Thesis (Ph. D. in Physics)--Vanderbilt University, May 2006. / Title from title screen. Includes bibliographical references.

Thesis (M.S.)--University of Delaware, 2005. / Principal faculty advisor: Norman J. Karin, Dept. of Biological Sciences. Includes bibliographical references.

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

The sarcoplasmic reticulum is an internal membrane system that controls the Ca²⁺ concentration inside muscle cells, and hence the contractile state of both skeletal and cardiac muscle. A key protein that that regulates the Ca²⁺ concentration in this membrane is known as the calcium release channel (CRC). The effects on Ca²⁺ dependent activation is of major importance in the study of CRC since other channel modifiers cannot effect the channel in the absence of Ca²⁺, or they require Ca²⁺ for maximum results. In this study of the high-affinity Ca²⁺ binding site, expected increases in total binding and shifts in the sensitivity of the channel to Ca²⁺ were observed when the pH increased or the solution redox status became more oxidative. Ranolazine, a drug used for treating Angina Pectoris (chest pain), desensitized the cardiac CRC activation but had no effect on the skeletal CRC. This selective desensitization may be the cause of Ranolazine's beneficial therapeutic effects. Both Ranolazine, and homocystein thiolactone (HCTL), a naturally occurring derivative of homocysteine, alters Ca²⁺ dependent activation by calcium without changing the number of channels found in the open state. Surprisingly the effect of HCTL was observed only in a reduced redox potential which leads to speculation that the formation of an alpha-carbon radical by HCTL on the cardiac CRC only occurs if select thiols are in a reduced state.

The purpose of this study is the characterization of the membrane-water interfaces of both sarcoplasmic reticulum membrane (SR) and charged lipid bilayers under varied properties of the surrounding aqueous solution. In this work we studied the electrokinetic properties of liposomes and SR vesicles as well as the interaction of lipophilic ions with these membranes. The study of electrokinetic properties is based on the measurements of electrophoretic mobility of SR membrane vesicles and PC/PG liposomes. Electrophoretic mobility of SR vesicles was measured as a function of ionic strength for six pH values (pH 4.0, 4.7, 5.0, 6.0, 7.5, and 9.0). Electrophoretic mobility of single-layered and multi-layered PC/PG liposomes was measured at neutral pH as a function of ionic strength. For interpretation of electrophoretic mobility studies, SR vesicles (at pH 4, 7, and 9) and multi-layered and single-layered liposome sizes were determined using photoelectron microscopy. The study of the interaction of lipophilic ions with these membranes is based on (1) measurements of their partition coefficients described in terms of an ion partition model based on the Langmuir adsorption model and (2) electrophoretic mobility measurements of SR vesicles and PC liposomes in suspension with varied concentration of lipophilic ions. SR-water and PC-water partition coefficients were measured as a function of concentration for two anions tetraphenylborate (TePB-) and pentabromophenol (PBP-) and two cations (Imipramine+, and Clomipramine+). The anions belong to a class of pesticides and the cations are drugs once prescribed as anti-depressants. Partition into the SR membrane was shown to be significantly greater for all lipophilic ions except TePB-, which only showed this effect at the higher lipophilic ion range of the data. The PC-water partition coefficient was also measured for TePP+. Since the lipid bilayer of SR is not significantly different than that of PC liposomes, we believe the differences in partition are due to excess lipophilic ions being absorbed to the proteins of SR. The electrokinetics of charged PCPG liposomes, and PC liposomes with absorbed lipophilic ions could be understood in terms of the charge being located below their surface and screened by counter-ions inside the polar head-group region. We call this model the "permeable surface model". The assumptions of this model are that (1) the charge exists on a plane at a depth, d, below the surface of the liposome within the lipid head-group region and (2) small ions (Na+, K+, Cl-) are able to penetrate the lipid head-group region with a molar membrane-water partition coefficient of 0.4. Using this model we were able to obtain the depth of sorption of lipophilic ions in PC liposomes. We found values of 0.13 nm for TePB-, 0.5 nm for PBP-, 0.12 nm for Imipramine+, 0.17 nm for Clomipramine and 0.25 nm for TePP+. The depth of lipophilic ions in PC is a valuable quantity for the study of the effect of lipophilic ions on membrane function. For PCPG mobility we found the charged plane due to PG lipids was 0.2 nm for single-layered liposomes and 0.1 nm for multi-layered liposomes. This is consistent with the relative size of PC and PG head groups The dependence of SR mobility on pH was found to be directly correlated with the total charge of the A, P, and N domains of the Ca2+-ATPase as determined by the amino acid residues and their corresponding pKa values in water. We found that detached charged plane model, a new model developed in our group, could be fit to the mobility of SR as a function of ionic strength while other soft particle models failed. The assumptions of this model are that (1) the friction caused by protruding proteins on the surface of SR can be represented by a homogeneous retardation layer of thickness D and softness parameter λRL, and (2) the charge of the APN domain can be represented as a plane of charge embedded in the retardation layer at a distance s from the membrane surface. The best-fit values for λRL, and s were not consistent for different pH value studies. The detached charged plane model was unable to predict the mobility of SR vesicles in the presence of lipophilic ions if we assumed that the lipophilic ions were sorbing to the detached charged plane that represents the native charge of the APN domains of SR. At high lipophilic ion concentration the experimental mobilities consistently were greater in magnitude than the values predicted by the model. We concluded that there is significant absorption of lipophilic ions to the proteins in SR membrane, and that the lipophilic ion sorption sites are not the same as the detached plane of charge that represents the native charge of the APN domain.

