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Les opioïdes sont les analgésiques les plus efficaces dans le traitement des douleurs sévères. Ils produisent leurs effets en ciblant spécifiquement les récepteurs opioïdes localisés tout le long de la voie de perception de la douleur où ils modulent la transmission de l'information douloureuse. La plupart des études dans ce domaine essaient de caractériser les récepteurs opioïdes à l'état isolé de tout partenaire de signalisation. Cette thèse, par contre, montre que le récepteur opioïde delta (DOR) peut former un complexe avec sa protéine G et l'un de ses effecteurs impliqués dans la production de l'effet analgésique, le canal potassique à rectification entrante activée par les protéines G (Kir3 ou GIRK). Après avoir établi la présence de ce complexe constitutif, on a ensuite caractérisé sa stabilité, modulation et régulation suite à une stimulation avec des agonistes opioïdes. En premier lieu, on a caractérisé la transmission de l'information du récepteur DOR, suite à son activation par un agoniste, vers le canal Kir3. On a remarqué que cette transmission ne suit pas le modèle de collision, généralement accepté, mais nécessite plutôt un simple changement dans la conformation du complexe préformé. Ensuite, on a déterminé que même suite à l'activation prolongée du récepteur DOR par un agoniste complet, le complexe DOR/Kir3 maintenait son intégrité et a été reconnu par la βarrestine (βarr) comme une seule unité signalétique provoquant ainsi l'internalisation de DOR et Kir3 par un mécanisme clathrine et dynamine-dépendant. Ainsi, prises ensemble, ces données montrent que l'activation du récepteur DOR déclenche non seulement l'activation de l'effecteur Kir3 mais également un mécanisme de régulation qui élimine cet effecteur de la membrane plasmique. / Opioids are the most effective analgesics in the treatment of severe pain. They produce their effects by specifically targeting opioid receptors located all along the pain perception pathway where they modulate the transmission of pain information. Most studies in this area try to characterize the opioid receptor in isolation from any signaling partner. This thesis, on the other hand, shows that the delta opioid receptor (DOR) can form a complex with its G protein and one of its effectors involved in the production of the analgesic effect, the G protein coupled inward rectifying potassium channel (Kir3 or GIRK). Having established the presence of this constitutive complex, we then characterized its stability, modulation and regulation following stimulation with opioid agonists. First, we characterized the transmission of information from DOR, following its activation by an agonist, to the Kir3 channel. We have noticed that this transmission does not follow the collision model, generally accepted, but rather requires a simple change in the conformation within the preformed complex. Then, we have determined that even following prolonged DOR activation by a full agonist, the DOR/Kir3 complex maintained its integrity and was recognized by βarrestin (βarr) as a single signaling unit producing the internalization of DOR and Kir3 by a clathrin and dynamin-dependent mechanism. Thus, taken together, these data show that DOR activation triggers not only activation of the Kir3 effector but also a regulatory mechanism that removes this effector from the plasma membrane.

Doctor of Philosophy(PhD) / Two recently identified transient receptor potential (TRP) channels, TRPM8 and TRPA1, have been proposed to play an important role in mammalian cool and cold peripheral sensory transduction. When expressed in cell-lines the cloned TRPM8 and TRPA1 receptors have distinct pharmacological and temperature response characteristics. Although these receptors are also transported to the central terminals of primary afferents, little is known about their centrally mediated actions. In this thesis, I use an in vitro electrophysiological approach to investigate the dorsal horn processing of cool afferent modalities and the role of TRP ion channels. The results of this thesis provide further information on thermal processing, indicate direction for further research and suggest possible therapeutic targets for the management of abnormal cold sensory processing. Initial experiments demonstrate that the cooling agents and known TRPM8 and TRPA1 agonists, menthol and icilin, inhibit primary afferent evoked excitatory postsynaptic currents (EPSCs) in rat spinal cord dorsal horn neurons. In addition, temperature reduction, menthol and icilin increase the frequency of miniature EPSCs without affecting amplitude distribution or kinetics. Little or no direct postsynaptic effect on dorsal horn neurons, GABAergic or glycinergic