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Los resultados permiten concluir que la abundancia de isoformas rápidas de MyHC en el miocardio de la región cardiaca de entrada y de isomiosinas lentas en la región cardiaca de salida es una sinapomorfía de los gnatostomados. Además, se han identificado dos isomiosinas (MYH2 y MYH7B) adicionales a las MyHC cardiacas convencionales descritas en tetrápodos y teleósteos (MYH6 y MYH7) y se propone un modelo sobre la evolución de su expresión en distintos segmentos del corazón de los vertebrados. La distribución de estas isoformas en el miocardio del segmento atrioventricular, interpuesto entre el miocardio de las regiones de entrada y de salida, sugiere su función en la sincronización de la contracción cardiaca en vertebrados que carecen de un sistema de conducción especializado, como es el caso de S. canicula. La tercera y última cuestión trata sobre el desarrollo del miocardio ventricular en el corazón primitivo de los gnatostomados. Hasta la fecha se han descrito dos modelos de formación de las trabéculas cardiacas (trabeculación) durante el desarrollo embrionario: uno en teleósteos, que en general poseen un miocardio ventricular trabeculado, y el otro en tetrápodos, con una mioarquitectura compacta. Los resultados obtenidos han puesto de manifiesto que en S. canicula, con un miocardio mixto, su trabeculación se asemeja a la de los tetrápodos, con mioarquitectura compacta. Por lo tanto, se propone que el mecanismo de trabeculación se ha conservado desde grupos basales de vertebrados hasta aves y mamíferos, mientras que, en los teleósteos, con mioarquitectura trabeculada, este mecanismo es derivado. Actualmente se acepta que el corazón de los condrictios refleja fielmente la condición primitiva de los gnatostomados. En la presente tesis se han estudiado diferentes aspectos de la morfología, la composición molecular y el desarrollo embrionario del corazón de los condrictios. Adicionalmente, se ha comparado cada uno de estos aspectos con respecto a otros grupos de vertebrados, con el objetivo de indagar en la evolución de la mioarquitectura y la vascularización del corazón de los gnatostomados. La primera cuestión versa sobre la histomorfología del miocardio ventricular en los gnatostomados. Se describió la mioarquitectura ventricular de cuatro especies de holocéfalos: Chimaera monstrosa, Hydrolagus affinis, Harriotta raleighana y Rhinochimaera atlantica. Los holocéfalos constituyen el grupo hermano de los elasmobranquios, junto con los que conforman los condríctios, el grupo hermano del resto de gnatostomados. Los resultados obtenidos confirman que la mioarquitectura mixta debe ser considerada como la condición primitiva en gnatostomados. Por lo tanto, las mioarquitecturas ventriculares compacta y trabeculada son condiciones derivadas. Además, la peculiar vascularización cardiaca en algunas especies estudiadas ha permitido formular nuevas hipótesis sobre la asociación entre mioarquitectura e irrigación coronaria y sobre la variabilidad de tipos morfológicos de mioarquitectura en los gnatostomados. La segunda cuestión pivota sobre la composición en isoformas de cadena pesadas de miosina (MyHC) del corazón de los gnatostomados. Se realizó, por una parte, análisis proteómicos del miocardio del elasmobranquio Scyliorhinus canicula, y por otra, estudios inmunohistoquímicos en dicha especie y en otras pertenecientes a grupos representativos de gnatostomados.

Neste trabalho foi isolado e caracterizado parcialmente uma fração de testículo de coelho em fosfocelulose enriquecida em atividade ATP/GTPase. Os testículos foram homogeneizados em tampão I (Imidazol-Cl 50 mM, EDTA 10 mM, EGTA 10 mM, DTT 1.0 mM, PMSF 0.3 mM e benzamidina 1 mM) pH 8.5 e centrifugado a 15.000g x 40 min. O sobrenadante foi aplicado em coluna de DEAE-Sepharose. Proteínas retidas foram eluídas com tampão Imidazol-Cl 50 mM, pH 6.5 e o eluato foi aplicado em coluna de fosfocelulose. A fração enriquecida em atividade ATPase foi obtida eluindo-se a coluna com tampão Imidazol-Cl, pH 8.0. Esta atividade não foi retida por uma coluna de GTP-aAga frroasçeã.o isolada de testículo de coelho possui as seguintes propriedades. 1) Em altas concentrações (mM) de substrato, a atividade GTPase é maior do que a ATPase. Em baixas concentrações os dois nucleotídeos são hidrolisados indistintamente. 2) Ambas as atividades ocorrem como de Ca++. Nos dois casos, a atividade Ca/Nucleotidase foi maior do que a atividade Mg/Nucleotidase. 3) Diferentemennte de uma fração correspondente obtida a partir de cérebro de rato (Coelho, 1988), a atividade MgATPase não Ca++/CaM. 4) A atividade ATPase é estável em baixa temperatura (4 «C/7 dias), mas é totalmente perdida quando aquecida (75 °C/ 5') ou congelada (-20 »C). 5) Não possui atividade (K-EDTA)ATPase 6) A atividade MgATPase não foi sensível a altas concentrações de sal (NaCl ou KC10 3 M) Triton X-100 0.2%, azida 1 mM ou NaVO, 250 pM. Contudo, foi inibida cerca de 80% por A1F3 (NaF 2.4 mM/ A1C1, 0.1 mM) e 35% na Presença de Na VO, 1 mM ou NaF 5 mM. No gel, observou-se que os polipeptídeos: 160, 100, 74 e 48 kDa apareceram no "flow through" da coiuna de GTP-agarose juntamente com a r • cAmplhante a atividade da fração PC. No eluato atividade MgATPase que foi semelhan r «ujpnq- 32 e 16 kDa, que não apresentaram detectou-se sómente dois polipeptídeos. apresentou estimulação poratividade MgGTPase. Estes dois polipeptídeos podem corresponder às pequenas proteínas G citoplasmáticas, que participam na organização do citoesqueleto e controle do transporte vesicular. Os resultados aqui apresentados mostram a caracterização parcial de uma Mg/Ca nucleotidase trifosfatásica de testículo de mamíferos e levanta a possibilidade de estudo de proteínas relacionadas as vias de mecanotransdução e transdução de sinal. In this work we isolated and partially characterized a fraction of rabbit testis containing the ATPase/GTPase activity by using a phosphocellulose column. Testis was homogenated in 50mM Imidazole pH 8.5 with 10 mM EDTA/EGTA, 1.0 mM DTT, 0.3 mM PMSF, 1.0 mM benzamidine, and then centrifuged by 15.000g for 40 min. The supematant was loaded in DEAE-Sepharose column. Bound proteins were eluted with 50 mM Imidazole pH 6.5 and the eluate was directly loaded in a phosphocellulose column. The fraction containing ATPase activity was then eluated with 50 mM Imidazole pH 8.0. No proteins were retained in GTP-agarose column. The isolated fraction presented the following properties. 1) At high concentrations (mM) of ATP or GTP, GTPase larger than ATPase activity. At low substrate concentrations both nucleotides are indistinctly hidrolyzed. 2) Both activities were dependent on the presence of Mg++ or Ca++. In both cases, the Ca++-nucleotidase was larger of than Mg++-nucleotidase activity. 3) Ehfferenttly from the corresponding fraction isolated from rat brain (Coelho, 1988), Mg++ATPase activity presented no stimulation by Ca++-CaM. 4) ATPase activity is well preserved when stored at 4 to 10 «C by one week, but not at 75 »C by 5 minutes or in a freezer at -20 °C. 5) (K+-EDTA)ATPase activity was not detected. 6) Mg-ATPase activity was not sensitive to high salt concentrations (0.3 M KC1 or NaCl), 0.2 % Triton X-100, 1.0 mM azide w 250 nM NaVO3. However, it was 80% inhibited by A1F3 (2.4 mM NaF/ 0.1 mM AlClj) and 35 % in presence ofthe 1 mM NaVO3 or 5 mM NaF. . 1 4. tn GTP-agarose column were analyzed in Some polypeptides not bound to Utr agaiu , O0 J60 100 74 e 48 kDa bands. The column SDS-PAGE and caractenzed as 160, . ^tained just two polypeptides, with 32 and 16 kDa, that didn’t e i ite SMÍ.Í ilial ” lhe vesicle transport. The proteins here described may have a participation in mechanotransduction and signalization events, for what they still need more investigation. Dissertação (Mestrado)