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第20059号 / 薬科博第66号 / 新制||薬科||8(附属図書館) / 京都大学大学院薬学研究科薬科学専攻 / (主査)教授 竹島 浩, 教授 中山 和久, 教授 根岸 学 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

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No description available.

Feeding high levels of distillers grains increases polyunsaturated fatty acid (PUFA) levels in beef. It is well stablished that beef with higher concentrations of PUFA is more likely to have increased lipid and myoglobin oxidation. This is important because lipid and myoglobin oxidation lead to off-flavor development and discoloration of retail-displayed beef, reducing display life. In the first study, the effects of feeding different dietary fat sources with modified distillers grains plus solubles (MDGS) on beef display life were evaluated. Results suggest that feeding MDGS to cattle reduces color and lipid stability in addition to increasing C18:2 and PUFA content of beef in comparison to the corn diet. Thus, feeding MDGS to cattle has the potential to reduce beef display life. Perhaps, MDGS in feedlot diets increases PUFA concentration in the sarcoplasmic reticulum (SR) membrane, thereby altering membrane integrity, resulting in more rapid calcium leakage and improved tenderness. Therefore, the second study was dedicated to evaluate the effects of dietary fat source on the basic mechanism of beef tenderization. Feeding MDGS to cattle increased 18:2 and tended to increase PUFA concentration in the SR membrane, in addition to increase free calcium at d 2 postmortem in comparison to the corn diet. Beef from cattle finished on de-oiled MDGS and de-oiled MDGS plus oil had lower Warner-Bratzler shear force values than beef from cattle finished on corn at 2 d postmortem. No differences among dietary treatments were found for sarcomere length and troponin T degradation at 2 d postmortem. The results from this study suggest that feeding MDGS may increase tenderness, possibly by increasing free calcium in muscle early post-mortem. However, the true mechanism by which dietary fat source may accelerate the beef tenderization process is still unclear and should be further explored. / De acordo com a literatura, a adição de resíduo de destilaria com solúveis em dietas de terminação de bovinos de corte aumenta a quantidade de ácidos graxos poliinsaturados (PUFA) na carne bovina. É sabido que quanto maior a concentração de PUFA na carne, maior será a probabilidade de ocorrência da oxidação lipídica e da mioglobina. Isso é importante porque o aumento da oxidação lipídica e da mioglobina acelera o desenvolvimento de odores indesejáveis e da descoloração da carne, reduzindo assim sua vida útil. O objetivo do primeiro estudo foi avaliar os efeitos do uso de diferentes resíduos de destilaria parcialmente desidratados com solúveis (MDGS) na vida útil da carne. Os resultados deste estudo sugerem que a inclusão de 40% de MDGS na dieta de terminação de bovinos pode reduzir a estabilidade lipídica e da cor, além de aumentar a quantidade de ácido linoléico (18:2) e PUFA da carne em comparação à carne de animais terminados com milho, reduzindo a vida útil da carne. No segundo estudo, nós trabalhamos com a hipótese de que a adição de elevadas quantidades de MDGS em dietas de terminação poderia aumentar a concentração de PUFA na membrana do retículo sarcoplasmático, e assim, alterar a integridade e permeabilidade dessa membrana, antecipando a liberação de cálcio postmortem. Com mais cálcio disponível, a proteólise muscular seria favorecida, aumentando a maciez da carne nos primeiros dias após o abate. Portanto, o objetivo do segundo estudo foi avaliar os efeitos da fonte de gordura dietética contida em diferentes tipos de MDGS na maciez da carne bovina. A inclusão de 40% de MDGS na dieta de terminação de novilhos aumentou a concentração de 18:2 e tendeu a aumentar a concentração de PUFA na membrana do retículo sarcoplasmático em comparação ao gado terminado com milho (sem inclusão de MDGS). Carne de animais terminados com MDGS apresentou maior quantidade de cálcio livre no sarcoplasma 48 horas após o abate. Carne de novilhos alimentados com MDGS desengordurado apresentou menor força de cisalhamento 48 horas após o abate quando comparada à carne de animais terminados com milho. Não houve diferença significativa entre nenhum dos tratamentos dietéticos 48 horas após o abate para comprimento de sarcômero e degradação de troponina T. Os resultados deste estudo sugerem que a inclusão de 40% de MDGS desengordurado na dieta de terminação de novilhos aumenta a maciez da carne 48 horas após o abate, possivelmente por aumentar a quantidade de cálcio livre no sarcoplasma. No entanto, o mecanismo pelo qual a fonte de gordura dietética acelerou a proteólise muscular ainda não está completamente elucidado.

