Made available in DSpace on 2015-03-26T13:22:04Z (GMT). No. of bitstreams: 1 text completo.pdf: 757671 bytes, checksum: 435de7db4aaa7b1bdaf1bc5260f57dd9 (MD5) Previous issue date: 2014-03-11 / O objetivo do presente estudo foi investigar os efeitos do treinamento físico sobre as propriedades morfológicas e mecânicas de cardiomiócitos de camundongos knockout (KO) para receptores os β1- e β2 adrenérgicos. Camundongos C57BL/6J, KO para os receptores β1-adrenérgicos, FVB/N e KO para os receptores β2-adrenérgicos com 4 meses de idade, foram inicialmente separados aleatoriamente em 8 grupos: C57c, C57t, KO β1c, KO β1t, FVBc, FVBt, KO β2c e KO β2t. Os animais dos grupos treinados (C57t, KO β1t, FVBt, KO β2t) foram submetidos a um protocolo de treinamento aeróbico de 8 semanas, 5 dias por semana, 1hora por dia, com intensidade de 60% da velocidade máxima de corrida. 3 a 4 animais de cada grupo foram separados para a análise de contração celular com perfusão do agonista β-adrenérgico isoproterenol (ISO) [100nM]. Após a eutanásia, os cardiomiócitos do ventrículo esquerdo foram isolados por dispersão enzimática. O comprimento e a largura dos cardiomiócitos foram medidos utilizando um sistema de captação de imagens e o volume celular foi calculado. As contrações celulares foram medidas através da técnica de alteração do comprimento dos cardiomiócitos, após estimulação elétrica a 1 Hz, em temperatura controlada à 37oC, usando-se um sistema de detecção de bordas. Os resultados mostraram que os camundongos KO β1 apresentaram menor comprimento celular comparados aos C57. O grupo KO β2t apresentou maior comprimento celular comparado ao grupo KO β2c. O protocolo de treinamento aumentou a amplitude de contração, a velocidade máxima de contração e a velocidade máxima de relaxamento dos cardiomiócitos dos animais KO β1t e KO β2t. O ISO não alterou a amplitude de contração, a velocidade máxima de contração e de relaxamento nos cardiomiócitos dos camundongos KO β1+ISO, já os animais KO β2+ISO apresentaram aumento nas mesmas propriedades mecânicas. Conclui-se que o protocolo de treinamento aeróbico: a) aumentou as propriedades mecânicas dos cardiomiócitos dos animais KO β1 e KO β2; b) aumentou o comprimento dos cardiomiócitos dos camundongos KO β2; c) associado ao ISO não alterou as propriedades mecânicas nos cardiomiócitos dos camundongos KO β1 e reduziu nos cardiomiócitos dos camundongos KO β2. / The aim of this study was to investigate the effects of endurance training on cardiovascular parameters and morphological and mechanical properties of isolated cardiomyocytes from the left ventricle of β1-and β2 adrenergic receptors knockout mice. 4 months old C57BL/6J, β1- adrenergic receptor knockout (KO 1), FVB/N and β2-adrenergic receptor knockout (KO 2) mice were randomly allocated into one of the 8 groups: C57c, C57t, KO β1c, KO β1t, FVBc, FVBt, KO β2c e KO β2t. The animals of the trained groups (C57t, KO β1t, FVBt, KO β2t) underwent a protocol of aerobic training 8 weeks, 5 days/week, 1 hour/day, intensity of 60% of maximal speed. 3 to 4 animals in each group were divided for the analysis of cellular contraction with infusion of -adrenergic agonist isoproterenol (ISO) [100nM]. At sacrifice, the heart was removed and left ventricular myocytes were enzymatically dispersed. Cell length and width were measured using an image capture system and cell volume was calculated. Myocytes were stimulated at 1 Hz at controlled temperature (37oC) and cell contractility was measured by using an edge detection system. The results showed that KO 1 mice showed lower cell length compared to C57. The KO β2t group showed higher cell length compared to the KO β2c group. The training protocol increased the contraction amplitude, maximum contraction velocity and maximum relaxation velocity of cardiomyocytes of KO β1t and KO β2t animals. The ISO did not alter the amplitude of contraction, maximum contraction velocity and maximum relaxation velocity of cardiomyocytes from KO β1+ISO animals, while the KO β2+ISO animals showed an increase in the same mechanical properties. It is concluded that the aerobic training protocol: a) increased the mechanical properties of cardiomyocytes from KO β1t and KO β2t animals; b) increased the length of cardiomyocytes from β2 KO mice; c) associated with the ISO did not alter the mechanical properties of cardiomyocytes from β2 KO 1mice and reduced of cardiomyocytes from KO β2 mice.