Recentemente obtivemos uma fração enriquecida em atividade Mg2+-ATPásica a partir de citosol congelado de cérebro de rato. Nosso objetivo neste trabalho foi analisar esta ATPase e caracterizá-la. Nossos procedimentos foram extração de cérebro de rato, homogeneização imediata em tampão Imidazol-HCI 50 mM pH 8,0; EDTA 10 mM; DTT 1,0 mM e inibidores de proteases (Aprotinina 1% e Benzamidina 1 mM), centrifugação a 45000xg por 40 minutos a 4 °C e congelamento da fração solúvel a -20 °C. Após um mínimo de 48 horas descongelou-se essa fração e seguiu-se uma nova centrifugação a 45000xg por 40 minutos a 4 °C. Ressuspendeu-se a fração precipitada e realizou-se uma nova centrifugação, sob as mesmas condições supracitadas. A fração precipitada é enriquecida em atividade Mg2+-ATPásica e análise em SDS-PAGE mostrou três principais polipeptídeos, um com alta massa molecular similar a cadeia pesada de miosina e outros dois com massa molecular corrrespondente a 57 e 45 kDa. O polipeptídeo de alta massa molecular foi marcado com anticorpo especifico para miosina V. O polipeptídeo de 57 kDa foi solubilizado com Triton X-100 0,2%, encontrando-se no precipitado os polipeptídeos de alta massa molecular e o polipeptídeo de 45 kDa. A atividade Mg2+-ATPásica desse precipitado é três vezes menor que o precipitado obtido sem triton X-100. Nossa fração não mostrou estimulação da atividade Mg2+-ATPásica por Ca2+ e Calmodulina, tendo apenas uma baixa atividade K+/EDTA-ATPásica, quando comparada com a atividade Mg2+-ATPásica. A atividade Ca2+-ATPásica foi cerca de 80% da atividade Mg2+-ATPásica. Também não ocorreu alteração da atividade Mg2+-ATPásica por cloreto de alumínio, fluoreto de sódio ou fluoreto de alumínio. Vanadato (50, 200 e 1000 pM) ou azida (1 mM) também não inibiu essa atividade. Essa fração apresentou preferência de hidrólise para ATP, entretanto hidrolisou também ADP (40%) e GTP (60%), mas não AMP, AMP-PNP, PPj, ADP2’5'3,5 AMP-PNP inibiu a atividade Mg2+-ATPásica. A atividade Mg2+-ATPásica foi inibida cerca de 50% por Fe2+ numa concentração menor que 1 mM, mas não pelos cátions cobre, cobalto ou zinco. A digestão protéica por tripsina causou uma perda da atividade e uma degradação do polipeptídeo de alta massa molecular, mantendo intacto o polipeptídeo de 45 kDa em nossa fração. Nesse trabalho descrevemos um método simples para obter miosina V a partir de uma fração solúvel de cérebro de rato no entanto não foi estimulada por Ca2+/Calmodulina e não expressou atividade K+/EDTA-ATPásica. We recently obtained a fraction enriched in Mg2 + -ATPasic activity from frozen rat brain cytosol. Our goal in this paper was to analyze this ATPase and characterize it. Our procedures were rat brain extraction, immediate homogenization in 50 mM Imidazole-HCI pH 8.0 buffer; 10 mM EDTA; 1.0 mM DTT and protease inhibitors (1% Aprotinin and 1 mM Benzamidine), centrifugation at 45000xg for 40 minutes at 4 ° C and freezing the soluble fraction at -20 ° C. After a minimum of 48 hours this fraction was thawed and then centrifuged again at 45000xg for 40 minutes at 4 ° C. The precipitated fraction was resuspended and centrifuged again under the same conditions as above. The precipitated fraction is enriched in Mg2 + -ATPase activity and SDS-PAGE analysis showed three main polypeptides, one with high molecular weight similar to myosin heavy chain and two with corresponding molecular weight at 57 and 45 kDa. The high molecular weight polypeptide was labeled with myosin V specific antibody. The 57 kDa polypeptide was solubilized with 0.2% Triton X-100, with the high molecular weight polypeptide and 45 kDa polypeptide being precipitated. The Mg2 + -ATPasic activity of this precipitate is three times lower than the precipitate obtained without triton X-100. Our fraction showed no stimulation of Mg2 + -ATPasic activity by Ca2 + and Calmodulin, having only a low K + / EDTA-ATPasic activity when compared to Mg2 + -ATPasic activity. Ca2 + -ATPasic activity was about 80% of Mg2 + -ATPasic activity. There was also no change in Mg2 + -ATPasic activity by aluminum chloride, sodium fluoride or aluminum fluoride. Vanadate (50, 200 and 1000 pM) or azide (1 mM) did not inhibit this activity either. This fraction showed hydrolysis preference for ATP, however it also hydrolyzed ADP (40%) and GTP (60%), but not AMP, AMP-PNP, PPj, ADP2'5'3,5 AMP-PNP inhibited Mg2 + -ATPasic activity. . Mg2 + -ATPasic activity was inhibited about 50% by Fe2 + at a concentration of less than 1 mM, but not by copper, cobalt or zinc cations. Trypsin protein digestion caused a loss of activity and degradation of the high molecular weight polypeptide, keeping the 45 kDa polypeptide intact in our fraction. In this paper we describe a simple method for obtaining myosin V from a soluble rat brain fraction however was not stimulated by Ca2 + / Calmodulin and did not express K + / EDTA-ATPasic activity. Dissertação (Mestrado)

The α-helix of myosin decreased and the diameter of the myosin clusters increased monotonically with increasing heating time at 50 and 90°C. The clusters that formed during heating at 90°C were much larger than those that formed at 50°C. With increasing heating time, the G′ of the myosin heated at 50°C increased, but it decreased in the myosin heated at 90°C. The rheology data suggested that myosin heated at 50°C exhibited better gel formation than myosin that was heated at 90°C. The myosin gel network formed at 50°C was more consistent and homogeneous, whereas the gel formed 90°C was irregular and sturdy. Myosin heated at 50°C tended to form small, open clusters via heads, and the tails were outstretched, whereas the clusters that formed via myosin heads and tails at 90°C were larger and closed. Al aplicar un tratamiento de calentamiento a temperaturas de 50 °C y 90 °C se constató que, a medida que aumentó la duración del calentamiento, se redujo la hélice-α de miosina y se incrementó monotónicamente el diámetro de los clústeres de miosina. Los clústeres que se produjeron durante el calentamiento a 90 °C fueron más largos que los formados en el calentamiento a 50 °C. A medida que se incrementó la duración del tratamiento térmico aumentó la G’ de la miosina sometida a calentamiento a 50 °C, disminuyendo cuando la miosina fue calentada a 90 °C. Los datos reológicos sugieren que la miosina calentada a 50 °C produjo una mejor formación de gel que aquella que se calentó a 90 °C. Además, la red de gel de miosina formada a 50 °C fue más consistente y homogénea, evidenciándose que el gel formado a 90 °C era irregular y macizo. La miosina sometida a calentamiento a 50 °C tendió a formar clústeres pequeños y abiertos en las cabezas y las colas se presentaron alargadas, en tanto los clústeres formados mediante cabezas y colas de miosina calentada a 90 °C fueron más grandes y cerrados.

Treballs Finals de Grau de Física, Facultat de Física, Universitat de Barcelona, Curs: 2020, Tutors: Valeria Venturini , Stefan Wieser i Jaume Casademunt, In this project we have characterised the dynamics of single myosin II molecules in stem cells under mechanical stress combining high spatial and time resolution microscopy with image analysis and simulations. We show that in the bleb, intensity of myosin spots increases as they assemble into bipolar thick filaments. In comparison with the myosin II under the nucleus, in the bleb they undergo a super dffusive motion which could drive the bleb contraction. We have analysed their spatial distribution using DBSCAN clustering analysis and we have performed Cytosim simulations to study the myosin II dynamics in the retraction bleb. This analysis sets the framework and the computational pipeline for further experiments.

Recycling endosomes regulate plasma membrane recycling. Recently, recycling endosome–associated proteins have been implicated in the positioning and orientation of the mitotic spindle and cytokinesis. Loss of MYO5B, encoding the recycling endosome–associated myosin Vb, is associated with tumor development and tissue architecture defects in the gastrointestinal tract. Whether loss of MYO5B expression affects mitosis is not known. Here, we demonstrate that loss of MYO5B expression delayed cytokinesis, perturbed mitotic spindle orientation, led to the misorientation of the plane of cell division during the course of mitosis, and resulted in the delamination of epithelial cells. Remarkably, the effects on spindle orientation, but not cytokinesis, were a direct consequence of physical hindrance by giant late endosomes, which were formed in a chloride channel–sensitive manner concomitant with a redistribution of chloride channels from the cell periphery to late endosomes upon loss of MYO5B. Rab7 availability was identified as a limiting factor for the development of giant late endosomes. In accordance, increasing rab7 availability corrected mitotic spindle misorientation and cell delamination in cells lacking MYO5B expression. In conclusion, we identified a novel role for MYO5B in the regulation of late endosome size control and identify the inability to control late endosome size as an unexpected novel mechanism underlying defects in cell division orientation and epithelial architecture., Loss of the recycling endosome-associated motor protein myosin Vb causes the formation of giant late endo-lysosomes; these in turn hinder the orientation of the mitotic spindle and chromosome segregation. Deregulated endosome size thus hampers faithful cell division.

KIT signaling; Adaptor molecules; Cell migration and adhesion Senyalització KIT; Molècules adaptadores; Migració i adhesió cel·lular Señalización KIT; Moléculas adaptadoras; Migración y adhesión celular Mast cell chemotaxis is essential for cell recruitment to target tissues, where these cells play an important role in adaptive and innate immunity. Stem cell factor (SCF) is a major chemoattractant for mast cells. SCF binds to the KIT receptor, thereby triggering tyrosine phosphorylation in the cytoplasmic domain and resulting in docking sites for SH2 domain-containing molecules, such as Lyn and Fyn, and the subsequent activation of the small GTPases Rac that are responsible for cytoskeletal reorganization and mast cell migration. In previous works we have reported the role of 3BP2, an adaptor molecule, in mast cells. 3BP2 silencing reduces FcεRI-dependent degranulation, by targeting Lyn and Syk phosphorylation, as well as SCF-dependent cell survival. This study examines its role in SCF-dependent migration and reveals that 3BP2 silencing in human mast cell line (LAD2) impairs cell migration due to SCF and IgE. In that context we found that 3BP2 silencing decreases Rac-2 and Cdc42 GTPase activity. Furthermore, we identified Myo1f, an unconventional type-I myosin, as a new partner for 3BP2. This protein, whose functions have been described as critical for neutrophil migration, remained elusive in mast cells. Myo1f is expressed in mast cells and colocalizes with cortical actin ring. Interestingly, Myo1f-3BP2 interaction is modulated by KIT signaling. Moreover, SCF dependent adhesion and migration through fibronectin is decreased after Myo1f silencing. Furthermore, Myo1f silencing leads to downregulation of β1 and β7 integrins on the mast cell membrane. Overall, Myo1f is a new 3BP2 ligand that connects the adaptor to actin cytoskeleton and both molecules are involved in SCF dependent mast cell migration.

