The beta2-adrenergic receptor is a G-protein coupled receptor, involved in several physiological processes, which enables signaling through the cell membrane. To study the effect of dopamine and adrenaline binding on the receptor structure and function, we used machine learning methods applied to data from molecular dynamics simulations. We found that the three machine learning methods Random Forest, Kullback-Leibler divergence, and Principal Component Analysis generated results that correspond to previous studies. When comparing the active state of the receptor with or without a ligand bound, we found that residues around Ser203 and Asn301 of the orthosteric binding pocket and residues around Ala91 of the TM2 differed. When instead comparing the active state of the receptor with adrenaline or dopamine bound, we found that residues around Thr68 differed. Additionally, we also found that adrenaline and dopamine cause different structural changes in the intracellular parts of TM5 and TM6. These findings indicate ligand-specific effects on the receptor, providing potentially useful information for the understanding of the interaction of adrenaline and dopamine with the beta2-adrenergic receptor.

The cost of lamb mortality caused by cold exposure has been estimated at approximately 40 million dollars per year. This value is probably conservative as it does not include the cost due to the reduction in productivity in hypothermic lambs that manage to survive or the cost of reduced selection potential incurred by fewer lambs surviving until selection. The objectives of this research was to investigate whether sire-line variation exists in neonatal lamb cold tolerance and whether polymorphism in the β₃ adrenergic receptor gene can be used as a genetic marker for lamb cold tolerance and lean muscle growth. The influence of the climate, birthweight, age of dam at lambing, gender and birth rank on neonatal lamb cold tolerance was also analysed. Neonatal lamb mortality due to cold exposure was analysed in four field trials that used neonatal lamb morality from cold exposure as a predictor of neonatal lamb cold tolerance. Sire-line variation in neonatal lamb morality was observed in all trials, though it appeared that this effect was largely mediated through sire-line variation in lamb birth weight. Variation in lamb birth weight between birth rank classed was also found to be responsible for the influence of birth rank on neonatal lamb mortality due to cold exposure. The age of dam at lambing and the lamb gender was not observed to influence neonatal lamb mortality due to cold exposure. The sires from the cold tolerance study and the progeny of the lean muscle growth study were genotyped for the β₃ adrenergic receptor locus. Other studies have found evidence that a major gene exists in the catecholamine stimulation of brown adipose thermogenesis and evidence that the β₃-AR gene is a likely candidate. However, this hypothesis and the hypothesis that polymorphism in the β₃-AR gene is also linked to lean muscle growth in lambs was not confirmed in this study. So while it appears that the results were confounded by experimental design, there is evidence that influence of polymorphism in the ovine β₃ AR gene on neonatal lamb mortality and/or lean muscle growth is not sufficient to be considered a major gene effect. The implications of this experiment on the sheep industry and sheep farmers in general are huge. While completely eliminating lamb deaths due to inadequate cold tolerance is impossible, this study shows that large gains in lamb survival could be possible through selective breeding.

The cost of lamb mortality caused by cold exposure has been estimated at approximately 40 million dollars per year. This value is probably conservative as it does not include the cost due to the reduction in productivity in hypothermic lambs that manage to survive or the cost of reduced selection potential incurred by fewer lambs surviving until selection. The objectives of this research was to investigate whether sire-line variation exists in neonatal lamb cold tolerance and whether polymorphism in the β₃ adrenergic receptor gene can be used as a genetic marker for lamb cold tolerance and lean muscle growth. The influence of the climate, birthweight, age of dam at lambing, gender and birth rank on neonatal lamb cold tolerance was also analysed. Neonatal lamb mortality due to cold exposure was analysed in four field trials that used neonatal lamb morality from cold exposure as a predictor of neonatal lamb cold tolerance. Sire-line variation in neonatal lamb morality was observed in all trials, though it appeared that this effect was largely mediated through sire-line variation in lamb birth weight. Variation in lamb birth weight between birth rank classed was also found to be responsible for the influence of birth rank on neonatal lamb mortality due to cold exposure. The age of dam at lambing and the lamb gender was not observed to influence neonatal lamb mortality due to cold exposure. The sires from the cold tolerance study and the progeny of the lean muscle growth study were genotyped for the β₃ adrenergic receptor locus. Other studies have found evidence that a major gene exists in the catecholamine stimulation of brown adipose thermogenesis and evidence that the β₃-AR gene is a likely candidate. However, this hypothesis and the hypothesis that polymorphism in the β₃-AR gene is also linked to lean muscle growth in lambs was not confirmed in this study. So while it appears that the results were confounded by experimental design, there is evidence that influence of polymorphism in the ovine β₃ AR gene on neonatal lamb mortality and/or lean muscle growth is not sufficient to be considered a major gene effect. The implications of this experiment on the sheep industry and sheep farmers in general are huge. While completely eliminating lamb deaths due to inadequate cold tolerance is impossible, this study shows that large gains in lamb survival could be possible through selective breeding.

