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New data on morphine, its catabolism and its binding protein in the central nervous system
No full textLa morphine constitue l'un des analgésiques les plus utilisés en milieu hospitalier pour soulager les douleurs aiguës et chroniques. Elle exerce ses effets analgésiques en se liant aux récepteurs opioïdes μ (MOR) présents au niveau central et périphérique et possède des effets secondaires, incluant la tolérance, qui limitent son usage à long terme. La première partie de mes travaux avait pour objectif d'étudier le phénomène de tolérance à la morphine et d'en déterminer les mécanismes sous-jacents. A l'heure actuelle, ce phénomène est décrit comme résultant d'une désensibilisation des MOR conduisant à leur endocytose. Nos résultats montrent qu'en cas de tolérance, le catabolisme de la morphine est exacerbé au niveau du système nerveux central. Mes travaux ont également porté sur la caractérisation de la Créatine Kinase (CK) comme étant une protéine liant la morphine à très haute affinité. Nos résultats montrent que la CK possède 2 sites de liaison pour la morphine avec des affinités similaires. En étudiant l'effet potentiel de la CK sur l'analgésie induite par la morphine in vivo, nous avons mis en évidence, que les peptides correspondant aux 2 sites de liaison à la morphine étaient par eux-mêmes analgésiques et que cette analgésie semblait dépendre des récepteurs opioïdes.Morphine is one of the most used analgesics in hospitals to relieve acute and chronic pain. Morphine exerts its analgesic effects by binding central and peripheral μ opioid receptors (MORs) and has many side effects that limit its long-term use including tolerance. The first part of my thesis was aimed to study the phenomenon of morphine tolerance and to determine the underlying mechanisms. Previously, this phenomenon was explained as resulting !rom MORs desensitization by endocytosis. However, our results show that in case of tolerance, the catabolism of morphine is exacerbated in the central nervous system in particular in astocytes. The second part of my work has focused on the characterization of the Creatine Kinase (CK) as a novel protein that binds morphine with a high affinity. Our results showed that CK has two binding sites with a similar affinity for morphine. Surprisingly, by studying the potential effect of CK on morphine-induced analgesia in vivo, we noticed that the two peptides corresponding to morphine binding sites are analgesics and such analgesia seems to be mediated by opioid receptors
New data concerning endogenous morphine: description of its localization in the central nervous system and development of biotechnological and therapeutic tools
No full textC’est depuis les années 80 que de nombreuses études ont démontré la présence de morphine endogène (ME), de structure identique à la morphine du pavot, dans le système nerveux central (SNC) de mammifères. Cependant, déterminer ses rôles au sein du SNC est difficile puisqu’il existe que très peu de données la concernant (localisation cellulaire, protocoles de quantification). Dans ce contexte, mon objectifs était fournir de nouveaux outils permettant l’étude et la compréhension des rôles de la ME au sein du SNC. La première partie de mes résultats décrit la localisation de la ME au sein du SNC de souris adulte. Après avoir caractérisé l’anticorps anti-morphine que j’ai utilisé, j’ai montré que la ME et ses dérivés sont principalement retrouvés dans les interneurones GABAergiques et les astrocytes du SNC. La seconde partie de mes résultats présente les études réalisées sur deux protéines liant les alcaloïdes endogènes/exogènes : (i) la
PhosphatidylEthanolamine Binding Protein, capable de lier la M6G et la M3G avec une affinité identique à son ligand de référence, la PhosphatidylEthanolamine. (ii) La créatine kinase, qui lie à haute affinité la ME et ses dérivés. Du plus, j’ai décrit une dissociation de ce complexe par le lithium. Enfin ma dernière partie décrit que l’utilisation du lithium lors de prélèvements sanguins permet d’augmenter jusqu’à quatre fois le rendement de détection de la ME.