ilustraciones Este estudio tuvo como objetivo determinar el efecto crioprotector del sorbitol incorporado al músculo de yamú (Brycon amazonicus), sometido a diferentes tiempos y temperaturas de almacenamiento. La metodología consistió en analizar cambios microestructurales, perfil proteico y propiedades fisicoquímicas (textura, capacidad de retención de agua y pH) de la carne bajo dos temperaturas (2 ± 2°C y -18 ± 2°C), dos tiempos de almacenamiento (24 y 48 horas) y la incorporación o no de 5 % (p/p) de una solución de sorbitol al 60%. Los cambios microestructurales fueron analizados por medio de microscopia óptica y electrónica de barrido y el perfil proteico se analizó por medio de electroforesis SDS PAGE. El análisis de varianza mostró diferencia significativa entre tratamientos para las tres variables analizadas. El tratamiento con mayor textura fue el tratamiento 4 (-18°C con sorbitol durante 24 horas) y el de menor textura fue el tratamiento 7 (-18°C sin sorbitol durante 48 horas). La mayor capacidad de retención de agua fue para el tratamiento 1 (2°C sin sorbitol durante 24 horas) y la menor para el tratamiento 7 (-18°C sin sorbitol durante 48 horas). El pH más alto lo obtuvo el tratamiento 2 (2°C con sorbitol durante 24 horas) y el más bajo el tratamiento 7 (-18°C sin sorbitol durante 48 horas). Las propiedades fisicoquímicas evaluadas en la carne de yamú se vieron afectadas principalmente por la interacción entre la temperatura y el tiempo de almacenamiento. Las proteínas miofibrilares sufrieron una degradación parcial y se evidenciaron cambios en el tejido conectivo, relacionados con la perdida de textura, especialmente cuando la carne no fue tratada con sorbitol a temperatura de congelación (-18 ± 2 °C). La utilización de sorbitol minimizó los efectos negativos de la congelación sobre las características del músculo de yamú, manteniendo una mayor integridad de la microestructura muscular, generando un efecto crioprotector en comparación a la carne no tratada. (Texto tomado de la fuente). The aim of this study was to determine the cryoprotective effect of sorbitol incorporated into the yamú muscle (Brycon amazonicus), exposed to different storage times and temperatures. The methodology consisted of analyzing microstructural changes, protein profile and physicochemical properties (texture, water holding capacity and pH) of the meat under two temperatures (2 ± 2 °C or -18 ± 2 °C), two storage times (24 and 48 hours) and the incorporation or not of 5% (w/w) of a 60% sorbitol solution. The microstructural changes were analyzed by optical microscopy and scanning electron microscopy; the protein profile was analyzed by SDS PAGE electrophoresis. The treatment with the highest texture was treatment 4 (-18 ° C with sorbitol for 24 hours) and the one with the least texture was treatment 7 (-18 ° C without sorbitol for 48 hours). The highest water holding capacity was for treatment 1 (2 ° C without sorbitol for 24 hours) and the lowest for treatment 7 (-18 ° C without sorbitol for 48 hours). The highest pH was obtained by treatment 2 (2 ° C with sorbitol for 24 hours) and the lowest by treatment 7 (-18 ° C without sorbitol for 48 hours). The physicochemical properties evaluated in yamú meat were affected mainly by the interaction between temperature and storage time. The myofibrillar proteins unerwent a partial degradation and changes in the connective tissue were observed, related to the loss of texture especially when the meat was not treated with sorbitol at freezing temperature (- 18 ± 2 °C). Sorbitol minimized the negative effects of freezing on the characteristics of the yamú muscle, maintaining the integrity of the muscular microstructure, generating a cryoprotective effect in comparison to untreated meat. Maestría Magíster en Ciencia y Tecnología de Alimentos Aseguramiento de la calidad de los alimentos y desarrollo de nuevos productos Alimentos y Agroindustria

SUMMARY: Exposure to mercury in the environment continues to be a significant worldwide concern, especially for developing embryos and fetuses. While extensive research effort has focused on the effects of mercury on the developing nervous system, much less is known concerning adverse effects of mercury on other organ systems, including the development of skeletal muscle. We exposed developing zebrafish embryos to a range of concentrations of mercuric chloride (100 to 400 µg/liter or ppb) and compared them to control embryos (0 µg/L mercuric chloride). Embryos were examined at 48 hours post fertilization (hpf) for morphometry and morphological deformities of skeletal muscle fibers in the trunk and tail. Embryos exposed to 400 ppb mercuric chloride showed decreased trunk and tail areas compared to control embryos. A dose-dependent reduction in muscle fiber length was observed, and exposure to all concentrations of mercuric chloride used in this study resulted in decreased muscle fiber immunohistochemical staining with anti-myosin antibodies. Irregular muscle fiber diameters, twisted muscle fibers, and degenerated muscle fibers were observed in sections of embryos stained with eosin at the higher exposure concentrations. Evidence presented in this study suggests that exposure to even low concentrations of mercuric chloride adversely affects skeletal muscle fiber development or muscle fiber integrity, or both. RESUMEN: La exposición al mercurio en el medio ambiente sigue siendo una preocupación mundial importante, especialmente para el desarrollo de embriones y fetos. Si bien un amplio esfuerzo de investigación se ha centrado en los efectos del mercurio en el sistema nervioso en desarrollo, se sabe mucho menos sobre los efectos adversos en otros sistemas orgánicos, incluido el desarrollo del músculo esquelético. Expusimos embriones de pez cebra en desarrollo a un rango de concentraciones de cloruro de mercurio (100 a 400 mg / l o ppb) y los comparamos con embriones de control (0 mg / L de cloruro de mercurio). Los embriones se examinaron a las 48 horas después de la fertilización (HPF) pararealizar la morfometría y verificar las deformidades morfológicas de las fibras del músculo esquelético en el tronco y la cola. Los embriones expuestos a 400 ppb de cloruro de mercurio mostraron una disminución de las áreas del tronco y la cola en comparación con los embriones de control. Se observó una reducción dependiente de la dosis en la longitud de la fibra muscular, y la exposición a todas las concentraciones de cloruro de mercurio utilizadas en este estudio, dio como resultado una tinción inmunohistoquímica de fibra muscular disminuida con anticuerpos anti-miosina. Se observaron diámetros irregulares de fibras musculares, fibras musculares retorcidas y fibras musculares degeneradas en secciones de embriones teñidos con eosina en las concentraciones de exposición más altas. La evidencia presentada en este estudio sugiere que la exposición incluso a bajas concentraciones de cloruro mercúrico afecta negativamente el desarrollo de la fibra del músculo esquelético o la integridad de la fibra muscular, o ambas.

In extant vertebrates, myosin heavy chain (MyHC) 6 and 7 are the main isoforms of atrial and ventricular myocardium respectively, whereas MyHC7b has been proposed to be an ancient cardiac isoform only expressed during embryonic development in modern species. In preliminary immunohistochemical studies of the heart of the lesser spotted dogfish (Scyliorhinus canicula; Chondrichthyes), we have observed that while MF20 labels homogeneously all the myocardium, A4.1025 labels the inflow cardiac segments (sinus venosus and atrium) but not the outflow segments (ventricle and conus arteriosus). In order to interpret these results, we have performed western and slot blots from samples of dogfish and hamster (Mesocricetus auratus) hearts, HPLC-ESI-MS/MS from dogfish samples, and immunohistochemistry in hearts of representative species of vertebrates, namely elasmobranchs, polypteriforms, acipenseriforms, teleosts and mammals, using MF20 and A4.1025 antibodies. Western and slot blot results confirmed the specificity of MF20 and A4.1025 for MyHC in dogfish, as well as their differential reactivity against different myocardial segments. HPLC-ESI-MS/MS using protein databases from Callorhinchus milii (Chondrichthyes) and Chordata revealed the presence of MyHC6 and 7 in all the dogfish myocardial segments, and of MyHC7b only in the outflow segments. Immunohistochemistry showed that while MF20 signals were homogeneous in all the myocardial segments of all the species studied, A4.1025 signals were restricted to the inflow myocardial segments in elasmobranchs, homogeneous in teleosts and acipenseriforms, and low in the ventricle of polypteriforms. It can be inferred that the A4.1025 antibody, as opposed to MF20, has a low affinity for MyHC7b, at least in the dogfish. In addition, we show that MyHC distribution in the cardiac chambers has changed during the evolution of gnathostomes. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech.Study supported by grants CGL2017-85090-P and CGL2014-52356-P (Ministerio de Economía y Competitividad), FPU15/03209 (Ministerio de Educación, Cultura y Deporte), contract UMAJI75 (Junta de Andalucía, European Social Found), FEDER and Universidad de Málaga.

Introduction and objective: Hereditary causes are responsible for half of cases of sensorineural hearing loss in young people. MYH14 mutation is autosomal dominant. Description: A 33 years-old patient with moderate-to-severe sensorineural hearing loss. Genetic study revealed MYH14 mutation. Discussion: This is a case of post-lingual sensorineural deafness, compatible with autosomal dominant inheritance. MYH14 mutation seems to increase susceptibility to acoustic trauma, which may justify the late onset of hearing loss. Conclusions: MYH14 mutation is, probably, a cause for hearing loss. Genetic study has, therefore, a growing importance. Introducción y objetivo: Las causas hereditarias son responsables por mitad de los casos de hipoacusia neurosensorial en jóvenes. La mutación del gen MYH14 es autosómica dominante. Descripción: Paciente de 33 años con hipoacusia neurosensorial bilateral de moderada a severa. El estudio genético ha revelado mutación del MYH14. Discusión: Es un caso de sordera neurosensorial post-lingual, compatible con una herencia autosómica dominante. La mutación del MYH14 puede aumentar la susceptibilidad para el trauma acústico, justificando el aparecimiento tardío de la sordera. Conclusiones: La mutación del MYH14 es, probablemente, una causa de sordera. El estudio genético tiene, así, una creciente importancia.