This research developed validated QSPKR and PK-AS models for predicting human systemic PK properties of three, preselected, pharmacological classes of drugs, namely opioids, β-adrenergic receptor ligands (β-ARL) and β-lactam antibiotics (β-LAs) using pertinent human and animal systemic PK properties (fu,, CLtot, Vdss, fe) and their biologically relevant unbound counterparts from the published literature, followed by an assessment of the effect of different molecular descriptors on these PK properties and on the PK-AS slopes for CLtot and Vdss from two species (rat and dog). Lipophilicity (log (D)7.4) and molecular weight (MW) were found to be the most statistically significant and biologically plausible, molecular properties affecting the biologically relevant, systemic PK properties: For compounds with log (D)7.4 > -2.0 and MW < 350 D (e.g., most opioids and β-ARL), increased log (D)7.4 resulted in decreased fu and increased Vdssu, CLtotu and CLnonrenu, indicating the prevalence of hydrophobic interactions with biological membrane/proteins. As result, the final QSPKR models using log (D)7.4 provided acceptable predictions for fu, Vdssu, CLtotu and CLnonrenu. CLnonrenu and CLtotu. For both the datasets, inclusion of drugs undergoing extrahepatic clearance worsened the QSPKR predictions. For compounds with log (D)7.4 < -2.0 and MW > 350 D (e.g., β-LA), increased MW (leading to more hydrogen bond donors/acceptors) resulted in a decrease in fu, likely indicating hydrogen bonding interactions with plasma proteins. In general, it was more difficult to predict PK parameters for β-LAs, as their Vdssu approached plasma volume and CLrenu and CLnonrenu were low - as a result of their high hydrophilicity and large MW, requiring specific drug transporters for distribution and excretion. The PK-AS analysis showed that animal body size accounted for most of the observed variability (r2> 0.80) in systemic PK variables, with single species methods, particularly those using dog, gave the best predictions. The fu correction of PK variables improved goodness of fit and predictability of human PK. There were no apparent effects of molecular properties on the predictions. CLren, CLrenu, CLnonren, and CLnonrenu were the most difficult variables to predict, possibly due to the associated interspecies differences in the metabolism, renal and hepatobiliary drug transporters.

Dados mostram que a estimulação do Sistema Nervoso Simpático (SNS) induz osteopenia via receptor &beta;2-adrenérgico, que é expresso no osso. Porém, um estudo recente demonstrou que camundongos knockouts (KO) para os receptores adrenérgicos &alpha;2A e &alpha;2C (&alpha;2A/&alpha;2C-AR-/-) apresentam alta massa óssea, mesmo com hiperatividade simpática. Além disso, esses camundongos são resistentes à osteopenia induzida pelo excesso de hormônio tireoidiano. Esses achados sugerem que o &alpha;2A e/ou &alpha;2C-AR também possam mediar as ações do SNS no esqueleto e que esses receptores estão envolvidos na interação do HT com o SNS para regular o metabolismo ósseo. Neste estudo, tivemos como objetivo: avaliar se o &alpha;2CAR interfere na fisiologia óssea e se a ação do HT no tecido ósseo depende do &alpha;2CAR, avaliando o efeito do HT na fisiologia óssea dos camundongos &alpha;2CAR-/- tratados com dose suprafisiológica de T3. A microtomografia computadorizada mostrou que o volume de osso trabecular foi menor e maior nos animais &alpha;2CAR-/-, no fêmur e na vértebra respectivamente. Os animais KO também apresentaram diminuição da resistência óssea quando comparados com os selvagens. Além disso, vimos que os animais KOs foram resistentes aos efeitos deletérios da tirotoxicose no osso, tanto no fêmur quanto na vértebra. Esses achados sugerem que o &alpha;2C-AR apresenta um papel na mediação dos efeitos da ativação do SNS no osso e que o HT interage via &alpha;2C-AR, para regular massa e resistência óssea. / Data demonstrates that sympathetic nervous system (SNS) activation causes osteopenia via &beta;2-adrenoceptor signaling. A recent study showed that mice with gene inactivation of the adrenergic