Дисертації: "Endocannabinois"

1

Serzysko, Malgorzata. "Endocannabinoids and excitotoxicity: lessons from hypoglossal motoneurons." Doctoral thesis, SISSA, 2015. http://hdl.handle.net/20.500.11767/3908.

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Brainstem hypoglossal motoneurons (HMs) exclusively innervate tongue muscles and are severely damaged in the neurodegenerative disease called amyotrophic lateral sclerosis (ALS). One mechanism leading to such cell death is proposed to be glutamate-mediated excitotoxic stress. HMs are particularly vulnerable to excitotoxicity due to their expression of calcium-permeable α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors and scarcity of intracellular Ca2+ binding proteins like parvalbumin and calbindin. Indeed, blocking glutamate uptake in medullary slices can lead to pathological bursting and motoneuron damage. The endocannabinoid system is widely distributed in the brain and is believed to be an important regulator of synaptic transmission. Several studies reported neuroprotection mediated by the endocannabinoid system in such pathological insults like brain ischemia, traumatic brain injury or excitotoxicity. Moreover, in ALS animal models, up-regulation of the endocannabinoid system has been detected, suggesting it can play a role during disease development. Thus, detailed information on how the endocannabinoid system can affect cells during pathological insults like excitotoxicity is a valuable asset for future investigations of novel therapy approaches for ALS. The objective of this work was to investigate the effect of modulation of the endocannabinoid system during excitotoxic stress in hypoglossal motoneurons in vitro. Thin medullary slices (for electrophysiology and viability assay) or whole brainstem isolates (for Western Blot) from postnatal Wistar rats were used. Each slice/brainstem containing hypoglossal nuclei was transferred to a recording/incubation chamber and superfused with oxygenated Krebs solution. Excitotoxic stress was evoked by application of DL-TBOA (DL-threo-β-benzyloxyaspartic acid, 50 μM), a potent and selective inhibitor of excitatory amino acid transporters, with consequent build-up of extracellular glutamate. It was observed that modulation of endocannabinoid CB1 receptor (CB1R) function affected TBOA-evoked bursting, an event previously correlated with TBOA toxicity. Co-application of the endocannabinoid anandamide (AEA, 10 μM), a CB1R agonist, with TBOA resulted in lowered probability of the occurrence of pathological bursting, whereas co-application of the CB1R antagonist AM251 (10 μM) disrupted TBOA-induced bursts, leading to their “fragmentation”. Furthermore, AEA significantly decreased the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) isolated by co-application of bicuculline and strychnine (10 μM and 0.4 μM, respectively) and caused occurrence of biphasic activity in spontaneous inhibitory postsynaptic currents (sIPSCs; isolated by co-application of DNQX and APV at 10 μM and 50 μM, respectively) in some of the recorded cells. AM251 caused a decrease in the frequency of sIPSCs, but during application of bicuculline and strychnine it evoked activity which partly resembled bursting observed during TBOA application. Moreover, co-application of AEA with TBOA significantly decreased the number of damaged propidium iodide-positive cells with respect to counterstained Hoechst 33342-positive cells, which suggests a protective effect of this CB1R agonist against TBOA-induced toxicity. In addition, Western blot analysis showed a significant increase in CB1R protein levels after only 4 hours of TBOA incubation, indicating that the endocannabinoid system is activated during this excitotoxic insult. We suggest that a likely role of the endocannabinoid system in our brainstem preparation is to counteract the effects and consequences of elevated glutamate levels in the extracellular compartment.

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2

Mateu, Codina Gerard Àngel. "Factores genéticos en patología dual." Doctoral thesis, Universitat Autònoma de Barcelona, 2019. http://hdl.handle.net/10803/667189.

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El término patología dual describe la coexistencia de una o más enfermedades mentales y trastornos por el uso de sustancias, la cual es muy común y, a menudo, se asocia con una mayor gravedad psiquiátrica y peores condiciones médicas y sociales. Últimamente ha adquirido relevancia la investigación relacionada con el papel del sistema endocannabinoide (eCB) en las bases neurobiológicas de diversas afecciones neuropsiquiátricas, tales como la ansiedad, los trastornos del estado de ánimo o la esquizofrenia, así como los trastornos relacionados con el uso de sustancias. El propósito de este estudio es investigar la asociación entre varios polimorfismos de los genes que codifican algunas proteínas del sistema endocannabinoide (receptor de cannabinoides 1 [CNR1] y amida hidrolasa de ácido graso [FAAH]) de acuerdo con (1) la presencia o ausencia de uso de sustancias trastornos en toda la muestra y (2) según la presencia o ausencia de patología dual en el grupo de sujetos diagnosticados con un trastorno por uso de sustancias. Es un estudio de casos y controles con un tamaño muestral de 675 sujetos, de los cuales 362 son controles (C; individuos sin diagnóstico psiquiátrico) y 313 casos (Cs; individuos con un trastorno por uso de sustancias sin ningún trastorno neuropsiquiátrico comórbido [ TCS] así como individuos con un trastorno de uso de sustancias con una enfermedad neuropsiquiátrica comórbida [PD]). Se compararon los grupos C y Cs y los grupos TCS y PD en base a datos demográficos, antecedentes familiares y personales de trastornos psiquiátricos y trastornos por uso de sustancias (mediante la versión española validada de la Psychiatric Research Interview for Substance and Mental Disorders (PRISM-IV)) y los rasgos de personalidad (evaluada con la versión española del Cloninger’s Temperament and Character Inventory (TCI-R). Se genotiparon 768 polimorfismos de nucleótido único (SNP) que se utilizaron como marcadores genéticos en una aproximación de estudio de gen candidato. Se realizaron análisis estadísticos simples de polimorfismos, genes y haplotipos así como un posterior análisis multivariante. Finalmente se relacionaron los resultados del análisis genético con las características sociodemográficas y clínicas de la muestra. Los sujetos Cs mostraron una mayor prevalencia de antecedentes familiares de enfermedades mentales y trastornos por uso de sustancias, así como una puntuación más baja en dependencia de la recompensa, autodirección, cooperación y autotrascendencia respecto a los sujetos C. Asimismo, los sujetos PD tenían mayor prevalencia de trastornos por consumo de alcohol y cocaína, menor prevalencia de trastornos por consumos de cannabis y mayor prevalencia de antecedentes familiares de enfermedades mentales y trastornos por uso de sustancias frente a los sujetos TCS. Además, los sujetos con TCS obtuvieron puntuaciones más altas en evitación del daño y autotrascendencia, aunque el resultado en autodirección fue significativamente más bajo en los sujetos con PD. Se halló una prevalencia significativa de las variantes AA/AC de SNP rs324420 y TT de SNP rs11576941 (ambos SNP que pertenecen al mismo haplotipo del gen FAAH) en el grupo Cs en el estudio de asociación de CS vs. C. La prevalencia fue también significativa en el caso de la variante TT del SNP rs11576941 que pertenece al gen FAAH en el grupo TCS en el estudio de asociación de TCS frente a C. Además, se detectó un subgrupo de sujetos del grupo de DP con puntuaciones significativamente más bajas en la dimensión de la personalidad evitación del daño que se caracterizan por ser homocigotos GG para el SNP rs806380 del gen CNR1. Esta variación alélica se ha asociado con una mayor sensibilidad para los estímulos positivos y podría representar un marcador endofenotípico para aquellos sujetos con mayor inestabilidad emocional y, por lo tanto, con mayor gravedad clínica potencial.
The term dual pathology describes the coexistence of one or more mental illnesses and substance use disorders which is very common and is often associated with higher psychiatric severity, and worse medical conditions and social status. Likewise, there is now an important body of research on the important role that the endocannabinoid system (eCB) could play on the neurobiological bases of various neuropsychiatric conditions, such as anxiety, mood disorders or schizophrenia, in addition to several substance use disorders. The purpose of this study is to investigate the association between various polymorphisms of the genes encoding for some endocannabinoid system proteins (cannabinoid receptor 1 [CNR1] and fatty acid amide hydrolase [FAAH]) according to (1) the presence or absence of substance use disorders in the whole sample and (2) according to the presence or absence of dual pathology within the group of subjects diagnosed with a substance use disorder. This is a case-control study with a total sample size of 675 subjects, of whom 362 are controls (C; individuals without any psychiatric diagnosis) and 313 cases (Cs; individuals with a substance use disorder without any co-morbid neuropsychiatric condition [TCS] and those individuals with a substance use disorder with co-morbid neuropsychiatric condition [PD]). A comprehensive comparison including demographics, parental and personal background of psychiatric and substance use disorders (according to DSM-IV-R and performed by using the Spanish validated version of Psychiatric Research Interview for Substance and Mental Disorders (PRISM-IV)) and personality characteristics (evaluated using the Spanish version of the Cloninger’s Temperament and Character Inventory (TCI-R))was performed between both Cs and C groups and between TCS and PD groups. 768 single-nucleotide polymorphisms (SNPs) were genotyped wich were used as genetic markers in an approximation of candidate gene study. Simple statistical analysis of polymorphisms, genes and haplotypes and subsequent multivariate analysis were performed. We will relate the results of genetic analysis with sociodemographic and clinical characteristics. The phenotypical results of the comparison between both Cs and C groups showed that Cs subjects had greater prevalence of family background of mental illness and substance use disorders and a lower score on reward dependence, self-directness, cooperativeness and self-transcendence as regarding on personality traits. Likewise, TCS and PD groups contrast show that PD subjects had higher prevalence of alcohol and cocaine use disorders and lower prevalence of cannabis use disorder, as well as a higher prevalence of family background of mental illness and substance use disorders. In addition, TCS subjects had higher score on harm avoidance and self-transcendence but, interestingly, PD subjects score significantly lower on self-directness. We have found a significant prevalence of the AA/AC variants of SNP rs324420 and TT of SNP rs11576941 (both SNPs belonging to the same haplotype of the FAAH gene) in the Cs group in the association study of CS vs. C. The prevalence was significant in the case of the TT variant of the SNP rs11576941 belonging to the FAAH gene in the TCS group in the association study of TCS vs. C. Furthermore, a subgroup of subjects of the PD group with significantly lower scores in the personality dimension harm avoidance that are characterized by being homozygous GG for the SNP rs806380 of the CNR1 gene have been detected. This allelic variation has been associated with a greater sensitivity for positive stimuli and could represent an endophenotypic marker for those subjects with higher emotional instability and, therefore, with greater clinical potential severity.

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3

Strohm, Daniela. "Modulation der Insulinsignalgebung durch Prostaglandin E2 und Endocannabinoide." Phd thesis, Universität Potsdam, 2010. http://opus.kobv.de/ubp/volltexte/2011/4967/.

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Die adipositasbedingte Insulinresistenz geht mit einer unterschwelligen Entzündungsreaktion einher. Als Antwort auf dieses Entzündungsgeschehen wird PGE2 unter anderem von Kupffer Zellen der Leber freigesetzt und kann seine Wirkung über vier PGE2-Rezeptorsubtypen (EP1-EP4) vermitteln. In vorangegangenen Arbeiten konnte gezeigt werden, dass PGE2 in Rattenhepatozyten über den EP3 R ERK1/2-abhängig die intrazelluläre Weiterleitung des Insulinsignals hemmt. Über die Modulation der Insulinrezeptorsignalkette durch andere EP-Rezeptoren war bisher nichts bekannt. Daher sollte in stabil transfizierten Zelllinien, die jeweils nur einen der vier EP-Rezeptorsubtypen exprimierten, der Einfluss von PGE2 auf die Insulinrezeptorsignalkette untersucht werden. Es wurden HepG2-Zellen, die keinen funktionalen EP-Rezeptor aufwiesen, sowie HepG2-Zellen, die stabil den EP1-R (HepG2-EP1), den EP3β-R (HepG2 EP3β) oder den EP4-R (HepG2 EP4) exprimierten, sowie die humane fötale Hepatozytenzelllinie, Fh hTert, die den EP2- und den EP4-R exprimierte, für die Untersuchungen verwendet. Die Zellen wurden für 330 min mit PGE2 (10 µM) vorinkubiert, um die pathophysiologische Situation nachzustellen und anschließend mit Insulin (10 nM) für 15 min stimuliert. Die insulinabhängige Akt- und ERK1/2-Phosphorylierung wurde im Western-Blot bestimmt. In allen Hepatomzelllinien die EP-R exprimierten, nicht aber in der Zelllinie, die keinen EP R exprimierte, hemmte PGE2 die insulinstimulierte Akt-Phosphorylierung. In allen drei stabil transfizierten Zelllinien, nicht jedoch in den Fh-hTert-Zellen, steigerte PGE2 die basale und insulinstimulierte Phosphorylierung der Serin/Threoninkinase ERK1/2. In den HepG2 EP1- und den HepG2-EP3β-Zellen steigerte PGE2 mutmaßlich über die ERK1/2-Aktivierung die Serinphosphorylierung des IRS, welche die Weiterleitung des Insulinsignals blockiert. Die Hemmung der Aktivierung von ERK1/2 hob in EP3 R-exprimierenden Zellen die Abschwächung der Insulinsignalübertragung teilweise auf. In diesen Zellen scheint die ERK1/2-Aktivierung die größte Bedeutung für die Hemmung der insulinstimulierten Akt-Phosphorylierung zu haben. Da durch die Hemmstoffe die PGE2-abhängige Modulation nicht vollständig aufgehoben wurde, scheinen darüber hinaus aber noch andere Mechanismen zur Modulation beizutragen. In den Fh hTert-Zellen wurde die Insulinrezeptorsignalkette offensichtlich über einen ERK1/2-unabhängigen, bisher nicht identifizierten Weg unterbrochen. Eine gesteigerte PGE2-Bildung im Rahmen der Adipositas ist nicht auf die peripheren Gewebe beschränkt. Auch im Hypothalamus können bei Adipositas Zeichen einer Entzündung nachgewiesen werden, die mit einer gesteigerten PGE2-Bildung einhergehen. Daher wurde das EP R-Profil von primären hypothalamischen Neuronen und neuronalen Modellzelllinien charakterisiert, um zu prüfen, ob PGE2 in hypothalamischen Neuronen die Insulinsignalkette in ähnlicher Weise unterbricht wie in Hepatozyten. In allen neuronalen Zellen hemmte die Vorinkubation mit PGE2 die insulinstimulierte Akt-Phosphorylierung nicht. In der neuronalen hypothalamischen Zelllinie N 41 wirkte PGE2 eher synergistisch mit Insulin. In durch Retinsäure ausdifferenzierten SH SY5Y-Zellen waren die Ergebnisse allerdings widersprüchlich. Dies könnte darauf zurückzuführen sein, dass die Expression der EP Rezeptoren im Verlauf der Kultur stark schwankte und somit die EP R-Ausstattung der Zellen zwischen den Zellversuchen variierte. Auch in den primären hypothalamischen Neuronen variierte die EP R-Expression abhängig vom Differenzierungszustand und PGE2 beeinflusste die insulinstimulierte Akt-Phosphorylierung nicht. Obwohl in allen neuronalen Zellen die Akt-Phosphorylierung durch Insulin gesteigert wurde, konnte in keiner der Zellen eine insulinabhängige Regulation der Expression von Insulinzielgenen (POMC und AgRP) nachgewiesen werden. Das liegt wahrscheinlich an dem niedrigen Differenzierungsgrad der untersuchten Zellen. Im Rahmen der Adipositas kommt es zu einer Überaktivierung des Endocannabinoidsystems. Endocannabinoidrezeptoren sind mit den EP Rezeptoren verwandt. Daher wurde geprüft, ob Endocannabinoide die Insulinsignalweiterleitung in ähnlicher Weise beeinflussen können wie PGE2. Die Vorinkubation der N 41-Zellen für 330 min mit einem Endocannabinoidrezeptoragonisten steigerte die insulinstimulierte Akt-Phosphorylierung, was auf einen insulinsensitiven Effekt von Endocannabinoiden hindeutet. Dies steht im Widerspruch zu der in der Literatur beschriebenen endocannabinoidabhängigen Insulinresistenz, die aber auf indirekte, durch Endocannabinoide ausgelöste Veränderungen zurückzuführen sein könnte.
The obesity related insulin resistance is accompanied by a low grade inflammation. In response to inflammatory stimuli, PGE2 is released from Kupffer cells and signals through four G-Protein coupled PGE2-receptors (EP1-EP4). Previous work showed that PGE2 attenuated insulin signaling in rat hepatocytes through an EP3ß- and ERK1/2-dependent mechanism. Since EP-receptor expression on hepatocytes varies between species and physiological conditions, the effect of the individual EP receptor subtypes on insulin signaling was studied in hepatoma cell lines expressing individual EP receptor subtypes. HepG2 cells lacking functional EP-receptors, and derivatives stably expressing either EP1 receptor (HepG2-EP1), EP3ß receptor (HepG2-EP3ß) or EP4 receptor (HepG2-EP4) and Fh-hTert cells expressing EP2- and EP4-receptor were pre-incubated with PGE2 for 330 min to mimic the sub-acute inflammation. The cells were subsequently stimulated with insulin for 15 min. Akt and ERK1/2 activation was determined by Western Blotting with phospho-specific antibodies. PGE2 inhibited insulin stimulated Akt phosphorylation in all cell lines expressing EP receptors, except in HepG2 cells which are lacking functional EP receptors. PGE2 increased insulin stimulated phosphorylation of the serine/threonine kinase ERK1/2 in all EP R expressing HepG2 cell lines except in Fh-hTert cells. In HepG2-EP1 and HepG2 EP3ß cells PGE2 increased the serine phosphorylation of the insulin receptor substrate, presumably through an ERK1/2 activation. This IRS-serine phosphorylation leads to attenuation of insulin signal transduction. Inhibiting ERK1/2 activation with a specific inhibitor attenuated the PGE2-dependent inhibition of insulin signal transmission in HepG2 EP3ß cells to some extent. ERK1/2 activation in these cells seems to be of major importance for the observed attenuation of insulin stimulated Akt phosphorylation. Application of inhibitors in the other cell lines stably expressing EP receptors provided evidence that other mechanisms contributed to the attenuation of insulin signaling. Insulin signal transduction in Fh-hTert cells by PGE2 was apparently blocked by an ERK1/2-independent mechanism. Increased PGE2 production during obesity is not limited to the periphery. Signs of inflammation have been detected in the hypothalamus, which might be associated with an increased PGE2 production. Therefore, the EP receptor profile of primary neurons as well as neuronal cell models was characterised in order to investigate, whether PGE2 attenuates insulin signal transduction in neuronal cells similar to what was observed in hepatocytes. Pre-incubation with PGE2 did not attenuate insulin stimulated Akt phosphorylation in all neuronal cells. The EP receptor profile in SH SY5Y cells and in primary neurons varied depending on the differentiation status of the cells. Although Akt-kinase was phosphorylated in response to insulin stimulation in all neuronal cells studied, gene expression of insulin target genes (POMC, AgRP) was not modulated by insulin. This might be due to the low level of differentiation of the investigated cells. In the course of obesity, an over-activation of the endocannabinoid system is detected. Since endocannabinoid receptors are related to EP receptors, it was investigated whether endocannabinoids can interfere with insulin signaling in a similar way as PGE2. Pre-incubation of the neuronal cell line N 41 for 330 min with an endocannabinoid receptor agonist, increased insulin stimulated Akt phosphorylation. This implies an insulin sensitising effect of endocannabinoids. This is contradictory to the endocannabinoid-dependent insulin resistance described in the literature and might be caused by indirect endocannabinoid-triggered mechanisms.

