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

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.

No description available.

In this dissertation, I explored the effects of developmental hypoxia on heart contractility in three separate species of ectotherms: the common snapping turtle (Chelydra serpentina), the American alligator (Alligator mississippiensis), and the zebrafish (Danio rerio). I began with the common snapping turtle and tested whether the utilization of the sarcoplasmic reticulum was altered in response to developmental hypoxia. In the next two chapters, developmental hypoxia of the American alligator was explored studying how the cardiac tissue was affected, specifically in physiological stressors, sarcoplasmic reticulum utilization and sensitivity to pharmacological increases in contractility. The last chapter explored how zebrafish heart contractility was altered in response to chronic hypoxia from egg to adult. Findings from these chapters suggest that while developmental hypoxia did alter cardiac contractility, it did not alter the response of the heart to physiological stressors such as increased heart rate or under hypoxia. Overall, these findings contribute to increasing the current understanding of how developmental hypoxia alters the cardiovascular system but with an emphasis on the cardiac tissue level.

La contraction du muscle squelettique est initiée par une libération de Ca2+ du réticulum sarcoplasmique (RS) en réponse à une dépolarisation du sarcolemme. Celle-ci induit un changement de conformation du récepteur des dihydropyridines (DHPR) localisé dans les tubules T entraînant l'ouverture du récepteur de la ryanodine de type 1 (RyR1), canal calcique du RS, et la libération du Ca2+ accumulé dans le RS. Au repos, RyR1 serait maintenu fermé par une action répressive du DHPR. Néanmoins, un efflux de Ca2+ continu se développe à travers la membrane du RS, constamment compensé par l'activité des pompes Ca2+-ATPases. Des études suggèrent que cet efflux pourrait être impliqué dans la perturbation de l'homéostasie calcique dans une des pathologies musculaires des plus fréquentes et sévères, la myopathie de Duchenne. Le travail présenté vise à caractériser l'efflux de Ca2+ du RS dans les fibres musculaires squelettiques de souris normales et mdx, modèle murin de la myopathie de Duchenne, en couplant la technique de potentiel imposé et la mesure fluorimétrique du Ca2+ intracellulaire. La mise au point d'une mesure directe des variations de Ca2+ du RS à l'aide du Fluo-5N a permis de révéler dans les fibres mdx une fuite calcique du RS exacerbée. Cette approche a permis de démontrer que l'efflux calcique du RS dans la fibre musculaire squelettique au repos n'est pas un phénomène incontrôlé à travers RyR1 mais un efflux étroitement contrôlé par le DHPR. Enfin, on s'est intéressée à l'efflux de Ca2+ du RS lors d'une stimulation musculaire prolongée. Nos résultats montrent que le déclin du signal calcique cytosolique dans ces conditions résulterait de la déplétion calcique du RS / Contraction of skeletal muscle is triggered by the release of Ca2+ from the sarcoplasmic reticulum (SR) in response to depolarization of the sarcolemma. Depolarization elicits a conformational change of the dihydropyridine receptor (DHPR) localized in the tubular membrane that controls the opening of the type 1 ryanodine receptor (RyR1), the SR Ca2+ release channel. At rest, RyR1s are kept in a closed state imposed by the repressive action of DHPRs. Yet, a resting Ca2+ efflux occurs across the SR membrane, constantly balanced by the pumping activity of SR Ca2+-ATPases. Several studies suggest that this SR Ca2+ efflux, considered as purely passive, may contribute to the alteration of Ca2+ homeostasis in one of the most common and severe skeletal muscle disease, namely the Duchenne Muscular Dystrophy. The present work aims at characterizing the SR Ca2+ efflux in skeletal muscle fiber from normal and mdx mice, the murine model of Duchenne Muscular Dystrophy, by combining voltage-clamp and intracellular Ca2+ measurements. The development of a methodology allowing direct monitoring of Ca2+ changes in the SR using the Fluo-5N led us to reveal an elevated SR Ca2+ leak in mdx fibers, which may contribute to the alteration of Ca2+ homeostasis. Still using this approach, we demonstrate that the resting SR Ca2+ efflux in normal skeletal muscle fiber is not, an uncontrolled process through RyR1 but is tightly controlled by DHPR. Finally, we investigates the SR Ca2+ efflux during long-lasting stimulation. Our data indicate that the decline of SR Ca2+ release in these conditions results from SR Ca2+depletion and does not involve voltage-dependent inactivation of SR Ca2+ release

Made available in DSpace on 2016-06-02T19:22:10Z (GMT). No. of bitstreams: 1 5716.pdf: 1902370 bytes, checksum: 9a5b25a55be97bb37553eeb1f8130b09 (MD5) Previous issue date: 2014-01-28 / Financiadora de Estudos e Projetos / Phenomena as feeding and thermoregulation can challenge both the cardiac output and heart rate (fH). Postprandial changes in many physiological patterns are typically recorded for ectotherms, although predominantly studied in snakes. Furthermore, thermoregulation is a critical process to ensure different survival and reproduction strategies from reptiles. Ventricular preparations in vitro were used to investigate the inotropic plasticity at 30 ºC and 25 ºC and during fasting (30 days fasting group) and after feeding (2 days digesting group). No significant differences were verified in the relative ventricular mass (RVM) after feeding. The peak tension (Fc) was significantly higher and rates (dFc/dt) of contraction (TC) and relaxation (TR) were faster at 25° C than at 30° C for both experimental groups. However, feeding caused significant increase in peak tension (PT) at both temperatures (396.7 % at 30 ºC and 416.7 % at 25 ºC). A significant post-rest potentiation was recorded for digesting group at 25 ºC (121.0 ± 6.4 %), which was inhibited by ryanodine (86.0 ± 2.9 %). At 30 ºC, myocytes from fasting group were able to support higher frequencies (up to 1.6 Hz) than at 25 ºC (up to 1.0 Hz), even with the sarcoplasmic reticulum (SR) blocked by 10 &#956;M ryanodine (RYA). Data from digestion group showed greater values of the Fc and reached higher frequencies than fasting group, at both temperatures. However, pre-treatment with RYA decreased PT in frequencies between 1.4 and 1.8 Hz for digesting group at 30 ºC. Sarcolemma mechanisms were more representative in the fasting group at 25 ºC, but the SR must contribute to increased speed of contractility under those conditions. During SDA, the SR seem representative at 25 ºC, however the positive inotropic effect caused by SDA enlarged sarcolemma mechanisms representativeness. The SR significance at 30 °C seems critical during high frequencies. At this temperature, the SDA increases the contractility efficiency, even with the RS blocked, despite the relaxation and Fc development at high frequencies been present impaired. Our results indicate that Caiman latirostris myocytes show large inotropic plasticity at different temperatures, frequencies and metabolic states. / A digestão e a termorregulação são processos que podem desafiar tanto o débito quanto a frequência cardíaca (fH). Alterações pós-prandiais em diferentes padrões fisiológicos são comumente identificadas para ectotérmicos, embora sejam predominantemente estudados em serpentes. Além disso, a termorregulação é um processo crítico para garantir diferentes estratégias de sobrevivência e reprodução nos répteis. Preparações ventriculares in vitro foram utilizadas para investigar a plasticidade inotrópica dos miócitos ventriculares do Caiman latirostris a 25 e 30 °C, durante o período pós- absortivo (30 dias - após a alimentação) e após a alimentação (2 dias - grupo digestão). A massa ventricular