receptor &alpha;2A and &alpha;2C (&alpha;2A/&alpha;2C-AR-/-) have a high bone mass phenotype, even presenting SNS hyperactivity. Also, these knockout (KO) mice are resistant to the thyroid hormone-induced osteopenia. These findings suggest that SNS interacts with thyroid hormone (TH) to regulate bone mass and &alpha;2A and/or &alpha;2C adrenoceptors may have an important role in mediating the actions of the SNS in the skeleton. In this study, we had the following objectives: (i) to evaluate whether the isolated inactivation of &alpha;2CAR interferes with the bone metabolism and to evaluate whether the action of HT on bone tissue depends on &alpha;2CAR, treated with 20 times the physiological dose of T3. The microtomography analysis showed that the trabecular bone volume of the femur and of the sixth lumbar vertebra (L6) were, respectively, lower and higher in &alpha;2C-AR-/- mice, when compared with WT animals. Furthermore, we showed a resistance of KO animals on the deleterious effects of TH on bone. These findings suggest: (i) &alpha;2C-AR is involved with bone longitudinal growth; (ii) &alpha;2C-AR may mediates the effects of the SNS in the bone in a skeletal site specific manner, (iii) the actions of thyroid hormone on bone metabolism involves interactions with the SNS via &alpha;2C adrenergic receptors.

Introduction : Le cancer colorectal (CCR) est responsable de 500 000 morts par an dans le monde et représente la 2ème cause de mortalité par cancer dans les pays industrialisés. En dépit des progrès réalisés, il existe un réel besoin de développer de nouvelles thérapies pour améliorer la survie des patients. Un des principaux facteurs favorisant la survenue et la progression du CCR est le stress se traduisant notamment par la sécrétion de catécholamines activant les récepteurs β-adrénergiques (β1-, β2- et β3-AR) au niveau de la tumeur. Diverses études et observations ont démontré que l’activation des β-AR pouvait favoriser la prolifération tumorale de manière directe (via différents mécanismes comme la prolifération cellulaire) ou indirecte (via une action sur la composante immunitaire). Parmi les cellules immunitaires présentes au sein de la tumeur, les macrophages tumoraux (TAM) peuvent représenter jusqu’à 50% du volume tumoral. Ces derniers y sont retrouvés sous leurs différents phénotypes (M1-like antitumoral et M2-like pro-tumoral). Divers travaux ont fait état de la présence des β-AR à la surface des macrophages où leur effet semble être en faveur d’une polarisation vers un phénotype pro-tumoral. En outre, parmi les 3 sous-types de récepteurs, de nombreux arguments soulignent une implication majoritaire du β3-AR dans ces effets par rapport aux β1- et β2-AR, tandis que seule une surexpression du β3-AR a été observée dans les biopsies de tumeurs du côlon. Objectifs/Méthodes : Nous nous sommes donc attachés à mettre au point une stratégie méthodologique pour l’étude du β3-AR dans le macrophage en condition d’inflammation tissulaire présentant une importante composante macrophagique. Nous avons ensuite étudié les effets du β3-AR sur la prolifération de lignées de cancer colorectal puis sur la polarisation de macrophages humains ainsi que de TAM. Enfin, après avoir étudié la signalisation de ce récepteur chez les macrophages M1 et M2, nous avons observé les effets d’une inhibition pharmacologique du β3-AR sur la polarisation de TAM et sur la progression de tumeurs murines et humaines in vivo. Résultats : Nous avons confirmé que le β3-AR est présent et fonctionnel à la surface des macrophages où son activation résulte en un puissant effet antioxydant et anti-inflammatoire via l’inhibition de NOX2 et l’induction de l’expression de la catalase. Ces effets semblent passer par une signalisation Gs/PKA/Src/Erk1/2 induisant l’activation de PPARγ. Dans nos travaux, nous avons aussi pu voir que le β3-AR n’a pas d’effet prolifératif sur des lignées humaines de CCR. Nous avons également démontré que le β3-AR favorise la polarisation des macrophages vers un phénotype M2 et diminue la polarisation de ces derniers vers un phénotype M1. L’étude