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4

LISAI, SARA. "Nutritional factors influencing tissue omega-3 metabolism and endocannabinoids levels in experimental models and humans." Doctoral thesis, Università degli Studi di Cagliari, 2016. http://hdl.handle.net/11584/266644.

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The nutritional role of dietary fatty acids is quite controversial and often the object of mediatic campaigns either as panacea against all diseases or detrimental for human health. The most recent data are instead showing that it should be considered, in an optimal dietary regimen, the balance among all dietary fatty acids based on the current physiological needs, applying what it is currently known as personalised nutrition. It has been paid particular attention to the ratio between highly polyunsaturated fatty acids (HPUFAs) n-6, mainly arachidonic acid (ARA, 20:4n-6;) and n-3 HPUFAs, mainly eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) in tissues, since an inverse correlation between circulating n-3 HPUFAs and several chronic pathological states has been observed, possibly mediated by modulation of the endocannabinoid system (ECS). However, to assess this balance and controlling it by dietary means remains a great challenge for nutritionists. In addition, other nutritional factors may influence their tissue concentration. It is therefore compelling to individuate the mechanism(s) of action and what are the most efficient nutritional conditions to maximize n-3 HPUFAs health-related beneficial activities. The aim of this thesis was to evaluate the effects of some nutritional factors, such as conjugated linoleic acid (CLA), naturally occurring in the diet or produced by the probiotic Bifidobacterium breve (B. breve), in influencing n-3 PUFA metabolism to optimize the n-3 HUFA score (a biomarker of n-3 status in tissues) and possible interactions with the ECS in rodents and humans. We evaluated whether CLA, from different dietary sources, was able to enhance DHA biosynthesis, from its precursor alpha linolenic (ALA), in experimental models and humans. We found that different dietary ALA/CLA ratios affected n-3 PUFA and EC profile with a graded decrease of ALA and EPA and corresponding increase of DHA, while the EC arachidonoylethanolamine (AEA) decreased parallel to ratio ALA/CLA, whereas dietary ALA, in absence of CLA, was not able to increase significantly circulating DHA levels. We also evaluated whether supplementation with B. Breve as probiotic, along with ALA, in mice, as a nutritional factor that may increase dietary CLA, modulated n-3 fatty acid metabolism. We found that dietary probiotics increased DHA biosynthesis in liver and epididymal adipose tissue. Actually, we showed an increased of DHA biosynthesis in liver, but not an increase of CLA, while the ratio CD 16:2/CLA, a biomarker of peroxisomal beta oxidation, increased significantly but not associated to a higher PPAR α gene expression. The observed parameters suggested that these effects may be related to a pro-inflammatory event possibly triggered by the activation of toll like receptors by B. breve. In fact, in an ancillary experiment on rats challenged with LPS, we obtained similar results on fatty acid metabolism. In addition, dietary probiotics decreased AEA and its congener palmitoylethanolamine (PEA) levels in liver, while in adipose tissue we found a significant increased levels of AEA, PEA and the other AEA congener oleoylethanolamide (OEA). Our studies strongly suggest that background diet may play an important role in modulating fatty acid metabolism and in particular in modulating n-3/n-6 fatty acid balance, thereby differently affecting the ECS. Therefore, the synergistic effects of different nutritional factors, as it occurs in daily life in humans are somewhat difficult to predict and may explain the contradictory results in the literature on the effects of dietary fatty acids, in particular when they are singularly considered without taking into account the whole dietary regimen. The discover that gut microbiota may directly interfere and modify fatty acid metabolism opens to novel nutritional strategies in shaping the optimal milieu for contrasting several metabolic disorders by modulating the ECS. Future studies in humans will be focus on evaluating possible synergistic effects of CLA-enriched products with B. breve in maintaining and/or re-equilibrating energy and lipid metabolism, by modulating EC and congeners tissue profile, acting on energy metabolism and EPA and DHA related molecules, and also to assess if there is an optimal level of dietary EPA and DHA to allow a synergistic activity in different physio-pathological conditions.

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5

Patel, Annie. "Endocannabinoid turnover and function." Thesis, University of Nottingham, 2010. http://eprints.nottingham.ac.uk/12625/.

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The therapeutic benefits of cannabis have been known for centuries of years. Yet it has only been in the last 40 years that an understanding of the system by which its works in our bodies has begun to be defined. This has in turn led to the discovery and understanding of the endogenous cannabinoid (eCB) system, alongside its main synthesizing and hydrolysing enzymes as well as the endogenous ligands. The use of synthetic cannabinoid receptor (CBR) ligands for therapeutic use has provided problems regarding the natural endogenous regulatory tone of the eCBs, which in turn has resulted in unwanted side effects. Part of the reason of this is due to synthetic agonists producing the well documented psychotropic effects at CB t receptors. Alternative targets for the manipulation of the eCB system for therapeutic benefits have been explored. One remains to be the use of FAAH inhibitors, which in turn potentially increase levels of eCBs in the system, hence potentiating their effects at the CBRs, or at other receptor sites. Therefore we have developed two HTS assays for the identification of potential inhibitors of FAAH and MAGL. They prove to be robust, cheap and facile and provide a clear indication of inhibitable levels of FAAH and MAGL activity. The FAAH assay can be further used to establish concentration-response curves of initial `hit' compounds. Yet, the HTS MAGL assay requires further characterization for use in construction of concentration-response curves, as they are not assays specific for MAGL acitivity and include hydrolysis of the substrate 4-NPA by non-specific esterases. Z-factor scores were calculated for both assays, indicating excellent assays, which can potentially be applied to industrial lab robotics for screening of compound libraries.

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6

Kilaru, Aruna. "Endocannabinoid System in Plants?" Digital Commons @ East Tennessee State University, 2012. https://dc.etsu.edu/etsu-works/4781.

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7

Norris, Leonie. "Endocannabinoid modulation of nociceptive processing." Thesis, University of Nottingham, 2010. http://eprints.nottingham.ac.uk/28701/.

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The analgesic effects of cannabis-based medicines are widely described and elevation of the endocannabinoids (ECLs), by inhibition of endocannabinoid metabolism, also produces analgesia. Measurement of endocannabinoids (ECLs) and metabolites, in low weight biological tissue, provides an important tool for investigating the potential therapeutic effects of this receptor system. The main aims of this thesis were to develop an analytical method to measure ECLs, and cyclooxgenase-2 (COX-2) metabolites of anandamide (AEA) and 2-arachidonoylglycerol (2-AG), and to apply the method to determine their role in nociceptive processing in models of acute and chronic inflammatory pain. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which allowed for the simultaneous measurement of AEA, oleoylethanolamide (OEA), palmitoylethanolamine (PEA) and 2-AG, as well as COX-2 metabolites of AEA and 2-AG was developed and validated. This method was demonstrated to be able to quantify ECLs and related compounds in rat hindpaw, brain, spinal cord and cell samples. The effects of TRPV1 activation on levels of intracellular calcium [Ca2+] and ECLs in sensory nerves were investigated. The TRPV1 agonist, capsaicin, dose dependently increased [Ca2+], which was blocked by the TRPV1 antagonist iodoresiniferatoxin (IRTX). Although synthesis of ECLs is activity and calcium dependent, there was no relationship between the TRPV1-mediated increases in [Ca2+], and levels of intracellular or extracellular ECLs. The mechanism of sensitization of primary afferent nociceptors by the proinflammatory cytokine tumor necrosis factor-α (TNFα), which is heavily implicated in inflammatory responses, was also studied. TNFα facilitated capsaicin-evoked calcium responses, an effect which was mediated by p38MAPK-induced phosphorylation. The ability of inhibition of fatty acid amide hydrolase (FAAH) to modulate inflammatory pain responses, and the sites at which ECLs were elevated, was studied in the carrageenan model of inflammatory pain. This model was considered ideal for the investigation of the role of alternative metabolism of ECLs by COX-2 as inflammatory pain is associated with an up-regulation of COX-2. The effects of acute and repeated administration of URB597 (0.3 mgkg-1), a selective inhibitor of FAAH metabolism of ECLs, on carrageenan-evoked changes in weight bearing and hindpaw oedema were determined. Behavioural effects were considered in parallel with the changes in levels of ECLs and COX-2 metabolites of AEA and 2-AG in the hindpaw and spinal cord. Acute administration of URB597 delayed the onset and attenuated carrageenan-induced hyperalgesia, an effect which was associated with increased levels of AEA, OEA and PEA in the spinal cord. By contrast, carrageenan-induced hyperalgesia was not altered by repeated administration of URB597 and levels of AEA in the spinal cord were not significantly increased by this treatment. There was no evidence for the metabolism of ECLs via COX-2 in the hindpaw, or spinal cord, of carrageenan-treated rats, under basal conditions or following inhibition of FAAH. The role of the endocannabinoid system in a model of osteoarthritis (OA) pain was also investigated. Intra-articluar injection of the chondrocyte inhibitor monosodium iodoacetate (MIA, 1 mg/50µl) produced histological changes reminiscent of joint damage in OA patients, and a time dependant decrease in paw withdrawal thresholds and weight bearing on the injured (ipsilateral) side. These behavioural pain responses were accompanied with an increase in ECLs in the spinal cord, suggestive of a role of ECLs in modulation of OA-induced pain.

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8

Chilufya, Jedaidah Y., Shivakumar P. Devaiah, Richard R. Sante, and Aruna Kilaru. "Endocannabinoid-Like Lipids in Plants." Digital Commons @ East Tennessee State University, 2015. https://dc.etsu.edu/etsu-works/4747.

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Classically, endogenous fatty acid ethanolamides and their derivatives that bind to the cannabinoid receptors and trigger a signalling pathway are referred to as endocannabinoids. Although derivatives of arachidonic acid, including arachidonylethanolamine or anandamide, are the known endogenous ligands for cannabinoid receptors, other fatty acid ethanolamides or N-acylethanolamines (NAE) that vary in carbon chain length and saturation occur ubiquitously in eukaryotic organisms and play an important role in their physiology and development. The metabolic pathway for NAEs is highly conserved among eukaryotes and well characterised in mammalian systems. Although NAE pathway is only partly elucidated in plants, significant progress has been made in the past 20 years in understanding the implications of the metabolism of saturated and unsaturated endocannabinoid-like molecules in plant development and growth. The latest advancements in the field of plant endocannabinoid research are reviewed. Key Concepts Endocannabinoids are endogenous ligands of cannabinoid receptors in mammalian systems. Endocannabinoids belong to a class of small bioactive lipid molecules that are derivatives of fatty acids including their ethanolamides, referred to as N-acylethanolamines. N-Acylethanolamines are ubiquitous and their metabolic pathway is highly conserved among eukaryotes. In higher plants, only 12–18C N-acylethanolamines have been identified and their metabolic pathway is partly elucidated. The endocannabinoid-like lipids play an important role in seed germination, seedling development, flowering and cellular organisation. In plants, N-acylethanolamines also participate in mediating responses to biotic and abiotic stress.

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9

Lenz, Frederike. "The endocannabinoid system and autistic behavior in the Fmr1- KO mouse." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2018. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-231397.

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Background: Background of this work was the investigation of the endocannabinoid system (ECS) in the Fmr1 knock- out (KO) mouse. The Fmr1- KO mouse is a mouse model for fragile X syndrome (FXS). FXS is the leading monogenic cause for autism spectrum disorders (ASD) in humans. The Fmr1- KO mouse displays autistic behavior such as an impaired social interaction, repetitive behavior, cognitive deficits, increased anxiety and aggressiveness. Alterations of the ECS have been suggested to play a key role in the etiopathology of a variety of neuropsychiatric disorders. Until today, little has been described about the involvement of the ECS in ASD. Interrogation: 1. Evaluating the manifestation of typical cannabinoid- induced effects in the Fmr1- KO mouse 2. Investigating the influenceability of autistic symptoms with THC treatment in the Fmr1- KO mouse 3. Analyzing the signaling cascade of the stimulated and unstimulated ECS in different brain regions of the Fmr1- KO mouse Material and Methods: Experiments were carried out on adult (12±1 weeks old) male Fmr1- KO and Fmr1- wild- type (WT) mice from the C57BL/6J- (B6)- background. N= 15 mice received THC (10mg/kg bodyweight) and N= 16 received WIN55,212 (3mg/kg bodyweight). 30min after injection, the body temperature was measured and the distance animals moved in an open field during 15min was recorded (locomotion). Then, animals were placed with their forepaws onto a horizontally fixed bar and the time remaining in this position (catalepsy) was measured. Finally animals were placed on a preheated plate and the temperature at which a pain stimulus occurred was determined (testing analgesia). All 4 experiments are called tetrad experiment. Afterwards changes in body temperature, locomotion, catalepsy and analgesia of the animals was evaluated. To explore long-term effects of THC after the tetrad, N= 15 animals were tested in a social interaction test with a female contact mouse, 10 and 20 days after THC treatment. Therefore, the tested mouse and the contact mouse were placed together into a cage and the time mice spent in social interaction (nose, body and anogential sniffing, allogrooming and body contact) was manually quantified during 6min of recorded testing time. Another group of N= 19 received a premedication of rimonabant (Cannabinoid- receptor 1 (CB1) antagonist, 3mg/kg bodyweight) 30min prior to THC treatment. Rimonabant prevents THC from binding to CB1 and therefore allows the assessment of the involvement of CB1 in mediating social behavior. Furthermore the suggestibility of context-dependent fear conditioning with THC treatment has been tested on N= 13 mice. Animals were placed into a conditioning chamber that delivered 6 short electric shocks with a 30sec pause to their paws (conditioning phase). Immediately afterwards mice received THC or placebo. 24h later contextdependent fear was evaluated by quantification of the time mice spent freezing in the conditioning-chamber (fear) without receiving foot shocks. Intraneuronal signaling of the ECS was analyzed with N= 29 animals using western blots. Quantities of phosphorylated (“activated”) protein kinases (ERK, AKT and S6) from different brain homogenates (hippocampus, striatum, cortex and cerebellum) were therefore measured after THC or placebo injection (30 minutes prior to sacrificing). Results: Cannabinoids induced hypothermia, hypolocomotion, analgesia and catalepsy in WTmice. These effects were significantly less detectable in Fmr1- KO mice. Effects of both cannabinoids, THC and WIN55,212, were comparable with a slightly greater but not significant efficiency of THC. THC treated WT- mice exhibited further reduced social interaction 10 days after treatment, an effect that was partially prevented by premedication with rimonabant. THC increased social interaction in Fmr1- KO mice comparable to the level of untreated WT- mice. THC had no effect on behavior of WT- mice in context-dependent fear conditioning. Fmr1- KO mice showed significant less contextdependent fear conditioning compared to WT- mice. THC facilitated the recognition of an anxiety-correlated context in Fmr1- KO mice comparable to untreated WT- mice. In western blots significant changes in the THC- induced signaling cascade were detectable and depending on genotype, brain-region and analyzed protein-kinase. In the hippocampus there were no changes in untreated Fmr1- KO mice compared to WT- mice. THC had no effect on activation of protein-kinases in WT- and Fmr1- KO mice. In the striatum there were no changes in untreated Fmr1- KO mice compared to WTmice. THC significantly increased activity of ERK, AKT and S6 in WT-mice and not in Fmr1- KO mice. In the cortex of untreated Fmr1- KO mice AKT showed a significantly increased activity compared to WT- mice. THC significantly increased AKT activity in WT- mice without having an effect on KO- mice. In the cerebellum there were no changes in untreated Fmr1- KO mice compared to WT- mice. THC significantly increased ERK- activity in Fmr1- KO mice but had no effect on protein kinase activity in WT- mice. Conclusion: We observed physiological cannabinoid effects in WT- mice after treatment with THC and WIN55,212. These effects are significantly attenuated in Fmr1- KO mice. This may be interpreted as a desensitization of the ECS in the Fmr1- KO mouse. At the same time it was demonstrated that THC has the potential to improve context dependent memory consolidation and to increase social interaction in the Fmr1- KO mouse. In particular the influence of THC on impaired social interaction should be a target of further investigations to find possible therapeutic options for this typical symptom of Autism. Underlying molecular mechanisms remain unclear and the analysis of THC stimulated intraneuronal signaling gave no clear indication of possible molecular alterations in the Fmr1- KO mouse.

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Bonneville, Marika. "Endocannabinoid Modulation of Post-Ischemia Depression." Thesis, Université d'Ottawa / University of Ottawa, 2016. http://hdl.handle.net/10393/35056.

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Post-ischemia depression (PID) is a condition that affects approximately 30% of survivors from stroke or cardiac arrest and has an important impact on patients’ quality of life. Previous studies support important roles of the endocannabinoid (eCB) system in depression and brain ischemia. This study attempts to link all three variables together by investigating the role and mechanism of eCB signaling in the development of PID. A global ischemia + hypotension model was used to induce a PID phenotype in CD1 mice. Three ischemic time frames were tested, and even though all three could induce significant cell death in the CA1 region of the hippocampus, only the 15-minute time point led to an increased immobility time on the forced swimming test (FST). The main goal of this study was to investigate the effect of a cannabinoid type-I receptor (CB1R) antagonist/inverse agonist, AM281, on the development of two depressive symptoms: anhedonia, measured with the sucrose preference test (SPT), and behavioral despair, measured with the FST. AM281 administration was able to significantly reduce the symptoms of anhedonia and behavioural despair. Subsequently, the mechanism behind this antidepressant-like effect was investigated. Administration of bicuculine with AM281 did not significantly affect the antidepressant effect on the FST, therefore suggesting that AM281 does not act on GABAergic synapses. A similar protocol was adopted with NVP-AM077, where its administration combined with AM281 was able to block the effect of AM281, thus confirming the importance of glutamatergic synapses for the antidepressant effect of AM281. Furthermore, the administration of a TAT-GLUR2 peptide did not significantly affect the effect of AM281, implying that the astroglial cell-mediated LTD (long-term depression) at glutamatergic synapses is not involved in the antidepressant effects of AM281. Finally, a bilateral intra-BLA (basolateral nucleus of the amygdala) administration of AM281 was able to reduce the immobility time on the FST. In conclusion, these results highlight the important contribution of BLA glutamatergic synapses to the antidepressant-like effect conferred by AM281.

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11

Bambico, Francis. "Cannabinoids and Endocannabinoids in mood regulation." Thesis, McGill University, 2010. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=95031.

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Cannabinoids (CB) are compounds structurally derived from Δ(9)-THC, the main pharmacologically active component of cannabis and marijuana. CBs produce psychoactive effects by binding the cerebral CB1 receptor (CB1R). The mammalian brain also naturally produces endocannabinoids (eCBs), intrinsic ligands of CB1R. At the start of this doctoral project, the role of the eCB system in mood regulation and the interaction between CBs and monoamines remained largely unexplored. The main aim of this project was to characterize the effects of CBs and eCBs on depression-like reactivity in murids. Since serotonin (5-HT) and norepinephrine (NE) are the major monoamine neurotransmitters implicated in depression pathophysiology and in the mechanism of action of antidepressants, we employed electrophysiological techniques to assess the in vivo effect of CBs and eCBs on 5-HT neurons of the dorsal raphe, on NE neurons of the locus coeruleus, and on postsynaptic limbic areas. The direct CB1R agonist WIN55,212-2 produced a bell-shaped effect in the rat forced swim test (FST), with low doses eliciting antidepressant-like activity while high doses being inert. This behavioural pattern was congruent with electrophysiological data. Indeed, a rapid increase of 5-HT neural activity was observed at low-doses while an attenuation below basal levels was observed at high doses. The enhanced 5-HT activity was instigated by an excitatory feedback driven by CB1Rs in the ventromedial prefrontal cortex, a limbic structure involved in stress controllability. Chronic exposure to low or high doses of WIN55,212-2 in adolescence but not in adulthood precipitated depressive- and anxiety-like reactivity that persisted in adulthood. These emotional impairments were associated with 5-HT hypoactivity and NE hyperactivity. URB597 is a novel compound that inhibits fatty acid amide hydrolase (FAAH), an enzyme that degrades anandamide, one of the major eCBs. URB597 is thus able to markedly enhance brain ananda
Les cannabinoïdes (CBs) sont des composés dérivés structurellement du Δ(9)-THC, le principe actif du cannabis. Ces drogues produisent leurs effets en se liant aux récepteurs CB1 (CB1R). Le cerveau produit également des endocannabinoïdes (eCBs) naturellement, et ceux-ci constituent les ligands intrinsèques du CB1R. Le rôle du système eCB dans la régulation de l'humeur et l'interaction entre les CBs et les monoamines étaient des sujets encore largement inexplorés. L'objectif était de caractériser l'impact de la modulation du système eCB par les CBs/eCBs sur des modèles de dépression chez le rongeur. Comme la sérotonine (5-HT) et la norépinéphrine (NE) sont les neurotransmetteurs impliqués dans la pathophysiologie et le traitement de la dépression, nous avons utilisé des techniques électrophysiologiques pour isoler in vivo l'effet des CBs/eCBs sur l'activité neurones 5-HT du raphé dorsal, des neurones NE du locus coeruleus et sur les aires postsynaptiques du système limbique. L'agoniste du CB1R, le WIN55,212-2, produit un effet bi-phasique lors du test de nage forcée (FST). Les faibles doses ont entraîné une réponse de type anti-dépresseur, alors que les doses élevées sont restées sans effet. La modulation de la 5-HT a également montré une réponse bidirectionnelle, les faibles doses ayant stimulé l'activité neuronale 5-HT, et les doses élevées la réduisant sous le niveaude base. L'augmentation de l'activité 5-HT semble mettre en action une boucle excitatrice engendrée par la stimulation des CB1R du cortex préfrontal ventromédial, qui est impliqué dans le contrôle du stress. À l'adolescence, l'exposition chronique au WIN55,212-2, a engendré une perturbation semblable à la dépression et l'anxiété qui persiste à l'âge adulte. Ces troubles émotionnels semblent être associés à une hypoactivité de la 5-HT et à une hyperactivité de la NE. L'URB597 est un composé inhibant l'acide gras amide hydrolase (HAAG),

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12

Mustonen, Katie Lynn. "Endocannabinoid System in a Planarian Model." Thesis, University of North Texas, 2010. https://digital.library.unt.edu/ark:/67531/metadc33188/.

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In this study, the presence and possible function of endocannabinoid ligands in the planarian is investigated. The endocannabinoids ananadamide (AEA) and 2-arachidonoylglycerol (2-AG) and entourage NAE compounds palmitoylethanolamide (PEA), stearoylethanolamide (SEA) and oleoylethanolamide (OEA) were found in Dugesia dorotocephala. Changes in SEA, PEA, and AEA levels were observed over the initial twelve hours of active regeneration. Exogenously applied AEA, 2-AG and their catabolic inhibition effected biphasic changes in locomotor velocity, analogous to those observed in murines. The genome of a close relative, Schmidtea mediterranea, courtesy of the University of Utah S. med genome database, was explored for cannabinoid receptors, none were found. A putative fatty acid amide hydrolase (FAAH) homolog was found in Schmidtea mediterranea.

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13

Costa, Lia Filipa Alvarez Pereira da Mota e. "Cannabinoids impact on pregnancy: effects in trophoblast cells." Master's thesis, Universidade de Aveiro, 2015. http://hdl.handle.net/10773/15948.

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Mestrado em Biologia Molecular e Celular
Cannabinoids (CBs) can be classified as: phytocannabinoids, the constituents of the Cannabis sativa plant; synthetic cannabinoids lab-synthesized and the endocannabinoids that are endogenous lipid mediators. Cannabinoid compounds activate cannabinoid receptors – CB1 and CB2. The most prevalent psychoactive phytocannabinoid is Δ9tetrahydrocannabinol (THC), but more than 60 different CBs were already identified in the plant. The best characterized endocannabinoids (eCBs) are anandamide (AEA) and 2arachidonoylglycerol (2-AG), that are involved in several physiological processes including synaptic plasticity, pain modulation, energy homeostasis and reproduction. On the other hand, some synthetic cannabinoids that were initially designed for medical research, are now used as drugs of abuse. During the period of placental development, highly dynamic processes of remodeling occur, involving proliferation, apoptosis, differentiation and invasion of trophoblasts. It is known that a tight control of eCBs levels is required for normal pregnancy progression and that eCBs are involved in trophoblast cells turnover. Therefore, by sharing activation of the same receptors, exposure to exocannabinoids either by recreational or medicinal use may lead to alterations in the eCBs levels and in the endocannabinoid system homeostasis In this work, it was studied the impact of CBs in BeWo trophoblastic cells and in primary cultures of human cytotrophoblasts. Cells were treated for 24 hours with different concentrations of THC, the synthetic cannabinoid WIN‐55,212 (WIN) and 2-AG. Treatment with THC did not affect BeWo cells viability while WIN and 2-AG caused a dose-dependent viability loss. Morphological studies together with biochemical markers indicate that 2-AG is able to induce apoptosis in cytotrophoblasts. On the other hand, morphological studies after acridine orange staining suggest that autophagy may take part in WIN-induced loss of cell viability. All cannabinoids caused a decrease in mitochondrial membrane potential (Δψm) but only 2-AG led to ROS/RNS generation, though no changes in glutathione levels were observed. In addition, ER-stress may be involved in the 2-AG induced-oxidative stress, as preliminary results point to an increase in CCAAT-enhancer-binding protein homologous protein (CHOP) expression. Besides the decrease in cell viability, alterations in cell cycle progression were observed. WIN treatment induced a cell cycle arrest in G0/G1 phase, whereas 2-AG induced a cell cycle arrest in G2/M phase. Here it is reinforced the relevance of cannabinoid signaling in fundamental processes of cell proliferation and cell death in trophoblast cells. Since cannabis-based drugs are the most consumed illicit drugs worldwide and some of the most consumed recreational drugs by pregnant women, this study may contribute to the understanding of the impact of such substances in human reproduction.
Os canabinóides (CBs) podem ser classificados como: fitocanabinóides, os constituintes da planta Cannabis sativa L.; canabinóides sintéticos, sintetizados em laboratório e os endocanabinóides, que são mediadores lipídicos endógenos. Os compostos canabinóides ativam recetores canabinóides – CB1 e CB2. O composto psicoativo mais prevalente é o Δ9-tetrahidrocanabinol (THC), mas mais de 60 diferentes CBs foram já identificados a partir da planta. Os endocanabinóides (eCBs) melhor caracterizados são a anandamida (AEA) e o 2-araquidonoilglicerol (2-AG), que estão envolvidos em vários processos biológicos, incluindo plasticidade sináptica, modulação da dor, homeostasia energética e reprodução. Por outro lado, alguns canabinóides sintéticos, inicialmente projetados para investigação médica, são agora usados como drogas de abuso. Durante o período de desenvolvimento placentário ocorrem processos de remodelação que envolvem proliferação, apoptose, diferenciação e invasão dos trofoblastos. Sabe-se que um controlo rigoroso dos níveis de eCBs é necessário para uma progressão normal da gravidez e que os eCBs estão envolvidos no turnover celular dos trofoblastos. Assim sendo, ao partilharem a ativação dos mesmos recetores, a exposição a exocanabinóides, seja pelo uso recreativo ou medicinal, pode levar a alterações nos níveis de eCBs e na homeostasia do sistema endocanabinóide (ECS). Neste trabalho foi estudado o impacto dos CBs em células trofoblásticas BeWo e em culturas primárias de citotrofoblastos humanos. As células foram tratadas durante 24 horas com diferentes concentrações de THC, do canabinóide sintético WIN-55,212 (WIN) e de 2AG. O tratamento com THC não afetou a viabilidade das células BeWo, enquanto que o WIN e o 2-AG causaram uma perda de viabilidade dependente da dose. Estudos morfológicos, juntamente com marcadores bioquímicos, indicam que o 2-AG é capaz de induzir apoptose em citotrofoblastos. Por outro lado, estudos morfológicos realizados com laranja de acridina sugerem que a autofagia pode estar envolvida na perda de viabilidade induzida pelo WIN. Todos os canabinóides induziram perda de potencial de membrana mitocondrial (Δψm), mas apenas o 2-AG levou a um aumento na formação de ROS/RNS, sem terem sido observadas diferenças nos níveis de glutationa. O stress reticular pode estar envolvido no stress oxidativo induzido pelo 2-AG, visto que resultados preliminares apontam para um aumento na expressão de CCAAT-enhancer-binding protein homologous protein (CHOP). Para além da diminuição da viabilidade celular, os resultados sugerem alterações na progressão do ciclo celular. O tratamento com WIN induziu retenção do ciclo celular em fase G0/G1, enquanto que o 2-AG levou a uma retenção em fase G2/M. Neste trabalho é reforçada a importância da sinalização canabinóide em processos importantes de proliferação e morte celular de células trofoblásticas. Visto que as drogas canabinóides são as mais consumidas a nível mundial, e umas das drogas recreativas mais consumidas pelas mulheres grávidas, este estudo pode contribuir para a compreensão do impacto destas substâncias na reprodução humana.

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SATTA, VALENTINA. "Possibile ruolo del sistema endocannabinoide nel disturbo d’alimentazione incontrollata (binge eating disorder): studi comportamentali, farmacologici e biochimici." Doctoral thesis, Università degli Studi di Cagliari, 2014. http://hdl.handle.net/11584/266471.

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According to the Diagnostic and Statistical Manual of Mental Disorders (DSM-V), binge eating disorder (BED) is an eating disorder characterized by repetitive episodes of uncontrolled and excessive food consumption (binge eating), in a short period of time, without the inappropriate compensatory behaviors for limiting weight gain. BED is a stable condition that is associated with elevated psychiatric comorbidity, including depression and anxiety. A large body of evidence supports a contribution of the endocannabinoid system in the modulation of energy balance by controlling food intake through central and peripheral mechanisms. The cannabinoid CB1 receptor is abundantly expressed in the central nervous system and induces inhibition of neurotransmission through modulation of presynaptic neurotransmitter release. CB1 receptors inverse agonists inhibit food intake through both central and peripheral mechanisms while, in contrast, cannabinoid agonists stimulate food intake in humans and induce beneficial effects in acquired immune deficiency syndrome related to anorexia, suggesting altered endocannabinoid neurotransmission in anorectic conditions. A possible involvement of the endocannabinoid system in the pathogenesis of BED and other eating disorders has recently been supported by independent experimental evidence. Elevated plasma levels of anandamide were found in women affected by AN and BED. Moreover, anandamide levels were inversely correlated with plasma leptin concentrations. On the basis of these evidence, the aim of this thesis was to study whether and which elements of the endocannabinoid system might be correlated with the binge eating behavior, which brain areas are specifically involved, and if pharmacological treatments specific for the endocannabinoid system (i.e. agonists, antagonists, inverse agonists of cannabinoid receptors, inhibitors of endocannabinoid metabolisms) are be able, besides to modify the state of BED induced in laboratory animals, to restore a correct functionality of the endocannabinoid system. Furthermore an analysis of the behavioral profile of animals with BED has been evaluated before the start of drug treatment by means of different mazes in which is possible to study anxiety and depression. Binge eating behavior was induced in animals by giving them a sporadic (3 days/week) and limited (2h) access to a high fat diet (margarine) in addition to a continuous access to chow and water. In these animals, the intake of margarine becomes significantly greater than in animals with limited daily access to margarine and remains stable over prolonged periods of time. As revealed by forced swim test, animals with binge eating behavior did not show a depressive like behavior compared to control animals. However, using the elevated plus maze, an anxiety like behavior was highlighted in those animals before access to margarine (Pre - binge phase) that it was significantly reduced after the consumption of this palatable food (Post - binge phase). Results showed that an increase of the endocannabinoid signaling by CB1 agonists or by inhibitors of endocannabinoid metabolism did not modify the binge eating behavior presents in our animals. On the contrary, a decrease of the endocannabinoid signaling by CB1 receptors inverse agonist/antagonist rimonabant, was able to alter this behavior when given chronically. As regards the CB1 receptor density, no difference has been highlighted between animals showing binge eating behavior and control group. In conclusion, negative modulation of the endocannabinoid signal may represent an important strategy in the treatment of Binge Eating Disorder.

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MATURO, PAOLO. "Infiammazione parodontale e sistema endocannabinoide." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2008. http://hdl.handle.net/2108/425.

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Анотація:

Gli endocannabinoidi, vale a dire ammidi, esteri ed eteri di acidi grassi polinsaturi a catena lunga, sono agonisti endogeni dei recettori cannabici di tipo 1 e 2 (CB1R e CB2R; Cannabinoid Binding Receptor type 1/2), in grado di mimare gli effetti farmacologici del Δ9-tetraidrocannabinolo (Δ9-THC), principio attivo della Cannabis sativa. Di questa classe di mediatori lipidici, i composti maggiormente studiati sono l’N-arachidonoiletanolammina (AEA) ed il 2-arachidonilglicerolo (2-AG). Le vie di sintesi ed il rilascio di AEA e 2-AG, sono la NAPE-PLD (N-acil-fosfatidiletanolammina fosfolipasi D) e la DAG lipasi (1,2-diacilglicerolo lipasi). Molte evidenze sperimentali hanno dimostrato che l’attività di questi composti a livello dei recettori viene limitata dal loro trasporto all’interno della cellula, attraverso uno specifico trasportatore (AMT, AEA Membrane Transport) e, successivamente, dalla loro degradazione intracellulare. Una idrolasi specifica, la FAAH (Fatty Acid Amide Hydrolase) è il principale enzima che degrada l’AEA mentre la MAGL (Mono Acyl Glycerol Lipase) è l’enzima chiave nella degradazione del 2-AG. Insieme all’AEA, al 2-AG e ad altri analoghi, queste proteine formano il “Sistema Endocannabinoide” (SE). L’AEA è in grado di legare ed attivare anche il recettore vanilloide di tipo 1 (Transient Receptor Potential channel Vanilloid subunit 1), pertanto è considerato anche un “endovanilloide”. Molti sono gli effetti biologici derivati dal legame degli endocannabinoidi ai loro target molecolari. Infatti, questa classe di mediatori lipidici esercita attività immunosoppressiva, ipotensivante, bradicardizzante, antinfiammatoria, mancato impianto embrionale e induzione dell’apoptosi nelle cellule neuronali e cerebrali. Le malattie parodontali sono un gruppo di patologie infiammatorie ad eziologia batterica, che portano alla distruzione del tessuto di supporto del dente. In Italia, circa il 60% della popolazione è affetta da malattie parodontali, e circa il 10-14% manifesta forme abbastanza avanzate (parodontiti). La risposta immunitaria all’infezione batterica svolge un ruolo fondamentale nella patogenesi di queste malattie. La ricerca scientifica si sta sempre più orientando verso lo studio molecolare di queste patologie e dei mediatori chimici responsabili dei processi infiammatori. Abbiamo voluto studiare il ruolo del sistema endocannabinoide nei processi infiammatori parodontali. ipotizzando che l’azione batterica possa stimolare il rilascio di endocannabinoidi nel tessuto parodontale ed in particolare nel fluido crevicolare gengivale prodotto al fine di svolgere la sua azione anti-infiammatoria. Recentemente è stata dimostrata la presenza in fibroblasti gengivali umani (HGFs) dei recettori cannabici di tipo 1 e 2 i quali, in condizioni patologiche, vengono sovraespressi. Inoltre sono stati rilevati livelli endogeni di AEA nel fluido crevicolare gengivale (GCF). L’AEA riduce in maniera significativa la produzione di citochine (IL-6, IL-8 and MCP-1) indotte dall’azione batterica in HGFs, attraverso i recettori cannabici. I nostri studi dimostrano che il GCF è in grado di idrolizzare l’Anandamide attraverso la FAAH, inoltre in pazienti affetti da periodontite tale attività sembrerebbe aumentare significativamente. Questi dati dimostrano per la prima volta la presenza della FAAH nel fluido gengivale che insieme all’identificazione dei recettori cannabici e dei livelli endogeni di AEA nei tessuti parodontali confermano il coinvolgimento del sistema endocannabinoide nei processi infiammatori causati dalla periodontite.
Endocannabinoids, which include amides, esters and ethers of long chain polyunsaturated fatty acids are the main endogenous agonists of cannabinoid receptors type 1 and 2 (CB1R and CB2R) able to mimic several pharmacological effects of Δ-9-tetrahydrocannabinol, the active principle of Cannabis sativa. In this class of lipid mediators Anandamide (N-arachidonoylethanolamine; AEA) and 2-arachidonoylglycerol (2-AG) are the most studied molecules. The pathways leading to the synthesis and release of AEA and 2-AG are the NAPE-PLD (N-acyl-phosphatidylethanolamine-specific phospholipase D) e la DAG lipasi (1,2-diacilglicerolo lipasi) . Instead, it is known that the activity of AEA is limited by cellular uptake through a specific membrane transporter (AMT), followed by intracellular degradation. A fatty acid amide hydrolase (FAAH) is the principal enzyme for AEA degradation, while MAGL (Mono Acyl Glycerol Lipase) is the enzyme for 2-AG. Together with AEA, 2-AG and congeners, these proteins form the “endocannabinoid system”. AEA can also activate the Vanilloid receptor type 1 (Transient Receptor Potential channel Vanilloid subunit 1), therefore it is consdered also an endovanilloid. There are different biological consequences of the endocannabinoids binding to their molecular target. This class of lipid mediators has activity on immunosuppression, ipotension, inflammation, failed embryonic implantation and apoptosis induction in neuronal and cerebral cells. Periodontal diseases are a group of inflammatory pathologies with bacterial ethiology that leeds to the loss of tooth supporting tissues. The prevalence of periodontal diseases in Italy is around 60%, and 10-14% of the population are afflicted by a severe form (periodontitis). Immunitary response to the bacterial infection plays an important role in the pathogenesys of these pathologies. Scientific research is going towards the molecular analysis of these pathologies and the chemical mediators involved in this inflammatory process We studied the role of the endocannabinoid system in the periodontal inflammation process. We hypothesize that bacterial infection can stimulate the release of endocannabinoids in the periodontal tissue and in particular into the gingival crevicular fluid producted for anti-inflammatory reasons. The presence in human gingival fibroblast (HGFs) of the cannabic receptors type 1 and 2 has been recently demonstrated, those receptors are overexpressed in pathologic conditions. Endogenous levels of AEA in the gingival crevicular fluid (GCF) has been noticed. Cytokine production (IL-6, IL-8 and MCP-1), caused by bacterial action in HGFs is significantly reduced by AEA through cannabic receptors. Our studies demonstrate that GCF can hydrolize AEA through FAAH, moreover in patiences with periodontitis this activity seems significantly increased. These data demonstrate for the first time the presence in the gingival fluid of FAAH, together with the identification of the cannabic receptors and of the endogenous levels of AEA in the periodontal tissues confirm the involvment of the endocannabinoid system in the inflammatory process caused by periodontitis.

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16

Busquets, Garcia Arnau 1985. "Targeting the endocannabinoid system for therapeutic purposes." Doctoral thesis, Universitat Pompeu Fabra, 2013. http://hdl.handle.net/10803/119617.

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The endocannabinoid system is an endogenous neuromodulatory system that regulates a plethora of physiological functions, including the modulation of memory, anxiety, pain, synaptic plasticity and neuronal excitability, among others. The activation of this system through exogenous or endogenous cannabinoid agonists has been proposed as a therapeutic strategy in different pathological states, although an important caveat to their use is the possible central adverse effects, such as memory impairment, anxiety and tolerance. The activity of the endocannabinoid system has been recently found involved in the pathophysiological conditions leading to obesity and fragile X syndrome, and the blockade of this system has also been investigated as a possible therapeutic approach. This thesis mainly focuses on the behavioral, paying more attention on the cognitive effects, cellular and molecular effects of exogenous and endogenous cannabinoids in order to identify potential therapeutic effects minimizing the negative consequences associated to the cannabinoid activation. This experimental research has been centered on the modulation of the positive and negative effects of Δ9-tetrahydrocannabinol, the main psychoactive component of the Cannabis sativa plant, the possibility to enhance the endogenous tone of specific endocannabinoids to improve certain therapeutic applications of cannabinoids, and the effects of inhibiting the endocannabinoid system in the amelioration of different traits associated to fragile X syndrome. The combination of behavioral, cellular and molecular approaches allowed the elucidation of different important aspects of the endocannabinoid system as an interesting therapeutic target.
El sistema endocannabinoid és un sistema neuromodulador endogen que regula diferents funcions fisiològiques com la memòria, l’ansietat, el dolor i l’excitabilitat neuronal entre altres. L’activació d’aquest sistema per agonistes exògens o endògens ha estat usada com a estratègica terapèutica en diferents estats patològics tot i que els efectes adversos, com la pèrdua de memòria, l’ansietat o la tolerància, són el principal problema pel seu ús. El sistema endocannabinoid també s’ha trobat alterat en malalties com la obesitat o la síndrome del cromosoma X fràgil i, per tant, el bloqueig d’aquest sistema també s’ha emprat com a aproximació terapèutica. Aquesta tesis es centra en els efectes comportamentals i moleculars de l’administració exògena del Δ9-Tetrahydrocannabinol, el component principal de la planta Cannabis sativa, i en la modulació endògena del sitema endocannabinoid per tal de potenciar els efectes terapèutics minimitzant els efectes adversos dels cannabinoids. A més, en aquesta tesis també hem estudiat els posibles efectes terapèutics del bloqueig dels receptors cannabinoides en la síndrome del cromosoma X fràgil. La combinació d’aproximacions moleculars, farmacològiques, electrofisiològiques i comportamentals han permès el descobriment de diferents aspectes importants que permeten demostrar que el sistema endocannabinoid és una diana terapèutica molt interessant.

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17

Ozdurak, Rabia Hurrem. "Exercise Induced Endocannabinoid And Immune System Alterations." Phd thesis, METU, 2009. http://etd.lib.metu.edu.tr/upload/12611362/index.pdf.

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Endocannabinoid and immune system alterations at moderate (18 m/min) and endurance (32 m/min) exercise intensities were assessed and compared to controls. Rats were exercised for 60 minutes/day, 5 days/week for 16 weeks. Immune effector cell proportions (T cell subtypes, B cells, NK cells, and neutrophils) and endocannabinoid serum levels were determined. Anandamide (ANA) and 2 arachidonyl-glycerol (2-AG) serum levels increased with endurance type of exercise. mRNA expression of the CB1 receptor increased together with ANA in the same group. Apoptotic index increased while immune effector cells responded divergently. B lymphocyte percentage decreased while T lymphocyte and NK cell percentage increased in blood. CD8+ subtypes increased whereas CD11b+ cell and CD25+ cell numbers decreased in the spleen in the endurance type of exercise group. Rats were grouped as the control, the endurance type of exercise, the AM281 (CB1 receptor antagonist) and the AM281+AM630 (CB2 receptor antagonist) groups in the second part of the study. Flow cytometry and microarray analyses of the spleen and the thymus were conducted. Endurance type of exercise associated significantly to immunological changes particularly to that of the T lymphocytes. T lymphocytes increased whereas cytolytic T lymphocytes decreased in blood. T cell and double positive T cell percentages significantly increased in the spleen. Activated T cells and NK like T cells furthermore decreased in the spleen. AM281 and/or AM630 could partially reverse the effect of exercise in blood but not in the spleen. Alterations in the thymus were not observed. Exercise altered 302 genes, some of them related with the immune system. Up-regulation of heat-shock protein coding genes was the most significant ones.

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18

Ababio, Frank James Kweku. "The endocannabinoid system in inflammatory bowel system." Thesis, Nelson Mandela Metropolitan University, 2014. http://hdl.handle.net/10948/d1020338.

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Crohn’s disease (CD) and ulcerative colitis (UC) constitute the two major forms of inflammatory bowel disease (IBD), which are disorders of chronic inflammation in the gastrointestinal tract that are associated with significant morbidity and socioeconomic burden. IBD patients with long-standing intestinal inflammation are more prone to developing colorectal cancer (CRC). Until now, none of the existing IBD treatments is able to heal the mucosal ulcerations satisfactorily. The endocannabinoid system (ECS), which comprises of endogenous cannabinoid ligands, their receptors, and metabolic enzymes, has been implicated in gut homeostasis, visceral sensation, inflammation and gastrointestinal motility. Available studies in rodent models of IBD suggest that enhancing the ECS tone may reduce inflammation and improve mucosal integrity. This evidence indicates that the components of the ECS seem well positioned to exert a protective role in IBD and also to offer a great opportunity for therapeutic exploitation. Despite the role of the ECS in the gut, the presence and function of the components of the ECS is not well characterised in human IBD. The primary aim of the study was to investigate the state of the major components of the ECS in human IBD and to establish whether IBD is associated with any changes of the components of the ECS. Cannabinoid CB1 and CB2 receptors, enzymes for endocannabinoid biosynthesis PLC, “LRAT”, NAPE-PLD and DAGL, and endocannabinoid metabolic enzymes FAAH and MAGL were analysed from colonic tissue samples of CD, UC and control patients by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) to determine the relative mRNA expression of the above genes. The RT-qPCR analysis showed that the mRNA expression of PLC, LRAT, and NAPE-PLD were unchanged in both CD and UC, whiles DAGL mRNA was decreased in UC but was unchanged in CD. The endocannabinoid degradation enzymes, FAAH mRNA expression was also unchanged in CD but decreased in UC, whereas the mRNA expression of MAGL was significantly decreased in both CD and UC. NAPE-PLD/FAAH and DAGL/MAGL ratios, an estimation of the balance of AEA and 2-AG levels, showed that AEA and 2-AG levels could be increased and unchanged, respectively, in IBD. The mRNA expression of CB1 was significantly decreased in CD and UC whilst CB2 mRNA expression was unchanged in both forms of IBD. The study demonstrated that the components of the ECS which were investigated were present in colonic tissues of both IBD patients and healthy individuals, but they appear to be off balance in CD and UC patients. The decreased CB1 receptors in IBD patients could be an important modifier in the disease and could also provide a possible pathoaetiological mechanism linking IBD and CRC. Although these findings look promising, more studies with larger sample size are required to characterise the components of the ECS in human IBD.

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19

Klein, Carolin. "The endocannabinoid system and female sexual arousal." Thesis, University of British Columbia, 2011. http://hdl.handle.net/2429/35739.