transmission was found. In combination, these observations demonstrate that temperature reduction, menthol and icilin act presynaptically to increase the probability of glutamate release from primary afferent fibres. Further examination of the changes in glutamatergic synaptic transmission induced by temperature reduction, menthol and icilin reveals a subset of neurons sensitive to innocuous cool (< 29 oC) and low concentrations of icilin (3-10 µM) which closely match the temperature activation and pharmacological profile of TRPM8. In addition, the majority of lamina I and II neurons displayed characteristics partly consistent with TRPA1-activation, including a concentration-dependent response to icilin and blockade by ruthenium red. The present experiments did not allow thermal characterisation of these TRPA1-like responses. Together these observations indicate that the effects of menthol and icilin on glutamatergic synaptic transmission in the superficial dorsal horn are mediated by TRPM8 and possibly by TRPA1. Examination of the anatomical location of neurons activated by temperature reduction, menthol, icilin and capsaicin allowed the central termination pattern of thermoreceptive primary afferent fibres with specific TRP-like response characteristics to be determined. TRPM8-like presynaptic activation was confined to a subpopulation of neurons located in lamina I and outer lamina II, while the majority of neurons throughout laminae I and II received inputs sensitive to menthol, high concentrations of icilin and capsaicin. These findings suggest that innocuous cool sensation projects to a specific subpopulation of superficial dorsal horn neurons unlike other modalities (mediated by TRPV1, possibly TRPA1 and other receptors), which non-selectively engage circuits within the entire superficial dorsal horn. No morphological specificity was identified for recovered neurons after electrophysiological characterisation. Finally, mu-opioids were shown to inhibit basal glutamatergic synaptic transmission as well as menthol- and icilin-induced transmission in the superficial dorsal horn. Of particular interest, delta-opioids selectively inhibited icilin-induced synaptic transmission within the same location. The selective effect of delta-opioids suggests a possible role in modulating receptors activated by icilin (TRPM8 and TRPA1). Overall, this thesis provides further evidence that TRPM8 is responsible for the transduction of innocuous cold sensation in mammals and is a potential therapeutic target in humans with cold hyperaesthesia secondary to abnormal thermal processing. The use of delta-opioid agonists warrants further investigation in cold hypersensitivity states and potentially other forms of pain.

憂鬱症是盛行的精神疾病之一。慢性疼痛或是處在長期壓力情境的患者常與憂鬱症產生共病。在動物研究中，類鴉片delta受體制效劑能產生抗憂鬱效果，並且在發炎性疼痛的研究也指出類鴉片delta受體制效劑能展現抗痛覺過敏的效果。本研究主要利用大白鼠腦室內給予類鴉片delta受體制效劑SNC80以及三環抗憂鬱劑amitriptyline，來探討並比較其所產生的抗憂鬱效果在發炎性疼痛或長期壓力情境下與正常情境下的異同。大白鼠強迫游泳試驗被用來比較測試藥物的抗憂鬱效果；佛氏完全佐劑經由皮下注射至大白鼠右後腳掌底板來產生發炎性疼痛；腎上腺皮質酮經由皮下注射且持續21天來產生長期性壓力；西方墨點法用來檢驗在發炎性疼痛或長期壓力下，類鴉片delta受體蛋白質在大白鼠海馬迴的細胞膜上的改變。另外，拮抗劑實驗則用來確認類鴉片delta受體所產生的抗憂鬱效果。實驗結果顯示，大白鼠在正常情境下，SNC80及amitriptyline皆能產生抗憂鬱效果；然而在發炎性疼痛下，SNC80所產生的抗憂鬱效果有提高的表現，並且類鴉片delta受體蛋白質的數量在海馬迴的細胞膜上也隨著疼痛的時間增長而增加，amitriptyline則跟正常情境下的效果相似。另外，大白鼠在長期性壓力下，SNC80的抗憂鬱效果則沒有提高的表現，並且類鴉片delta受體蛋白質的數量在海馬迴的細胞膜上也未受到改變。本研究透過行為實驗提出類鴉片delta受體制效劑的藥理特性，並用分子生物學的方法來對應行為實驗的結果。本研究可做為未來類鴉片delta受體制效劑在治療慢性疼痛的憂鬱症患者上，可能發展為抗憂鬱藥的一個證據。 / Depression is one of the most prevalent mental illnesses all over the world. Patients with chronic pain or stress often have depression. Previous studies have shown that delta opioid receptor (DOR) agonists produced antidepressant-like effects in animal models and that antihyperalgesic effects of DOR agonists can be enhanced in rats under inflammatory pain. The aim of the study was to investigate and compare the antidepressant-like effects of a DOR agonist, SNC80, and a tricyclic antidepressant, amitriptyline, following intracerebroventricular (i.c.v.) administration in rats under different states. The forced swim test was used to determine the antidepressant-like effects of test compounds. Complete Freund’s adjuvant was injected subcutaneously into the right hind paw of rats to elicit inflammatory pain. Corticosterone was injected subcutaneously once per day for 21 days to induce chronic stress. The western blot was used to quantify the levels of DOR protein on plasma membrane in the hippocampus of rats under inflammatory pain or chronic stress. In addition, antagonist experiment was conducted to verify the receptor mechanism underlying the antidepressant-like effects of DOR agonist. Results indicated that i.c.v. SNC80 and amitriptyline dose-dependently produced antidepressant-like effects in rats under normal state. More importantly, the potency of SNC80-induced antidepressant-like effects, but not amitriptyline, was enhanced in rats under inflammatory pain. In addition, up-regulation of supraspinal DORs was time-dependently associated with enhanced antidepressant-like effects of SNC80 in rats under inflammatory pain. On the other hand, SNC80 did not produce enhanced antidepressant-like effects, and DOR density was not changed in rats under chronic stress. This study provides evidence of the DOR agonist’s state-dependent effects and suggests that DOR agonists may be more effective as potential antidepressants for patients with depression comorbid with chronic pain.

Doctor of Philosophy(PhD) / Two recently identified transient receptor potential (TRP) channels, TRPM8 and TRPA1, have been proposed to play an important role in mammalian cool and cold peripheral sensory transduction. When expressed in cell-lines the cloned TRPM8 and TRPA1 receptors have distinct pharmacological and temperature response characteristics. Although these receptors are also transported to the central terminals of primary afferents, little is known about their centrally mediated actions. In this thesis, I use an in vitro electrophysiological approach to investigate the dorsal horn processing of cool afferent modalities and the role of TRP ion channels. The results of this thesis provide further information on thermal processing, indicate direction for further research and suggest possible therapeutic targets for the management of abnormal cold sensory processing. Initial experiments demonstrate that the cooling agents and known TRPM8 and TRPA1 agonists, menthol and icilin, inhibit primary afferent evoked excitatory postsynaptic currents (EPSCs) in rat spinal cord dorsal horn neurons. In addition, temperature reduction, menthol and icilin increase the frequency of miniature EPSCs without affecting amplitude distribution or kinetics. Little or no direct postsynaptic effect on dorsal horn neurons, GABAergic or glycinergic transmission was found. In combination, these observations demonstrate that temperature reduction, menthol and icilin act presynaptically to increase the probability of glutamate release from primary afferent fibres. Further examination of the changes in glutamatergic synaptic transmission induced by temperature reduction, menthol and icilin reveals a subset of neurons sensitive to innocuous cool (< 29 oC) and low concentrations of icilin (3-10 µM) which closely match the temperature activation and pharmacological profile of TRPM8. In addition, the majority of lamina I and II neurons displayed characteristics partly consistent with TRPA1-activation, including a concentration-dependent response to icilin and blockade by ruthenium red. The present experiments did not allow thermal characterisation of these TRPA1-like responses. Together these observations indicate that the effects of menthol and icilin on glutamatergic synaptic transmission in the superficial dorsal horn are mediated by TRPM8 and possibly by TRPA1. Examination of the anatomical location of neurons activated by temperature reduction, menthol, icilin and capsaicin allowed the central termination pattern of thermoreceptive primary afferent fibres with specific TRP-like response characteristics to be determined. TRPM8-like presynaptic activation was confined to a subpopulation of neurons located in lamina I and outer lamina II, while the majority of neurons throughout laminae I and II received inputs sensitive to menthol, high concentrations of icilin and capsaicin. These findings suggest that innocuous cool sensation projects to a specific subpopulation of superficial dorsal horn neurons unlike other modalities (mediated by TRPV1, possibly TRPA1 and other receptors), which non-selectively engage circuits within the entire superficial dorsal horn. No morphological specificity was identified for recovered neurons after electrophysiological characterisation. Finally, mu-opioids were shown to inhibit basal glutamatergic synaptic transmission as well as menthol- and icilin-induced transmission in the superficial dorsal horn. Of particular interest, delta-opioids selectively inhibited icilin-induced synaptic transmission within the same location. The selective effect of delta-opioids suggests a possible role in modulating receptors activated by icilin (TRPM8 and TRPA1). Overall, this thesis provides further evidence that TRPM8 is responsible for the transduction of innocuous cold sensation in mammals and is a potential therapeutic target in humans with cold hyperaesthesia secondary to abnormal thermal processing. The use of delta-opioid agonists warrants further investigation in cold hypersensitivity states and potentially other forms of pain.