As doencas cardiovasculares sao a principal causa de morte no Brasil e estao fortemente associadas a disturbios metabolicos como a sindrome metabolica. A sindrome metabolica e caracterizada pela cocorrencia de fatores de risco cardiovasculares como hipertensao, hiperinsulinemia, a glicose, obesidade central e dislipidemia. Campomanesia xanthocarpa, conhecida no Brasil como Guabiroba, tem sido utilizada empiricamente devido ao seu potencial efeito na reducao dos niveis de colesterol sanguineos, e para varias condicoes, incluindo doencas inflamatorias, renais, digestivas, dislipidemias e outras. O objetivo deste estudo foi analisar a composicao quimica e investigar os efeitos do extrato aquoso de C. xanthocarpa na pressao arterial de ratos normotensos agudamente, bem como seu possivel mecanismo de acao, in vivo e in silico. Alem disso, tambem se avaliou os efeitos do extrato aquoso de C. xanthocarpa sobre a atividade da ATPase miosinica e expressao genica de marcadores cardiacos em ratos com sindrome metabolica induzida por uma dieta rica em frutose. O extrato foi analisado quanto aos compostos fenolicos totais e ao teor total de flavonoides. Os componentes quimicos foram determinados por analise de HPLC. Para a avaliacao aguda, a pressao arterial sistolica e diastolica e a frequencia cardiaca foram medidas na presenca de extrato e com administracao de farmacos. Para avaliacao dos animais com sindrome metabolica, os animais foram tratados durante 30 dias com extrato aquoso das folhas preparado por infusao. A extracao de miosina cardiaca foi realizada com centrifugacao de alta forca ionica, a medida da atividade da ATPase miosinica foi quantificada pelo fosfato inorganico liberado atraves de metodo colorimetrico e expresso em nmolPi/min/mg de proteina. A expressao genica de α-MHC, β-MHC, fator natriuretico atrial e α-actina esqueletica foi avaliada por PCR em tempo real. As folhas de C. xanthocarpa apresentaram conteudo relevante de fenois e flavonoides, e sugerimos a presenca de acido clorogenico, acido galico, quercetina e teobromina. A administracao aguda de extrato aquoso de C. xanthocarpa tem um efeito hipotensor dose-dependente em ratos normotensos, sugerindo que o mecanismo de acao pode ser mediado pelo sistema renina-angiotensina atraves do bloqueio do receptor AT1 (AT1R) e resposta simpatica autonomica. Estudos “docking” mostraram modelos que indicaram uma interacao entre acido clorogenico e quercetina com o sitio de ligacao do AT1R. Os achados desses estudos de acoplamento sugerem o potencial dos constituintes de C. xanthocarpa para uso como agentes preventivos para a pressao sanguinea. No tratamento de ratos com sindrome metabolica o extrato aumentou a atividade de ATPase miosinica e impediu a reexpressao da isoforma fetal da MHC, auxiliando na prevencao do dano cardiaco. Cardiovascular diseases are the leading cause of death in Brazil and are strongly associated with metabolic disorders such as the metabolic syndrome. Metabolic syndrome is characterized by the co-occurrence of cardiovascular risk factors such as hypertension, hyperinsulinemia, glucose intolerance, central obesity and dyslipidemia. Campomanesia xanthocarpa, known in Brazil as Guabiroba, has been used empirically because of its potential effects on reduce blood cholesterol levels, and for various conditions including inflammatory, renal, digestive, dyslipidemia, and other diseases. The objective of this study was to analyze the chemical composition and to investigate the effects of the aqueous extract of C. xanthocarpa on blood pressure of normotensive rats acutely, as well as its possible mechanism of action, using in vivo and in silico procedures. In addition, the effects of C. xanthocarpa aqueous extract on myosin ATPase activity and gene expression of cardiac markers in rats with metabolic syndrome induced by a high fructose diet were also evaluated. The extract was evaluated for total phenolic compounds and total flavonoid content. The chemical components were determined by HPLC analysis. For acute evaluation, systolic and diastolic blood pressure and heart rate were measured in the presence of extract and with drug administration. For the evaluation of animals with metabolic syndrome, the animals were treated for 30 days with aqueous extract of the leaves prepared by infusion. Cardiac myosin extraction was performed with high ionic strength centrifugation, the myosin ATPase activity measurement was quantified by the ionorganic phosphate released through a colorimetric method and expressed in nmolPi/min/mg of protein. Gene expression of α-MHC, β-MHC, atrial natriuretic factor and skeletal α-actin was evaluated by real-time PCR. C. xanthocarpa leaves presented a relevant content of phenols and flavonoids, and we suggest the presence of chlorogenic acid, gallic acid, quercetin and theobromine. Acute administration of C. xanthocarpa aqueous extract has a dose-dependent hypotensive effect in normotensive rats, suggesting that the mechanism of action may be mediated by the renin-angiotensin system through AT1 receptor blockade (AT1R) and autonomic sympathetic response. Docking studies showed models that indicated an interaction between chlorogenic acid and quercetin with the active site of the AT1R. The findings of these coupling studies suggest the potential of the C. xanthocarpa constituents for use as preventive agents for blood pressure. In the treatment of rats with metabolic syndrome the extract increased the myosin ATPase activity and prevented reexpression of fetal MHC isoform, helping to prevent cardiac damage.

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No. of bitstreams: 1 enriquezvalencia_ce_dr_jabo.pdf: 1054537 bytes, checksum: 7dec2790aa8a6f7dc9e2010c1e059d9f (MD5) Previous issue date: 2016-06-24 Pró-Reitoria de Extensão Universitária (PROEX UNESP) Com o intuito de estudar o potencial de aplicação de genes candidatos na seleção animal para o melhoramento genético da qualidade de carne de bovinos Nelore (Bos indicus), o presente trabalho teve como objetivos principais: (1) Avaliar em bovinos Nelore e cruzamentos Nelore x Bos taurus a ocorrência de associações entre o SNP g.98535683A>G:BTAU7 do gene CAST e as características da carne produzida e (2) quantificar a expressão gênica e proteica da cadeia pesada da miosina em bovinos Nelore com características fenotípicas contrastantes de crescimento e maciez da carne. Para o desenvolvimento do primeiro objetivo, 500 animais foram genotipados para o SNP g.98535683A>G:BTAU7 e fenotipados para força de cisalhamento (FC), índice de fragmentação miofibrilar (MFI), área de olho de lombo (AOL), espessura de gordura subcutânea (EGS) e lipídeos totais (LIP). Associação significativa entre o SNP e a maciez da carne foi observada. O genótipo AA apresentou os melhores valores em relação a esta característica. As diferenças encontradas entre os genótipos AA e AG (AA - AG) foram de - 0,19 kg e 5,31 para FC e MFI, respectivamente. O SNP não mostrou associação significativa com AOL, EGS e LIP. Para a execução do segundo objetivo, 90 bovinos Nelore terminados em confinamento foram utilizados. Levando em consideração o peso final ao abate (PF) e a FC dos 90 animais, 24 indivíduos com características contrastantes de peso e maciez da carne foram selecionados e divididos em quatro grupos experimentais (6 animais/grupo). Os grupos corresponderam a animais leves com carne macia (PF = 493,50 kg e FC = 3,76 kg), animais leves com carne dura (PF = 515,66 kg e FC = 8,06 kg), animais pesados com carne macia (PF = 604,33 kg e FC = 3,99) e animais pesados com carne dura (PF = 605,33 kg e FC = 7,83 kg). Em cada grupo, foram avaliadas características fenotípicas como MFI, AOL, EGS, LIP, índice de marmorização (IM), coloração instrumental (L*, a*, b*) e perdas por cozimento (PT). A análise de expressão dos genes MYH7, MYH2 e MYH1 foi feita por PCR em tempo real (RT-qPCR). A caracterização e quantificação proteica das isoformas da MyHC (MyHC-I, MyHC-IIa e MyHC-IIx/d) foi realizada por eletroforese SDS-PAGE. Em todas as análises (qualidade da carne e expressão gênica e proteica) não foi observada interação significativa entre crescimento e maciez da carne. Nas características AOL, EGS, IM e LIP não foi observado efeito significativo do crescimento e a maciez da carne. Porém, os animais pesados apresentaram maiores valores de L*, a* e b* em relação aos animais leves. Na maciez, o grupo de carne macia mostrou maiores valores de L* (32,29 ± 2,78) que o grupo de carne dura (29,10 ± 2,61). Por outro lado, maiores PT foram observadas em animais com carne dura em relação a animais com carne macia. Nos resultados de expressão gênica, o gene MYH7 não mostrou diferenças entre animais leves e pesados. Entretanto, a expressão dos genes MYH2 e MYH1 foi significativamente menor nos animais pesados em relação aos animais leves. Quanto à maciez da carne, a expressão dos três genes não diferiram significativamente entre grupos de carne dura e macia. Na expressão proteica, as três isoformas (MyHC-I, IIa e IIx), não mostraram diferença significativa entre animais leves e pesados. No entanto, as proporções da MyHC-I foram significativamente maiores no grupo de carne dura (16,51 ± 5,47) em relação ao grupo de carne macia (9,12 ± 3,16) e pelo contrário, as proporções da MyHC-IIa foram maiores no grupo de carne macia (73,78 ± 4,45) em relação ao grupo de carne dura (65,45 ± 8,79). A isoforma MyHC-IIx/d não mostrou efeito significativo sobre a maciez. Os resultados deste trabalho mostraram o potencial de aplicação do SNP g.98535683A>G:BTAU7 do gene CAST e da isoforma MyHC-IIa na seleção de animais para o melhoramento da maciez da carne de bovinos Nelore, além de, manifestar o diferencial de expressão dos genes MYH1 e MYH2 entre animais pesados e leves. In order to study the potential of application of candidate genes in animal selection for breeding meat of Nellore cattle, this work had as main objectives: (1) Assess in Nellore cattle (Bos indicus) and crosses Nellore x Bos taurus the occurrence of associations between the g.98535683A>G:BTAU7 SNP in the CAST gene and traits of the meat produced and (2) quantify the gene and protein expression of myosin heavy chain in Nellore cattle with contrasting phenotypic traits of growth and meat tenderness. For the development of first objective, 500 animals were genotyped for the g.98535683A>G:BTAU7 SNP and phenotyped for shear force (SF), myofibrillar fragmentation index (MFI), ribeye area (RA), backfat thickness (BT) and total lipids (TL). Significant association between the SNP and meat tenderness was observed. The AA genotype showed the best values in relation to this trait. The differences found between genotypes AA and AG (AA - AG) were – 0.19 kg and 5.31 for SF and MFI, respectively. The SNP did not show significant association with RA, BT and TL. For the execution of the second aim, 90 feedlot Nellore cattle were used. Considering the final slaughter weight (FW) and the SF of the 90 animals, 24 individuals with contrasting characteristics of weight and tenderness of meat were selected and divided into four experimental groups (6 animals / group). The groups corresponded to lightweight animals with tender meat (FW = 493.50 kg and SF = 3.76 kg), lightweight animals with tough meat (FW = 515.66 kg and SF = 8.06 kg), heavy animals with tender meat (FW = 604.33 kg and SF = 3.99) and heavy animals with tough meat (FW = 605.33 kg and SF = 7.83 kg). In each group, were evaluated phenotypic characteristics as MFI, RA, BT, TL, marbling índex (MI), instrumental color (L*, a*, b*) and cooking losses (CL). The expression analysis of the MYH7, MYH2 and MYH1 genes was performed by Real-Time PCR (RT-qPCR). The characterization and quantification of the MyHC isoforms (MyHC-I, MyHC-IIa and MyHC-IIx/d) was carried out by SDS-PAGE. In all analyzes (meat quality and gene and protein expression) there was no significant interaction between growth and meat tenderness. In the traits MFI, RA, BT, IM and TL it was not observed significant effect of the growth and the tenderness of meat. However, the heavy animals showed higher values of L*, a* and b* than lightweight animals. In the tenderness, the tender meat group showed higher values of the L* (32.29 ± 2.78) than the tough meat group (29.10 ± 2.61). On the other hand, higher CL were observed in animals with tough meat than animals with tender meat. In the results of gene expression, the MYH7 gene did not show differences between lightweight and heavy animals. However, the expression of the MYH2 and MYH1 genes was significantly lower in heavy animals than lightweight animals. As for meat tenderness, the expression of the three genes did not differ significantly between tough and tender meat groups. In the protein expression, the three isoforms (MHC-I, IIa and IIx), did not show significant difference between lightweight and heavy animals. Nevertheless, the MyHC-I proportions were significantly higher in the tough meat group (16.51 ± 5.47) than tender meat group (9.12 ± 3.16) and on the contrary, the proportions of the MyHC-IIa were higher in tender meat group (73.78 ± 4.45) than tough meat group (65.45 ± 8.79). The MyHC-IIx isoform did not show significant effect on tenderness meat. The results of this work showed the potential of application of the g.98535683A>G:BTAU7 SNP in gene CAST and the MyHC-IIa isoform in animal selection to improve meat tenderness of Nellore cattle, in addition, it showed the differential expression of MYH1 and MYH2 genes between heavy and lightweight animals.