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Evidence from several lines of research points to the potential role of the endocannabinoid system in female sexual functioning. This evidence includes results from studies describing the subjective effects of exogenous cannabinoids on sexual functioning in humans and the observable effects of exogenous cannabinoids on sexual functioning in other species, as well as the results from studies investigating the location of cannabinoid receptors in the brain and periphery, and the effects of cannabinoid receptor activation on neurotransmitters implicated in sexual functioning. However, while these lines of research are suggestive of a role of the endocannabinoid system in female sexual functioning, no studies investigating the relationship between levels of endogenous cannabinoids (i.e., arachidonoylethanolamide [anandamide or AEA] and 2-arachidonoylglycerol [2-AG]) and sexual functioning, or investigating the effects of exogenous cannabinoid use on physiological measures of sexual functioning in humans, have been conducted. Experiments 1 and 2 of the present dissertation are the first known studies to measure and examine circulating endocannabinoid levels in relation to both subjective and physiological indices of sexual arousal in women. Experiment 3 is the first known study to examine the relationship between exogenous cannabinoid use and physiological sexual arousal in women. Physiological sexual arousal in all three studies was measured via the vaginal photoplethysmograph. Although the results of Experiment 1 did not reveal an association between endocannabinoid levels and female sexual arousal, the results of Experiment 2 revealed a significant relationship, whereby increases in both physiological and subjective indices of sexual arousal were significantly associated with decreases in endocannabinoid (AEA and/or 2-AG) levels. Experiment 3 revealed that women who use marijuana show significantly smaller increases in physiological sexual arousal in response to erotic film stimuli than women who do not use marijuana. The findings from these studies support the hypothesis that the endocannabinoid system plays a role in female sexual functioning. This line of research has broad clinical and research implications, not only in terms of furthering understanding of the biological mechanisms underlying female sexual functioning, but also in terms of finding effective treatments for sexual dysfunctions in women.

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20

Finney, Mark. "The role of endocannabinoids in early pregnancy." Thesis, University of Leicester, 2009. http://hdl.handle.net/2381/8686.

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The endocannabinoid N-arachidonoylethanolamine (anandamide; AEA) adversely affects early pregnancy initiation and maintenance in both animals and humans. Anandamide acting through the cannabinoid receptors CB1 and CB2 affects many aspects of reproduction and is formed on demand by the hydrolysis of N-arachidonoyl-phosphatidylethanolamine (NAPE), by a specific phospholipase D (PLD) NAPE-PLD. Furthermore, AEA is catabolised by Fatty Acid Amide Hydrolase (FAAH). The aim of this thesis was to investigate how variations in the various components of the endocannabinoid system might lead to different early pregnancy outcomes. Using a newly developed UPLC-MS/MS method, plasma AEA levels were measured in women throughout pregnancy, compared to previous data and shown to be comparable. Moreover, women presenting with a viable pregnancy (on USS) with a threatened miscarriage (bleeding), who went on to miscarry and women presenting with a confirmed miscarriage (on USS) when compared with women with a viable pregnancy at the time of USS in whom there were no symptoms of miscarriage or those that went to term, all had increased plasma AEA levels. Median plasma AEA levels in the miscarriage group were 3-fold (p<0.001) higher than the levels in the live birth group. Using an AEA level of 2.0nM as cut-off for predicting subsequent miscarriage gave a sensitivity of 100% and a specificity of 94%, (negative predictive value 100%; positive predictive value 82%). Although other markers of pregnancy success (progesterone, hCG and PAPP-A) all gave the expected concentrations in on-going, failed and failing pregnancies, there was no statistically significant correlation between these factors and plasma AEA levels. Immunohistochemical evaluation of CB1, CB2, FAAH and NAPE-PLD expression in first trimester pregnancy tissues (trophoblast and decidua) showed that FAAH staining in the miscarriage/non-viable group was significantly lower than that in the surgical termination of pregnancy group. In women undergoing medical termination of pregnancy, using an anti-progesterone (mifepristone), FAAH staining was actually increased compared with that from the surgical termination of pregnancy group; whilst CB2 had increased staining and CB1 had reduced staining in the miscarriage and the medical termination groups when compared with the surgical termination of pregnancy group. There was no significant difference in NAPE-PLD staining between all three groups. These data provide new evidence that the endocannabinoid system plays a significant role in early pregnancy failure/miscarriage and that plasma AEA may be used as a future predictive marker for miscarriage. The lack of correlation between AEA and progesterone and the finding of elevated FAAH in the medical miscarriage (anti-progesterone) group, but low FAAH in spontaneous miscarriage suggest that factors other than progesterone are responsible for increased AEA levels in cases of miscarriage.

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21

Robinson, Ian. "Effects of endocannabinoid metabolism on nociceptive behaviour." Thesis, University of Nottingham, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.537783.

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Chronic pain is a debilitating condition that is poorly treated in the clinic as conventional analgesics often lack efficacy and therapeutic doses can be limited by adverse side effects. The endocannabinoid (EC) system has been implicated in the modulation of pain and the antinociceptive effects of ECs can be enhanced by inhibiting catabolic enzymes. The main aims of this thesis were to investigate the roles that EC metabolism play in nociceptive behaviour. Theses aims were addressed by measuring tissue levels of ECs in inflammatory conditions, probing the mechanisms underlying EC mediated antinociception using pharmacological approaches and developing a sensitive analytical method to facilitate the analysis of ECs in microdialysate samples. t The key pharmacological tools used in the studies were the fatty acid amide hydrolase (FAAH) inhibitor URB597, the selective COX2 inhibitor nimesulide and the putative monoacylglycerol lipase (MAG lipase) inhibitor URB602. Intraplantar injection of URB597 (25 Ng), nimesulide (50 pg) or URB602 (70 pg) was analgesic in the carrageenan model of inflammatory pain, but only COX2 inhibition was shown to be antiinflammatory. A higher dose of URB597 (100 pg), was not, however, found to be analgesic in this model. Inhibition of metabolising enzymes resulted in changes in levels of ECs in spinal and paw tissue in the presence of carrageenan induced inflammationThe analgesic effects of URB597 (25 pg) and nimesulide (50 pg) were associated with an increase in peripheral levels of the EC-like acylethanolamine palmitoylethanolamide and were found to be mediated by the peroxisome proliferator-activated receptor alpha (PPARa). The higher dose of URB597 (100 pg) was associated with an increase of the EC anandamide, which has been shown to be a ligand for the pro-nociceptive transient receptor potential channel type V1 (TRPV1). However, the disappearance of an analgesic response at the higher dose of URB597 was independent of TRPV1 activation. Treatment with URB602 (70 pg) resulted in an increase in all ECs and related compounds ipsilateral hindpaw tissue suggesting that the inhibitor blocks FAAH as well as MAG lipase activity and so is not a selective inhibitor. Two high performance liquid chromatography-coupled tandem mass spectrometry (HPLC-MS/MS) methods, nano-HPLC-MS/MS and micro- HPLC-MS/MS, were developed to quantitatively analyse ECs and related compounds in low fmol concentrations. The micro-HPLCMS/ MS was then used to assess the time-related effects of systemic URB597 treatment on EC levels in the hippocampus of the awake, freely-moving rat and post-mortem EC levels in hippocampal tissue. Whilst intraperitoneal injection of URB597 (1 mg kg-1) appeared to reduce release of ECs in the hippocampus compared to vehicle controls, a positive correlation between dialysate and tissue EC levels was seenThe results of these studies show that EC metabolism is a key limiting factor in the analgesic action of the EC system in inflammatory pain and highlights EC metabolising enzymes as potential targets for new analgesic drugs

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22

Rettori, Valeria, Javier Fernandez-Solari, Juan P. Prestifilippo, Claudia Mohn, Laurentiis Andrea De, Stefan R. Bornstein, Monika Ehrhart-Bornstein, Juan C. Elverdin та Samuel M. McCann. "Endocannabinoids in TNF-α and Ethanol Actions". Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-135765.

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During marijuana and alcohol consumption as well as during inflammation the reproductive axis is inhibited, mainly through the inhibition of luteinizing hormone-releasing hormone release. In male rats, this inhibitory effect is mediated, at least in part, by the activation of hypothalamic cannabinoid type 1 receptors (CB1). During inflammation, this activation of the endocannabinoid system seems to be mediated by an increase in TNF-α production followed by anandamide augmentations, similarly the effect of intragastric administration of ethanol (3 g/kg) seems to be due to an increase in anandamide. On the other hand, a number of different actions mediated by the endocannabinoid system in various organs and tissues have been described. Both cannabinoid receptors, CB1 and CB2, are localized in the submandibular gland where they mediate the inhibitory effect of intrasubmandibular injections of the endocannabinoid anandamide (6 × 10–5M) on salivary secretion. Lipopolysaccharide (5 mg/kg/3 h) injected intraperitoneally and ethanol (3 g/kg/1 h) injected intragastrically inhibited the salivary secretion induced by the sialogogue metacholine; this inhibitory effect was blocked by CB1 and/or CB2 receptor antagonists. Similar to the hypothalamus, these effects seem to be mediated by increased anandamide. In summary, similar mechanisms mediate the inhibitory actions of endocannabinoids and cannabinoids in both hypothalamus and submandibular gland during drug consumption and inflammation
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23

Maroof, Nazia. "The role of endocannabinoids in Alzheimer's disease." Thesis, University of Nottingham, 2013. http://eprints.nottingham.ac.uk/30577/.

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The endocannabinoid system (ECS) comprises the endocannabinoids (ECs), including anandamide (AEA) and 2-arachidonoyl glycerol (2AG), which interact with the G protein-coupled type-1 and type-2 cannabinoid receptors(CB1 and CB2 respectively). The ECS is thought to have a role in a number of central processes including neuroinflammation, neurogenesis, neuroprotection, learning and memory. Due to its influence on a diverse number of processes, it has been suggested that modifying the ECS may be therapeutically beneficial in Alzheimer's disease (AD). AD is an age-related neurodegenerative disorder characterised by the presence of extracellular amyloid beta (Ab) plaques and intracellular neurofibrillary tangles (NFTs) resulting in impairments in learning in memory. The aim of this thesis was to determine the status of the brain ECS in the APPswe/PS18E9 mouse model of AD and wild type littermates at 4, 6 and 8 months of age and the performance of these animals in a behavioural test battery. The results of this study indicated that APPswe/PS18E9 animals were hyperactive compared to their wildtype counterparts at all ages and that they also displayed deficits in behavioural flexibility. EC levels increased with age in both wild type and APPswe/PS18E9 mice. Cannabinoid receptor coupling was increased in the frontal cortex and striatum of APPswe/PS18E9 mice relative to wildtype. This study concluded that the status of the brain ECS is altered in AD. Modifications to the performance of the ECS were made in the form of chronic administration of a CB1 receptor antagonist (SR141716A1rimonabant) and a CB2 receptor agonist (JWH133). Chronic administration of SR141716A was able to reverse some learning impairments in APPswe/PS18E9 animals. In contrast, chronic administration of JWH133 resulted in impaired memory extinction in both wildtype and APPswe/PS18E9 mice. The results support the potential benefit of modulating the endocannabinoid system in the treatment of memory impairment in AD.

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24

Rettori, Valeria, Javier Fernandez-Solari, Juan P. Prestifilippo, Claudia Mohn, Laurentiis Andrea De, Stefan R. Bornstein, Monika Ehrhart-Bornstein, Juan C. Elverdin та Samuel M. McCann. "Endocannabinoids in TNF-α and Ethanol Actions". Karger, 2007. https://tud.qucosa.de/id/qucosa%3A27675.

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During marijuana and alcohol consumption as well as during inflammation the reproductive axis is inhibited, mainly through the inhibition of luteinizing hormone-releasing hormone release. In male rats, this inhibitory effect is mediated, at least in part, by the activation of hypothalamic cannabinoid type 1 receptors (CB1). During inflammation, this activation of the endocannabinoid system seems to be mediated by an increase in TNF-α production followed by anandamide augmentations, similarly the effect of intragastric administration of ethanol (3 g/kg) seems to be due to an increase in anandamide. On the other hand, a number of different actions mediated by the endocannabinoid system in various organs and tissues have been described. Both cannabinoid receptors, CB1 and CB2, are localized in the submandibular gland where they mediate the inhibitory effect of intrasubmandibular injections of the endocannabinoid anandamide (6 × 10–5M) on salivary secretion. Lipopolysaccharide (5 mg/kg/3 h) injected intraperitoneally and ethanol (3 g/kg/1 h) injected intragastrically inhibited the salivary secretion induced by the sialogogue metacholine; this inhibitory effect was blocked by CB1 and/or CB2 receptor antagonists. Similar to the hypothalamus, these effects seem to be mediated by increased anandamide. In summary, similar mechanisms mediate the inhibitory actions of endocannabinoids and cannabinoids in both hypothalamus and submandibular gland during drug consumption and inflammation.
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25

Kilaru, Aruna. "Discovery and Implications of Endocannabinoids in Moss." Digital Commons @ East Tennessee State University, 2019. https://dc.etsu.edu/etsu-works/7726.

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26

Booker, Lamont. "Targeting the endocannabinoid system to reduce nociception." VCU Scholars Compass, 2011. http://scholarscompass.vcu.edu/etd/2419.

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Pain of various etiologies (e.g., visceral, inflammatory) can be a debilitating disorder that presents a problem of clinical relevance. While it is known that ∆9-tetrahydrocannabinol (THC) the primary psychoactive constituent found in marijuana produces analgesia in various rodent models of pain, its pharmacological properties are overshadowed by its psychomimetic effects. THC is the primary phytocannabinoid found in marijuana though other prevalent constituents such as the phytocannabinoids (e.g., cannabidiol (CBD), cannabinol (CBN), cannabichromene (CBC), tetrahydrocannabivarin (THCV)) may possess antinociceptive actions without the psychomimetic effects associated with THC. Indeed, these phytocannabinoids act upon the endocannabinoid system (ECS) that is comprised of the CB1 and CB2 cannabinoid receptors, endogenous ligands (anandamide (AEA), 2-arachidonoyolglycerol (2-AG)), and endocannabinoid biosynthetic and catabolic enzymes. We hypothesize that phytocannabinoids as well as endocannabinoid catabolic enzyme inhibitors reduce nociception preclinical models of pain. In the first series of studies, the antinociceptive effects of prevalent phytocannabinoids were evaluated in the acetic acid stretching test, a rodent visceral pain model. While CBN and THC both produced antinociceptive effects via a CB1 mechanism of action, CBC, and CBD had no effect on nociception. Conversely, THCV antagonized the antinociceptive effects of THC. These results suggest that various constituents of marijuana may interact in a complex manner to modulate pain. Since the THC and CBN displayed their effects via specific endogenous cannabinoid receptors, we investigated whether increasing endocannabinoids block nociceptive behavior. Blockade of the catabolic enzyme fatty acid amide hydrolase (FAAH) elevates AEA levels and elicits antinociceptive effects, without psychomimetic issues associated with THC. Similarly, blockade of another endocannabinoid catabolic enzyme monoacylglycerol lipase (MAGL) elevates (2-AG) and elicits antinociceptive effects. Therefore, we tested the hypothesis that FAAH and/or MAGL inhibition blocks nociception in the acetic acid abdominal stretching model, and the LPS-induced allodynia (i.e. painful response to a non-noxious stimuli) model of inflammation. Genetic deletion or pharmacological blockade of FAAH or pharmacological blockade of MAGL significantly reduced the total number of abdominal stretches in the visceral pain model. Additionally, blockade of both enzymes simultaneously produced an enhanced antinociceptive effect versus blocking the enzymes individually. These effects were mediated through CB1 receptors. However, in the LPS-induced allodynia model, FAAH inhibited anti-allodynic effects through a CB1 and CB2 receptor mechanismn. In both assays other potential targets of FAA substrates (i.e., mu-opioid, TRPV1, and PPAR-alpha receptors) did not play an apparent role in FAAH inhibited antinociceptive responses. Taken together, these results illustrate that targeting the endocannabinoid system via direct acting agonists such as the phytocannabinoids, or indirect methods (i.e. inhibiting degradative enzymes of the endogenous cannabinoids), represents a promising strategy to treat pain.

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Kilaru, Aruna. "Marijuana to Moss: Discovery of Plant Endocannabinoids." Digital Commons @ East Tennessee State University, 2015. https://dc.etsu.edu/etsu-works/4772.

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The elucidation of the binding of marijuana’s psychoactive compound, (-)-D9- tetrahydrocannabinol (THC), to specific membrane receptors, in the early 1990s, led to the identification of endogenous arachidonate-based lipids that activate cannabinoid receptors in mammals. While the metabolic and signaling pathway for these 20 carbon N-acylethanolamines (NAE) and their derivatives has been well characterized in mammals, thus far, only 12-18 carbon NAEs have been identified in plants and their metabolic pathway has been partly characterized. In plants, NAEs have been shown to modulate a number of physiological processes, including seed and seedling development and ability to respond to stress; however, the mechanisms by which they function remain to be elucidated. Our recent identification of a 20C NAE (arachidonylethanolamide) in moss provided us with an exciting possibility to identify receptor-mediated endocannabinoid signaling responses in plants that is akin to mammals. In this seminar, I will provide insights into the past, present and future aspects of plant endocannabinoid research.

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Kilaru, Aruna. "Marijuana to Moss: Discovery of Plant Endocannabinoids." Digital Commons @ East Tennessee State University, 2013. https://dc.etsu.edu/etsu-works/4775.

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Ford, Lorna. "An investigation into the effects of endocannabinoids and the COX-2 metabolite of 2-Arachidonyl glycerol on bone cells." Thesis, Available from the University of Aberdeen Library and Historic Collections Digital Resources, 2009. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?application=DIGITOOL-3&owner=resourcediscovery&custom_att_2=simple_viewer&pid=33596.