relativa (MVR) após 2 dias da alimentação não apresentou diferenças significativas em relação ao grupo pós-absortivo. A força de contração (Fc) foi significativamente maior e as taxas (dFc/dt) de contração (TC) e relaxamento (TR) foram mais rápidas a 25 °C do que a 30 °C, para ambos os grupos experimentais. No entanto, a alimentação causou aumento significativo na força de contração (Fc) em ambas as temperaturas (316.7% a 30 °C e a 296.7% 25 °C). Uma significativa potenciação pós-pausa foi identificada no grupo digestão a 25 °C (121.0 ± 6.4 %), que foi inibida pela rianodina (86.0 ± 2.9 %). No grupo pós-absortivo a 30 °C, os miócitos foram capazes de suportar frequências de estimulação superiores (até 1.6 Hz) às suportadas a 25 °C (até 1.0 Hz), mesmo com a função do retículo sarcoplasmático (RS) bloqueada pela adição de 10 &#956;M de rianodina (RYA). As preparações ventriculares do grupo digestão apresentaram valores maiores de Fc e alcançaram frequências superiores do que o grupo pós-absortivo, em ambas as temperaturas. Contudo, o pré-tratamento com RYA reduziu a Fc em frequências entre 1,4 e 1,8Hz no grupo digestão a 30 °C. Os mecanismos sarcolemais são mais representativos no grupo pós-absortivo a 25 °C, porém o RS pode contribuir com o aumento da velocidade da contratilidade nessas condições. Durante a SDA, o RS parece representativo nessa condição de temperatura, porém a SDA causou efeito inotrópico positivo e ampliou a representatividade dos mecanismos sarcolemais. A importância do RS a 30 °C parece crítica apenas em altas frequências de estimulação. Nessa temperatura, a SDA garante a eficiência da contratilidade mesmo com o RS bloqueado, apesar do prejuízo significativo no desenvolvimento da Fc e do relaxamento em altas frequências. Nossos resultados indicam que os miócitos ventriculares do Caiman latirostris apresentam ampla plasticidade inotrópica, capaz de assegurar o débito cardíaco em diferentes temperaturas, frequências e estados metabólicos.

Abstract Skeletal muscle cells (myofibers) are huge multinucleated cells responsible for muscle contraction and hence for the everyday movements of the joints. The structure of these voluminous cells differs greatly from that of the mononucleated cells – the characteristic features of the myofibers include dozens of peripherally located nuclei, tightly packed contractile apparatus and a sophisticatedly organized endomembrane system. The basic physiology involving myofibers is quite well known, but scarce data exist on the membrane biology of the myofibers. The purpose of this study was to examine the localization of mRNA and the site of protein synthesis in the myofibers. The characterization of the membrane dynamics in muscle cells was also performed. In this study we utilized a primary cell culture model obtained from the rat flexor digitorum brevis (FDB) muscle. Also frozen sections from the rat extensor digitorum longus muscle were used. The precursor cells of the myofibers – myoblasts and myotubes – were also utilized in some experiments. Furthermore, methods of immunohistochemistry and molecular biology were applied extensively in this study. We found that in FDB myofibers the mRNA lies just under the plasma membrane. Protein synthesis seemed to be concentrated in the vicinity of nuclei locating beneath the plasma membrane but also in interfibrillar dot-like structures. Protein products moved hundreds of micrometers away from the nuclei of origin. Moreover, there were no barriers for protein movement into the core regions of the myofibers. Movement of proteins was found to be rapid in the cytosol and in the endomembrane system, too. Interestingly, when examining exocytic trafficking we observed that ER-to-Golgi trafficking significantly differed from that of mononucleated cells. Finally, myofibers were found to be able to generate lipid bodies under stress conditions. The dynamics of lipid bodies seemed to deviate from the