Epithelial invagination in many model systems is driven by apical cell constriction, mediated by actin and myosin II contraction regulated by GTPase activity. Here we investigate apical constriction during chick lens placode invagination. Inhibition of actin polymerization and myosin II activity by cytochalasin D or blebbistatin prevents lens invagination. To further verify if lens placode invaginate through apical constriction, we analyzed the role of Rho-ROCK pathway. Rho GTPases expression at the apical portion of the lens placode occurs with the same dynamics as that of the cytoskeleton. Overexpression of the pan-Rho inhibitor C3 exotoxin abolished invagination and had a strong effect on apical myosin II enrichment and a mild effect on apical actin localization. In contrast, pharmacological inhibition of ROCK activity interfered significantly with apical enrichment of both actin and myosin. These results suggest that apical constriction in lens invagination involves ROCK but apical concentration of actin and myosin are regulated through different pathways upstream of ROCK. genesis 49:368–379, 2011. © 2011 Wiley-Liss, Inc.

CNPq Objetivo: Avaliar os efeitos do treinamento resistido de salto livre com sobrecarga sobre a composição muscular e as propriedades biomecânicas do complexo gastrocnêmio-plantar de ratos induzidos ao diabetes experimental tipo 1. Métodos: Foram utilizados oitenta e oito ratos da linhagem Wistar, com 60 dias de vida, distribuídos em quatro grupos: Grupo Controle Sedentário – GCS (n=15), Grupo Diabético Sedentário – GDS (n=27), Grupo Controle Treinado – GCT (n=17), Grupo Diabético Treinado – GDS (n=29). A indução ao diabetes foi feita por estreptozotocina. O protocolo de exercício resistido de salto teve duração de 9 semanas. Foram avaliadas glicemia e peso corpóreo dos animais do início ao fim do experimento. Com o término do protocolo de exercício o complexo gastrocnêmio-plantar direito dos animais foram coletados, com parte da amostra (n=65) sendo encaminhada para avaliação das propriedades mecânicas, e a outra parte da amostra (n=22) sendo encaminhada para a imunohistoquímica. Resultados: Os animais de todos os grupos diabéticos apresentaram pesos corporais finais inferiores (p 0.05). com relação ao efeito do treino resistido no diabetes, ao comparar os músculos dos animais do GDT com o GDS observamos aumento para o parâmetro Força Máxima e Rigidez (p0,05). Foi observado que os pesos dos músculos gastrocnêmios laterais mostraram-se reduzidos no GDS quando comprado ao GCS (p0,05). Conclusão: O Diabetes experimental promove prejuízos em parâmetros mecânicos no complexo muscular gastrocnemio-plantar e o exercício resistido exerce papel protetor na Força Máxima e na Rigidez extrínseca muscular dos animais treinados. Com relação à composição muscular, a miopatia diabética não altera a distribuição normal da miosina rápida e lenta no músculo gastrocnemio lateral, e o treino resistido de força não apresenta qualquer efeito sobre a distribuição dessa proteína. Objective: To evaluate the effects of a resistance training protocol on muscle composition and biomechanical properties of the gastrocnemius-plantaris complex of rats induced to experimental diabetes. Methods: At the age of 60 days eighty-eight Wistar rats were divided into four groups: Sedentary Control Group - SCG (n = 15), Sedentary Diabetic Group - SDG (n = 27), Trained Control Group - TCG (n = 17), Trained Diabetic Group - TDG (n = 29). Diabetic animals were induced to diabetes by streptozotocin. Exercise protocols had a 9 weeks duration. We evaluated animal’s blood glucose and body weight in the beginning and in the end of the experiment. At the end of the exercise protocol the right gastrocnemius-plantaris complex was collected, then part of the sample (n = 65) was referred for evaluation of mechanical properties, and the other portion of the sample (n = 22) was referred to the immunohistochemistry. Results: Animals of all diabetic groups showed lower final body weight (p 0.05). Trained diabetic animals showed muscles with increased Maximum Force and Stiffness compared to SDG animals (p 0.05). It was observed lower lateral gastrocnemius muscle weights in SDG animals compared to SCG animals (p 0.05). Conclusion: Experimental diabetes promoted damage in mechanical parameters in gastrocnemius-plantaris complex and resistance exercise had a protective role in Maximum Strength and muscle stiffness of Trained Diabetic Group. With respect to muscle composition, diabetic myopathy did not alter the normal distribution of the fast and slow myosin in the lateral gastrocnemius muscle, and the resistance training had no effect on the distribution of this protein.

Microtubules (MTs) are part of the cytoskeleton in eukaryotic cells and are nucleated by the γ-tubulin ring complex (γTuRC), at the centrosome, which is also called the MTOC (microtubule organizing centers). The subunit Mozart2/GCP8 is not required for γTuRC assembly/stability and has no obvious role in mitotic spindle assembly. As part of my PhD thesis project I tried to shed light on the role of GCP8 and it’s interphase function in cytoskeleton organization. I was able to further characterise GCP8 as a subunit and it’s γTuRC independent role. I provided a deeper insight into non-centrosomal/non-mitotic γTuRC functions and the organization/regulation of the interphase microtubule network. Further more I might implicate an additional function of GCP8 in cell attachment/migration and cancer cell migration., Los Microtúbulos (MTs) son parte del citoesqueleto en las células eucariotas y son nucleados por el complejo de la γ-tubulina (γTuRC) en el centrosoma, el cual es conocido como MTOC (centro organizador de microtúbulos). La subunidad Mozart2/GCP8 no se necesita para la formación/estabilidad del γTuRC y no parece jugar un papel evidente durante la formación del uso mitótico. Como parte de mi proyecto de tesis he caracterizado el papel de la GCP8 en la organización del citoesqueleto en interfase. También, he descrito la función de GCP8 tanto asociada al γTuRC como independiente del complejo. Así como las funciones no centrosomales/no mitóticas del γTuRC y la organización/regulación de la red del citoesqueleto durante la interfase. Además demostré que GCP8 esta relacionada con la función de adhesión y migración celular, incluida la migración celular en cáncer.

The binding of red pigment concentrating hormone (RPCH) to membrane receptors in crustacean chromatophores triggers Ca 2+ /cGMP signaling cascades that activate cytoskeletal motors, driving pigment granule translocation. We investigate the distributions of microfilaments and microtubules and their associated molecular motors, myosin and dynein, by confocal and transmission electron microscopy, evaluating a functional role for the cytoskeleton in pigment translocation using inhibitors of polymer turnover and motor activity in vitro . Microtubules occupy the chromatophore cell extensions whether the pigment granules are aggregated or dispersed. The inhibition of microtubule turnover by taxol induces pigment aggregation and inhibits re-dispersion. Phalloidin-FITC actin labeling, together with tannic acid fixation and ultrastructural analysis, reveals that microfilaments form networks associated with the pigment granules. Actin polymerization induced by jasplaquinolide strongly inhibits RPCH-induced aggregation, causes spontaneous pigment dispersion, and inhibits pigment re-dispersion. Inhibition of actin polymerization by latrunculin-A completely impedes pigment aggregation and re-dispersion. Confocal immunocytochemistry shows that non-muscle myosin II (NMMII) co-localizes mainly with pigment granules while blebbistatin inhibition of NMMII strongly reduces the RPCH response, also inducing spontaneous pigment dispersion. Myosin II and dynein also co-localize with the pigment granules. Inhibition of dynein ATPase by erythro-9-(2-hydroxy-3-nonyl) adenine induces aggregation, inhibits RPCH-triggered aggregation, and inhibits re-dispersion. Granule aggregation and dispersion depend mainly on microfilament integrity although microtubules may be involved. Both cytoskeletal polymers are functional only when subunit turnover is active. Myosin and dynein may be the molecular motors that drive pigment aggregation. These mechanisms of granule translocation in crustacean chromatophores share various features with those of vertebrate pigment cells.