Orientadores: José Wilson Magalhães Bassani, Rosana Almada Bassani / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Elétrica e de Computação / Made available in DSpace on 2018-08-17T02:35:28Z (GMT). No. of bitstreams: 1 Ricardo_RafaeldeAlmeida_D.pdf: 1783057 bytes, checksum: 51a4ed51fbdbcd3a024bf9f16c44793c (MD5) Previous issue date: 2010 / Resumo: A relação força-freqüência é uma característica intrínseca do músculo cardíaco, e se manifesta de diferentes formas, dependendo da espécie, e seus mecanismos ainda não estão totalmente esclarecidos. Um possível mecanismo é a modificação da liberação de Ca2+ pelo retículo sarcoplasmático (RS), principal fonte do íon para ativar a contração em miocárdio de mamíferos. O principal objetivo deste trabalho foi determinar a relação entre a liberação fracional de Ca2+ do RS (FR) e a freqüência estimulatória em miócito ventricular isolado de rato. O potencial de ação (PA) representativo medido em cada freqüência de interesse foi registrado com a técnica perforated patch clamp em temperatura ambiente (23°C). A corrente de Ca2+ tipo L (ICaL) e o transiente de Ca2+ (D[Ca2+]i, medido com indo-1) foram registrados simultaneamente por meio da técnica action potential clamp. A FR em cada freqüência foi considerada como a relação entre a diferença entre variação total de [Ca2+] e a integral de ICaL, e a carga de Ca2+ do RS ([Ca2+]RS). A duração do PA a 90% de repolarização (APD90) aumentou de 76 ± 9 ms em 0,2 Hz para 130 ± 11 ms em 2 Hz (p<0,05). Em 0,2 Hz, o pico de ICaL foi -4,5 ± 0,7 pA/PF e, com o aumento da freqüência para 2 Hz, foi reduzido para -2,7 ± 0,4 pA/pF (p<0,05). A D[Ca2+]i caiu de 603 ± 56 para 408 ± 32 nM (p<0,05), em 0,2 e 2 Hz, respectivamente. A FR também diminuiu com o aumento da freqüência, de 0,81 ± 0,02 em 0,2 Hz para 0,54 ± 0,02 em 2 Hz (p<0,01), enquanto a [Ca2+]RS permaneceu constante para todas as freqüências utilizadas. O aumento da freqüência estimulatória reduziu a D[Ca2+]i possivelmente devido à redução da FR. É possível que a redução do pico de ICaL, que é o principal trigger de liberação de Ca2+ do retículo sarcoplasmático, seja a principal causa da redução da FR. / Abstract: The force-frequency relationship is an intrinsic characteristic of cardiac muscle. It is manifested in different ways depending on the species, and its mechanisms still remain to be completely understood. A possible mechanism is the variation of Ca2+ release by the sarcoplasmic reticulum (SR), the main source of the ion for contraction activation in mammalian myocardium. The main goal of this work was to determine the relationship between the fractional SR Ca2+ release (FR) and stimulatory frequency in isolated rat ventricular myocytes. Action potentials (AP) were recorded at 23ºC using the perforated whole-cell patch-clamp technique at different rates, and the representative AP at each rate was used later as stimulus waveforms (action potential clamp). L-type Ca2+ current (ICaL) and intracellular Ca2+ transient (D[Ca2+]i, measured with indo-1) were recorded simultaneously using action potential clamp. FR at a twitch was assumed to be the ratio of the systolic variation in total [Ca2+] minus ICaL integral, and the SR content ([Ca2+]SR). AP duration at 90% repolarization (APD90) increased from 76 ± 9 ms at 0.2 Hz to 130 ± 11 ms at 2 Hz (p<0.05). At 0.2 Hz, ICaL peak was -4.5 ± 0.7 pA/pF, and it decreased to -2.7 ± 0.4 pA/pF with increasing frequency to 2 Hz (p<0.05). D[Ca2+]i decreased from 603 ± 56 nM to 408 ± 32 nM (p<0,05), at 0.2 and 2 Hz, respectively. FR also decreased with frequency, from 0.81 ±0.02 at 0.2 Hz to 0.54 ±0.02 at 2 Hz (p<0.01), whereas [Ca2+]SR did not change significantly. The negative relationship between Ca2+ transient amplitude and stimulation rate is likely to be due to the observed decrease in SR Ca2+ release. Lower FR at higher rates might be due to decrease in ICaL peak, which is the main trigger of SR Ca2+ release. / Doutorado / Engenharia Biomedica / Doutor em Engenharia Elétrica