dynamics found in other cells types. Nowadays not much muscle research with primary myofibers is done worldwide, and therefore dilemmas involving myofibers such as insulin resistance and myotoxicity of statins are mostly unresolved. The knowledge gained from this study may be used in the future to solve clinical problems related to the cell biology of the myofibers. / Tiivistelmä Luurankolihassolut eli myofiiberit ovat jättimäisiä monitumaisia soluja, jotka vastaavat lihassupistuksen aikaansaamisesta ja siten mahdollistavat jokapäiväisen liikkumisemme. Näiden suurten solujen rakenne poikkeaa selkeästi yksitumaisten solujen rakenteesta: myofiiberien tunnusomaisia piirteitä ovat kymmenet solun reunoille sijoittuneet tumat, tiiviisti pakkautunut supistumiskoneisto ja monimutkaisesti järjestynyt solukalvostojärjestelmä. Vaikka myofiiberien perusfysiologia tunnetaankin hyvin, niin tiedetään itse myofiiberien kalvostobiologiasta sangen vähän. Kokonaisuutena tämän tutkimuksen tarkoituksena oli tarkastella mRNA:n ja proteiinisynteesin sijaintia myofiibereissä. Lisäksi selvitimme lihassolujen kalvostodynamiikkaa. Tässä tutkimuksessa käytimme rotan flexor digitorum brevis (FDB) -lihaksesta saatua primääristä soluviljelymallia. Lisäksi hyödynsimme rotan extensor digitorum longus -lihaksesta hankittuja jääleikkeitä. Joissakin kokeissa käytimme myös myofiiberien esiastesoluja (myoblasteja ja myotuubeja). Immunohistokemian ja molekyylibiologian menetelmiä sovellettiin tutkimuksessa laajasti. Havaitsimme, että FDB –myofiibereissä mRNA sijaitsee aivan solukalvon alla. Proteiinisynteesi vaikutti olevan keskittynyt solukalvon alla sijaitsevien tumien ympärille, mutta myös solusisäisiin pistemäisiin rakenteisiin. Proteiinituotteet ylsivät satojen mikrometrien päähän alkuperäisestä tumastaan. Lisäksi proteiineille ei ilmennyt leviämisestettä myofiiberin sisäosiin. Leviämisen havaittiin olevan nopeaa sekä solulimassa että solulimakalvostoissa. Tutkiessamme solun eritystoimintaa huomasimme, että kuljetus ER:stä Golgin laitteeseen eroaa huomattavasti yksitumaisten solujen vastaavasta kuljetuksesta. Lopuksi havaitsimme myofiiberien pystyvän muodostamaan rasvapisaroita rasitusolosuhteissa. Rasvapisaroiden käyttäytyminen näytti myös poikkeavan siitä, mitä muissa soluissa on havaittu. Nykyään lihastutkimusta primäärisoluilla ei juuri tehdä maailmalla, minkä vuoksi myofiibereihin liittyvät lääketieteelliset pulmat kuten insuliiniresistenssi ja statiinien lihashaitat ovat suurelta osin ratkaisematta. Tästä tutkimuksesta saatuja tuloksia voitaneen jatkossa käyttää myofiiberien solubiologiaan liittyvien kliinisten ongelmien selvittämiseen.

Abstract The function of skeletal muscle is to generate force and produce movement. These tasks are carried out by long multinucleated cells, the skeletal myofibres. The membrane system and the cytoskeleton of these cells are uniquely organized to respond rapidly to neuronal stimuli and to achieve efficient contraction. In the present study the organization and distribution of selected protein/lipid based microdomains that reside in the plasma membrane and sarcoplasmic reticulum of isolated rat skeletal myofibres, were investigated. Aquaporin 4 (AQP4) water channels are arranged as higher order oligomers of several sizes in the sarcolemma and in the T tubules. These oligomers, however, were absent from many specialized micro- and- macrodomains. The distribution of AQP4 coincided with that of a highly organized protein assembly, the dystrophin glycoprotein complex (DGC), in the sarcolemma. A chimaeric venus-AQP4 was equally mobile in the T tubules and sarcolemma, but the anchoring mechanisms of the protein appeared to be different. In contrast to AQP4, the proteins resident in cholesterol and sphingolipid-based microdomains, known as rafts, also occupied DGC deficient areas, which surround the T tubule openings. Indeed, flotillin-1 rafts were located in the neck portions of the T