Brucella spp. and Trypanosoma cruzi are two intracellular pathogens that have no evolutionary common origins but share a similar lifestyle as they establish chronic infections for which they have to circumvent the host immune response. Both pathogens have a virulence factor (prpA in Brucella and tcPrac in T. cruzi) that induces B-cell proliferation and promotes the establishment of the chronic phase of the infectious process. We show here that, even though PrpA promotes B-cell proliferation, it targets macrophages in vitro and is translocated to the cytoplasm during the intracellular replication phase. We observed that PrpA treated macrophages induce the secretion of a soluble factor responsible for B-cell proliferation and identified non-muscular myosin IIA (NMM-IIA) as a receptor required for binding and function of this virulence factor. Finally, we show that the Trypanosoma cruzi homolog of PrpA also targets macrophages to induce B-cell proliferation through the same receptor, indicating that this virulence strategy is conserved between a bacterial and a protozoan pathogen. Fil: Spera, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Herrmann, Claudia Karina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Roset, Mara Sabrina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Comerci, Diego José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina Fil: Ugalde, Juan Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina. Comisión Nacional de Energía Atómica; Argentina

Myosin V (MyoV) motors have been implicated in the intracellular transport of diverse cargoes including vesicles, organelles, RNA-protein complexes, and regulatory proteins. Here, we have solved the cargo-binding domain (CBD) structures of the three human MyoV paralogs (Va, Vb, and Vc), revealing subtle structural changes that drive functional differentiation and a novel redox mechanism controlling the CBD dimerization process, which is unique for the MyoVc subclass. Moreover, the cargo- and motor-binding sites were structurally assigned, indicating the conservation of residues involved in the recognition of adaptors for peroxisome transport and providing high resolution insights into motor domain inhibition by CBD. These results contributetounderstanding the structural requirements for cargo transport, autoinhibition, and regulatory mechanisms in myosin V motors. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc Published in the U.SA. © 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Doutor em Genética e Bioquímica CHAPTER II: Ca+2/calmodulin complex interacts with and regulates various enzymes and target proteins, known as calmodulin-binding proteins (CaMBPs). This group of proteins includes molecular motors such as myosins. Non-muscle myosin-IIB was shown in our study to be overexpressed in diabetic rat brains. In this study, we compared CaMBPs, purified by immobilized-calmodulin, affinity chromatography, isolated from non-diabetic and streptozotocin-induced diabetic rat brains. Proteins eluted with EGTA and urea were separated on a SDS-PAGE gel, digested and submitted to peptide mass fingerprinting analysis. There were fifteen and sixteen intense bands for non-diabetic and diabetic brains, respectively. Two proteins were exclusively in non-diabetic brains, four were found exclusively in diabetic brains and thirteen were found in both. Protein sequences were then analyzed for the presence of the calmodulin-binding sites. A large fraction of the eluted proteins (83%) had putative IQ motifs or calmodulin-binding sites. The myosin-IIB affinity chromatography elution in association with western blot, immunohistochemistry and RT-PCR analyses strongly suggest that myosin-IIB protein and mRNA are highly expressed levels in diabetic rat brains. This is the first study that identified and showed calmodulin-binding proteins differential expression in non-diabetic and diabetic rat brains through a comparative proteomic analysis, and it opens up a new field of study relating myosin-IIB expression in the brain, diabetes mellitus and intracellular calcium regulation. CHAPTER III: Diabetes mellitus is a chronic disease characterized by persistent hyperglycemia, which may lead to brain tissue damage due to oxidative stress. The enhancement of free radicals during hyperglycemia contributes to increased neuronal death and changes in synaptic transmission. This study evaluated the effect of oxidative stress and the use of antioxidants (calcium, zinc and vitamin E) supplementation on myosins expression levels in brain tissues of streptozotocin-induced diabetes experimental rat model. Lipid peroxidation, antioxidant enzymes activities and myosins IIB and Va expressions at transcriptional and protein levels were examined after 5 and 90 days post streptozotocin induction. The chronic effect of the streptozotocininduced diabetes led to upregulation of glutathione peroxidase (GPx) and malondialdehyde (MDA) levels, and downregulation of superoxide dismutase (SOD) and catalase (CAT) activities. These alterations were accompanied by increased myosin-IIB and decreased myosin-Va expressions in diabetic rat brains. The antioxidant supplementation protected against hyperglycemia damage by reducing CAT activity and increasing GPx and SOD activities, although it had no influence on MDA levels. Interestingly, only diabetic rats restored myosin-IIB expression at both transcriptional and protein levels in brain tissues after supplementation, and no differences were observed in myosin-Va expression in comparison to non-diabetic rat brains. The functional alterations of antioxidant enzymes and myosins IIB and Va in rat brains of experimentallyinduced diabetes were strongly associated with the disease and aging during acute and chronic stages, but only myosin-IIB expression could be reversed by antioxidants. These data highlight the supplementation (calcium, zinc and vitamin E) antioxidants effects on the oxidative stress and the involvement of myosins IIB and Va in the diabetes stages; however, their mechanisms of action on enzymes activities, lipid peroxidation and myosins expression remain to be elucidated.

FUNDAMENTO: A cardiomiopatia hipertrófica (CH) é a doença cardíaca hereditária mais frequente, causada por mutações nos genes codificadores para proteínas do sarcômero. Embora mais de 430 mutações tenham sido identificadas em vários continentes e países, não há relato de que isso tenha sido estudado no Brasil. OBJETIVO: Conduzir um estudo genético para identificar mutações genéticas que causam a CH em um grupo de pacientes no estado do Espírito Santo, Brasil. MÉTODOS: Usando a técnica SSCP, 12 exons dos três principais genes envolvidos com a CH foram estudados: exons 15, 20, 21, 22 e 23 do gene da cadeia pesada da β-miosina (MYH7), exons 7, 16, 18, 22 e 24 do gene da proteína C ligada à miosina (MYBPC3) e exons 8 e 9 do gene da troponina T (TNNT2). RESULTADOS: 16 alterações foram encontradas, incluindo duas mutações, uma delas possivelmente patogênica no gene MYBPC3 gene (p. Glu441Lys) e a outra patogênica já descrita no gene TNNT2 (p.Arg92Trp); 8 variações de seqüência raras e 6 variações de seqüência com frequência alélica maior do que 1% (polimorfismos). CONCLUSÃO: Com esses dados, é possível concluir que a genotipagem dos pacientes é factível em nosso meio. É possível que a variante p.Glu441Lys no exon 16 do gene MYBPC3 seja patogênica, resultando em um fenótipo mais leve do que o encontrado em associação com outras mutações. A variante p.Arg92Trp no exon 9 do gene TNNT2 não resulta em um fenótipo tão homogêneo como descrito anteriormente e pode levar à hipertrofia grave. BACKGROUND: Hypertrophic cardiomyopathy (HC) is the most frequent cardiac hereditary disease, caused by mutations in sarcomere protein coding genes. Although more than 430 mutations have been identified in several continents and countries, there have been no reports of mutations in Brazil. OBJECTIVE: To carry out a genetic study to identify genetic mutations that cause HC in a group of patients in Espirito Santo, Brazil. METHODS: Using the SSCP technique, 12 exons from the three main genes involved in HC were studied: exons 15, 20, 21, 22 and 23 of the β-myosin heavy chain gene (MYH7), exons 7, 16, 18, 22 and 24 of the myosin binding protein C gene (MYBPC3) and exons 8 and 9 of troponin T gene (TNNT2). RESULTS: 16 alterations were found, including two mutations, one of them possibly pathogenic in the MYBPC3 gene (p. Glu441Lys) and another pathogenic one, previously described in the TNNT2 gene (p.Arg92Trp), 8 rare sequence variations and 6 sequence variations with allelic frequency higher than 1% (polymorphisms). CONCLUSION: These data allow the conclusion that the genotyping of patients is feasible in our country. It is possible that the isolated p.Glu441Lys variant identified in exon 16 of the MYBPC3 gene is pathogenic, promoting a milder phenotype than that found when in association with other mutations. The p.Arg92Trp variant in the exon 9 of TNNT2 gene does not promote such a homogeneous phenotype as previously described and it can lead to severe hypertrophy. FUNDAMENTO: La cardiomiopatía hipertrófica (CH) es la enfermedad cardíaca hereditaria más frecuente, causada por mutaciones en los genes codificadores para proteínas del sarcómero. Aunque se hayan identificado más de 430 mutaciones en varios continentes y países, no hay relato de que esto se haya estudiado en Brasil. OBJETIVO: Conducir un estudio genético para identificar mutaciones genéticas que causan la CH en un grupo de pacientes en el estado de Espírito Santo, Brasil. MÉTODOS: Usando la técnica SSCP, se estudiaron 12 exones de los tres principales genes involucrados con la CH: exones 15, 20, 21, 22 y 23 del gen de la cadena pesada de la β-miosina (MYH7), exones 7, 16, 18, 22 y 24 del gen de la proteína C unida a la miosina (MYBPC3) y exones 8 y 9 del gen de la troponina T (TNNT2). RESULTADOS: Se encontraron 16 alteraciones, incluyendo dos mutaciones, una de ellas posiblemente patogénica en el gen MYBPC3 gen (p. Glu441Lys) y otra patogénica ya descrita en el gen TNNT2 (p. Arg92Trp); 8 variaciones de secuencia raras y 6 variaciones de secuencia con frecuencia alélica mayor que el 1% (polimorfismos). CONCLUSIONES: Con estos datos, es posible concluir que el genotipaje de los pacientes es factible en nuestro medio. Es posible que la variante p.Glu441Lys en el exón 16 del gen MYBPC3 sea patogénica, resultando en un fenotipo más leve que el encontrado en asociación con otras mutaciones. La variante p.Arg92Trp en el exón 9 del gen TNNT2 no resulta en un fenotipo tan homogéneo como el descrito anteriormente y puede llevar a hipertrofia grave.

