Made available in DSpace on 2016-07-18T17:53:09Z (GMT). No. of bitstreams: 1 dissertacao.pdf: 1231508 bytes, checksum: 25c34b6cb3fd325bb34877b212f30d24 (MD5) Previous issue date: 2011-09-29 / This study aimed to evaluate the sedative, antinociceptive and cardiopulmonary effects of detomidine alone and in combination with morphine or methadone to the oral cavity approach. Six adult mares were evaluated using a crossover design with at least 7 days between treatments. All the horses were medicated with detomidine (20µg/kg, IV), detomidine (10µg/kg, IV) in combination with morphine (0,1mg/kg, IV) or methadone (0,1mg/kg, IV). Behaviors alteration, sedation degree, oral cavity sensibility, muscle tone of tongue, heart rate, mean arterial blood pressure, arterial blood gases were investigated during 120 minutes. The parametric data were analyzed with ANOVA and Tukey s test, and non parametric data were analyzed with Kruskall-Wallis and Fridman s test with post-Dunn test (P<0,05). In all the treatment groups, the maximum sedative effect was observed between 5 and 60 minutes after drugs administration. The must sedative effect was observed in detomidine horses treated. Respiratory stability was observed in all the groups, with more pronounced cardiovascular changes in DET group. It was concluded that all treatments were satisfactory for oral cavity approach. However, the combination of opioids did not potentiate the sedative effect and, neither increased or prolonged the antinoceptive effect mediate by detomidine. / Objetivou-se avaliar os efeitos sedativos, antinociceptivos e cardiorrespiratórios da administração da detomidina isolada e associada à morfina e metadona para abordagem da cavidade oral de equinos em posição quadrupedal. Foram avaliadas seis éguas, mestiças, adultas, com peso médio de 428±42kg, que foram distribuídas em delineamento de quadrado latino. Cada um dos animais foi tratado com intervalo mínimo de sete dias entre cada avaliação, sendo submetidos aos protocolos de sedação com detomidina (20&#61549;g/kg, IV) (DET), detomidina (10&#61549;g/kg, IV) associada a 0,1mg/kg (IV) de morfina (MORF) e detomidina (10&#61549;g/kg IV) associada a 0,1mg/kg (IV) de metadona (MET). Foram avaliados, através do sistema de escore durante 120 minutos: alteração comportamental, grau de sedação, sensibilidade da cavidade oral, tônus da língua, frequência cardíaca, pressão arterial média, frequência respiratória e variáveis hemogasométricas. A estatística foi realizada com análise de variância, teste de Tukey e análise de medidas repetidas, para as variáveis paramétricas. Para as variáveis não paramétricas foram empregados os testes de Kruskall-Wallis e de Friedmanm com contrastes pelo método de Dunn (P<0,05). O efeito sedativo foi predominante entre 5 e 60 minutos após a administração dos fármacos, em todos os grupos, sendo mais pronunciado no grupo DET. A sensibilidade da cavidade oral e o tônus da língua não diferiram entre os tratamentos. Estabilidade respiratória foi observada em todos os tratamentos, com alterações cardiovasculares mais pronunciadas no grupo DET. Conclui-se que os três tratamentos permitiram a abordagem da cavidade oral, no entanto a associação dos opioides não potencializou o efeito sedativo, bem como não incrementou ou prolongou o efeito antinociceptivo mediado pela detomidina.