Orientador: Cláudia Herrera Tambeli / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba / Made available in DSpace on 2018-08-24T16:27:32Z (GMT). No. of bitstreams: 1 Favaro-Moreira_NadiaCristina_D.pdf: 14440949 bytes, checksum: 19ac7e9f7196c13dfd756bd4150b1456 (MD5) Previous issue date: 2014 / Resumo: Disfunção temporomandibular (DTM) é um termo coletivo que abrange uma série de problemas clínicos que envolvem os músculos mastigatórios e a articulação temporomandibular (ATM) e está comumente associada à inflamação. Apesar de medicamentos anti-inflamatórios não esteróides (AINEs) serem frequentemente utilizados no controle de dor inflamatória, sabe-se que a dor inflamatória possui um componente simpático que pode predominar em casos menos responsivos a tratamentos com AINEs. Portanto, os objetivos deste estudo foram: (i) avaliar se os hormônios gonadais modulam a resposta antinociceptiva ao bloqueio dos ?-adrenoreceptores (ARs) na ATM de ratos, (ii) avaliar se um e três dias de tratamento com o ?-bloqueador não seletivo para AR ?1 e ?2 nadolol reduzem a dor em pacientes com DTM significativamente mais do que o placebo, (iii) avaliar se as mulheres experimentam relativamente maior benefício com o tratamento com nadolol que os homens dependendo do seu estado hormonal, e (iv ) comparar o efeito do nadolol com o efeito do ibuprofeno. O primeiro objetivo foi desenvolvido em ratos machos e fêmeas, intactos ou gonadectomizados (com ou sem reposição hormonal) através da coadministração de formalina e antagonista específico para AR ?1 , ?2 e ?3 na região da ATM. O comportamento nocieptive foi quantificado e utilizado para a análise estatística. Para o segundo objetivo Nadolol (40 mg/dia), ibuprofeno (400 mg/dia) ou placebo foi administrado em 27 mulheres que não usam contraceptivo oral (CO), 28 mulheres usando CO e 29 homens, que estavam de acordo com os Critérios Diagnósticos para Pesquisa (Research Diagnostic Chriteria) para DTM. Eles completaram um estudo cruzado, aleatorizado, duplo-cego e com controle placebo. As mulheres participaram durante três meses (durante a fase menstrual e durante a fase peri-ovulatória em mulheres que não usam CO, e durante a fase menstrual e durante a fase de uso do CO em mulheres usando CO), em um total de 6 etapas de análise ( 2 por mês), e homens participaram durante um mês com três etapas de análise com um período de 6 dias entre cada etapa. Cada etapa consistiu de uma avaliação de basal e duas avaliações durante o tratamento, uma no primeiro e o outra no terceiro dia de tratamento. A dor foi avaliada por meio da utilização da escala visual analógica (VAS) e as comparações foram feitas através do pré-tratamento (basal), o primeiro e o terceiro dia de intervenção (pós-tratamento). O bloqueio dos ARs ? reduz significativamente a nocicepção da ATM em ratos machos e fêmeas, mas as fêmeas são mais sensíveis. Os hormônios gonadais reduziram a resposta ao bloqueio de ARs ? na ATM de machos e fêmeas. No estudo em humanos um e três dias de tratamento com nadolol ou ibuprofeno produz uma analgesia em mulheres e homens significativamente maior que placebo, mas as mulheres são mais sensíveis independente do estado hormonal. Em resumo, estes dados demonstram que os hormonios sexuais podem modular o efeito analgésico de bloqueadores de ARs ? dependendo dos níveis séricos dos hormonios gonadais, do subtipo de ARs ? ativado e da dose de droga administrada. A maior eficácia no tratamento da dor em mulheres é clinicamente relevante uma vez que a DTM é mais prevalente e mais severa em mulheres do que em homens / Abstract: Temporomandibular disorder (TMD) is a collective term embracing a number of clinical problems that involve the masticatory muscles, and the temporomandibular joint (TMJ), commonly associated with inflammation. Despite anti-inflammatory drugs (NSAIDs) are frequently used in the control of inflammatory pain, it is well known that inflammatory pain has a sympathetic component that might predominate in the cases less responsive to NSAIDs treatments. Therefore, the aims of this study were: (i) to evaluate whether gonadal hormones modulate the antinociceptive responsiveness to the blockade of ?