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30

Pastor, Bosch Antoni. "La formació d´esters i amides dels àcids grassos naturals com un mecanisme per explicar els seus efectes en la salut humana." Doctoral thesis, Universitat Autònoma de Barcelona, 2016. http://hdl.handle.net/10803/381084.

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Els efectes dels cannabinoids en la gana en humans es coneixen des de l´antiguitat. Les experiències dels consumidors moderns de cannabis descriuen un augment en les propietats sensorials i hedòniques del menjar, que els porta a un consum impulsiu de menjar saborós. Aquests efectes són deguts a l´activació per delta9-tetrahidrocannabinol (THC), el principal component psicoactiu present en la resina de la planta de marihuana, dels receptors de cannabinoids del cervell. Els lligands endògens dels receptors cannabinoids s´anomenen endocannabinoids (eCBs), els més coneguts són 2-arachidonoyl glycerol (2-AG) i N-arachidonoylethanolamine (AEA o anandamida). 2-AG i AEA son esters o amides derivats d´àcid araquidònic, que pertanyen respectivament, a dues famílies de compostos lipídics cannabimimètics, els 2-monoacilglicerols (2-MG) i les N-aciletanolamines (NAE), que són derivats de tots els acids grassos naturals de cadena llarga. El sistema endocannabinoid té un paper clau en la regulació homeostàtica i hedònica de la ingesta de menjar. L´expressió elevada de receptors cannabinoids en àrees del cervell que regulen no només la gana sinó també el dolor, l´emoció, la cognició i les funcions motores ressalten el paper que juguen els eCBs en molts processos fisiopatològics. El sistema endocannabinoid està excessivament activat en l´obesitat. Recentment, s´ha demostrat que el sistema endocannabinoid estimula la ingesta de menjar en ratolins mitjançant un augment en la capacitat de detecció olfactiva. Un augment en l’índex de massa corporal (BMI) s´ha associat a dèficits en la capacitat olfactiva en humans. Aquestes investigacions suggereixen una possible interacció entre el sistema endocannabinoid, l´olfacte i l´obesitat. ECBs i cannabimimètics s´estan avaluant en l´actualitat com a biomarcadors d´una sèrie de condicions fisiopatològiques. El fet que les concentracions de eCBs en matrius biològiques no són intercanviables entre laboratoris, limita la seva utilitat per recerca clínica i traslacional. Per superar aquests problemes els investigadors han de prendre una sèrie de mesures en l´obtenció de la mostra, la preservació, el processament i l´anàlisi. La generació artefactual de 2-MG en plasma, la isomerització química de 2-MG i la generació artefactual de NAE en sang són alguns dels reptes a superar. Els objectius principals d´aquesta tesi són i) desenvolupar i validar metodologia analítica per quantificar eCBs i cannabimimètics en plasma humà per utilitzar com a biomarcadors en estudis clínics i traslacionals; ii) avaluar la interacció entre el sistema endocannabinoide, la capacitat olfactiva i la obesitat. En la primer part d´aquesta tesi s´ha validat un mètode analític per la determinació d´una sèrie de 2-MG i NAE en plasma i on s´han superat i/o controlat la major part de factors que contribueixen a la variabilitat en la quantificació de eCBs i compostos relacionats. La troballa més important és que la generació artefactual de 2-MG en plasma es pot controlar utilitzant l´inhibidor de lipases Orlistat. En la segona part d´aquesta tesi s´ha avaluat la funció olfactiva i s´han mesurat les concentracions plasmàtiques d´eCBs i cannabimimètics en un estudi observacional de 161 dones amb un rang de BMI des de baix pes a obesitat mòrbida. Els resultats principals d´aquest estudi mostren que les persones amb obesitat tenen una menor capacitat olfactiva que les persones no obeses i que concentracions plasmàtiques elevades de 2-AG estan associades a una menor capacitat olfactiva. Confirmant estudis anteriors, les concentracions elevades de 2-AG estan associades a un BMI elevat i a obesitat.
The effects of cannabinoids on appetite in humans have been known since ancient times. Following cannabis smoking modern users report an intensification of the sensory and hedonic properties of food, which impels the over-consumption of palatable food. These effects are mediated by activation of brain cannabinoid receptors by delta9-tetrahydrocannabinol (THC), the main psychoactive compound present in the resin of marijuana plant. The endogenous ligands of cannabinoid receptors are called endocannabinoids (eCBs), the most known are 2-arachidonoyl glycerol (2-AG) and N-arachidonoylethanolamine (AEA or anandamide). 2-AG and AEA are ester or amide derivatives of arachidonic acid, and they belong respectively, to two families of cannabimimetic lipid compounds, the 2-monoacylglycerols (2-MG) and N-acylethanolamines (NAE). 2-MG and NAE are respectively ester and amide derivatives of all natural long-chain fatty acids. The eCB system is a key regulator of both the homeostatic and hedonic regulation of food intake. The high expression of eCB receptors in brain areas that modulate not only appetite but also pain, emotion, cognition and motor functions highlight the role of the eCB in many pathophysiological processes. The eCB system is over-active in obesity. It has been recently demonstrated that the eCB system can promote food intake by increasing odor detection in mice. An increase in the body mass index (BMI) in humans has been associated to deficits in olfactory capacity. These findings suggest a possible interaction between the eCB system, olfaction and obesity. ECBs are under evaluation as biomarkers of number physiopathological conditions. The lack of interchangeability of eCB concentrations in biological matrices between laboratories, limit their usefulness in clinics and translational research. To overcome these problems researchers should take into account a number of issues concerning sample procurement, preservation, processing and analysis. The artefactual 2-MG generation in plasma, the chemical isomerization of 2-MG and the artefactual generation of NAE in blood are some of the challenges to be confronted. The main aim of this thesis are i) to develop and validate analytical methodology for the quantification of eCBs and cannabimimetic compounds to be used as biomarkers in clinical and translational studies; ii) to evaluate the interaction between the eCB system, olfactory capacity and obesity. In the first part of this thesis we have validated a method for the determination of a range of NAE and 2-MG in human plasma. We have been able to overcome and/or control most factors contributing to eCB quantification variability. The most relevant finding is that the artefactual generation of 2-MG can be controlled by using the lipase inhibitor Orlistat. In the second part of this thesis we evaluated the olfactory function and we measured the plasma concentrations of eCBs and cannabimimetics in a cross sectional study of 161 females with BMI ranging from under-weight to morbidly obese. The main results of this study show that obese subjects have a lower olfactory capacity than non-obese ones and that elevated circulating 2-AG plasma concentrations are linked to a lower olfactory capacity. In agreement with previous studies we show that elevated 2-AG concentrations are linked to high BMI and obesity.

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DI, SIENA SARA. "Meiotic progression of mouse spermatogonia in vitro." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2009. http://hdl.handle.net/2108/208718.

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La spermatogenesi è un processo complesso attraverso il quale le cellule germinali mitotiche vanno incontro a proliferazione, differenziamento, divisione meiotica e cambiamenti morfologici per produrre spermatozoi maturi. L’entrata in meiosi delle cellule germinali mitotiche, gli spermatogoni, è un evento critico per il loro differenziamento in condizioni di La spermatogenesi è un processo complesso attraverso il quale le cellule germinali mitotiche vanno incontro a proliferazione, differenziamento, divisione meiotica e cambiamenti morfologici per produrre spermatozoi maturi. L’entrata in meiosi delle cellule germinali mitotiche, gli spermatogoni, è un evento critico per il loro differenziamento in condizioni di abbiamo osservato che SM attiva il signaling della fosfatidilinositolo 3 kinasi (PI3K) e favorisce l’ultimo round di replicazione del DNA nelle cellule allo stadio di preleptotene permettendone il loro ingresso in meiosi. L’effetto positivo della microgravità sul differenziamento delle cellule germinali è stato osservato anche su culture di cellule spermatogoniali indifferenziate Kit negative che in RCCS acquisiscono il recettore Kit ed esprimono Stra8. In conclusione il presente studio dimostra che la microgravità è una condizione ambientale che può giocare un ruolo importante nel differenziamento delle cellule germinali e fornisce uno strumento per lo studio dei meccanismi molecolari che regolano lo switch dal ciclo cellulare mitotico a quello meiotico nei mammiferi. 2) per quanto riguarda il secondo approccio, il ruolo esatto del sistema endocannabinoide (ECS) nella spermatogenesi non è stato ancora bene identificato. Abbiamo usato frazioni di cellule germinali purificate rappresentative di tutte le fasi della spermatogenesi e culture primarie di spermatogoni. Questo metodo garantisce la precisa quantificazione dei livelli dei ligandi dei recettori dei cannabinoidi, l’anandamide e il 2-‐arachidonoilglicerolo, e l’espressione a livello trascrizionale e tradizionale dei loro enzimi metabolici e dei recettori. I nostri dati indicano che le cellule germinali maschili di topo possiedono un attivo e completo ECS, il quale è modulato durante la meiosi, e suggerisce la presenza di un segnale endocannabinoide autocrino durante la spermatogenesi. Le cellule mitotiche possiedono livelli più elevati del 2-‐arachidonoilglicerolo, il quale diminuisce negli spermatociti e negli spermatidi. Di conseguenza, gli spermatogoni esprimono livelli più alti dell’enzima che sintetizza il 2-‐arachidonoilglicerolo e livelli più bassi dell’enzima che lo degrada, rispettivamente , in confronto alle cellule meiotiche e postmeiotiche. Questo endocannabinoide gioca probabilmente un ruolo fondamentale nel promuovere la progressione meiotica delle cellule germinali attivando i recettori CB2. In effetti, abbiamo constatato che l’agonista specifico per il recettore CB2, JWH133, induce la fosforilazione delle Erk1/2 MAPK negli spermatogoni e la loro progressioneverso la meiosi, perché incrementa il numero di cellule SCP3 positive, un marker della profase meiotica, e l’espressione di geni precosi della profase I della meiosi.
Spermatogenesis is a complex process by which mitotic germ cells undergo proliferation, differentiation, meiotic division and cell morphological changes to produce mature sperm. The entry into meiosis of mitotic germ cells, spermatogonia, is a critical step of germ cell differentiation in cell culture conditions. Up to now, only two agents, all-‐trans retinoic acid (ATRA) and Kit ligand (KL) have been postulated to have a role in the induction of meiotic entry in male mitotic germ cells. I report two new approaches that we have developed to induce meiotic entry of spermatogonia: 1) in microgravity conditions and 2) after activation of the endocannabinoid system. 1) concerning the first approach we report that mouse mitotic spermatogonia cultured under Simulated Microgravity (SM) with a rotary cell culture system (RCCS) enter into meiosis in the absence of any added exogenous factor or contact with somatic cells. We found that isolated Kit-‐positive spermatogonia under RCCS conditions enter into the prophase of the first meiotic division (leptotene stage), as monitored by chromosomal organization of the synaptonemal complex 3 protein (Scp3) and up-‐regulation of several pro-‐meiotic genes. SM was found to activate the phosphatidyl inositol 3 kinase (PI3K) pathway and to induce in Kit-‐positive spermatogonia the last round of DNA replication, typical of the preleptotene stage. A positive effect of SM on germ cell differentiation was also observed in undifferentiated (Kit-‐negative) spermatogonia, in which RCCS conditions stimulate the expression of Kit and Stra8. In conclusion, SM is an artificial environmental condition which promotes postnatal male germ cell differentiation; it might provide a tool for studying the molecular mechanisms underlying the switch from mitosis to meiosis in mammals. 2) concerning the second approach, the endocannabinoid system (ECS) during spermatogenesis has not been clarified. We used purified germ cell fractions representative of all phases of spermatogenesis and primary cultures of spermatogonia. This approach allowed the precise quantification of the cannabinoid receptor ligands, anandamide and 2-arachidonoylglycerol, and of the expression at transcriptional and transductional levels of their metabolic enzymes and receptors. Our data indicate that male mouse germ cells possess an active and complete ECS, which is modulated during meiosis, and suggest the presence of an autocrine endocannabinoid signal during spermatogenesis. Mitotic cells possess higher levels of 2-‐arachidonoylglycerol, which decrease in spermatocytes and spermatids. Accordingly, spermatogonia express higher and lower levels of 2-‐arachidonoylglycerol biosynthetic and degrading enzymes, respectively, as compared to meiotic and postmeiotic cells. This endocannabinoid likely plays a pivotal role in promoting the meiotic progression of germ cells by activating CB2 receptors. In fact, we found that the selective CB2 receptor agonist, JWH133, induced the Erk 1/2 MAPK phosphorylation cascade in spermatogonia and their progression towards meiosis, because it increased the number of cells positive for SCP3, a marker of meiotic prophase, and the expression of early meiotic prophase genes.

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DI, NISIO VALENTINA. "The endocannabinoid system in female reproduction: characterization of major endocannabinoid-binding receptors expression and function during mouse oocyte meiotic maturation." Doctoral thesis, Università degli Studi dell'Aquila, 2020. http://hdl.handle.net/11697/144327.

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Introduction Endocannabinoid system (ECS) includes lipid messengers termed endocannabinoids (eCBs), their receptor (CBRs) targets type-1 (CB1) and type-2 (CB2) cannabinoid receptors, G-protein coupled receptor 55 (GPR55), transient receptor potential vanilloid type 1 channel (TRPV1) and a number of metabolic enzymes. ECS has a key-role in virtually all steps of female reproduction. To date, among the 4 main receptors, only 2 receptors have been extensively studied in the mammalian oocytes, i.e. CB1 and CB2, both modulated during meiotic maturation. The aim of this thesis was (a) to determine expression levels of all these CBRs and (b) to investigate their role in the process of mouse oocyte meiotic maturation. Methods Adult CD1 female mice were primed with 5 IU PMSG and sacrificed: (a) 44 h later to obtain GV oocytes, (b) 8 or 12h after hCG (5 IU) injection to obtain MI or MII oocytes. CBRs mRNA and protein contents were assessed by qRT-PCR and Western blot; CBRs localization by confocal microscopy. CB1, CB2 and GPR55 roles during meiotic resumption (germinal vesicle breakdown, GVBD) or maturation up to MI and MII stages were assessed by incubating oocytes in the presence/absence of receptor antagonists (SR1, SR2, and ML193, respectively). cAMP concentration was assessed by EIA kit; MI/MII spindle morphology by appropriate immunofluorescent antibodies. Experiments were repeated at least 3 times. Results Despite a significant decrease of CB1, CB2 and GPR55 mRNAs occurring after GVBD, CB2 and GPR55 protein contents increased significantly from GV to MI and MII. At GV, only CB1 was localized in the oolemma, although it disappeared at MI. TRPV1 (mRNA/protein) was always undetectable. When oocytes were in vitro matured with CB1 and CB2 antagonists, a significant delay of GVBD was recorded, sustained by higher intraoocyte cAMP concentration. Although CBRs antagonists did not affect polar body I emission nor chromosome alignment at metaphase I or II plates, ML193 impaired the formation of normal MI or MII spindles in about 70% of oocytes. Indeed, ML193-incubated oocytes showed a significant reduction of spindle length as compared with control. Conclusion In mouse oocytes, all the major eCB-receptors are differentially expressed during meiotic maturation. CB1 and CB2 have a prominent role in the control of meiosis resumption, while GPR55 could be involved in the assembly of MI and MII spindles. These findings offer potentially novel biomarkers involved in the physiological maturation of mouse oocytes and could be a starting point for investigating female fertility issues also in women.

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Naccarato, Marcello. "Endocannabinoidi e stroke: uno studio nell'uomo." Doctoral thesis, Università degli studi di Padova, 2008. http://hdl.handle.net/11577/3427196.

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Background: Endocannabinoids have shown neuroprotective properties in murine and neuronal coltural models of stroke, acting on NMDA-mediated excitotoxicity and probably modulating the subsequent neuroinflammatory response. Methods: 10 patients with a first ischemic stroke in the MCA territory with at least hand movement impairment and 8 age-matched control subjects were included. Groups were matched for body mass index (BMI) and plasmatic fasting glucose, cholesterol and triglyceride levels. All control subjects underwent a blood sample collection for anandamide (AEA), 2-arachidonoylglycerol (2-AG) and palmitoylethanolamide (PEA) measurement; patients admitted within 6 hours since stroke onset underwent the same procedure on admittance (T0), after 12 (T1) and after 24 (T2) hours since symptom onset. Patient’s neurological impairment was assessed using NIHSS and Fugl-Meyer Scale arm subitem (FMS); stroke volume was determined on 48-hours follow-up brain CT scans. Blood samples were analyzed by liquid chromatography-atmospheric pressure chemical ionization-mass spectrometry. Statistical analysis was performed with SPSS 12.0, using Mann-Whitney U test and Spearman correlation analysis. Results: 1)The T0 levels of AEA, but not 2-AG and PEA, were significantly higher in stroke patients compared to control subjects. 2) There was a correlation between BMI, cholesterol, glucose and triglyceride plasmatic levels and 2-AG, but not AEA, levels. 3) Stroke patients showed a significant inverse correlation between T0 AEA levels and FMS score: the higher the AEA levels, the worse the neurological arm impairment. PEA T0 levels showed a significant direct correlation with NIHSS score. Finally, a positive correlation trend was found between T0 AEA levels and stroke volume at 48-hours follow-up brain CT scans. Conclusions: This is the first demonstration of elevated peripheral AEA levels in stroke patients; moreover, like in previous murine studies, there seems to be a significant relationship between AEA levels and neurological impairment at 6 hours since stroke onset.