des signalisations de ce récepteur chez ces deux phénotypes a indiqué que les voies Gs/PKA/Src/ERK1/2 (M1) et Src/PI3K/ERK1/2 (M2) étaient impliquées. Enfin, l’inhibition du β3-AR a prévenu la progression de tumeurs murines (CT-26) et humaines (SW480) in vivo, via un effet anti-M2-like et pro-M1-like sur les TAM. En conclusion, ces résultats suggèrent que l’inhibition du β3-AR, à travers ses effets sur la polarisation des macrophages, pourrait être une stratégie prometteuse afin d’améliorer la prise en charge des patients souffrant de CCR. / Background: Colorectal cancer (CRC) is responsible for 500.000 deaths per year worldwide and represents the 2nd cause of death by cancer in industrialized countries. Despite the progress made, there is a real need for new therapies to increase patients’ survival. Stress is one of the main factors, which contributes to the occurrence and the progression of CRC, by secreting catecholamines that activate β-adrenergic receptors (β1-, β2- and β3-AR) within the tumor. Several studies and observations have showed that the activation of β-ARs could directly increase tumor proliferation (via mechanisms such as cell proliferation), or indirectly (via an action on immune cells). Among immune cells within the tumor, tumor-associated macrophages (TAMs) represent up to 50% of the tumor mass where they exhibit their different phenotypes (M1-like anti-tumor and M2-like pro-tumor). Several studies report the presence of β-ARs in macrophages where they seem to favour a pro-tumor polarization. Furthermore, among the three subtypes of β-ARs, most of the studies seem to describe a major implication of the β3-AR compared to β1- and β2-AR. Moreover, only the β3-AR was found to be overexpressed in CRC biopsies. Objectives/Methods: We thus aimed to develop a model to study the β3-AR in macrophages within inflammatory macrophage-dependent conditions. Then, we studied the effects of the β3-AR on colorectal cancer cells’ proliferation and human macrophages and TAMs polarization. Finally, after the study of the signaling pathways of this receptor within M1 and M2 macrophages, we assessed the effects of a pharmacological inhibition of the β3-AR on TAM polarization and tumor progression. Results: We confirmed that the β3-AR is expressed and functional in human macrophages where its activation leads to potent antioxidant and anti-inflammatory effects through NOX2 inhibition and catalase expression. These effects appear to be subsequent to a Gs/PKA/Src/Erk1/2 signaling leading to the activation of PPARγ. In this work, we also saw that the β3-AR does not produce any effect on human CRC cell lines’ proliferation. We also showed that the β3-AR increases macrophage polarization towards the M2 phenotype while it decreases the M1 polarization. The study of β3-AR signaling in M1 and M2 macrophages exhibited Gs/PKA/Src/ERK1/2 and Src/PI3K/ERK1/2 pathways respectively. Finally, a pharmacological inhibition of the β3-AR prevented murine (CT-26) and human (SW480) tumors progression in vivo, through anti-M2-like and pro-M1-like effects on TAM polarization. In conclusion, these results suggest that the inhibition of the β3-AR, through its effects on macrophages polarization, could represent a promising strategy in order to improve CRC patient care.

Stress activates a number of endocrine pathways that alter an animal's physiology in a manner which can result in undesirable meat quality. Animals frequently exhibit meat quality defects, including ecchymosis, at slaughter due to the stress of slaughter. This thesis explores how stress related hormones interact with adrenergic receptors to alter muscle and vascular physiology. Fallow deer were exposed to either a transciptional regulator (hydrocortisone), a beta adrenergic recptor agonist (clenbuterol) or a beta adrenergic receptor antagonist (propranolol). The administration of hydrocortisone resulted in a negative feed-back type reduction in circulating cortisol. Animals treated with propranolol and clenbuterol displayed less severe eccymosis. These results indicated that the beta 2 adrenergic