-adrenoreceptors (ARs) in the TMJ of rats, (ii) to evaluate whether one and three days of treatment with the nonselective ?1 and ?2-AR antagonist nadolol would reduce clinical pain symptoms in TMD patients significantly more than placebo, (iii) to evaluate whether women would experience relatively greater benefit from nadolol than men depending on their hormonal status, and (iv) compared the effect of nadolol with the effect of ibuprofen. The first aim was developed in male and female rats, intact or gonadectomized (with or without hormone replacement), by coadministration of formalin and specific antagonist for ?1, ?2 and ?3-ARs in the TMJ region. The nocieptive behavior was quantified and used for estatistical analysis. For the second aim Nadolol (40 mg/day), ibuprofen (400 mg/day) or placebo was administrated in 27 women not using oral contraceptive (OC), 28 women using OC, and 29 men which met the Research Diagnostic Criteria for TMD. They completed a randomized, crossover, double¿blind, placebo controlled study. Women participated for three months (during menstrual phase and during peri-ovulatory phase in women not using OC, and during menstrual phase and during OC using phase in women using OC) in a total of 6 stages of analysis (2 per month), and men participated for one month whith three stages of analysis and 6 days of wash out. Each stage consisted of a baseline evaluation and two evaluations during treatment, one on the first and the other on the third day of treatment. Clinical pain ratings were obtained by Visual Analog Scale (VAS) and comparisons were made across the pre-treatment (baseline), the first and the third day of intervention (post-treatment). Blockade of ?-ARs significantly reduce the TMJ nociception in male and female rats, but females are more responsive. The gonadal hormones reduce the responsiveness to the blockade of ?-ARs in the TMJ of males and females rats. In the human study one and three days of treatment with nadolol or ibuprofen produces analgesia in TMD women and men significantly more than placebo, but women are more responsive independent of their hormonal status. In summary, these data demonstrate that gonadal hormones can modulate the analgesic effect of ?-ARs blockers depending on the gonadal hormones serum levels, on the ?-ARs activated subtype and on the dose of drug administered. The greater treatment efficacy in women is of clinical relevance since TMD is more prevalent and severe in women than in men / Doutorado / Fisiologia Oral / Doutora em Odontologia

Made available in DSpace on 2016-05-02T13:55:34Z (GMT). No. of bitstreams: 1 JussaraMaria ReisCalixto-dissertacao.pdf: 678492 bytes, checksum: 69d16272c951188cf230141c93f2978b (MD5) Previous issue date: 2012-11-23 / The objective of this study was to evaluate the effect of arginine and ractopamine on performance, carcass characteristics, lipid profile and liver histological features in growing and finishing pigs. The experimental design was randomized blocks with three treatments, eight repetitions, two animals per experimental unit, for a total of 24 animals (12 barrows and 12 gilts) with 65 days of age and live weight of 32 ± 0,75 quilogramas .The treatments were divided into: T1: control (C) (isocaloric meal and isocaloric), T2: C + 4 ppm of ractopamine and T3: C + 0.8% arginine.The additives ractopamine and arginine were added to the mash feed and these ad libitum for 89 days and 46 days of growth phase and 43 days from the termination phase. On reaching 150 days of age, the animals were slaughtered. We evaluated animal performance (average daily feed intake, average daily gain in weight and feed conversion) weighing the animals at the beginning and end of each phase and the rations offered. At slaughter blood samples were collected for biochemical analysis of lipid profile and liver slices for histological examination. The carcasses were hot and heavy after 24 hours refrigerated, carcass length, backfat thickness and loin eye area, were measured in hot carcass. Ractopamine supplementation improved the average feed intake during the growing phase (P <0.05), and average daily weight gain in crescimemto and finishing phases (P <0.05), without however changing other variables, carcass characteristics (P> 0.05) and lipid profile (P> 0.05). The addition of arginine did not affect performance (P> 0.05), carcass traits (P> 0.05) and lipid profile (P> 0.05).Therefore, it was characterized the beneficial effect of ractopamine on reducing the average consumption ration in the growth phase and increased average daily weight gain and decreased average daily consumption of feed during the finishing phase . / O objetivo deste trabalho foi avaliar o efeito da ractopamina e arginina sobre o desempenho, características de carcaça, perfil lipídico e característica histológica de fígado em suínos em crescimento e terminação. O delineamento experimental foi em blocos casualisados, com três tratamentos, oito repetições, dois animais por unidade experimental, perfazendo um total de 24 animais(12 suínos machos castrados e 12 fêmeas), com 65 dias de idade e peso vivo médio 32 ± 0,75 