VKORC1 est une enzyme membranaire du réticulum endoplasmique, responsable de la réduction de la vitamine K époxyde en vitamine K quinone, activant la synthèse des facteurs de coagulation. VKORC1 constitue ainsi une cible thérapeutique privilégiée des anticoagulants de type anti-vitamine K (AVKs). Cependant, certaines mutations de VKORC1 induisent une dérégulation physiologique et/ou une résistance aux AVKs. En l’absence de données structurales, plusieurs modèles topologiques de VKORC1 ont été proposés à partir des données biochimiques et biophysiques, fréquemment contradictoires. La topologie de VKORC1 ainsi que l'implication des résidus de cystéine (strictement conservés chez toutes les VKORs) dans le mécanisme enzymatique de la protéine restent incertaines. Nous avons construit, par des méthodes in silico, un modèle 3D de la VKORC1 humaine sauvage (hVKORC1WT) à l'échelle atomique. Des simulations de dynamique moléculaire de la protéine, en considérant tous les résidus de cystéine sous leur forme oxydée (SH), nous ont permis d'identifier les résidus de cystéine les plus susceptibles de former des ponts disulfures. Nous avons par conséquent décrit les conformations métastables de hVKORC1WT mimant les états fonctionnels de la protéine. L’étude de la reconnaissance des formes époxyde et réduites de la vitamine K par les conformations prédites de hVKORC1WT a mis en évidence leur sélectivité réciproque. Les conformations de hVKORC1WT ciblées par chaque forme de la vitamine K et leur rôle dans le mécanisme de réduction de la molécule ont ainsi été caractérisés. Nous avons postulé à partir de ces résultats le mécanisme enzymatique détaillé de hVKORC1WT et proposé la structure-cible des AVKs. Les interactions entre la cible prédite - hVKORC1WT à l'état actif - et trois AVKs différents ont été décrites en termes d’énergie libre de liaison. Ces résultats ont été corrélés avec les constantes d'inhibition mesurées in vivo, validant nos prédictions théoriques. Notre protocole, développé pour hVKORC1WT, est applicable aux formes mutées de l’enzyme. Il permettra la description des mécanismes modifiant l'activité réductase de hVKORC1 et/ou provoquant une résistance aux AVKs. Cette description ouvre la voie à la conception d'une nouvelle génération d'inhibiteurs surmontant les résistances aux AVKs. Notre concept et le protocole établi pour l’étude de hVKORC1 peuvent être étendus à l'analyse des VKORs mammaliennes et aux autres enzymes de la famille des oxydoréductases. / VKORC1 is an endoplasmic reticulum membrane-resident enzyme, responsible for vitamin K epoxide reduction to vitamin K quinone that activates coagulation factors synthesis. VKORC1 is thus a prominent target of vitamin K agonists (VKAs) in anticoagulant therapies. However, some VKORC1 mutations lead to physiological dysregulation and/or VKAs resistance. No VKORC1 structural data is available, and postulated topological models are based on biochemical and biophysical experimental observations frequently contradicting. Topology of VKORC1 and involvement of cysteines residues (highly conserved in VKORs) in the protein enzymatic mechanism remain unclear. We built an in silico 3D model of the wild-type human VKORC1 (hVKORC1WT) at the atomistic scale. Molecular dynamics simulations of the protein model, carrying all the cysteines residues in their oxidized form (SH), were used for identification of cysteines residues which may plausibly form disulfide bridges. We thus described hVKORC1WT metastable conformations depicting the functionally relevant states of protein. Study of the vitamin K (in epoxide and in reduced forms) recognition by the predicted conformations of hVKORC1WT revealed their reciprocal selectivity. The hVKORC1WT conformations targeted by each vitamin K form were established and their role in the reduction mechanism of this molecule was explained. Using our results, we postulated the comprehensive enzymatic mechanism of hVKORC1WT and we proposed the 3D structure as the VKAs target. Interactions between the predicted target - hVKORC1WT active state - and three different VKAs were characterized. The obtained affinities (free binding energy) were in good correlation with in vivo measured inhibition constants (Ki), thus validating our theoretical predictions. Our protocol developed for hVKORC1WT is suitable for a study of its mutants. Description of the enzymatic mechanisms of mutated hVKORC1 will lead to understanding of the modified reductase activity or/and to explaining of its resistance to VKAs. Such data are crucial for the development of novel strategies in the design of a new generation of inhibitors overcoming VKAs resistance. Our concept and the established in silico protocol can be extended to analysis of mammalian VKORs and other oxidoreductases family proteins.

Background Past studies from this laboratory have shown that quinpirole administration from postnatal day (P) 1–21 produces persistent supersensitization of the dopamine D2 receptor that persists throughout the animal's lifetime. Methods In Experiment 1, both male and female rats were treated with quinpirole or saline from P1–21 and tested on the place and match-to-place versions of the Morris water task (MWT) from P22–28. In Experiment 2, both male and female rats were administered either acute or chronic injections of quinpirole (1 mg/kg) or saline beginning on P1 until analysis for corticosterone (CORT) on P7, 14, or 21. Results Neonatal quinpirole treatment produced deficits on both versions of the MWT compared with saline control. One day after behavioral testing, brain tissue was harvested, and the hippocampus was analyzed for nerve growth factor (NGF) and brain-derived nerve growth factor (BDNF); NGF was found to be significantly decreased by neonatal quinpirole treatment. Acute or chronic quinpirole treatment on P14 produced a larger increase in CORT than controls and produced larger increases in CORT than control rats on P21. Conclusions These results demonstrate that neonatal quinpirole treatment produces cognitive deficits that could be related to decreases in hippocampal NGF and increases in CORT, resulting in abnormalities in hippocampal development.

This study was designed to analyze the effects of nicotine on yawning behavior and neurotrophin content in the hippocampus and frontal cortex of D2-receptor primed female adult Sprague-Dawley rats. Animals were neonatally treated with quinpirole, a dopamine (DA) D2/D3 agonist, from postnatal day 1-21 (P1-21) and raised to P60 and administered nicotine tartarate (0.3 mg/kg free base) or saline twice daily for 14 days. One day after nicotine treatment had ceased, the number of yawns was recorded for 1 h in response to an acute injection of quinpirole (i.p., 100 microg/kg). Yawning is a D2-receptor mediated event. D2-primed rats demonstrated a significant increase in yawning in response to acute quinpirole compared with that of controls, but nicotine did not alleviate this effect. Neonatal quinpirole treatment produced a significant decrease of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) in the hippocampus that was alleviated by adulthood nicotine treatment. Interestingly, nicotine treatment to controls produced a significant increase of NGF in the frontal cortex, but a significant decrease of both NGF and BDNF in the hippocampus and BDNF in the frontal cortex. The decreases shown in NGF and BDNF is contrary to past findings that have shown nicotine to produce significant increases of hippocampal NGF and BDNF, but these past studies utilized male rats or mice or were performed in vitro. This study shows that nicotine has complex interactions with NGF and BDNF in D2-primed and control animals, and emphasizes the importance of gender differences when analyzing nicotine's effects on neurotrophins.