Made available in DSpace on 2014-06-11T19:27:43Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-09-08Bitstream added on 2014-06-13T20:36:10Z : No. of bitstreams: 1 francisco_cl_me_botfmvz.pdf: 298670 bytes, checksum: 07cf29969b291dc9657e6127f73f1e6f (MD5) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Universidade Estadual Paulista (UNESP) Sabendo-se do grande potencial dos bubalinos como produtores de carne e da importância da análise das fibras musculares para a caracterização da qualidade da carne, objetivou-se neste estudo avaliar as isoformas da miosina de cadeia pesada (MyHC) e os tipos de fibras dos músculos Longissimus dorsi (LD) e Semitendinosus (ST) de 28 bubalinos Mediterrâneo não-castrados e a possível modulação de acordo com os diferentes pesos de abate (450, 480, 510 e 540 kg). Amostras dos músculos foram coletadas e submetidas às técnicas de eletroforese (SDS-PAGE), m-ATPase e NADH-TR. Foram analisadas a porcentagem relativa das isoformas de MyHC, assim como, frequência, área e diâmetro das fibras musculares. Constatou-se que bubalinos Mediterrâneo não possuem a isoforma MyHC IIb, apresentando somente três isoformas de MyHC (IIa, IIx/d e I), cujas porcentagens não variaram significativamente (P > 0,05) entre os pesos de abate. A presença de fibras musculares híbridas (IIA/X) nos músculos LD e ST permitiu a classificação dos tipos de fibras pela atividade contrátil, em fast e slow. Para ambos os músculos, a frequência foi maior para as fibras fast em todas as categorias avaliadas, porém houve diferença (P < 0,05) na frequência dos tipos de fibras dos músculos LD e ST de acordo com os pesos de abate. Os animais abatidos aos 540 kg obtiveram maiores valores para área e diâmetro das fibras, tanto para o LD quanto para o ST. O músculo ST apresentou maiores valores de área e diâmetro que o músculo LD para todos os pesos de abate avaliados, revelando o crescimento mais tardio. Knowing of the great productive potential of the water buffalo as meat producer and of the importance of the research of muscle fiber types for the characterization of the meat quality, the aim of the study was to evaluate Myosin Heavy Chain Isoforms (MyHC) and the muscle fiber types of Longissimus dorsi (LD) and Semitendinosus (ST) muscles of 28 Mediterranean buffaloes not-castrated and its possible modulation according with the different slaughter weights (450, 480, 510 and 540 kg). Samples of the muscles were collected and submitted the electrophoresis (SDS-PAGE), m- ATPase and NADH-TR techniques. The relative percentage of isoforms MyHC were measured, as well as, frequency, area and diameter of muscle fiber types. The absence of MyHC IIb isoform in Mediterranean buffaloes was evidenced, which had only three MyHC isoforms (IIa, IIx/d and I), whose percentage had not varied significantly (P > 0.05) between the slaughter weights. The presence of hybrid fibers (IIA/X) in muscles LD and ST allowed to the classification of the fiber types for a contraction activity, in fast and slow. For both muscles, the frequency was higher for fiber fast in all the evaluated categories, however had differences (P < 0.05) in the frequency of muscle fiber types LD and ST in the slaughter weights evalueted. The animals of slaughter weight 540 kg showed higher values for area and diameter of the fibers, as much for the LD as well as the ST. Muscle ST presented greater values of area and diameter then muscle LD for all slaughtered weights evaluated, disclosing the more delayed growth.

Nemaline myopathy (NM) is a congenital disease that leads to hypotonia and feeding difficulties in neonates. Some cases have a more benign course, with skeletal abnormalities later in life. We analyzed a series of eight patients with NM obtained from a retrospective analysis of 4300 muscle biopsies. Patients were classified as having the typical form in five cases, intermediate form in two cases and severe form in one case. Histochemical analysis showed mixed rods distribution in all cases and predominance of type I fibers in five cases. Immunohistochemical analysis showed abnormal nebulin expression in all patients (four heterogeneous and four absent), homogeneous desmin expression in four cases, strongly positive in three and absent in one, fast myosin expression in a mosaic pattern in six cases and absent in two cases. There was no specific relation between these protein expression patterns and the clinical forms of NM. Miopatia nemalínica (NM) é uma doença congênita que leva a hipotonia e dificuldade de sugar em neonatos. Alguns casos possuem uma evolução benigna, com deformidades ósseas tardias. Nós analisamos uma série de oito pacientes com NM obtidos da análise retrospectiva de 4300 biópsias musculares. Os pacientes foram classificados como forma típica em cinco casos, forma intermediária em dois casos e forma severa em um caso. Análise histoquímica mostrou distribuição mista dos rods em todos os casos e predominância de fibras tipo I em cinco casos. Análise imuno-histoquímica mostrou expressão anormal da nebulina em todos os pacientes (quatro heterogênea e quatro ausente), expressão homogenea da desmina em quatro casos, fortemente positiva em tres e ausente em um, expressão da miosina (rápida) com padrão em mosaico em seis casos e ausente em dois casos. Não há relação específica entre a expressão destas proteínas e as formas clínicas da NM.

El treball consta de 4 capítols diferents, dos dels quals ja han estat publicats en revistes científiques.El primer treball se centra en el paper d'una proteïna estructural del múscul, la cadena pesada de la miosina, en l'ensemblatge de les miofibril.les musculars. L'espècie escollida per l'estudi, el peix zebra, ofereix avantatges importants sobre altres ja que el seu desenvolupament és àmpliament conegut i existeix suficient informació genètica, amb eines moleculars ben dissenyades. Els resultats obtinguts indiquen un paper essencial de la cadena pesada de la miosina tipus 1 especìfica de mùscul vermell, ja que el seu knockdown implica l'alteració dels filaments gruixuts de miosina i prims d'actina del sarcòmer. Pel contrari, les línies M i els discs Z no resulten afectats, confirmant que aquestes estructures són determinades amb anterioritat a l'ensemblatge de l'actina i la miosina.En el segon capítol, s'examina el paper de l'IGF-II durant el procés de miogènesi en truita irisada, d'especial interès ja que les funcions de l'IGF-II en peixos teleostis difereix de la seva funció en mamífers. Per aquest estudi s'ha utilitzat el model de cultiu primari de cèl.lules musculars i s'ha analitzat el rol de l'IGF-II en processos com la proliferació cel.lular, captació de glucosa i activació de les principals vies de senyalització en múscul. Els resultats presentats indiquen que l'IGF-II poseeix efectes tant mitogènics com metabòlics en cèl.lules musculars de truita irisada i que aquests efectes són equivalents als trobats en resposta a l'IGF-I. Ambdós IGFs realitzen aquests efectes a través de les mateixes vies de senyalització intracel.lulars (MAPK i PI3K/Akt).El tercer treball està dedicat a la clonació i caracterizació d'un gen clau en el procés de miogènesi en orada, una espècie d'importància cabdal per l'aqüicultura del Mediterrani. En aquest capítol s'ha aïllat les seqüències del gen i del promotor de la miogenina, i s'han caracteritzat elements funcionals. A la vegada, s'ha determinat la seva expressió tissular i en embrions. S'ha vist que la seqüència de la miogenina d'orada comparteix elements reguladors amb les miogenines d'altres espècies, destacant el domini bHLH important per la dimerització de la molècula. El cDNA de miogenina és present tant en múscul blanc com en múscul vermell, i també en cèl.lules en desenvolupament. En embrions, la seva expressió segueix un patró anterio-posterior a mesura que les somites es van formant. L'anàlisi del promotor va revelar que els llocs d'unió a MEF2 i MEF3 són essencials per la seva especificitat muscular.El quart i darrer capítol intenta correlacionar l'expressió de diferents factors miogènics amb l'IGF-II. Aquesta expressió es va analitzar durant el procés de proliferació i diferenciació de les cèl.lules musculars en cultiu, així com en embrions en desenvolupament, mitjançant tècniques de PCR quantitativa i tècniques alternatives quan fou possible. En els embrions, el trànscrit de miogenina no es va detectar fins el començament de la miogènesi, i la màxima expressió es va trobar 27hpf. El patró d'expressió d'IGF-II suggereix una major importància durant la fase larvària. En cèl.lules musculars en cultiu, MyoD2 s'expressà principalment en la fase proliferativa, mentre que els nivells de miogenina van experimentar un increment durant la diferenciació. L'expressió d'IGF-II fou màxima en estadis primerencs, suggerint una major implicació en el procés de proliferació cel.lular. Els nivells de miostatina es van mantenir baixos durant tot el procés de diferenciació des de cèl.lules satèl.lit fins a miotubs., The present work consists of four results chapters, two of which have already been published in peer-reviewed scientific journals. The first one deals with the role of a structural protein, myosin heavy chain, in the myofibrille assembly. The species chosen for the study offers important advantages over others as its development is very well characterised, an important volume of genetic resources is available as well as specialized molecular tools for developmental studies. In the second chapter, the role of IGF-II in trout myogenesis is examined. The topic is of special interest since IGF-II functions in teleost deviate from those known in mammals. Trout primary muscle cell cultures is an established experimental tool that ensures controlled experimental conditions for the study of physiological factors. Thus, taking advantage of existing methodology, a multi-faceted study was performed to reveal novel functions of IGF-II in muscle proliferation and differentiation in fish as well as the signal transduction system mediating these functions.The third chapter focuses on the generation on genomic tools and the study of myogenesis in the gilthead sea bream, an important species for Mediterranean aquaculture that reaches large final sizes. Myogenin is a crucial myogenic factor and the chapter follows all the way from the isolation of the promoter of the gene, characterization of functional elements to its expression in primary cell cultures and embryos. The fourth chapter brings together molecular players and physiological factors of myogenesis in sea bream in an attempt to correlate the expression of different myogenic factors and IGF-II levels. The expression of all factors was followed during proliferation and differentiation of muscle cell cultures as well as in developing embryos. Real-time RT-PCR methods were developed and a thorough study on the appropriate housekeeping gene to-be-used was conducted. Results were verified with alternative methods where possible.

La estructura de los filamentos gruesos de músculo estriado en el estado relajado ha sido finalmente comprendida a nivel molecular. La estructura revela interacciones intra- e intermoleculares que mantienen las cabezas de miosina unidas formando hélices adosadas a la superficie del filamento grueso. La fosforilación de las cadenas ligeras reguladoras de la miosina induce el debilitamiento de estas interacciones permitiendo la activación de los filamentos gruesos, produciendo el desorden y la liberación de las cabezas de miosina, y permitiendo su interacción con los filamentos delgados. Estos resultados abren las puertas para la comprensión del mecanismo molecular de la regulación ligada a miosina de la contracción muscular, de relevancia ya que las mutaciones asociadas a la cardiomiopatía hipertrófica medioventricular están ubicadas cercanas al sitio de fosforilación en las cadenas ligeras reguladoras de miosina. The structure of the thick filaments of striated muscle has been finally understood at the molecular level. The structure reveals intra- and inter-molecular interactions that held the myosin heads forming helices on the thick filament surface. The phosphorylation of the myosin regulatory light chains induces the weakening of these interactions, allowing the activation of thick filaments, and enabling their interaction with the thin filaments. These results open the possibility to the understanding of the molecular mechanism of the myosinlinked regulation of muscle contraction. This is of relevance as the mutations associated with mid ventricular cardiomyopathy are located near the phosphorylation site in the regulatory light chain.