quilogramas.Os tratamentos foram: T1: controle (C): dieta isoenergética e isocalórica; T2: C + 4 ppm de ractopamina e T3: C + 0,8% de arginina. Os aditivos ractopamina e arginina foram adicionados às rações fareladas e estas fornecidas ad libitum por 89 dias sendo 46 dias da fase crescimento e 43 dias da fase terminação. Ao atingirem 150 dias de idade, os animais foram abatidos. Avaliou-se o desempenho animal (consumo médio diário de ração, ganho médio diário em peso e conversão alimentar) pesando-se os animais no início e final de cada fase e as rações oferecidas. No momento do abate foram colhidas amostras de sangue para análises bioquímicas de perfil lipídico e cortes de fígado para exame histológico. As carcaças foram pesadas quentes e após 24 horas de resfriadas, foram medidos o comprimento de carcaça, espessura de toucinho e a área de olho-de-lombo. A suplementação de ractopamina diminuiu o consumo médio de ração na fase de crescimento e terminação (P<0,05) e o ganho médio diário de peso nas fases de terminação(P<0,05) sem, contudo alterar as outras variáveis como as características de carcaça (P>0,05) e perfil lipídico (P>0,05). A inclusão de arginina não alterou o desempenho (P>0,05), característica da carcaça (P>0,05) e perfil lipídico(P>0,05). Portanto, ficou caracterizado o efeito benéfico da ractopamina sobre a diminuição do consumo médio de ração na fase de crescimento e terminação e o aumento do ganho médio de peso diário na fase de terminação

L’AMPc est un second messager exerçant un rôle vasculoprotecteur majeur, par ses effets relaxants et inhibiteurs de la prolifération et de la migration cellulaires. Les concentrations intracellulaires d’AMPc sont finement régulées par leur synthèse via les adénylates cyclases et leur dégradation par les phosphodiestérases (PDEs). Nous avons évalué l’impact de l’environnement cellulaire sur la voie de signalisation couplée au récepteur β-adrénergique (β-AR/AMPc/PDE) dans les cellules musculaires lisses vasculaires (CMLVs), ainsi que les altérations potentielles de celle-ci en situation pathologique d’insuffisance cardiaque (IC).Notre première étude montre que dans les CMLs d’aorte de rat en culture, adoptant un phénotype synthétique, la voie de signalisation β-AR/AMPc/PDE est hautement modulée par la densité cellulaire Ainsi, une faible densité cellulaire est associée à une régulation négative de l’expression fonctionnelle du récepteur β1-AR, à une activité hydrolytique des PDEs-AMPc plus faible et à des concentrations d’AMPc intracellulaire plus élevées que celles observées dans des cellules confluentes.Notre deuxième étude montre que dans l’aorte de rat, l’IC est associée à une dysfonction endothéliale (DE), une hyperréactivité aux agents contractants et une altération de la fonction et de l’expression des PDEs-AMPc. Nos résultats suggèrent que l’altération de la voie du NO/GMPc suite à la DE conduit à une hyper-activation de la PDE3, qui masque la fonction de la PDE4 et altère la relaxation β-AR.L’ensemble de ce travail met en évidence le rôle critique de l'environnement cellulaire dans le contrôle de la voie β-AR/AMPc/PDE des CMLVsMots clés : Muscle lisse vasculaire, récepteur β-adrénergique, AMPc, phosphodiestérases, densité cellulaire, insuffisance cardiaque / CAMP is second messenger which plays a prominent vasculoprotective role by its relaxing effects and inhibition of cell proliferation and migration. Intracellular cAMP level is regulated by its synthesis by adenylate cyclase and its degradation by phosphodiesterases (PDEs). We evaluated the influence of cellular environment on signaling pathway coupled to β-adrenoceptors (β-AR/cAMP/PDE) on vascular smooth muscle cells (VSMCs), as well as potential alterations in heart failure (HF).The first study showed that in cultured rat aortic SMCs exhibiting synthetic phenotype, the β-AR/cAMP/PDE signaling pathway is highly modulated by the cellular density.Thus, the low density state being associated to a downregulation of the β1-AR, a lower cAMP-PDE activity and a higher basal [cAMP]i compared to confluent cells.Our second study showed that in rat aorta, HF is associated with endothelial dysfunction, hyper-reactivity to contractile agents and an alteration of function and expression of cAMP-PDEs. Our results suggest that NO/cGMP pathway alteration following the ED in HF leads to hyper-activation of PDE3 which masks PDE4 function and alters β-adrenoceptor relaxationThus, our works highlights the critical role of the cellular environment in controlling the vascular β-AR signaling.Keywords: Vascular smooth muscle, β-adrenoceptor, cAMP, phosphodiesterases, cellular density, heart failure.