En conclusion, ce travail de thèse présente de nouveaux outils et de nouvelles pistes de recherche permettant l’étude et la compréhension des rôles de la ME au sein du SNC (cartographie, protéine de liaison, amélioration de la quantification).Since 80s, endogenous morphine (EM), structurally identical to the morphine from poppy, has been found in mammalian central nervous system (CNS). Only few informations are available about EM (cellular localization, models and quantification protocols) and thus, studies of its roles and implications in the brain physiology is difficult. During my thesis, my goal was to developed new tools in order to study and understand the roles of EM in the CNS.
First, I have described the localization of EM in the CNS of adult mouse. Using a well characterized antibody, I have demonstrated that EM and its derived compounds are mainly found in astrocytes and GABAergic interneurons throughout the mouse CNS. Secondly, I described two endogenous / exogenous alkaloids binding proteins. (i) The PhosphatidylEthanolamine Binding Protein that is able to bind M6G and M3G in a similar manner as its reference ligand PhosphatidylEthanolamine. (ii) The creatine kinase (CK) that is able to bind EM and its derived compounds with high affinity. Such CK-EM complex is dissociated by a lithium treatment. Finally, in the last part of my results, I have described that the presence of lithium in the collection tube allows a better measuring of EM and exogenous morphine. To conclude, during my thesis I set up new tools and new research lines (EM CNS mapping, binding proteins and better quantification yield) that will allow to study and understand the roles of EM in the CNS of mammals
Involvement of microglia and astrocytes in the central metabolism of morphine
No full textLa morphine est l’opiacée de référence utilisée en clinique pour lutter contre la douleur. Néanmoins, dans certains cas, comme lors de douleurs neuropathiques, celle-ci est inefficace. La douleur neuropathique se caractérise par une neuroinflammation qui peut impacter l’activité des cellules gliales au sein du système nerveux central. Mes travaux de thèse montrent que ces cellules gliales, notamment les astrocytes réactifs, métabolisent la morphine de façon plus intense lors d’une neuroinflammation. Ce phénomène est relié à l’augmentation de l’expression d’un facteur de transcription appelé AhR qui est capable d’augmenter l’expression des enzymes de métabolisme de la morphine. En inhibant l’activité de l’AhR via l’injection d’un inhibiteur appelé SR1, j’ai pu montrer, pour la première fois que l’effet analgésique de la morphine était augmenté dans un modèle de douleurs neuropathiques. Ces travaux ouvrent alors la voie à un potentiel mécanisme, jusqu’alors inexploité, dans lequel il est possible d’augmenter les effets de la morphine pour traiter la douleur neuropathique.Morphine is the standard opiate used clinically to combat pain. However, in some cases, such as neuropathic pain, it is ineffective. Neuropathic pain is characterized by neuroinflammation that can affect glial cell activity within the central nervous system. My thesis work shows that these glial cells, particularly reactive astrocytes, metabolize morphine more intensely during neuroinflammation. This phenomenon is linked to increased expression of a transcription factor called AhR, which is capable of increasing expression of morphine-metabolizing enzymes. By inhibiting AhR activity via injection of an inhibitor called SR1, I was able to show, for the first time, that the analgesic effect of morphine was increased in a model of neuropathic pain. This work paves the way to a potential, hitherto unexploited, mechanism for enhancing the effects of morphine to treat neuropathic pain
Metabolism as a potential origin of sex differences in morphine-induced analgesia and tolerance