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Pegorini, S. "Sistema endocannabinoide e endovanilloide nell'ischemia cerebrale." Doctoral thesis, Università degli Studi di Milano, 2007. http://hdl.handle.net/2434/63139.

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To furtherr investigate the role of endocannabinoid and endovanilloid system against neuronal injury in vivo, we injected exogenous compounds such as the cannabinoid agonist Delta9-tetrahydrocannabinol (THC) (0.05-2 mg/kg/i.p.), and the vanilloid agonist capsaicin (0.01-0.6 mg/kg/s.c.). The role of CB1 and VR1 receptors was investigated after treatment with rimonabant (0.05-3 mg/kg/i.p.) capsazepine (0.01-10 mg/kg/s.c.) respectively. Finally the effect of the anandamide transport inhibitor AM404 (0.03-2 mg/kg/i.p.) was studied. To quantify the ischemic damage we measured from 1 hour to 7 days after recirculation, electroencephalographic (EEG) mean total spectral power, spontaneous motor activity, cognitive function, rectal temperature and hippocampal neuronal count. These compounds showed a protective effect (versus vehicle treated group) against hyperlocomotion on Day 1, evaluated in an activity cage (p<0.01), memory impairment (passive avoidance test) on Day 3 (p<0.05) and EEG flattening (p<0.01) and neuronal loss (p<0.01) on Day 7. Taken together, these results showed that endocannabinoid and endovanilloid systems are closely connected suggesting that the modulation of AEA levels plays an important role in mechanisms underlying neuroprotection.

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La, Porta Carmen 1985. "Involvement of the endocannabinoid system in osteoarthritis pain." Doctoral thesis, Universitat Pompeu Fabra, 2015. http://hdl.handle.net/10803/398384.

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Chronic pain is a major clinical problem producing huge economic and social burdens. Currently, chronic pain treatment has limited efficacy and significant side effects. One of the reasons of this unmet clinical need is the insufficient knowledge of the exact mechanisms involved in the generation and maintenance of chronic pain and pain-related comorbidities, such as affective and cognitive disorders that can negatively affect the life quality of patients. It is an important challenge to treat not only the nociceptive symptoms, but also the comorbidities accompanying chronic pain. In the present Thesis, we have validated different behavioral outcomes to evaluate the nociceptive, affective and cognitive alterations promoted by chronic pain in mice. Our work mainly focuses on a particular type of chronic pain that is the osteoarthritis pain. Pain is the principal symptom of osteoarthritis, a degenerative joint disease characterized by articular cartilage degradation. The endocannabinoid system has recently emerged as a new potential therapeutic target for osteoarthritis pain. The endocannabinoid system regulates a wide range of physiopathological processes including articular metabolism, pain, emotions and cognitive functions, and a therapeutic intervention on this system could offer the potential advantage to treat multiple aspects of this disease. We have used behavioral, genetic, pharmacological and biochemical approaches to study the involvement of the endocannabinoid system in different osteoarthritis pain-related alterations in mice, and explored the potential usefulness of the endocannabinoid system components as biomarkers for human osteoarthritis.
El dolor crónico es un problema clínico grave con una enorme carga económica y social. Actualmente, el tratamiento del dolor crónico presenta eficacia limitada y efectos adversos significativos. Una de las razones de esta necesidad clínica insatisfecha es el escaso conocimiento de los mecanismos exactos que están involucrados en la generación y mantenimiento del dolor crónico y las comorbilidades relacionadas con el dolor, como son los trastornos afectivos y cognitivos. Estos tienen un impacto negativo sobre la calidad de vida de los pacientes y pueden agravar ulteriormente la percepción del dolor. Por ello, tratar no solamente los síntomas nociceptivos sino también las cormorbilidades que acompañan el dolor crónico representa un reto importante. En la presente Tesis, hemos validado diferentes modelos conductuales para evaluar las alteraciones nociceptivas, afectivas y cognitivas inducidas por el dolor crónico en ratones. Nuestro trabajo se centra principalmente en un tipo concreto de dolor crónico, el dolor osteoartrítico. El dolor es el principal síntoma de la osteoartritis, una enfermedad degenerativa de las articulaciones caracterizada por la degradación del cartílago. El sistema endocannabinoide ha emergido recientemente como una nueva diana terapéutica para el dolor osteoartrítico. Este sistema endógeno regula una vasta gama de procesos fisiopatológicos, incluyendo el metabolismo articular, el dolor y las funciones emocionales y cognitivas, y una intervención terapéutica sobre este sistema podría ofrecer la ventaja potencial de tratar diferentes aspectos relacionados con esta enfermedad. La combinación de aproximaciones comportamentales, genéticas, farmacológicas y bioquímicas nos han permitido determinar la participación de determinados componentes del sistema endocannabinoide en las diferentes alteraciones relacionadas con el dolor osteoartrítico en ratones. Además, hemos analizado la utilidad potencial de los componentes del sistema endocannabinoide como biomarcadores de la osteoartritis humana.

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Rüden, Eva-Lotta von. "Pharmacological and genetic modulation of the endocannabinoid system." Diss., Ludwig-Maximilians-Universität München, 2015. http://nbn-resolving.de/urn:nbn:de:bvb:19-180521.

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Epilepsy is one of the most common chronic neurological diseases worldwide and the prevention of epileptogenesis is so far unmet. A major challenge in epilepsy research is the development of new therapeutic approaches for patients with therapy-resistant epilepsies, for epilepsy prevention and for disease modification. The endocannabinoid system serves as a retrograde negative feedback mechanism and one of its key functions is regulating neuronal activity within the central nervous system. Thus, the endocannabinoid system can be considered a putative target for central nervous system diseases including epilepsies. The purpose of this thesis was to evaluate the impact of the endocannabinoid and endovanilloid systems on both epileptogenesis and ictogenesis. Therefore, I modulated the systems pharmacologically and genetically and analyzed the impact on the generation of a hyperexcitable neuronal network as well as on ictogenesis in the kindling model of temporal lobe epilepsy. In addition, the impact of seizures on associated cellular alterations, like CB1-receptor (CB1R) expression and neurogenesis, was evaluated. I established that the endocannabinoid system affects seizure and afterdischarge duration dependent on the neuronal subpopulation being modulated. Genetic deletion of CB1Rs from GABAergic forebrain neurons caused shorter seizure duration. Deletion of CB1R from principal neurons of the forebrain and pharmacological antagonism with rimonabant (5 mg/kg) resulted in the opposite effect. Along with these findings, the CB1R density was increased in mice with recurrent induced seizures. However, neither genetic knockout nor pharmacological antagonism had any impact on the development of generalized seizures. In contrast to genetic deletion or pharmacological blockade of CB1Rs, modulation of transient receptor potential vanilloid receptor 1 (TRPV1) neither genetically nor pharmacologically with SB366791 (1 mg/kg) had an effect on the duration of behavioral or electrographic seizure activity. Pharmacological blockade of the 2-arachidonoylglycerol degrading enzyme, monoacylglycerol lipase (MAGL) with JZL184 (8 mg/kg), delayed the development of generalized seizures and decreased seizure and afterdischarge durations whereas in fully-kindled mice JZL184 (4, 8 and 16 mg/kg) had no relevant effects on associated seizure parameters. In addition, I confirmed by the use of conditional CB1R knockout mice that these effects are CB1R mediated. In conclusion, my findings support the concept that the endocannabinoid system may be a therapeutic target for decreasing seizure duration and that it is involved in terminating seizures as an endogenous mechanism. Moreover, targeting MAGL may be a promising strategy for an antiepileptogenic approach. Respective strategies are of particular interest for the management of long-lasting refractory status epilepticus and cluster seizures as well as for the prevention of the development of symptomatic epilepsies after an initial insult.

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37

Dionisi, Mauro. "Endocannabinoid metabolism and peroxisome proliferator-activated receptor signalling." Thesis, University of Nottingham, 2010. http://eprints.nottingham.ac.uk/11384/.

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The fatty acid amides (FAAs) family includes endocannabinoids, such as anandamide, as well as endocannabinoid-like molecules, such as N-palmitoylethanolamine (PEA) and N-oleoylethanolamine (OEA). Members of the FAA family show agonist activity at transmitter-gated channels (TRPV1), as well as peroxisome poliferator-activated receptors (PPARs). Given that FAAs appear to be hydrolysed principally through the action of the enzyme fatty acid amide hydrolase, inhibition of FAAH should lead to accumulation of a variety of FAAs. Therefore, in this study it was investigated whether pharmacological inhibition of FAAH could influence PPAR activity in SH-SY5Y human neuroblastoma cells or HeLa human cervical carcinoma cells. FAAH activity was assessed by monitoring liberation of [3H]-ethanolamine from labelled anandamide. FAAH protein and RNA expression were measured by immunoblotting and qRT-PCR respectively. Endocannabinoid levels were measured by LC-MS/MS. In order to evaluate PPAR activation, a PPRE-linked luciferase construct was co-transfected with expression plasmids for either PPAR α, β or γ. Binding to PPAR receptors was assessed with a competitor displacement assay (Invitrogen). In intact SH-SY5Y cells, sustained FAAH inhibition by URB597 (~75 %) led to accumulation of AEA, 2AG and PEA, but not OEA. Treatment with URB597, OL135 or PF750, three structurally and functionally distinct FAAH inhibitors, induced activation of endogenously expressed PPARs, while no activation was observed in FAAH-1 negative HeLa cells. Furthermore, exposure to URB597, OL135 or PF750 led to activation of over-expressed PPARs in SH-SY5Y cells. To rule out direct activation of PPARs by the FAAH inhibitors, cell-free binding assays showed that URB597, OL135 and PF750 could not bind to PPARα, PPARβ or PPARγ. Surprisingly, treatment with URB597 in HeLa cells led to intracellular accumulation of PEA but not AEA, OEA or 2AG. This might be due to inhibition of either FAAH-2 or NAAA, both of which are expressed in HeLa cells. Moreover, the presence of either URB597 or OL135 led to activation of PPARγ receptors over-expressed in HeLa cells. In conclusion, data in this study showed activation of PPAR nuclear receptors in vitro by inhibition of FAAH activity and subsequent augmentation of endocannabinoid tone. These data suggest that, at least in a model setup, it is possible to modulate the endocannabinoid tone without any previous external stimulus of their synthesis and trigger a functional effect.

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38

Leggett, James Daniel. "Studies on the putative endocannabinoids oleamide and virodhamine." Thesis, University of Nottingham, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.408624.

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39

Ghosh, Sudeshna. "Targeting the Endocannabinoid System to Reduce Inflammatory Pain." VCU Scholars Compass, 2012. http://scholarscompass.vcu.edu/etd/313.

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The endogenous cannabinoids (endocannabinoids) anandamide (AEA) and 2-arachidonylglycerol (2-AG) exert their effects predominantly through cannabinoid CB1 and CB2 receptors, but these actions are short-lived because of rapid hydrolysis by fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL), respectively. Selective inhibition of either enzyme elevates CNS levels of the appropriate endocannabinoid and produces analgesic effects with fewer psychomimetic side effects than Δ9-tetrahydrocannabinol (THC), the primary active constituent of marijuana. While cannabinoid receptor agonists and FAAH inhibitors reliably produce anti-inflammatory and anti-hyperalgesic effects in the carrageenan test and other inflammatory pain models, much less is known about the consequences of inhibiting MAGL in these assays. Here, we tested whether the selective MAGL inhibitor JZL184 would reduce nociceptive behavior in the carrageenan test. JZL184 significantly attenuated carrageenan-induced paw edema and mechanical allodynia, whether administered before or after carrageenan. Complementary genetic and pharmacological approaches revealed that JZL184’s anti-allodynic effects required both CB1 and CB2 receptors, but only CB2 receptors mediated its anti-edematous actions. Importantly, the anti-edematous and anti-allodynic effects of JZL184 underwent tolerance following repeated injections of high dose JZL184 (16 or 40 mg/kg), but repeated administration of low dose JZL184 (4 mg/kg) retained efficacy. Interestingly, the anti-allodynic effects of the combination of low dose of JZL184 (4mg/kg) and high dose of the selective and long-acting FAAH inhibitor PF-3845 (10 mg/kg) was augmented compared with each drug alone. On the contrary, the combination treatment did not reduce edema more than either JZL184 or PR-3845 given alone. These results suggest that low doses of MAGL inhibitors alone or in combination with FAAH inhibitors, reduce inflammatory nociception through the activation of both CB1 and CB2 receptors with no evidence of tolerance following repeated administration.

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40

Kovacs, Flora [Verfasser], and Bela [Akademischer Betreuer] Szabo. "Endocannabinoid-vermittelte retrograde synaptische Übertragung im zentralen Nervensystem." Freiburg : Universität, 2010. http://d-nb.info/1123459665/34.

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41

Ramírez, López Ángela 1992. "Role of CB2 cannabinoid receptor in nociception and food intake control." Doctoral thesis, TDX (Tesis Doctorals en Xarxa), 2021. http://hdl.handle.net/10803/672620.

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The endocannabinoid system is a natural modulatory system that participates in multiple physiological processes, including nociceptive, emotional and rewarding responses. These central responses are mainly mediated by cannabinoid receptor 1 (CB1R)-dependent mechanisms, although the side effects associated to these central responses limit the therapeutic use of CB1R agonists. Recent research on the cannabinoid receptor 2 (CB2R) provides an alternative approach to avoid the central side effects associated with CB1R stimulation. The purpose of this Thesis was to investigate the involvement of CB2R in two different pathological conditions that currently lack effective treatment: neuropathic pain and food addiction. The results revealed that the pain-resistant phenotype of Fmr1KO mice against the nociceptive and emotional manifestations triggered by persistent nerve damage requires the participation of CB2R. We also demonstrated that CB2R are involved in the neurobiological substrate underlying the behavioral and affective alterations that arise from food addiction. Altogether, these data highlight the potential therapeutic interest of targeting CB2R for the treatment of neuropathic pain, food addiction disorders and their co-morbid emotional manifestations.
El sistema endocannabinoide es un sistema modulador natural que participa en múltiples procesos fisiológicos, incluidas las respuestas nociceptivas, emocionales y de refuerzo. Estas respuestas centrales están mediadas principalmente por mecanismos dependientes del receptor cannabinoide 1 (CB1R), aunque los efectos secundarios asociados a estas respuestas centrales limitan el uso terapéutico de agonistas de CB1R. La investigación reciente sobre el receptor cannabinoide 2 (CB2R) proporciona un enfoque alternativo para evitar los efectos secundarios centrales asociados con la estimulación de CB1R. El propósito de esta Tesis era investigar la implicación de CB2R en dos condiciones patológicas diferentes que actualmente carecen de tratamiento efectivo: el dolor neuropático y la adicción a la comida. Los resultados revelaron que el fenotipo resistente al dolor de los ratones Fmr1KO frente a las manifestaciones nociceptivas y emocionales desencadenadas por un daño nervioso persistente requiere la participación del CB2R. También demostramos que los CB2R están involucrados en el sustrato neurobiológico subyacente a las alteraciones conductuales y afectivas que surgen de la adicción a la comida. En conjunto, estos datos destacan el potencial interés de utilizar CB2R como diana terapéutica para el tratamiento del dolor neuropático, los trastornos de adicción a la comida y sus manifestaciones emocionales comórbidas.

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42

Goonawardena, Anushka V. "Cannabinoid effects on hippocampal neurophysiology and mnemonic processing." Thesis, Available from the University of Aberdeen Library and Historic Collections Digital Resources. Restricted: no access until Mar. 17, 2011, 2008. http://digitool.abdn.ac.uk:80/webclient/DeliveryManager?application=DIGITOOL-3&owner=resourcediscovery&custom_att_2=simple_viewer&pid=26047.

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43

PACIONI, SIMONE. "Endocannabinoid-mediated long-term depression of excitability and synaptic transmission in the neocortex." Doctoral thesis, Università degli Studi di Roma "Tor Vergata", 2010. http://hdl.handle.net/2108/209189.