No description available.

Einleitung: / Introduction:

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.

No description available.

An experiment was conducted using forty-one Bonsmara steers (age ± 7 months) to determine the effect of zilpaterol hydrochloride (ZH) on the growth performance and carcass characteristics. The trial was structured using a completely randomized design with two treatments, control and ZH group. The steers were fed ZH for 28 consecutive days at the end of the finishing period and ZH was withdrawn from the diet 2 days prior to slaughter of the animals. The steers were placed in individual pens and weighed fortnightly throughout the 4 months trial. Zilpaterol hydrochloride (ZH) was included in the diet at a rate of 8.3 mg/kg of DM. Feeding of ZH increased (P< 0.05) body weight (BW) gain and ADG (1.102 vs. 1.444) and tended to increase (P = 0.067) feed efficiency (F:G) during the last month of the finishing period. There were no significant differences (P> 0.05) in daily dry matter intakes (DMI). For the control group, high treatment weight gains were significantly associated with high initial weight (r = 0.424, P = 0.049) and also high pre-treatment body weight (r = 0.678, P= 0.001). Treatment weight gain increased as the initial and pre-treatment weight gain increased in the control group. For the steers that were fed ZH, there was no significant correlation between the treatment body weight gain with initial weight (r = 0.097, P = 0.694) and also pre- treatment live weight (r = 0.393, P = 0.096). Supplementation of ZH significantly increased (P < 0.0001) the dressing percentage (56.4% vs. 58.4%) and had no significant (P>0.05) effect on the carcass weight. The outcome of the study suggest that supplementation of ZH in the diet during the last month of the finishing period enhances growth performance and shows the repartitioning capacity of the feed additive as a beta- agonist. / Agriculture and  Animal Health / M. Sc. (Agriculture (Animal Science)

Récemment plusieurs récepteurs couplés aux protéines G (RCPGs) ont été caractérisés au niveau des membranes intracellulaires, dont la membrane nucléaire. Notre objectif était de déterminer si les sous-types de récepteurs β-adrénergiques (βAR) et leurs machineries de signalisation étaient fonctionnels et localisés à la membrane nucléaire des cardiomyocytes. Nous avons démontré la présence des β1AR et β3AR, mais pas du β2AR à la membrane nucléaire de myocytes ventriculaires adultes par immunobuvardage, par microscopie confocale, et par des essais fonctionnels. De plus, certains partenaires de signalisation comme les protéines GαS, Gαi, l’adénylate cyclase II, et V/VI y étaient également localisés. Les sous-types de βAR nucléaires étaient fonctionnels puisqu'ils pouvaient lier leurs ligands et activer leurs effecteurs. En utilisant des noyaux isolés, nous avons observé que l'agoniste non-sélectif isoprotérénol (ISO), et que le BRL37344, un ligand sélectif du β3AR, stimulaient l'initiation de la synthèse de l’ARN, contrairement à l'agoniste sélectif du β1AR, le xamotérol. Cette synthèse était abolie par la toxine pertussique (PTX). Cependant, la stimulation des récepteurs nucléaires de type B de l’endothéline (ETB) causaient une réduction de l'initiation de la synthèse d’ARN. Les voies de signalisations impliquées dans la régulation de la synthèse d’ARN par les RCPGs ont ensuite été étudiées en utilisant des noyaux isolés stimulés par des agonistes en présence ou absence de différents inhibiteurs des voies MAP Kinases (proteines kinases activées par mitogènes) et de la voie PI3K/PKB. Les protéines impliquées dans les voies de signalisation de p38, JNK, ERK MAP Kinase et PKB étaient présents dans les noyaux isolés. L'inhibition de PKB par la triciribine, inhibait la synthèse d’ARN. Nous avons ensuite pu mettre en évidence par qPCR que la stimulation par l’ISO entrainait une augmentation du niveau d'ARNr 18S ainsi qu’une diminution de l'expression d’ARNm de NFκB. En contraste, l’ET-1 n’avait aucun effet sur le niveau d’expression de l’ARNr 18S. Nous avons ensuite montré que la stimulation par l’ISO réduisait l’expression de