tubules. The rafts defined by the influenza haemagglutinin (HA) also resided in DGC deficient areas, but at the borders of the DGC area. Importantly, of the raft proteins, only the localization of caveolin 3 (CAV3) was dependent on the cholesterol enriched lipid environment, as evidenced by cholesterol depletion experiments and localization studies on a non-raft associated variant of HA. The organization and distribution of membrane associated rough ER (RER) proteins were also analysed. Biochemical detergent extraction analyses and immunofluorescence staining indicated that the ER proteins were assembled as microdomains within the sarcoplasmic reticulum (SR). The microdomains were distributed throughout the SR network and they were capable of protein translocation. Taken together, skeletal myofibres comprise visually distinct microdomains both in the plasma membrane and in the SR. In the plasma membrane, different types of microdomains are not homogenously distributed and function in diverse locations. This may have important physiological implications concerning, among other things, local regulation of ion concentrations and cell signalling cascades. Different constraints ranging from protein-protein interactions to the surrounding lipid environment are important for dictating the observed distribution patterns. / Tiivistelmä Luustolihaksen toimintojen perustana ovat supistumiskykyiset lihassolut, joiden kalvorakenne on järjestynyt erityisellä tavalla ohjaamaan supistusta. Tässä tutkimuksessa analysoitiin proteiini- ja lipidiperustaisten mikroalueiden järjestäytymistä ja tähän vaikuttavia tekijöitä luustolihassolun solukalvolla sekä lihassolun sisäisessä kalvojärjestelmässä, sarkoplasmisessa verkossa (SR). Ensin analysoitiin vesikanavatyyppiä 4 (AQP4), joka oligomerisoituessaan muodostaa erikokoisia mikroalueita. Havaittiin, että AQP4-mikroalueita esiintyy kaikkialla solukalvolla lukuun ottamatta eräitä erilaistuneita mikro- ja makroalueita. AQP4-oligomeerien jakauma solukalvon lateraalisessa osassa, sarkolemmalla, noudatti dystrofiini-glykoproteiinikompleksin jakaumaa. Fluoresoivan venus-AQP4-proteiinin avulla osoitettiin, että proteiinin liikkuvuus oli samanlainen solun sisään ulottuvissa poikkiputkistoissa ja sarkolemmalla, mutta liikkuvuutta rajoittavat tekijät olivat erilaisia näissä solukalvon osissa. Toiseksi analysoitiin kolesteroli- ja sfingolipidipitoisia mikroalueita, kalvolauttoja. Flotilliini-1- ja influenssaviruksen hemagglutiniini (HA) -proteiinia sisältäviä lauttoja esiintyi vain poikkiputkien suuaukkojen alueella, mutta lauttojen jakauma oli erilainen. Lauttojen lipidiympäristöllä ei ollut vaikutusta proteiinien sijaintiin. Tämä osoitettiin kolesterolin poistokokeilla sekä kokeilla, joissa käytettiin mutatoitua HA-proteiinia, joka ei hakeudu kolesteroliympäristöön. Kaveoliini-3-proteiinin sijainti poikkeaa edellä mainituista, ja kolesterolin poisto vaikutti merkittävästi sijainnin määräytymiseen. Kolmanneksi analysoitiin, miten karkean endoplasmakalvoston proteiinit ovat järjestäytyneet SR:ssä. Havaittiin, että endoplasmiset kalvoproteiinit eivät ole homogeenisesti levittäytyneet SR-kalvostoon vaan muodostavat pieniä mikroalueita. Detergenttiuuttoanalyysit osoittivat lisäksi, että näissä mikroalueissa on erilainen lipidikoostumus kuin SR:ssä yleensä. Huomattavaa oli myös, että mikroalueet olivat toiminnallisia kaikkialla SR-kalvostossa. Tulosten perusteella luustolihassolujen kalvojärjestelmä sisältää mikroalueita, joiden jakautuminen vaikuttaa hyvin organisoituneelta. Erityisesti solukalvon mikroalueet esiintyvät tietyillä spesifeillä alueilla, joissa niiden voidaan olettaa toimivan mm. erilaisissa solusignalointitapahtumissa ja paikallisessa ionipitoisuuksien säätelyssä. Eräissä tapauksissa lipidiympäristöllä on merkitystä mikroalueiden sijainnin määräytymisessä, mutta proteiinien sitoutuminen solukalvo- tai solukalvon alaisiin rakenteisiin saattaa myös olla määräävä tekijä.