5 Summary: Currently, the outcomes from traumatic exsanguination cardiac arrest (CA) show that over 50% of deaths due to trauma occur at the scene, where medical care is limited. Less than 10% of patients who become pulseless from trauma survive. However, in an appropriate setting, some of those traumatic injuries could be surgically repairable. Emergency preservation and resuscitation (EPR) is a novel approach for resuscitation of exsanguination CA victims. EPR uses deep hypothermic preservation for prolonged CA to buy time for transport, damage control surgery, and delayed resuscitation with cardiopulmonary bypass (CPB). Initially, we used a dog model to maximize clinical relevance. We showed that the efficacy of EPR is related to the depth of hypothermia and duration of CA. Pharmacologic adjuncts tested to augment hypothermia generally failed. Extended hemorrhagic shock did not prevent the success of EPR vs. conventional resuscitation if extended post-resuscitative hypothermia was provided. Oxygenation of the flush allowed extending of survivable duration of deep hypothermic CA. Because of the lack of molecular tools available for use in dogs, we developed a rat EPR model to study the cellular and molecular mechanisms underlying deep hypothermic neuroprotection to allow us to define specific targets for...

<p>The following thesis will focus on understanding the downstream behavioral effects of δORmediated β-arrestinsignaling. δORagonists have been implicated as effective targets for a variety of diseases, however detrimental side effects of opioid-targeting agonists limit their clinical use. δORagonists specifically can induce seizures, however the underlying mechanism contributing to this  behavior  is  unknown.  We  review  this  phenomenon  in  more  detail,  highlighting  current agonists known to induce seizures and potential circuits and pathways involved. Our work suggests β-arrestinsignaling  is  involved,  specifically β-arrestin2  mediated  signaling  may  be  largely contributing  to δORagonist-induced  seizure  behavior.  As  it  is  possible  the β-arrestinisoforms have unique roles in seizure behavior, we also analyzed methods in which to provoke β-arrestinisoform bias of δORtargeting compounds. Though the full mechanism relating δORagonists with seizures remains unknown, our work provides foundational detail of this behavior, implicating the importance of β-arrestinisoform signaling through δOR; allowing for future studies to full define this seizure pathway and develop δORsafer agonists.  </p>

Les opioïdes restent encore à l’heure actuelle les composés pharmacologiques les plus efficaces pour traiter les différentes formes de douleurs, et donc fournir une analgésie thérapeutique. Cependant, l’administration répétée de ces composés entraîne des effets secondaires majeurs comme la dépression respiratoire, la tolérance, mais également, il a été montré que certains de ces opioïdes pouvaient engendrer des états proépileptiques. D’un point de vue thérapeutique, il existe donc un réel besoin pour de nouveaux et meilleurs traitements analgésiques, n’élicitant pas ces effets secondaires. Notre laboratoire étudie la signalétique des récepteurs couplés aux protéines G comme les récepteurs opioïdes et leur capacité de sélectivité fonctionnelle depuis des années, et en particulier celle du récepteur delta opioïde (DOP). En effet, celui-ci présenterait moins d’effets indésirables que le récepteur mu opioïde (MOP) qui est la cible principale des opioïdes classiques comme la morphine. Cependant, il semblerait que le DOP justement soit à l’origine des états proépileptiques précédemment décrits. Ainsi malgré la promesse initiale des agonistes delta par rapport à la diminution des effets secondaires, les effets proépileptiques de certains ont notamment contribué à une baisse d’intérêt vers le DOP et aucun de ses agonistes n’a pu passer les phases de tests cliniques. Cependant, il a été démontré que certains agonistes delta n’entraînaient pas d’effet proépileptique; tandis que d’autres oui. Comment expliquer un tel phénomène ? Ceci est la question que pose la présente recherche. Ainsi notre objectif sera d’obtenir et de comparer les signatures pharmacologiques des agonistes connus pour être proépileptiques versus ceux qui ne le sont pas ; par rapport à la transduction de signal via le récepteur delta opioïde et sa protéine G hétérotrimérique ; et par rapport à un de ses effecteurs principaux pour l’analgésie, un canal potassique rectifiant entrant. Cette comparaison se fera selon les paramètres du modèle classique de la pharmacologie, comme l’efficacité et la puissance ; mais également avec un outil plus récent appelé modèle opérationnel, utilisant des paramètres comme l’affinité et le coefficient de transduction. Pour se faire, le transfert d'énergie par résonance de bioluminescence ou BRET sera utilisé afin de caractériser les différentes voies signalétiques impliquées. Cette recherche s’inscrit dans un vaste contexte de collaboration entre différents laboratoires, et au sein de chacun d’entre eux, dans l’espoir de pouvoir synthétiser un jour, de meilleurs composés pharmacologiques, capables de cibler uniquement les voies médiatrices des effets thérapeutiques voulus, ici l’analgésie ; sans éliciter celles entraînant les effets secondaires associés, ici, les états proconvulsifs. L’aboutissement de cette recherche permettrait donc d’impacter la vie de millions de gens en souffrance, et c’est pourquoi il nous semble plus qu’important de continuer à l’entreprendre. / Opioids are still nowadays the most efficacious pharmacological compounds available to treat the different types of pain, and therefore provide a therapeutic analgesia. However, repeated administration of those compounds lead to major secondary effects like respiratory depression, tolerance, but also it was shown that some opioid compounds could induce seizures. From a therapeutical point of view, there is a serious need for new and better analgesic treatments that do not elicit such adverse effects. Our lab has been studying for years the signaletics of G-protein coupled receptors like the opioid receptors, and their capacity for functional selectivity, especially more recently the one of the delta opioid receptor (DOP). Indeed, this receptor elicits fewer adverse effects compared to the mu opioid receptor (MOP) that is the main target of all clinically used opioids such as morphine. However, it seems like the DOP itself would be responsible for the pro-epileptic states previously described. Thus, despite initial promises of the delta agonists towards reducing adverse effects whilst providing analgesia, the pro-convulsive effects that some seem to elicit have induced a loss of interest towards the DOP, and so far none of its agonists have gone further than pre-clinical trials. However, it has been shown that not all of those DOP agonists had those pro-convulsive adverse effects. How to explain such a phenomenon? This is the question which the present research will be asking. Thus our goal is to obtain and compare pharmacological signatures of the agonists known for being pro-convulsive versus those that are not ; regarding the transduction of signals through the delta opioid receptor and its heterotrimeric G-Protein ; and also regarding one of its main effectors to induce analgesia, an inwardly rectifying potassium channel. This comparison will be done according to the classical parameters of pharmacology, such as efficacy and potency ; but also according to the newest operational model, with parameters such as affinity and transduction coefficients. In order to do so, bioluminescence resonance energy transfer or BRET, will be used in order to characterize and quantify the signalling pathways there implicated. This research is embedded in a vast collaboration context, in between laboratories around the world, and within those laboratories as well, in hope to be able to one day synthesize, better pharmacological compounds, capable of targeting only the pathways responsible for the desired effects, here analgesia ; without triggering the associated adverse effects, here pro-convulsive states. The culmination of this research could allow to impact the lives of millions of people throughout the world, and this is why it is more than important for us to keep on pursuing it.

Les opioïdes sont les analgésiques les plus efficaces mais leur utilisation est limitée par la tolérance, un processus lié en partie à la désensibilisation des récepteurs. Le rôle de la présente étude était de mieux caractériser le processus de désensibilisation des récepteurs et plus particulièrement, d’étudier le rôle de la tyrosine kinase Src sur la régulation de la signalisation des récepteurs delta opioïdes. Nos résultats démontrent que l’inhibition pharmacologique avec PP2 (à faible concentration : 20- 40µM) ou encore l’inhibition moléculaire de la kinase avec de faibles concentrations d’ADN d’un mutant dominant inactif de Src (0,2µg/ml) potentialise l’amplitude et la durée de l’activation de la cascade ERK lorsqu’un agoniste, DPDPE (1µM; 5 min), se lie aux récepteurs. Nous avons également démontré que de fortes concentrations d’inhibiteurs de Src (80 et 100µM de PP2 ou 1µg/ml d’ADN du mutant dominant négatif) bloquent la cascade des MAPK suivant la stimulation de DOR par l’agoniste DPDPE. Ces observations indiquent que Src a un effet biphasique sur l’activité de ERK : l’inhibition complète de Src inhibe l’activité de la cascade MAPK alors qu’une inhibition modérée potentialise cette même cascade. Nous pensons aussi que de fortes concentrations des bloqueurs de Src interfèrent avec l’activation de ERK alors que de faibles concentrations interfèrent avec la désensibilisation des récepteurs. Cette possibilité a été testée à l’aide d’essais d’accumulation d’AMPc qui visaient à évaluer l’effet des bloqueurs de Src (PP2, 20 µM; 1h) sur la désensibilisation induite par un agoniste. L'activation de DOR par DPDPE inhibe la production d’AMPc, préalablement stimulée par du forskolin, de façon dose-dépendante. Le maximum d'inhibition observé est de 61%, mais lors d’un prétraitement au DPDPE (1 µM, 30 min) l’inhibition maximale est réduite à 72% de l’inhibition initiale observée. Cependant, un prétraitement des cellules au PP2 (20µM pendant 1 heure) avant d’effectuer la désensibilisation protège contre cette désensibilisation. L’effet protecteur des bloqueurs de Src n’entraîne pas de changement au niveau de l’internalisation des DOR mais l’altération de leur internalisation via un mutant tronqué du DOR ou via un milieu sucré hypertonique (0.4M de saccharose) réduit cette protection. Ces données suggèrent alors que l’internalisation optimale du récepteur est nécessaire pour que l’effet protecteur prenne place. Nous concluons donc que Src contribue à la désensibilisation de DOR après que l’internalisation du DOR soit survenue. / Opioids are the most effective analgesics available but their use is limited by tolerance. Tolerance is related, at least in part, to receptor desensitization. Hence, the role of the present study was to better characterize the desensitization process, in particular concerning the role of the tyrosine kinase Src on regulation of delta opioid receptor signalling. Our results show that pharmacological inhibition with PP2 (administered at low concentration: 20-40µM) or molecular inhibition of the kinase with low expression levels of a dominant negative mutant of Src (0,2µg of DNA) potentiate the magnitude and duration of agonist-dependent (DPDPE; 1µM; 5 min) activation of the ERK pathway. We also showed that higher concentrations of Src inhibitors (80 and 100µM of PP2 or 1µg/ml of dominant negative mutant DNA) block the MAPK cascade following DOR stimulation by DPDPE. These observations indicate that Src has a biphasic effect on ERK activity, respectively potentiating or inhibiting agonist stimulation of the MAPK cascade at low and high levels of Src inhibition. We reasoned that high levels of Src blockers were interfering with ERK activation mechanism while low levels of inhibition were interfering with receptor desensitization. This possibility was tested by using cAMP accumulation assays to evaluate the effect of Src blockers (PP2, 20 µM; 1h) on agonist-induced desensitization. DOR stimulation by DPDPE inhibited forskolin stimulated cAMP production in a dose dependent manner with a maximal reduction of 61%. This inhibitory response was reduced by 72% following pre-exposure to DPDPE (1 µM, 30 min), an effect that was blocked by pre-treating cells with PP2 (PP2, 20 µM; 1 h) before desensitization. The protective effect of Src blockers did not involve changes in DOR internalization but interfering with internalization by using an internalization-deficient DOR mutant or hypertonic medium (0.4M sucrose) reduced this protection, indicating the need for optimal internalization in order for the protective effect of Src blockers to take place. Based on the latter observation it was possible to conclude that Src contribution to DOR desensitization is post-endocytic.

Les récepteurs couplés aux protéines GRCPG sont une des plus grandes familles de récepteur membranaire codifié par le génome humain et certainement la plus grande famille de récepteurs. Localisés au niveau des membranes plasmiques, ils sont responsables d’une grande variété de réponses cellulaires. L’activation de ces derniers par des ligands était traditionnellement associée à un changement de conformation de la protéine, passant d’un état inactif à un état actif. Toutefois, certaines observations entraient en contradiction avec cette théorie et laissaient supposer la présence de plusieurs conformations actives du récepteur. Ces différentes conformations pouvaient être actives pour certaines voies de signalisation ou de régulation et inactives pour d’autres. Ce phénomène, initialement appelé agoniste dirigé ou « biased agonism », est maintenant décrit comme étant la sélectivité fonctionnelle des ligands des RCPG. Cette sélectivité des voies de signalisation et de régulation permettrait en théorie de développer des ligands capables de cibler seulement les voies de signalisation et de régulation responsable des effets thérapeutiques sans activer les voies responsables des effets secondaires ou indésirables. Le récepteur delta opiacé (DOR) est un RCPG impliqué dans la gestion de la douleur chronique. L’action analgésique de ses ligands est toutefois soumise à un effet de tolérance produite lors de leur utilisation à long terme. Cet effet secondaire limite l’utilisation thérapeutique de ces médicaments. Cette thèse s’est donc intéressée à la sélectivité fonctionnelle des ligands du DOR afin d’évaluer la possibilité de réduire les effets de tolérance produits par ces molécules. En premier lieu, nous avons déterminé que le DOR peut être stabilisé dans plusieurs conformations actives dépendantes du ligand qui le lie et ces conformations possèdent différents profils d’activation des voies de signalisation et de régulation. En deuxième lieu, nous avons déterminé que les différents ligands du DOR stabilisent des conformations du complexe récepteur/protéine G qui ne concordent pas avec la théorie des récepteurs à deux états, suggérant plutôt la présence d’une multitude de conformations actives. Finalement, nous avons démontré que ces différentes conformations interagissaient de façon distincte avec les protéines de régulation des RCPG; le ligand favorisant le retour du récepteur à la membrane produisant moins de désensibilisation et moins de tolérance aiguë à l’analgésie que le ligand favorisant la séquestration du récepteur à l’intérieur de la cellule. Les résultats de cette thèse démontrent que la sélectivité fonctionnelle des ligands opiacés pourrait être utilisée dans le développement de nouveau analgésique produisant moins de tolérance. / G protein coupled receptors (GPCRs) are one of the largest families of membrane receptor coded by the human genome and certainly the biggest family of receptors. Localized at the plasma membrane, they are responsible for a wide variety of cellular responses. Activation of GPCRs by ligands has been traditionally associated with change in protein conformation, switching from an inactive to an active state. However, some observations were inconsistent with this theory suggesting the presence of several active conformations of the receptor. These different conformations could be active in some signalling or regulatory pathways and inactive in others. This phenomenon, originally called biased agonism, is now described as the functional selectivity of GPCRs ligands. In theory, this type of selectivity could lead to the development of drugs that therapeutic action without activating signalling pathways responsible for side effect. The delta opioid receptor (DOR) is a GPCR involved in chronic pain management. However, analgesic action of its ligands is subject to tolerance that develops over the course of long-term treatment, limiting their clinical use. This thesis focuses on functional selectivity of DOR ligands to assess the possibility of reducing the effects of tolerance produced by these molecules. First, we determined that DORs adopt ligand-specific active conformations with distinct ability to activate different signalling pathways and regulatory responses. Second, we established that DORs constitutively associate with Gαi heterotrimeric subunits, and that different receptor ligands induce distinct conformational changes within these signalling complexes. These observations are inconsistent with a two-state model of receptor activation, pointing instead the presence of multiple active conformations. Finally, we demonstrated that conformations stabilized by different agonists distinctively interact with βarrestin2, such that the agonist who promoted a transient receptor interaction with this regulatory protein produced more recycling, less desensitization and less acute analgesic tolerance than the ligand who promoted a stable DOR-βarrestin2 interaction. Taken together, the results presented in this thesis show DORs adopt ligand-specific conformations that support ligand-specific profiles of analgesic tolerance. This type of functional selectivity of opioid ligands could be eventually exploited in the development of novel therapeutic agents with longer analgesic actions.