plusieurs gènes impliqués dans l'activation de NFκB, tandis que l’inhibition de ERK1/2 et PKB renversait cet effet. Un microarray global nous a ensuite permis de démontrer que les βARs et les ETRs nucléaires régulaient un grand nombre de gènes distincts. Finalement, les βARs et ETRs nucléaires augmentaient aussi une production de NO de noyaux isolés, ce qui pouvait être inhibée par le LNAME. Ces résultats ont été confirmés dans des cardiomyocytes intacts en utilisant des analogues cagés et perméables d’ISO et de l'ET-1: l'augmentation de NO nucléaire détectée par DAF2-DA, causée par l'ET-1 et l'ISO, pouvait être prévenue par le LNAME. Finalement, l’augmentation de l’initiation de la transcription induite par l'ISO était aussi bloquée par le L-NAME ou par un inbitheur de PKG, le KT5823, suggérant que la voie NO-GC-PKG est impliquée dans la régulation de la transcription par les βAR. En conclusion, les βARs et les ETRs nucléaires utilisent des voies de signalisation différentes et exercent ainsi des effets distincts sur l’expression des gènes cardiaques. Ils représentent donc une avenue intéressante pour le développement de drogues pharmacologiques. / Recently several G protein-coupled receptors (GPCRs) have been shown to localize to intracellular membranes, in particular the nuclear membrane. As such, we sought to determine if the β-adrenergic receptor (βAR) subtypes and their associated signalling machinery are functionally localized to nuclear membranes. We demonstrated the presence of β1AR and the β3AR, but not the β2AR, in adult ventricular myocyte nuclei by western blotting, confocal microscopy and functional assays. Downstream signalling partners such as GαS, Gαi and adenylyl cyclase II and V/VI were also present. Nuclear-localized βARs were functional with respect to ligand binding and effector activation. In isolated nuclei, the non-selective βAR agonist isoproterenol (ISO) and the β3AR-selective ligand BRL37344, but not the β1AR-selective xamoterol, stimulated transcription initiation in a pertussis toxin (PTX)-sensitive manner. In contrast, stimulation of type B endothelin receptors (ETB), another GPCR family shown to be present on the nuclear membrane, decreased de novo RNA synthesis. To investigate the signalling pathway(s) involved in GPCR-mediated regulation of RNA synthesis, nuclei were isolated from intact adult rat hearts and treated with receptor agonists in the presence or absence of inhibitors of the PI3K/PKB and mitogen-activated protein kinase (MAPK) pathways. Components of p38, JNK, and ERK1/2 MAPK cascades as well as PKB were detected in nuclear preparations. Inhibition of PKB with triciribine converted the activation of the βAR from stimulatory to inhibitory with regards to transcription initiation. Analysis by qPCR indicated isoproterenol treatment increased 18S rRNA but decreased NFκB mRNA. In contrast, ET-1 had no effect on 18S rRNA expression. Further investigation using pathway-specific PCR arrays revealed that isoproterenol treatment also reduced the expression of several other genes involved in the activation of NFκB and that ERK1/2 and PKB inhibitors attenuated this effect. Subsequent genome-wide microarray analysis has revealed that nuclear βAR and ETB regulated a host of genes in an overlapping but distinct manner. Moreover, both ET-1 and ISO produced an L-NAME-sensitive increase in NO production in isolated cardiac nuclei. These observations were confirmed in intact cardiomyocytes using novel caged analogues of ISO and ET-1 and the cell-permable NO-sensitive fluorescent dye, DAF-2 DA. Briefly, both ET-1 and isoproterenol increased NO production, and this increase was prevented upon preincubation with L-NAME. Moreover, the ability of isoproterenol to increase transcription initiation in isolated nuclei was blocked by L-NAME or the PKG inhibitor KT5823, indicating the NO-GC-PKG pathway is involved in the regulation of gene expression by nuclear βARs. Hence, we have shown that βARs and ETRs in the nuclear membrane activate distinct signalling pathways, resulting in different effects on gene transcription and thus represent potentially important targets for drug development.