receptor (B2AR) is important in controlling ecchymosis severity. B2AR was also found to be important in mediating vascular dynamics, growth and energy pathways. To investigate how adrenergic receptors alter skeletal muscle gene expression and meat quality, an in vivo wistar rat model was developed in conjunction with in vitro muscle cell (L6) experiments. Gene expression of B2AR, its associated kinase (BARK) and collagen type III, prolyl- 4-hydroxylase (P4Hy) was measured in rat muscle and L6 cells. Following exposure to clenbuterol and hydrocortisone, growth and meat quality were determined. The L6 experiments revealed that gene expression following exposure to hydrocortisone and B2AR ligands paralleled the in vivo rat changes in B2AR, BARK, collagen type III, and P4Hy gene expression. In both L6 and wistar rat models the B2AR and BARK genes are similarly expressed following clenbuterol exposure. Both rats and deer exposed to clenbuterol had significant increases in growth rate and a reduction of intramuscular fat. The B2AR therefore appears to be a major mediator of many interrelated events including energy distribution, growth and vascular response to stress. Habituating animals to stress stimuli may increase their coping ability and improve welfare and meat quality. / Doctor of Philosophy (PhD)

Ο α2 –αδρενεργικός υποδοχέας διακρίνεται σε 3 γνωστούς υποτύπους (α2Α, α2Β, α2C) και μετά αλληλεπίδραση με G-πρωτεΐνες (GPCRs), διαμεσολαβεί μέρος των δράσεων των ορμονών- νευρομεταβιβαστών, επινεφρίνη και νορεπινεφρίνη σε πολλά όργανα, συμπεριλαμβανομένου και του νευρικού συστήματος. Πρότυπο μελέτης του νευρικού συστήματος in vitro, αποτελεί η κυτταρική σειρά PC12, που περιλαμβάνει κύτταρα από φαιοχρωμοκύττωμα αρουραίου, τα οποία υπό την επίδραση του Nerve Growth Factor (NGF) διαφοροποιούνται σε συμπαθητικούς νευρώνες. Μετά από διαμόλυνση, αυτά τα κύτταρα εκφράζουν τους υποτύπους των α2-αδρενεργικών υποδοχέων και βάσει δεδομένων από προηγούμενη εμπειρία του εργαστηρίου μας, μπορούν μετά από ενεργοποίηση με επινεφρίνη να οδηγήσουν στην ενεργοποίηση ενός καταρράκτη μεταγωγής σήματος, που περιλαμβάνει τις κινάσες Akt και ERK1/2. Δεδομένου ότι τα μόρια αυτά συμβάλλουν στην ενεργοποίηση του μεταγραφικού παράγοντα CREB (cAMP response element binding protein) θελήσαμε στην παρούσα εργασία να διερευνήσουμε κατά πόσο η ενεργοποίηση των α2-αδρενεργικών υποδοχέων προκαλεί την CREB φωσφορυλίωση. Βάσει προηγούμενων αποτελεσμάτων, που αποδείκνυαν την απελευθέρωση αραχιδονικού οξέος και διαφόρων μεταβολιτών του μετά από ενεργοποίηση των α2 –αδρενεργικών υποτύπων, μελετήσαμε εάν αυτή η απελευθέρωση αραχιδονικού οξέος, μπορούσε να προκαλέσει ενεργοποίηση μέσω φωσφορυλίωσης του CREB και μέσω ποιών μεταβολικών μονοπατιών μπορεί αυτό να πραγματοποιηθεί. Επιπλέον μελετήσαμε εάν αυτή η ενεργοποίηση του CREB ήταν παρούσα και στους 3 υποτύπους και εάν παρουσίαζε υποτυποειδικότητα. Χρησιμοποιήσαμε την τεχνική Western Blotting , σε εκχυλίσματα PC12 κυττάρων, κατάλληλα επεξεργασμένων με επινεφρίνη, παρουσία διαφόρων αναστολέων των μεταβολικών μονοπατιών του αραχιδονικού οξέος. Τα αποτελέσματά μας δείχνουν ότι η επινεφρίνη επάγει τη φωσφορυλίωση του CREB και στους 3 υποτύπους των α2-αδρενεργικών υποδοχέων σε PC12 κύτταρα. Επίσης η απελευθέρωση αραχιδονικού οξέος και η επακόλουθη φωσφορυλίωση του CREB διαμεσολαβείται από την PLC (φωσφολιπάση C) και την εποξυγενάση του κυτοχρώματος P450, αφού έχουμε αναστολή από τους ειδικούς αναστολείς U73122 και κετοκοναζόλη αντίστοιχα. Τα επίπεδα φωσφορυλίωσης ήταν ίδια στους α2A- και α2C-υποτύπους και σημαντικά μεγαλύτερα από τον α2Β-υπότυπο, αποδεικνύοντας ότι παρουσιάζεται σημαντική υποτυποειδικότητα. / α2-adrenergic receptor is divided into 3 known subtypes (α2A, α2Β, α2C) and after interaction with G-proteins (GPCRs) mediates part of actions of hormones-neurotransmitters, epinephrine and nor epinephrine in many organs, including Central Nervous System. Cell line PC12, which origins from cells of rats’ pheochromocytoma , consist a study model of nervous system in vitro and under the influence of Nerve Growth Factor (NGF) is differentiated