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Orientadores: José Wilson Magalhães Bassani, Rosana Almada Bassani / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Elétrica e de Computação / Made available in DSpace on 2018-08-19T11:34:49Z (GMT). No. of bitstreams: 1 Monteiro_MarinaCarneiro_M.pdf: 5677929 bytes, checksum: 5c42afe705a43b66a4505a6ecded7b7c (MD5) Previous issue date: 2011 / Resumo: A fração do conteúdo de Ca2+ do retículo sarcoplasmático (RS) liberada a cada contração (Fractional Release - FR) em miócitos cardíacos é regulada pela corrente de entrada de Ca2+ através da membrana celular pelos canais de Ca2+ tipo-L (ICa,L) e pelo conteúdo de Ca2+ do RS ([Ca2+]RS). Em trabalho anterior foi desenvolvido, no nosso laboratório, um modelo experimental denominado de modelo do RS funcionalmente isolado (MRSFI). Neste modelo, cardiomiócitos são perfundidos em solução sem Na+ e sem Ca2+, o que torna as suas membranas eletricamente inexcitáveis e inibe o transporte do íon pelo trocador Na+/Ca2+. As variações (transientes) da concentração intracelular de Ca2+ ([Ca2+]i) medidas com o indicador fluorescente Fluo-3 AM (5 ?M, 20 min, 24ºC) são evocadas por aplicação de pulsos rápidos (100 ms) de cafeína (10 mM). No presente trabalho, o MRSFI foi usado para estudo da relação entre FR e [Ca2+]RS na ausência do gatilho fisiológico (ICa,L) para liberação reticular de Ca2.... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digital / Abstract: The fraction of the sarcoplasmic reticulum (SR) Ca2+ content released at a twitch (Fractional Release - FR) in cardiac myocytes is regulated by the transmembrane inward Ca2+ current through the L-type Ca2+ channel (ICa,L) and by the SR Ca2+ content ([Ca2+]SR). In the experimental model of the functionally isolated SR model (FISRM), previously developed in this laboratory, cardiomyocytes are perfused with Na+, Ca2+-free solution, which makes the cells electrically unexcitable and thermodynamically inhibits the sarcolemmal Na+/Ca2+ exchanger. Variations in the intracellular Ca2+ concentration ([Ca2+]i) was measured with the Ca2+ indicator fluo-3 and Ca2+ transients due to SR release are evoked by pulse-like (100 ms duration) application of 10 mM caffeine. In the present work, the FISRM was used to study the relationship between FR and [Ca2+]SR in the absence of ICa,L, the physiological trigger for the release of Ca2+ from the SR.... Note: The complete abstract is available with the full electronic digital thesis or dissertations / Mestrado / Engenharia Biomedica / Mestre em Engenharia Elétrica

Membrane bound AChE from calf brain caudate nucleus was solubilized by use of ion-free media in presence of 10~ M EDTA and 10“5M tetracaine. The irreversible release of AChE was more effectively inhibited by divalent ions compared to monovalent ions added to the medium. EDTA appears to chelate divalent ions released from the tissue while tetracaine competes with the same ions at the membrane. The tetracaine effect is restricted to the procaine series of local anesthetics. Small amounts of soluble AChE are present in the cytosol fraction. In fresh preparations most of the enzyme appeared in a form having a molecular weight of 80.000 daltons as determined by gel filtration. The enzyme seems to be released in this form. It is proposed that this form represents the monomer form of the enzyme. In solution the AChE aggregates seemingly together with a factor that is released from the membrane in amounts stafchio- metric to the enzyme. By treatment with DEAE-Sephadex the enzyme preparation can be made non-aggregating. A highly purified nonaggregating monomeric AChE Specific activity 17150 micromoles acetylthiocholine hydrolyzed per minute at 27° C per mg protein) was obtained by affinity chromatography. Some anomalous binding phenomena was observed during the affinity chromatographic purification. The main observation was that edrophonium eluted crude enzyme preparation adsorbed to the affinity gel in a biphasic manner. It was found that in the crude preparation there is present besides