Made available in DSpace on 2014-06-11T19:32:59Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-07-05Bitstream added on 2014-06-13T20:04:48Z : No. of bitstreams: 1 morales_dc_dr_botfmvz.pdf: 1150740 bytes, checksum: 82142a24852f131d92e839bf0cd1454f (MD5) O objetivo do presente estudo foi identificar as isoformas da Cadeia Pesada de Miosina (Myosin Heavy Chain - MyHC) por eletroforese (SDS-PAGE), nos músculos Semitendinosus por biopsia durante o crescimento e ao abate e no músculo Longissimus dorsi ao abate. Foram utilizados 20 bovinos inteiros da raça Nelore submetidos ao modelo biológico superprecoce em um delineamento inteiramente casualizado. Foi realizada a caracterização morfológica das fibras musculares quanto ao tipo (SO, FOG e FG) e diâmetro nos mesmos músculos. Em relação ao músculo Semitendinosus somente foram identificadas duas bandas da cadeia pesada de miosina, a MyHC do tipo I e a MyHC do tipo II sem diferenciação da isoforma do tipo II e no músculo Longissimus dorsi foram identificadas as isoformas dos tipos I, II a e II x. Quanto a caracterização dos tipos de fibras no músculo Semitendinosus as mesmas estavam distribuídas em mosaico com predomínio de fibras glicolíticas FOG e FG e houve um aumento significativo (P

Fundação de Amparo a Pesquisa do Estado de Minas Gerais ABSTRACT - CHAPTER I Actomyosin from rat testis was obtained by freezing the soluble fraction and myosin II was isolated from this actomyosin. Testis was homogenized in a buffer extract, centrifuged, and the 40,000g x 40 supernatant frozen at -20°C for 48 hours. The supernatant was thawed at 4°C, centrifuged at 45.000g x 45 and the precipitate washed twice with imidazole buffer (pH 7.0 and 9.0, respectively). The resulting precipitate was enriched in 3 polypeptides: p205, p43 and one that migrated together with the front of the gel. These polypeptides were solubilized in pH 10.8 buffer at 27°C. When isolated in a Sephacryl S-400 column, p205 cosedimented with F-actin in the absence, but not in the presence, of ATP and was marked with anti-myosin II. Although the testis preparation expressed low K/EDTA-ATPase activity, a similar preparation obtained from skeletal muscle exhibited high K/EDTA-ATPase activity. In this study, myosin II was isolated from an actomyosin preparation obtained by freezing the soluble fraction of testis. ABSTRACT - CHAPTER II Actomyosin from swine, bovine and chicks brain was obtained by freezing the soluble fraction and myosin II and V was precipitate together, and myosin II from swine brain was partially isolated from this actomyosin. Brain was homogenized in a buffer extract, centrifuged, and the 40,000g x 40 supernatant frozen at -20°C for 48 hours. The supernatant was thawed at 4°C, centrifuged at 45.000g x 45 and the precipitate washed twice with imidazole buffer (pH 7.0 and 10.0, respectively). The resulting precipitate was enriched in 3 polypeptides: p205, p53, p43 and own high Mg2+-ATPase activity. These polypeptides were solubilized in pH 10.2 buffer at 27°C, with addition of ATP, Mg and NaCl. The p205 from swine brain was partially isolated in a Sephacryl S-500 column. This polypeptide of 205 kDa was marked with antibody anti-myosin II and V. Although the brain preparation expressed low K/EDTA-ATPase activity, we observed Co2+- ATPase activity and a stimulation of the Mg2+-ATPase activity by Tween 20. In this study, showed a precipitation together of myosin II and V, and partially isolated this myosin from swine brain from actomyosin preparation obtained by freezing the soluble fraction of brain. RESUMO GERAL - Miosinas são proteínas motoras que translocam sobre filamentos de actina e atualmente constituem uma superfamília com 20 classes. Algumas são conhecidas apenas a estrutura primária através de seqüência gênica, outras já estão bem caracterizadas bioquimicamente, como as miosinas I, II e V. São amplamente distribuídas em todas as células eucarióticas. Apresentam como características bioquímicas: alta atividade ATPase na presença de EDTA e alta concentração de potássio (atividade K/EDTA-ATPase) e uma baixa atividade Mg2+-ATPase, que é estimulada por F-actina. Preparação de actomiosina é obtida a partir da precipitação de fração solúvel de cérebro tanto em baixa quanto em alta força iônica e esse processo de precipitação constitui um passo importante para purificar e caracterizar miosina II e V a partir de fração solúvel de cérebro. Miosina II é precipitada quando a fração solúvel de cérebro é dializada contra tampão de baixa força iônica, enquanto que, miosina V é precipitada seletivamente em relação à miossina II, quando a fração solúvel de cérebro é tratada com alta força iônica, sendo postulado que essa precipitação de miosina (II e V) seja resultado de sua interação com actina e vesículas. Recentemente temos obtido a precipitação de actomiosina a partir do congelamento de fração solúvel de cérebro ou testículo de rato. Neste trabalho mostramos a purificação de miosina II de testículo de rato e a obtenção de actomiosina de cérebro de suino, bovino e pintainho através do congelamento de fração solúvel de cérebro e testículo. RESUMO - CAPÍTULO I Actomiosina de testículo de rato foi obtida através do congelamento de fração solúvel e miosina II foi isolada a partir dessa actomiosina. Testículos foram homogeneizados em tampão de extração, centrifugado e o sobrenadante 40.000g x 40 foi congelado a -20oC por 48 horas. O sobrenadante foi descongelado a 4oC, centrifugado a 45.000g x 45 e o precipitado foi lavado duas vezes com tampão imidazol (pH 7,0 e 9,0, respectivamente). O precipitado resultante é enriquecido em três polipeptídeos: p205, p43 e um que migra junto com a frente do gel. Esses polipeptídeos foram solubilizados em tampão pH 10,8 a 27oC. O p205, isolado em Sephacryl S-400, co-sedimenta com F-actina na ausência, mas não na presença, de ATP e foi marcado com anti-miosina II. Embora a preparação de testículo, praticamente, não expressou atividade K/EDTA-ATPásica, uma preparação similar obtida a partir de músculo esquelético apresentou alta atividade K/EDTA-ATPásica. Nesse trabalho, miosina II foi isolada a partir de uma preparação de actomiosina obtida pelo congelamento de fração solúvel de testículo. RESUMO - CAPÍTULO II Actomiosina de cérebro de suino, bovino e pintainho foi obtida através do congelamento de fração solúvel e miosina II e V foram precipitadas conjuntamente, sendo que miosina de cérebro de suino foi parcialmente isolada a partir dessa actomiosina. Os cérebros foram homogeneizados em tampão de extração, centrifugado e o sobrenadante 40.000g x 40 foi congelado a -20oC por 48 horas. O sobrenadante foi descongelado a 4oC, centrifugado a 45.000g x 45 e o precipitado foi lavado duas vezes com tampão imidazol (pH 7,0 e 10, respectivamente). O precipitado resultante é enriquecido em três polipeptídeos: p205, p53, p43 e possui alta atividade Mg2+-ATPase. Esses polipeptídeos foram solubilizados em tampão pH 10,2 a 27oC, com adição de ATP, Mg e NaCl. O p205 de cérebro de suino foi parcialmente isolado em Sephacryl S-500. Esse polipeptídeo de 205 kDa foi marcado com anticorpos anti-miosina II e V. Embora a preparação de cérebro, praticamente, não expressou atividade K/EDTAATPásica, apresentou atividade Co2+-ATPase e uma estimulação da atividade Mg2+-ATPase por Tween 20. Nesse trabalho mostramos a precipitação conjunta de miosina II e V e o isolamento parcial dessas miosinas de cérebro de suino a partir de uma preparação de actomiosina obtida pelo congelamento de fração solúvel de cérebro. Mestre em Genética e Bioquímica

"La producción in situ de ácido láctico a través de la fermentación láctica controlada con cepas lácticas seleccionadas, es un método barato y eficiente para reducir la contaminación microbiana en la superficie de la carne, sin embargo, esta tecnología puede alterar la calidad de la carne, entre otras causas, debido a la actividad proteolítica de las bacterias lácticas. En este trabajo se estudió el efecto de la fermentación láctica con Staphylococcus carnosus y Lactobacillus alimentarius sobre la fracción miofibrilar de proteínas contráctiles mayores (miosina pesada y actina) de carne de cerdo envasada al vacío y almacenada a 4°C y 19°C durante 96 h En ambas temperaturas de almacenamiento, en la muestra control y las inoculadas con bacterias lácticas, se observó a las 96 h de almacenamiento la aparición de 3 bandas identificadas como productos de degradación de miosina pesada, mientras que no se detectó degradación evidente en la molécula de activa."

Swine skeletal muscle fibers have traditionally been classified as types I, IIA, IIB and their hybrid forms by determining the activity of myosin ATPase (mATPase). More recently, molecular, electrophoretic and immunohistochemical approaches have allowed the identification of IIx isoform-containing fibers. In this study, myosin isoforms present in different types of muscle fibers were identified by combining histochemical and immunohistochemical methods. Samples from the M. longissimus dorsi were collected from primiparus pigs (mean body weight, 150 Kg) 45 minutes after slaughtering and subsequently frozen in dry ice-cooled acetone. Serial cross sections were processed for mATPase and NADH-TR activities and for immunohistochemistry. Five histochemical fiber groups were identified: type I (dark), type IIA (light) and three intermediate types. A panel of monoclonal antibodies identified the slow ßI, IIa, IIx and IIb isoforms present in types I, IIA, IIX and IIB fibers and the hybrid group IIAX Las fibras musculares esqueléticas del cerdo han sido tradicionalmente tipificadas como I, IIA y IIB, más sus formas híbridas, determinando la actividad de la enzima miosina ATPasa miofibrilar. Recientemente, la utilización de anticuerpos monoclonales contra isoformas de cadena pesada de miosina, electroforesis y estudios moleculares, han documentado la existencia, además, de la isoforma IIx. El objetivo de este estudio fue determinar la existencia de las isoformas de miosina, presentes en los distintos tipos fibrilares, utilizando técnicas histoquímicas e inmunohistoquímicas combinadas, dentro de una unidad experimental compuesta por muestras musculares del M. longíssimo del dorso, en cerdas de 150 kg promedio. Las muestras fueron obtenidas 45 minutos después de la faena, mediante escisión y congeladas en acetona enfriada con hielo seco. Cortes seriados de 10µm de espesor fueron tratados por la técnicas de mATPasa modificada por Nwoye (1982), a pH 4.6 , NADH-TR e inmunohistoquímica. Histoquímicamente fueron identificados cinco grupos de fibras: tipo I oscuras, IIA claras, y tres tipos intermedios. Los ensayos inmunohistoquímicos permitieron identificar las isoformas ß lenta I, IIa, IIx y IIb presentes en fibras de tipo I, IIA, IIX, IIB y un grupo híbrido IIAX