into sympathetic neurons. After transfection, these cells express the subtypes of α2-adrenergic receptors and based on data from previous experience, after activation with epinephrine, they activate a cascade of signal transduction , which includes kinases Akt and ERK1/2. Based on the fact that these molecules contribute to the activation of transcription factor CREB (cAMP response element binding protein) we study whether the activation of α2-adrenergic receptors can cause direct CREB phosphorylation. Based on previous results, which prove release of arachidonic acid and its metabolites after activation of α2-adrenergic subtypes, we study if the release of arachidonic acid could cause activation, through phosphorylation, of CREB and via which metabolic pathways this happens. Furthermore, we studied if the activation was present in all 3 subtypes and if it presented sub-specificity. We performed the Western Blotting technique in PC12 cells properly pro-incubated with epinephrine and addition of enzymic inhibitors of arachidonic acid metabolism. Our results figure that epinephrine induce CREB phosphorylation in all 3 subtypes of α2-adrenergic receptors in PC12 cells. The release of arachidonic acid and the following phosphorylation of CREB is mediated from phospholipase C (PLC) and cytochrome P450-dependent epoxygenase, as proved by inhibition with the specific inhibitors U73122 and ketokonazole, respectively. The levels of CREB phosphorylation were comparable between α2Α - and α2C- subtypes and higher than the α2Β- subtype, proving that this is an action which presents sub-specificity.

Die Kultivierung equiner Trachealepithelzellen stellt ein nützliches Modell dar, die (patho)-physiologischen Mechanismen der obstruktiven Atemwegserkrankungen des Pferdes auf zellulärer Ebene zu untersuchen. Ziel dieser Arbeit war es, Methoden für die Isolation, Charakterisierung und weitergehende Kultivierung equiner Trachealepithelzellen (ETEZ) zu etablieren und validieren und die Expression und Funktionalität der beta-adrenergen Rezeptoren an frisch isolierten ETEZ und deren Primärkulturen mittels pharmakologischer und biochemischer Verfahrenstechniken zu analysieren. Epithelzellen wurden durch Trypsinverdau aus der Trachea gesunder Pferde gewonnen, indem zuerst die Mukosa der Trachea freigelegt und diese dann vom daruntergelegenen Bindegewebe stumpf getrennt wurde. Das gewonnene Gewebe wurde zerkleinert und enzymatisch mit 0,25% Trypsin-EDTA-Lösung für 2 h bei 37°C verdaut. Durch Siebung und Zentrifugation wurden die Zellen gereinigt, vereinzelt und gesammelt, wobei kontaminierende Fibroblasten später durch differentielle Adhäsion von den Epithelzellen getrennt wurden. Die isolierten Zellen wurden sowohl licht- bzw. elektronenmikroskopisch charakterisiert, als auch immunzytochemisch hinsichtlich Zytokeratin (für Epithelzellen) und Vimentin (für Fibroblasten) gefärbt. Die durchschnittliche Zellausbeute wurde mit der Neubauer-Zählkammer bestimmt und betrug 6,10 ± 0,63×106 Zellen pro 500 mg zerkleinertem Gewebe (n = 11). Die Zellvitalität wurde mittels Trypanblau-Färbung ermittelt und betrug 94,70 ± 1,17% (n = 11). Immunfluoreszensfärbungen zeigten, dass 93,57 ± 1,67% (n = 11) der frisch isolierten Zellen und ca. 100% (n = 5) der Primärkulturen auf Zytokeratin 5/6/18 positiv reagierten. Auf Anti-Vimentin reagierten dagegen nur 9,83 ± 0,94% (n = 11) der Zellen positiv. Die Zellen wurden in einer Dichte von 6,90 x 104 Zellen/cm2 in serumfreiem AECGM ausgesät und bildeten innerhalb einer Woche einen konfluenten Monolayer. Die konfluenten Zellen wurden mittels Dispase II abgelöst. Die erste (P1) und die zweite (P2) Passage konnte erfolgreich in serumfreien AECGM kultiviert und auf der Stufe P2 30 Tage lang gehalten werden. Weitethin wurden die Expression und Funktionalität der b-adrenergen Rezeptoren in frisch isolierten und kultivierten Epithelzellen untersucht. Mittels Radioligandenbindungsstudien, Westernblot, Immunfluoreszensfärbung und cAMP-Assays konnten erstmalig die Dichte, Affinität, Subtypen, Proteinexpression und zelluläre Lokalisation der beta-adrenergen Rezeptoren sowie die Rezeptorfunktion bestimmt werden. Messungen an frisch isolierten ETEZ ergaben für die mittlere Dissoziationskonstante (KD) von 31,78 ± 6,57 pM (n = 7) und eine maximale b-adrenerge Rezeptordichte (BMax) von 12727 ± 883,6 Bindungsstellen/Zelle (n = 7) ermittelt aus Sättigungsexperimenten mit dem b-adrenergen Rezeptorantagonisten [125I] Iodocyanopindolol (ICYP) in Anwesenheit des nicht selektiven beta-Rezeptorantagonisten (±)-CGP 12177. Für Primärkulturen ergaben sich Werte für KD von 15,26 ± 3,37 pM (n =6) und für BMax von 3730 ± 212 Bindungsstellen/Zelle (n = 6). Bei Verdrängungsexperimenten wurde die ICYP konzentrationsabhängig durch den beta2-selektiven Rezeptorantagonisten ICI 118.551 und den beta1-selektiven Rezeptorantagonisten CGP 20712A verdrängt, wobei für ICI 118.551 eine 10.000-fach höhere Affinität (Ki = 1,74 ± 0,15 nM in frisch-isolierten Zellen und 1,19 ± 0,41 nM in Primärkultur) gezeigt wurde als für CGP 20712A (Ki = 17 ± 7,90 μM in frisch isolierten Zellen). Die cAMP-Bildung wurde in frisch isolierten ETEZ konzentrationsabhängig durch die beta-adrenergen Rezeptoragonisten Isoproterenol, Epinephrin und Norepinephrin in der Reihenfolge ihrer Potenz mit einer EC50 von 58 nM (n = 6), 13,60 μM (n = 6) bzw. 0,43 mM (n = 6) stimuliert. Diese cAMP-Bildung konnte durch Behandlung der Zellen mit 100 nM der beta2-selektiven ICI 118.551, nicht aber durch 300 nM des beta1-selektiven CGP 20712A blockiert werden. Mit einem beta2-adrenergen Rezeptorantikörper konnte eine 72 kDa Proteinbande und mit demselben Antikörper in der Fluoreszenzfärbung Rezeptorantigene auf der Zelloberfläche nachgewiesen werden. Zusammenfassend konnten mit dem etablierten Protokoll große Mengen equiner Trachealepithelzellen isoliert und kultiviert werden. Die Ergebnisse dieser Studie zeigen erstmalig, dass primäre equine Trachealepithelzellen funktionale beta2-adrenerge Rezeptoren exprimieren und das Protokoll zur Etablierung eines zellbasierten Modells geeignet ist, um in vitro verschiedene Funktionen und eine Pharmaka-induzierte Regulation der beta-adrenergen Signalkaskade hinsichtlich physiologischer und pathophysiologischer Zustände bei Atemwegserkrankungen des Pferdes und hierfür relevante pharmakologische und toxikologische Zielstrukturen untersuchen zu können.

Submitted by Maike Costa (maiksebas@gmail.com) on 2017-02-02T13:27:46Z No. of bitstreams: 1 arquivo total.pdf: 2209770 bytes, checksum: f1ff3ca285ff26a080821defd683aad9 (MD5) / Made available in DSpace on 2017-02-02T13:27:46Z (GMT). No. of bitstreams: 1 arquivo total.pdf: 2209770 bytes, checksum: f1ff3ca285ff26a080821defd683aad9 (MD5) Previous issue date: 2016-03-14 / Eugenol's ability to reduce nociception and act in the inflammatory process has gained great prominence in the scientific community, emerging interest in researching the ortho-eugenol also has these same activities. Researching ortho-eugenol’s antinociceptive and anti-inflammatory activity, and its possible mechanisms of action is therefore of interest. The administration of vehicle, ortho-eugenol (50, 75 and 100 mg/kg i.p), morphine (10 mg/kg, i.p) or dexamethasone (2 mg/kg, s.c) occurred thirty minutes before the completion of pharmacological tests. The experiments started with psychopharmacological screening and determination of the LD50. The effect of ortho-eugenol on motor coordination in mice was investigated in the rota-rod test. The antinociceptive activity was assessed using chemical tests (test of writhing induced by acetic acid, formalin, test and glutamate) and thermal (hot plate test). To study the role of the adrenergic system, was administered α2-adrenergic antagonist, yohimbine, fifteen minutes before the ortho-eugenol injection the test of writhing induced by acetic acid. The test vascular permeability induced by acetic acid and peritonitis test were used to assess the anti-inflammatory effects of ortho-eugenol in mice. In the test of peritonitis, the levels of pro-inflammatory cytokines and phosphorylated forms of NF-kB and p38 were analyzed in peritoneal fluid. In the behavioral pharmacological screening, the different doses tested ortho-eugenol (200, 225, 250, 300 e 400 mg/kg, i.p.)psicodepressoras showed behavioral changes such as decreased ambulation, and especially analgesia. The LD50 was 307.5 mg / kg, with a confidence interval between 212.1 and 446.0 mg / kg body weight for male and female mice. Pretreatment with ortho-eugenol did not change Rota-rod test coordination test results, but reduced the number of writhes and licking times in the glutamate assay. The reaction time from thermal stimulus was significantly increased in the hot plate test after administration of ortho-eugenol. Treatment with yohimbine reversed the antinociceptive effect of ortho-eugenol, suggesting involvement of the adrenergic system. In anti-inflammatory tests, ortho-eugenol inhibited acetic acid induced vascular permeability and leukocyte migration, reducing TNF-α and IL-1β by virtue of its suppression of NF-kB and p38 phosphorylated forms in the peritonitis test. From these results, ortho-eugenol antinociceptive effects mediated by the adrenergic system and anti-inflammatory activity through regulation of proinflammatory cytokines and phosphorylation of NF-kB and p38 become evident for the first time. / A capacidade do eugenol em reduzir a nocicepção e agir no processo inflamatório tem ganhado grande destaque na comunidade cientifica, surgindo o interesse em pesquisar se o orto-eugenol também possui essas mesmas atividades. Diante disso, surgiu interesse em pesquisar a atividade antinociceptiva e anti-inflamatória do orto-eugenol e seus possíveis mecanismos de ação. A administração do veículo (Tween 80 mais água destilada), orto-eugenol (50, 75 e 100 mg/kg, i.p.), morfina (10 mg/kg, i.p.) ou dexametasona (2 mg/kg, s.c.) aconteceram trinta minutos antes da realização dos testes farmacológicos. Os experimentos iniciaram com a triagem psicofarmacológica e determinação da DL50. O efeito do orto-eugenol sobre a coordenação motora de camundongos foi investigada no teste do Rota-rod. A atividade antinociceptiva foi avaliada utilizando testes químicos (teste das contorções abdominais induzidas pelo ácido acético, teste da formalina e teste do glutamato) e térmico (teste da placa quente). Para estudar a participação do sistema adrenérgico, administrou-se o antagonista adrenérgico α2, ioimbina, quinze minutos antes da injeção do orto-eugenol no teste das contorções induzidas pelo ácido acético. O teste da permeabilidade vascular induzida pelo ácido acético e o teste da peritonite foram utilizados para avaliar o efeito anti-inflamatório do orto-eugenol em camundongos. No teste da peritonite, os níveis de citocinas pró-inflamatórias e as formas fosforiladas de NF-κB e de p38 foram analisados no líquido peritoneal. Na triagem farmacológica comportamental, as diferentes doses testadas do orto-eugenol (200, 225, 250, 300 e 400 mg/kg, i.p.) apresentaram alterações comportamentais psicodepressoras, tais como ambulação diminuída e, principalmente, analgesia. A DL50 foi de 307,5 mg/kg, com intervalo de confiança entre 212,1 e 446,0 mg/kg de peso corporal para camundongos machos e fêmeas. O pré-tratamento com o orto-eugenol não alterou a coordenação motora no teste do Rota-rod, mas reduziu o número de contorções abdominais, o tempo de lambida no teste do glutamato e ambas as fases do teste da formalina. O tempo de reação ao estímulo térmico foi significativamente aumentando no teste da placa quente, após a administração do orto-eugenol. O tratamento com a ioimbina reverteu o efeito antinociceptivo do orto-eugenol, sugerindo ativação do sistema adrenérgico. Nos testes anti-inflamatórios, o orto-eugenol inibiu o aumento da permeabilidade vascular induzida por ácido acético e a migração de leucócitos via redução dos níveis de TNF-α e IL-1β em virtude da supressão das formas fosforiladas de NF-κB e p38 no teste da peritonite. Diante desses resultados, torna-se evidente, pela primeira vez o efeito antinociceptivo mediado pelo receptor adrenérgico α2 e atividade anti-inflamatória por meio da regulação de citocinas pró-inflamatórias e fosforilação de NF-κB e p38.