RATIONALE: Increases in dopamine D2-like receptor function are common in several psychological disorders that demonstrate a four to five fold increase in nicotine abuse compared to the general population.OBJECTIVE: The objective of this study was to analyze the interaction of sex differences and sensitization to nicotine in rats D2 receptor primed as neonates.MATERIALS AND METHODS: A total of 32 male and 32 female Sprague-Dawley rats derived from eight litters were ontogenetically treated with quinpirole (1 mg/kg) or saline from postnatal days (P) 1-21 and raised to adulthood. At P60, all animals were given an acute injection of quinpirole HCl (100 microg/kg) and yawns were counted for 1 h. Yawning has been shown to be a behavioral event mediated by D2-like receptors. Beginning on P61-65, animals were habituated to a locomotor arena and subsequently administered either nicotine (0.5 mg/kg free base) or saline (intraperitoneal) every second day for 3 weeks. Approximately 15 min after each injection, animals were placed into the arena and horizontal activity and vertical rears were recorded.RESULTS: A robust increase of yawning was observed at P60 in D2 primed as compared to saline controls. Priming of D2-like receptors increased the locomotor response to nicotine in horizontal activity in both males and females, but females demonstrated a more robust hypoactive locomotor response to initial nicotine treatment when compared to saline-treated females. Nicotine also produced a significant decrease of vertical rearing in both males and females.CONCLUSIONS: It appears that D2 receptor priming enhances sensitization to nicotine in adult rats, and females may be more behaviorally sensitive to nicotine than males.

Hyperactivity is thought to be associated with an alteration of dopamine (DA) neurochemistry in brain. This conventional view became solidified on the basis of observed hyperactivity in DA-lesioned animals and effectiveness of the dopaminomimetics such as amphetamine (AMP) in abating hyperactivity in humans and in animal models of hyperactivity. However, because AMPreleases serotonin (5-HT) as well as DA, we investigated the potential role of 5-HT in an animal model of hyperactivity. We found that a greater intensity of hyperactivity was produced in rats when both DA and 5-HT neurons were damaged at appropriate times in ontogeny. Therefore, previously we proposed this as an animal model of attention deficit hyperactivity disorder (ADHD) - induced by destruction of dopaminergic neurons with 6-hydroxydopamine (6-OHDA (neonatally) and serotoninergic neurons with 5,7-dihydroxytryptamine (5,7-DHT) (in adulthood). In this model effects similar to that of AMP(attenuation of hyperlocomotion) were produced by m-chlorophenylpiperazine (m-CPP) but not by 1-phenylbiguanide (1-PG), respective 5-HT2 and 5-HT3 agonists. The effect of m-CPP was shown to be replicated by desipramine, and was largely attenuated by the 5-HT2 antagonist mianserin. These findings implicate 5-HT neurochemistry as potentially important therapeutic targets for treating human hyperactivity and possibly childhood ADHD.

Thesis / Doctor of Philosophy (PhD)

No description available.

No description available.

Objective: The aim was to determine if isolated rabbit cardiomyocytes could be preconditioned. Methods: Cardiomyocytes isolated from rabbit hearts were subjected to 15 min oxygenated preincubation, with and without substrate, prior to concentration into an ischaemic slurry, with or without glucose present. The effects of an adenosine agonist (CCPA), an adenosine receptor blocker (SPT), and the protein kinase C blocker, calphostin C, on rates of ischaemic contracture and survival of the myocytes were determined after various times of ischaemia, following resuspension of the cells in hypotonic media. Results: A glucose-free preincubation period protected myocytes from subsequent ischaemic injury, with a 40% reduction of cell death at 90-120 min and 1-2 h delay in cell death. CCPA added during preincubation and during the ischaemic period also tended to protect from injury, but the differences were not significant and protection was less than with a glucose-free preincubation. Although preincubation with CCPA did not precondition, SPT added to the preincubation medium only, or to both the preincubation medium and the ischaemic pellet, inhibited the preconditioning effect of a glucose-free preincubation period. Calphostin C, added only into the ischaemic pellet, inhibited the preconditioning effect of glucose-free preincubation. Conclusions: Glucose-free preincubation protects ischaemic isolated myocytes from subsequent ischaemia. The degree of protection is great enough to account for protection seen in intact hearts, following preconditioning protocols. Protection is blocked by SPT and a highly specific protein kinase C inhibitor, calphostin C. Protection from ischaemic injury that seems to mimic ischaemic preconditioning can be induced in isolated cardiomyocytes, and appears dependent on adenosine receptors and activation of protein kinase C.Cardiovascular Research 1994;28:72-77.