basic protein residues is also emphasized. Moreover, the presented results demonstrate not only the consistently good degree of accuracy of protein pKa calculations with the second-degree POSSIM approximation of the polarizable calculations and the first-order approximation used in the Fuzzy-Border model for the continuum solvation energy, but also a high degree of transferability of both the POSSIM and continuum solvent Fuzzy Border parameters. Therefore, the FB model of solvation combined with the POSSIM force field can be successfully applied to study the protein and protein-ligand systems in water. "