unspecific material competing with the enzyme for the affinity gels a factor that increases the affinity of AChE to certain cholinergic ligands. Since the enzyme could be titrated by the factor it seems to have a very high affinity to the enzyme and the biphasic elution curve is explained by the presence of free as well as factor- bound enzyme in the preparation. In search for compounds having a similar effect it was found that fluoride ion too increased the affinity of AChE to the same ligands as the factor. The affinity of edrophonium to the site defined by the binding of AChE to MTA-CH (65x10“5m) is lower than that defined by the enzyme inhibitory constant (1.8xlO“7M). As an explanation of this finding it is proposed that the substrate induces a conformation having high affinity to edrophonium, a conformation that has a comparatively low relaxation rate. Thus acetylcholinesterase may be added to the list of enzymes that have hystere- tic properties. / <p>S. 1-54: sammanfattning, s. 55-100: 4 uppsatser</p> / digitalisering@umu

Orientadores: Eneida de Paula, Leonardo Fernandes Fraceto / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-16T01:05:29Z (GMT). No. of bitstreams: 1 Lima_RobertaAlineFrancode_M.pdf: 1479897 bytes, checksum: 3c9af4bda36adc886426ab930c957a40 (MD5) Previous issue date: 2010 / Resumo: A dor desempenha um papel biológico muito importante como indicador da presença de um estímulo nocivo, que pode representar dano real ou potencial ao organismo; entretanto, não é interessante que essa sinalização persista além das necessidades fisiológicas a ponto de tornar-se um incomodo, podendo dificultar o desempenho das atividades normais, comprometer a qualidade de vida do indivíduo e impedir a realização de práticas cirúrgicas curativas. Os anestésicos locais são fármacos empregados para alívio da dor aguda ou crônica, pois bloqueiam a condução do estímulo nervoso em membranas neuronais. Dentre eles, está a Tetracaína (TTC) um amino-éster de grande utilidade na prática clínica, empregado em procedimentos que requerem anestesia tópica, como aqueles oftalmológicos que necessitam de intervenções na córnea. As limitações do uso clínico da TTC está relacionadas à sua baixa estabilidade química (hidrólise da ligação éster por esterases plasmáticas) e sua toxicidade sistêmica. Sistemas de liberação modificada de fármacos, que empregam carreadores como ciclodextrinas, constituem uma alternativa para aumentar o tempo de ação anestésica e diminuir a toxicidade. As ciclodextrinas são capazes de formar complexos de inclusão com diversos fármacos, alterando propriedades como a solubilidade aquosa, o que torna esses oligosacarídeos cíclicos promissores para este tipo de aplicação. Neste trabalho, estudamos a complexação da TTC com beta-ciclodextrina (?-CD) e hidroxipropil-beta-ciclodextrina (HP-?-CD) em pH fisiológico e sua influência na toxicidade celular e efeito antinociceptivo, induzidos por aquele anestésico, objetivando a preparação de novas formas farmacêuticas. Para caracterização dos complexos de inclusão TTC:?-CD e TTC:HP-?-CD foram empregados métodos espectroscópicos (Ressonância Magnética Nuclear, fluorescência, absorção UV/VIS), difração de raios-X e calorimetria diferencial de varredura. A partir das análises realizadas, encontrou-se que a estequiometria de complexação da TTC com ambas as ciclodextrinas é de 1:1. A constante de associação aparente da TTC, determinada por DOSY-NMR, é de 777 M-1 e 2243 M-1, para ?-CD e HP-?-CD respectivamente, indicando uma forte interação entre as moléculas de anestésico e das ciclodextrinas, em pH fisiológico. Os dados de proximidade espacial, determinados por RMN (1DROESY) revelaram interações entre os hidrogênios Hh e Hi, (que fazem parte do anel aromático da TTC) e hidrogênios da cavidade interna das ciclodextrinas, permitindo assim propor a geometria dos complexos de inclusão da TTC com ambas ciclodextrinas. Ensaios in vitro, em cultura de fibroblastos da linhagem 3T3, mostraram aumento na viabilidade celular após complexação da TTC com ?-CD e HP-?