No full textLes effets de la morphine sont influencés par le sexe. Chez le rongeur, son métabolisme implique sa glucuronidation en morphine-3-glucuronide (M3G). La M3G provoque une hyperalgésie qui pourrait s’opposer aux effets antinociceptifs de la morphine. Nous avons constaté que l’antinociception induite par la morphine est plus forte chez la souris mâle et que la tolérance antinociceptive se développe plus rapidement chez la souris femelle. La M3G possède un effet pronociceptif qui ne semble pas dépendre du sexe. Nous avons quantifié la morphine et la M3G dans le sang et dans des régions cérébrales impliquées dans le contrôle de la douleur après une injection de morphine, et nous avons constaté que les ratios métaboliques M3G/morphine étaient largement supérieurs chez les femelles. Nous avons également observé un métabolisme central de la morphine in vivo. Nous avons conclu que le métabolisme périphérique et central était influencé par le sexe et que le métabolisme central de la morphine pourrait participer aux différences d’antinociception liées au sexe. Cependant, son implication dans la tolérance antinociceptive semble limitée.Morphine effects are influenced by sex. In rodents, morphine metabolism involves its glucuronidation to morphine-3-glucuronide (M3G). Interestingly, M3G has been shown to produce hyperalgesia. M3G pronociceptive effect could oppose morphine-induced antinociception. Morphine potency was higher in males than females and morphine antinociceptive tolerance developed earlier in females during the protocol. M3G pronociceptive effect did not seem to be influenced by sex. Morphine and M3G were quantified in the blood and some pain-related brain regions after morphine injection. The M3G/morphine metabolic ratios were dramatically higher in females than in males. In vivo central metabolism of morphine was observed. To conclude, peripheral and central metabolism of morphine are influenced by sex and central metabolism could participate in the sex differences observed in morphine antinociception. However, its implication seems limited during the induction of morphine antinociceptive tolerance
Istraživanje učinka trenutnih analgetika na metabolizam anastrozola
No full textThis research aimed to determine in vitro potential drug-drug interactions between anastrozole and commonly used analgesic drugs such as codeine and/or APAP, individually or in combination. Moreover, an additional goal in vivo was to clarify the potential impact of codeine and/or APAP on the metabolic pathway of anastrozole and, therefore, on anastrozole activity. Up to 80% of the diagnosed breast cancers express oestrogen receptors. Following their activation by oestrogens, oestrogen receptors induce proliferation of the tumour cells, therefore increasing cancer growth. Anastrozole is a third-generation aromatase inhibitor acting on the aromatase (CYP19A1), a key enzyme of the biosynthesis of oestrogens that is highly expressed by breast cancer cells. Paracetamol is the most used over-the-counter medication used either in single-compound form for mild pain or in multi-compound form as with an opioid for moderate to severe pain. Predominantly, paracetamol is combined with codeine. Codeine is a pro-drug, leading to morphine and its metabolites (morphine-3-glucuronide and morphine 6-glucuronide). Morphine acts on the mu-opioid receptors to produce analgesia. Mice livers were used to generate microsomes to determine the production of the anastrozole metabolite, hydroxy-anastrozole and impact of analgesic drugs on anastrozole metabolism in vitro using IC50 assay. For the in vivo experiments, female CB57BL/6J mice were treated with anastrozole (control condition) and codeine, paracetamol, or a combination of both in a single dose (acute treatment) or repeated injections over one week (chronic treatment). Both in vitro and blood samples were analysed and quantified by the liquid chromatography coupled to mass spectrometry (LCMS/ MS) using an isotope dilution method. In vitro, codeine and APAP showed an inhibitory effect on hydroxyanastrozole production in a dose-dependent matter. In vivo, all chronic treatments had a higher ratio of metabolite/parent drug than in the acute Page | 6 treatment, suggesting higher production of the hydroxy-anastrozole. Furthermore, differences between treatments of the same condition indicated that the combination of analgesic drugs and anastrozole had a lower metabolite/parent ratio, suggesting higher alternative metabolic pathways. These findings suggest that codeine and paracetamol impact anastrozole metabolism in mice and, therefore, could potentially impact the metabolism in humans. Further studies in humans are essential to assess the impact of potential interactions between breast cancer endocrine therapy and analgesic treatment with reducing prospective limitations.Svrha ovog istraživanja bila je utvrditi potencijalne interakcije između anastrozola, kodeina i paracetamola, pojedinačno ili u kombinaciji. Štoviše, dodatni cilj ovog istraživanja bio je razjasniti potencijalni utjecaj na metabolički put anastrozola, a time i na samu njegovu aktivnost. Do 80% dijagnosticiranih karcinoma dojke izražava estrogenske receptore, oviseći o estrogenu za proliferaciju. Anastrozol je inhibitor aromataze treće generacije koji djeluje na enzim aromatazu (CYP19A1). Aromataza je ključni enzim bio-sinteze estrogena koji je visoko izražen u stanicama karcinoma dojke. Paracetamol je najčešće korišten lijek dostupan bez recepta koji se koristi ili u obliku jednog spoja za blagu bol ili u obliku više spojeva za umjerenu do jaku bol. Pretežno, paracetamol se kombinira s kodeinom. Kodein je opijat i pro-lijek, koji dovodi do morfija i njegovih metabolita (morfij-3-glukuronid i morfij 6-glukuronid). Morfij djeluje na mu-opioidne receptore kako bi proizveo analgeziju. Jetre miševa korištene su za stvaranje mikrosoma za određivanje proizvodnje metabolita anastrozola, hidroksi-anastrozola i utjecaja analgetskih lijekova na metabolizam anastrozola in vitro s IC50 esejom. Za in vivo eksperimente, ženski CB57BL/6J miševi tretirani su anastrozolom (kontrolno stanje) i kodeinom, paracetamolom ili kombinacijom. Tretman je administriran u obliku jedne doze (akutno liječenje) i ponovljenih injekcija tijekom jednog tjedna (kronično liječenje). In vitro i uzorci krvi analizirani su i kvantificirani tekućom kromatografijom spojenom s masenom spektrometrijom (LC-MS/MS) metodom razrjeđivanja izotopa. In vitro, kodein i APAP pokazali su inhibitorni učinak na proizvodnju hidroksi-anastrozola u ovisnosti o dozi. In vivo, svi kronični tretmani imali su veći omjer metabolita/izvornog lijeka nego u akutnom liječenju. Ovaj rezultat upućuje na veću proizvodnju hidroksi-anastrozola. Nadalje, razlike između tretmana istog stanja pokazale su da kombinacija analgetskih Page | 8 lijekova i anastrozola ima niži omjer metabolita/izvornog lijeka, što potencijalno upućuje na alternativne metaboličke putove. Dobiveni rezultati sugeriraju da kodein i paracetamol utječu na metabolizam anastrozola kod miševa i stoga mogu potencijalno utjecati na metabolizam kod ljudi. Daljnja istraživanja na ljudima su neophodna za procjenu utjecaja potencijalnih interakcija između endokrine terapije raka dojke i analgetskog liječenja uz smanjenje potencijalnih ograničenja
Endogenous morphine in the mammalian central nervous system
No full textUn stress induit l’apparition de morphine sanguine et cérébrale, suggérant que la morphine endogène, et ses dérivés comme la M6G, représentent des médiateurs de la réponse au stress. Durant ma thèse j’ai utilisé la lignée SH-SY5Y et j’ai montré la présence de morphine et de M6G dans ces cellules. La morphine est secrétée de façon calcium-dépendante suite à une stimulation nicotinique. J’ai également quantifié la morphine endogène dans différentes zones du cerveau de souris. Dans le cervelet des études en microscopie électronique ont montré une immunoréactivité dans des cellules en corbeille et leurs terminaisons qui font des synapses sur les corps cellulaires des cellules de Purkinje. Des approches de cultures cellulaires ont confirmé la néosynthèse de morphine par des neurones, et ont montré que les astrocytes sont capables de recapter de la morphine. Nous travaux montrent que la morphine endogène cérébrale possède toutes les caractéristiques d’un neurotransmetteurUsing the adrenal chromaffin cell model, we have shown the presence of morphine-6-glucuronide (M6G) in secretory granules and their secretion, leading us to propose that this endogenous alkaloid represent new neuroendocrine factor. In the human neuroblastoma SH-SY5Y cells we have characterized morphine and M6G, and demonstrate the presence of the UGT2B7 enzyme responsible for the formation of M6G from morphine. Morphine is secreted in response to nicotine stimulation via a Ca2+-dependent mechanism. We also show that morphine and M6G at concentrations as low as 10−10 M evoke specific naloxone-reversible membrane currents. Microscopy and proteomic approaches were used to quantify endogenous morphine in different brain area like the hippocampus, the cortex or the cerebellum. In the cerebellum, morphine is present in basket cells and their termini, which form close contacts on Purkinje cell bodies. Our work shows that morphine has all the characteristics of a neurotransmitter
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
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