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Nel sistema nervoso centrale, gli endocannabinoidi sono rappresentati principalmente da due lipidi: l’anandamide, l’etanolamide dell’acido arachidonico, e il 2-arachidonilglicerolo (2-AG). E’ accertato che gli endocannabinoidi inibiscono in modo retrogrado il rilascio presinaptico di trasmettitori; è peraltro dimostrato il loro coinvolgimento nel fenomeno inibitorio detto slow self-inhibition (SSI) in interneuroni low-threshold spiking (LTS) della corteccia somatosensoriale. L’SSI è indotta in seguito a treni di potenziali d’azione ripetuti in cellule LTS, che esprimono colecistochinina o somatostatina. La SSI è generata dall’attivazione prolungata di un canale K+ ed è associata ad iperpolarizzazione nella stessa cellula. La sintesi di entrambi i cannabinoidi è dipendente dall’aumento della [Ca2+]i come accade durante una elevata attività neuronale. Per verificare se il 2-AG media la SSI in modo autocrino in cellule LTS, abbiamo bloccato la sua biosintesi a partire dalla fosfolipasi C (PLC) e da diacil-glicerolo lipasi (DAGLs). Queste manipolazioni hanno impedito l’insorgenza della SSI. Inoltre, l’attivazione di PLC mediata da recettori metabotropici del glutammato ha prodotto una prolungata iperpolarizzazione, la quale è stata inibita dall’antagonista del recettore cannabinoide di tipo 1 (CB1R) AM-251, e dagli inibitori di PLC e DAGL. La scomparsa della SSI in presenza di bloccanti intracellulari della DAGL conferma che la produzione di endocannabinoidi avviene nello stesso interneurone che va incontro a persistente iperpolarizzazione. Poiché le DAGLs non producono cannabinoidi se non 2-AG, questi risultati identificano tale composto come il mediatore autocrino responsabile della slow self-inhibition postsinaptica in interneuroni corticali LTS. Abbiamo inoltre dimostrato che la SSI è espressa anche da una significativa percentuale (~30%) di neuroni piramidali glutamatergici dello strato II/III della neocorteccia. La SSI è asssente in presenza di AM-251 e in topi CB-/-. Allo stesso modo, la somministrazione esogena di cannabinoidi mima la SSI in una percentuale equivalente di neuroni piramidali, provando un’espressione funzionale somatodendritica di CB1R in cellule glutammatergiche. Questa modulazione autoindotta dell’eccitabilità di neuroni piramidali è generata da un’azione autocrina di endocannabinoidi, poiché la SSI è inibita dal blocco intracellulare della loro sintesi. E’ interessante osservare che i neuroni piramidali che esprimono SSI mostrano un dendrite apicale più lungo e meno ramificato, suggerendo che la SSI può identificare un sottogruppo anatomicamente distinto di neuroni piramidali neocorticali. Risultati preliminari indicano l’esistenza della SSI anche in una frazione di neuroni piramidali dello strato V, e suggeriscono una bidirezionale plasticità a lungo termine della trasmissione sinpatica GABAergica perisomatica in neuroni piramidali corticali dello strato II/III in confronto a quelli dello strato V. In conclusione, i nostri risultati suggeriscono un’autoregolazione omeostatica di una rete di neuroni glutammatergici all’interno dei circuiti corticali, con possibili implicazioni che sono rilevanti per l’attività della neocorteccia sia in condizioni normali che patologiche.
In the central nervous system (CNS), endocannabinoids are identified mainly as two endogenous lipids: anandamide, the ethanolamide of arachidonic acid, and 2-arachidonoylglycerol (2-AG). Endocannabinoids are known to retrogradely inhibit presynaptic transmitter release; however it is demonstrated that they are also involved in slow self-inhibition (SSI) of layer V low-threshold spiking (LTS) interneurons in somatosensory cortex. SSI is induced by repetitive firing in LTS cells, which can express either cholecystokinin or somatostatin. SSI is triggered by an endocannabinoid-dependent activation of a prolonged somatodendritic K+ conductance and associated hyperpolarization in the same cell. The synthesis of both endocannabinoids is dependent on elevated [Ca2+]i such as occurs during sustained neuronal activity. To establish whether 2-AG mediates autocrine LTS-SSI, we blocked its biosynthesis from phospholipase C (PLC) and diacylglycerol lipases (DAGLs), preventing the SSI. Moreover, metabotropic glutamate receptor-dependent activation of PLC produced a long-lasting hyperpolarization which was prevented by the cannabinoid receptor type 1 (CB1R) antagonist AM-251, as well as byPLC and DAGL inhibitors. The loss of SSI in the presence of intracellular DAGL blockers confirms that endocannabinoid production occurs in the same interneuron undergoing the persistent hyperpolarization. Since DAGLs produce no endocannabinoid other than 2-AG, these results identify this compound as the autocrine mediator responsible for the postsynaptic slow self-inhibition of neocortical LTS interneurons. Moreover, here we show that SSI also occurs in a significant percentage (~30%) of neocortical layer II/III glutamatergic pyramidal neurons. SSI was prevented by AM-251 and in CB1-/- mice. Similarly, exogenously application of cannabinoids mimicked SSI in a corresponding percentage of pyramidal neurons, proving functional somatodendritic CB1R expression in glutamatergic cells. This self-induced endocannabinoid modulation of pyramidal neuron excitability resulted from an autocrine action of endocannabinoids, as SSI was prevented by intracellular blockade of endocannabinoid synthesis. Interestingly, pyramidal neurons exhibiting SSI showed a significant less branched and longer apical dendrite than SSI-negative neurons, suggesting that endocannabinoid-mediated SSI can identify an anatomical subtype of pyramidal neocortical neurons. Preliminary results indicate the existence of SSI also in a fraction of neocortical layer V pyramidal neurons, and suggest a bidirectional long-term plasticity of GABAergic perisomatic inhibition in neocortical layer II/III vs layer V pyramidal neurons. In conclusion, our results suggest a homeostatic self-regulation of a glutamatergic network within cortical circuits, with important possible implications for normal and pathological operations of the neocortex.

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MASSARENTI, Chiara. "Bile acid and indole scaffolds for the synthesis of new biologically relevant compounds." Doctoral thesis, Università degli studi di Ferrara, 2015. http://hdl.handle.net/11392/2389009.

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45

Heimendahl, Jenny von. "Changes of endocannabinoid plasma levels following type I trauma." Diss., lmu, 2012. http://nbn-resolving.de/urn:nbn:de:bvb:19-151060.

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46

Karasu, Tülay. "The role of the endocannabinoid system in fertility control." Thesis, University of Leicester, 2013. http://hdl.handle.net/2381/28526.

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Endocannabinoids are endogenous ligands for cannabinoid receptors that play a pivotal role in fertilisation, embryo development, transport, implantation and pregnancy maintenance. The endocannabinoid system (ECS) consists of ligands (e.g. anandamide, AEA), receptors (e.g. CB1, CB2 and TRPV1) and ligand modulating enzymes (e.g. NAPEPLD and FAAH). The ECS interacts with sex steroid hormones and cytokines to regulate reproduction. Progesterone (P4), essential for pregnancy maintenance, increases FAAH activity in human lymphocytes, keeping AEA levels low. An elevated plasma AEA level is detrimental for implantation and pregnancy maintenance and any ECS dysregulation adversely affects pregnancy outcome. The hypothesis therefore was that manipulating the ECS could be an effective way of interrupting implantation. (a) The effect of RU486, a P4 antagonist used to initiate medical termination of pregnancy (MTOP), on plasma AEA levels and levels of AEA and the ECS in trophoblast were investigated. These were examined using UHPLC-MS/MS, immunohistochemistry, qRT-PCR and Western Blotting. (b) The effect of exogenously administered AEA to female rats during the implantation window was studied. The results show that ethnicity and BMI can affect the ECS, increasing AEA levels. RU486 administration causes a rise in plasma (p=0.005) and trophoblast (p=0.0062) AEA levels. Trophoblast NAPE-PLD (p=0.0006), FAAH (p=0.021), TRPVR1 (p=0.042) and CB1 (p=0.03) are significantly elevated at the mRNA level but not at the protein level though protein levels were generally higher. Exogenous administration of AEA to rats, around the day of implantation causes a reduction in viable implantation sites and an increase in resorbed units. At gestation day 14 there was a significant correlation between number of viable embryos and plasma AEA levels (p=0.0091). The complete ECS was detected in implantation, interimplantation and resorbed units at day 14. These studies have shown that manipulating the ECS can interrupt implantation showing the importance of this system in pregnancy maintenance.

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Wojtalla, Alexandra [Verfasser], and Stefan [Akademischer Betreuer] Wölfl. "Liver fibrosis and endocannabinoids / Alexandra Wojtalla ; Betreuer: Stefan Wölfl." Heidelberg : Universitätsbibliothek Heidelberg, 2011. http://d-nb.info/1179782593/34.

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48

Xu, Hao. "Dopaminergic modulation of bidirectional endocannabinoid plastictity at corticostriatal synapse." Thesis, Paris 6, 2015. http://www.theses.fr/2015PA066182/document.

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Les ganglions de la base sont impliqués dans le contrôle adaptatif du comportement et l’apprentissage procédural. Il est bien établit que la plasticité synaptique cortico-striatale constitue le principal substrat de la mémoire procédurale, affectée lors de la maladie de Parkinson (MP). La plasticité cortico-striatale est modulée par différents neurotransmetteurs, tels que les endocannabinoïdes (eCB) ou la dopamine (DA). Il est donc crucial de caractériser l’implication des interactions eCB-DA dans la plasticité cortico-striatale en conditions physiologique mais aussi pathophysiologique. Nous avons étudié la « spike-timing dependent plasticity » (STDP), une règle synaptique d’apprentissage hebbien. Nous avons mis en évidence, grâce au patch-clamp sur tranches de cerveaux de rongeurs, une STDP potentiation (tLTP) médiée par les eCBs et la DA. Nous avons montré que :(1) Un faible nombre de potentiels d’action est suffisant pour induire une eCB-tLTP par l’activation du CB1R. (2) La eCB-tLTP est exprimée au niveau des neurones striataus de type D1 et D2. (3) La DA, via les D2R exprimé par les afférences présynaptiques, est nécessaire pour la eCB-tLTP. (4) La eCB-tLTP est altérée dans un modèle rongeur de la MP, et est restaurée par la L-DOPA. (5) L’exposition à un milieu enrichi de rongeurs « parkinsoniens » restaure la eCB-tLTP. En conclusion, ces résultats mettent en évidence l’existence d’une plasticité eCB bidirectionnelle modulée par la DA. Cela étend considérablement le spectre d’action des eCBs et en particulier leur implication dans l’engramme de l’apprentissage rapide
The basal ganglia (BG) are involved in the adaptive control of behavior and procedural learning. The striatum, the primary input nucleus of BG, integrates cortical and dopaminergic inputs constituing a major site of synaptic plasticity. Plasticity at corticostriatal synapses is a key substrate for procedural learning and is affected in Parkinson’s disease (PD). The corticostriatal transmission are modulated by the endocannabinoid (eCB) and dopaminergic (DA) systems. Thus it is pivotal to characterize their interactions in the striatal plasticity in physiological and pathophysiological conditions. Using electrophysiological recordings in rodent brain slices, we unraveled a homosynaptic spike-timing-dependent potentiation mediated by eCBs and DA. We show that at the single-cell level: (1) Few spikes are sufficient to induce eCB-tLTP through CB1R. (2) eCB-tLTP occurs in DA type 1 receptor (D1R)- and DA type 2 receptor (D2R)-expressing cells. (3) DA, through presynaptic D2R, is required for eCB-tLTP induction. (4) eCB-tLTP is impaired in a model of PD and is restored by L-DOPA treatment. (5) Enriched environment rescues eCB-tLTP in DA-deprived rats. In summary, this thesis confirms and further extends a new form of interplay between eCB and DA systems involved in physiological and pathophysiological plasticity processes

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Reisenberg, Melina. "Diacylglycerol lipaseα-dependent endocannabinoid signaling in neural stem cells". Thesis, King's College London (University of London), 2012. https://kclpure.kcl.ac.uk/portal/en/theses/diacylglycerol-lipasedependent-endocannabinoid-signaling-in-neural-stem-cells(19317ce6-b568-465d-a1de-540cb3392712).html.

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The diacylglycerol lipases (DAGL α and β) synthesise 2-AG, the major endocannabinoid (eCB) in the developing and adult brain. 2-AG acts most prominently on the CB1 and CB2 receptors and growing evidence points to a role of eCB signaling in adult neurogenesis as well as a wide range of different physiological roles in the brain. The focus of this thesis is on DAGL driven eCB signaling in neural stem cells (NSC). A role of the eCB system has previously been established in migration, proliferation, and neurogenesis. Here we investigated DAGLα and eCB signaling in the context of differentiation. The Cor-1 cells were adapted as a NSC model system and can be differentiated into glia and neurons. This differentiation was unaffected by CB signaling. While DAGLα expression is unaffected by glial differentiation, a rapid down regulation of DAGLα during neuronal differentiation was detected and we investigated the possibility of a specific degradation pathway being involved. DAGLα has a consensus motif for a putative destruction (d-) box that might target DAGL for ubiquitin-proteasome mediated degradation. No evidence was found to support that the d-box is involved in DAGLα degradation, however first indications towards degradation through the ubiquitin-proteasome pathway were identified. What is upstream of eCB signaling in NSCs remains an open question. The most likely candidates are EGF, FGF-2 and insulin and their effect on eCB signaling was investigated. Evidence was found indicating that neither EGF nor FGFR signaling is upstream of eCB signaling in Cor-1 cells. Microarray analysis indicates a potential common signaling node between EGFR and eCB signaling. mTOR was investigated in this context, but while we show that mTOR appears to be downstream the EGFR, no indication was found that it is downstream of the eCB receptors. Further experiments are needed to determine the relationship between insulin and eCB signaling. It is essential to be able to measure DAGL activity, and a surrogate substrate DAGL activity assay was adapted to Cor-1 cells in order to address what is driving eCB signaling.

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Lu, Leanne. "Investigating endocannabinoid signalling using a novel cell reporter assay." Thesis, King's College London (University of London), 2018. https://kclpure.kcl.ac.uk/portal/en/theses/investigating-endocannabinoid-signalling-using-a-novel-cell-reporter-assay(080152df-8a9b-4ba4-88de-05dde4f83f76).html.

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The endocannabinoid (eCB) system consists of cannabinoid receptors 1 and 2 (CB1 and CB2) that are activated by two well characterised lipid ligands, namely 2-arachidonoylglycerol (2-AG) and N-arachidonyl ethanolamine (AEA). 2-AG is synthesised by the diacylglycerol lipases (DAGLα and β), however these enzymes also synthesise other related lipids including 2-linoleoylglycerol (2-LG). The synthetic pathway for AEA synthesis is less clear. N-acylphosphatidylethanolamine-specific phospholipase D (NAPEPLD) is the most studied enzyme that can make AEA, however genetic knockout of NAPEPLD from animal models has little impact on AEA levels. More recently, a calcium activated N-acyl transferase (CaNAT) called PLA2G4E has been identified as a key regulatory enzyme in the AEA pathway. In this thesis, three key questions have been addressed. Firstly, a detailed study using a CB1 reporter cell assay was undertaken to test whether 2-LG can signal via the CB1 receptor and/or modulate the activity of eCBs. The results clearly showed that 2-LG is a novel CB1 receptor partial agonist capable of signalling on its own, and also capable of modulating eCB signalling. Using the same human osteosarcoma reporter cell assay, the hypothesis that a nine amino acid peptide Hemopressin is an antagonist for the CB1 receptor was tested. Hemopressin failed to inhibit CB1 activation stimulated by a synthetic CB1 agonist, or the response stimulated by 2-AG and AEA. Finally, the CB1 reporter cell assay was utilised to measure activation of the receptor in response to stimulation by calcium or by direct activation of PKA or PKC, presumably as a consequence of the regulated synthesis of one or more eCBs. The CRISPR/Cas9 system was then used to systematically knockout the DAGLs that are involved in the generation of 2-AG. The results from this section confirmed that the DAGLs are responsible for over 90% of 2-AG synthesis in the cells and that in some circumstances they can contribute to eCB signalling. However robust eCB signalling is still found in their absence, possibly mediated by AEA. Interestingly, genetic deletion of NAPEPLD had no impact on AEA levels or eCB signalling. The cells do not express detectable levels of PLA2G4E suggesting that an as yet to be discovered enzyme is responsible for AEA synthesis in the cells. I identified PLA2G4B as a possible candidate, however genetic deletion of this also had no impact on AEA levels or eCB signalling.

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