-CD, em relação à TTC em solução. Testes de atividade biológica, de bloqueio do nervo infraorbital em ratos, demonstraram aumento significativo do tempo de duração da anestesia nos animais tratados com os complexos TTC:?-CD e TTC:HP-?-CD (40 min e 40 min), em relação ao grupo tratado com 0,5% de TTC livre (30 min). Os resultados obtidos neste estudo demonstram a formação de complexos de inclusão com forte interação entre TTC e ?-CD e HP-?-CD; indicam ainda que a utilização destes complexos de inclusão é promissora como forma farmacêutica alternativa para este anestésico, devido ao aumento da duração da anestesia (pois os complexos apresentaram maior eficácia quando comparados à TTC livre nas mesmas doses), ou ainda a diminuição da toxicidade (atingindo a mesma eficácia da TTC livre, com menores concentrações de TTC complexada). / Abstract: Pain plays an important biological role as a sign of the presence of a noxious stimulus, that denotes actual or potential damage to the body. However, it is not interesting this signal to persist beyond the physiological point, to become a nuisance that impairs the performance of normal activities, the quality of life or prevents the procedure of healing surgical practices. Local anesthetics are used for the relief of acute or chronic pain since they block the conduction of the painful stimulation in neuronal membranes. Among them is Tetracaine (TTC) an amino ester useful in clinical procedures requiring local anesthesia such as those ophthalmic ones with intervention in the cornea. The clinical use of TTC is limited by its low chemical stability (hydrolysis of the ester bond by plasma esterases) and systemic toxicity. The use of drug-delivery systems, in carriers such as cyclodextrins is a good approach to improve the duration of action and to diminish the toxicity of local anesthetics. Cyclodextrins are suitable for this type of application since these cyclic oligosaccharides are able to form inclusion complexes with several drugs improving their water solubility. Here we have studied the complexation of TTC with beta (?-CD) and hydroxypropyl-beta-cyclodextrin (HP-?-CD) and its influence in cell toxicity and antinociceptive effects induced by that anesthetic, aiming the development of new pharmaceutical forms. TTC and ?-CD or HP-?-CD inclusion complexes were characterized using spectrophotometric methods (Nuclear Magnetic Resonance, fluorescence, UV-VIS absorption), X-ray Diffraction and Differential Scanning Calorimetry. A 1:1 stoichiometry of complexation was found for both complexes. The association constant determined by DOSY was 777 M-1 and 2.243 M-1 for TTC complexation with ?-CD and HP-?-CD, respectively, indicating a strong association between the anesthetic and both cyclodextrins, at physiological pH. The nuclear overhauser NMR data (ROESY) had disclosed trough the space proximities between hydrogens Hh and Hi - at the aromatic ring of TTC - and hydrogens from the inner cavity of the cyclodextrins, allowing us to suggest the geometry of TTC:?-CD and TTC:HP-?-CD complex formation. In vitro culture tests on 3T3 fibroblasts cells revealed an increase in cell viability after TTC complexation with ?-CD and HP-?-CD, in comparison to the effect of free TTC. Biological activity was assessed in rats, through the infraorbital nerve blockade test. The results revealed a significant increase in the duration of anesthesia in animals treated with TTC:?-CD and TTC:HP-?-CD (40 min and 40 min), in comparison to a solution of 0.5% tetracaine (30 min). The results presented here demonstrate the formation of strong associated inclusion complexes between TTC and ?-CD and HP-?-CD. The in vivo results allow us to consider those complexes as promising pharmaceutical forms to increase the duration of anesthesia induced by tetracaine (since both complexes showed higher anesthetic potency when compared to free TTC at equivalent doses) or to diminish its toxicity (since the same activity can be achieved with lower concentrations of the complexed TTC formulations). / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular