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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Caracterización de la entrada capacitativa de cationes en distintos sistemas celulares : estudio molecular de los componentes responsables de dicho proceso

Baldi, Carolina 14 November 2010 (has links)
Investigaciones en nuestro y otros laboratorios han demostrado que la hormona 1,25-dihidroxivitamina D3 (1,25(OH)2D3) modula los niveles de Ca2+ intracelular en células de músculo esquelético y tejido óseo por un mecanismo genómico, característico de hormonas esteroideas en el cual la hormona interacciona con un receptor intracelular específico (VDR: vitamin D receptor). Esta molécula exhibe una localización citoplásmica/nuclear, siendo el complejo hormona-receptor el responsable de la regulación de la expresión de genes portadores de elementos de respuesta a VDR. Sin embargo, 1,25(OH)2D3 modula también los niveles de Ca2+ intracelular a través de un mecanismo rápido, no genómico, independiente de la transcripción génica y de la síntesis proteica que implica acciones directas del esteroide sobre la membrana celular. Las acciones rápidas, no nucleares de 1,25(OH)2D3 requieren la activación de varios sistemas de señalización (adenilil ciclasa/AMPc/PKA; PLC/DAG/IP3/Ca2+/PKC), integrados en un mecanismo complejo que involucra proteínas G, interacción positiva entre las vías PKA y PKC e incremento en la fosforilación de proteínas de membrana. En conjunto, estos eventos conducen a una rápida y transitoria liberación de Ca2+ de los depósitos internos y activación de canales de Ca2+ dependientes de voltaje (VDCC) tipo L, con el consecuente influjo sostenido de Ca2+ desde el medio extracelular. En el presente trabajo empleando el indicador fluorescente de Ca2+ Fura-2, se caracterizaron los efectos de la hormona 1,25(OH)2D3 sobre los niveles de Ca2+ citosólico en cultivos de osteoblastos de rata. Estos estudios pusieron de manifiesto que la fase de influjo de Ca2+ inducido por 1,25(OH)2D3 es mediada por los canales VDCC ya caracterizados, indicando además que existe una contribución parcial a la entrada de Ca2+ mediada por canales no dependientes de voltaje operados por el nivel de Ca2+ de los depósitos internos. Se introduce así el novedoso concepto sobre la participación de canales SOC (Store Operated Channels; CCE: Capacitative Ca2+ Entry ) en la respuesta rápida de Ca2+ a la hormona. A partir de este hallazgo, estudiamos y caracterizamos esta entrada capacitativa de cationes en líneas celulares derivadas de epitelio mamario y de cáncer mamario, ambas de origen humano. Estos canales, activados por depleción y/o movilización de Ca2+ de depósitos internos, independientes de voltaje y regulados por múltiples eventos de señalización intracelular, estarían constituídos por proteínas de la familia de genes TRP, originalmente descriptos en células fotoreceptoras de Drosophila (mutantes trp: Transient Receptor Potential). Sus genes han sido clonados, secuenciados y su funcionalidad como canales responsables del influjo de Ca2+ tipo SOC, ha sido demostrada mediante expresión de proteínas TRP en sistemas heterólogos. Además, en el sistema fototransductor de Drosophila, TRP forma parte de un complejo de señalización multiproteico junto con RH1 (rodopsina), NORPA (PLC específica de células fotoreceptoras), INAC (PKC específica de células fotoreceptoras) y una proteína adaptadora (scaffold protein) denominada INAD (Inactivation No After potential D) responsable de asociar todos los componentes en un complejo. INAD posee dominios PDZ, responsables de su función adaptadora mediante interacciones proteína-proteína. Recientes estudios bioquímicos, moleculares y electrofisiológicos, sugieren que INAD representa una subunidad constitutiva del canal TRP con función adaptadora en la formación del complejo supramolecular de señalización. Esta organización multiproteica de los componentes de señalización proporciona las bases estructurales para una eficiente y rápida activación y regulación de la entrada de Ca2+ a través de canales TRP. Se describieron recientemente homólogos de INAD en distintos tipos de células de vertebrados, donde se observó que cumplen función adaptadora en la formación de complejos de señalización integrados por receptores hormonales, canales iónicos y quinasas. En este trabajo mostramos evidencias de una proteína homóloga a INAD en osteoblastos de rata y su participación en el influjo capacitativo. En síntesis, en la presente Tesis Doctoral se muestran evidencias de influjo capacitativo de calcio en distintos tipos celulares, habiendo caracterizado este influjo y estudiado exhaustivamente la modulación de cationes en el interior celular. Particularmente en osteoblastos, un sistema íntimamente relacionado con el metabolismo del calcio, demostramos la única evidencia existente sobre modulación de canales SOC por un esteroide y más precisamente, la participación de proteínas TRP e INAD asociadas a este mecanismo fisiológico vinculado a la homestasis de cationes intracelulares. / Evidences from our laboratory and others have demonstrated that the hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) modulates intracellular Ca2+ levels in skeletal muscle cells and bone tissue through a genomic mechanism, typical for steroidal hormones via specific an intracellular receptor (VDR: Vitamin D Receptor). Such molecule exhibits a cytoplasmatic-nuclear localization and the hormone-receptor complex is responsible for gene expression regulation of VDR responsive elements. Nevertheless, 1,25(OH)2D3 also modulates intracellular Ca2+ levels through a non genomic, rapid mechanism, independent of gene transcription and protein synthesis, implying direct steroid actions on the cellular membrane. Rapid or non genomic actions of 1,25(OH)2D3 requires the activation of diverse signal systems (adenylyl cyclase/cAMP/PKA; PLC/DAG/IP3/Ca2+/PKC), forming a complex mechanism that involves G proteins, positive interaction between PKA and PKC and increased protein phosphorylation. Altogether, these events lead to a rapid and transient Ca2+ release from internal stores and activation of Voltage Dependent Ca2+ Channels (VDCC) type L, followed by a sustained Ca2+ influx from the extracellular medium. In the present work by using the fluorescent Fura-2 Ca2+ dye, the effect of 1,25(OH)2D3 hormone on cytosolic Ca2+ levels in rat osteoblast cells were characterized. We have demonstrated that the 1,25(OH)2D3-induced Ca2+ influx phase is mediated by the well known VDCC channels and also that a Ca2+ partial entry contribution may occur by a non voltage dependent channels and operated by the status of internal Ca2+ stores. In this way we introduce a novel concept of SOC channels (Store Operated Channels; Capacitative Ca2+Entry) in rapid response of Ca2+ for the hormone. Accordingly, we have studied and characterized the capacitative cation entry in other cellular system derived from breast epithelium and breast cancer, both from human origen. These channels, activated by depletion and/or Ca2+ mobilization from internal stores, voltage independent and regulated by multiple intracellular signal events, involve proteins encoded by the trp family genes, first described in photoreceptor cells from Drosophila (mutants in trp: Transient Receptor Potential). Trp genes have been cloned and sequenced, their functionality as channels responsible from Ca2+ influx type SOC has been demonstrated through heterologous expression of TRP proteins. In addition, in Drosophila photoreceptor system, TPR is part of a multiproteic signal complex together with RH1 (rhodopsin), NORPA (PLC specific of photoreceptor cells), INAC (PKC specific of photoreceptor cells) and a scaffold protein named INAD (Inactivation No After potential D) responsible for the association of all components into a complex. INAD exhibits PDZ domains, responsible of its adaptor function through protein-protein interaction. Recently several biochemical, molecular and electrophysiologycal studies, suggests that INAD represents a constitutive subunit of TRP channels with an adaptor function in the supramolecular signaling complex assemble. This multiproteic organization of the signaling components provides the structural basis for an efficient and rapid activation and regulation of Ca2+ entry through TRP channels. INAD homologous sequences have been recently described in different vertebrate cells types, whereas it has been observed that plays scaffolding functions allowing the assembly of a complex composed by hormone receptors, ion channels and kinases. We demonstrated the presence of an INAD homologous protein in rat osteoblasts and its relationship to the capacitative Ca2+ influx. In summary, the present Doctoral Thesis describes the capacitative calcium entry in different cell types. We have characterized this influx and exhaustively studied the intracellular cation modulation. In particular, we have focused our efforts on osteoblast cells, a system closely dependant on calcium metabolism, and present the first evidence of SOC channels modulation by a steroid hormone and more precisely, the involvement of TRP and INAD proteins associated to this physiological mechanism in the control of intracellular Ca2+ homeostasis.
2

Avaliação imuno-histoquímica do marcado de proliferação celular KI-67 em glandula perianal normal e em neoplástica de cães/

Pereira, Rodrigo Storti. January 2011 (has links)
Orientador: Gisele Fabrino Machado / Banca: Felipe Augusto Ruiz Sueiro / Banca: Antonio Carlos Alessi / Resumo: A causa dos tumores da glândula perianal é desconhecida, entretanto eles podem ser modulados por influência de hormônios gonadais, basicamente a testosterona, uma vez que há uma alta prevalência (até 95%) de regressão da forma benigna dessa neoplasia após a castração dos machos. A proteína Ki-67 foi descrita pela primeira vez em 1983 é uma proteína nãohistona, que não é expressa em células na fase G0, mas que pode ser detectada nas fases ativas do ciclo celular, G1, S, G2 e mitose Verificou-se que o Ki-67 está associado com a proliferação celular, estando a população de células tumorais positivas ao Ki-67 correlacionada com a evolução clínica da doença. Foram estudados 42 casos de neoplasias da glândula perineal, sendo 15 adenomas, 15 epiteliomas, 12 carcinomas. Foram utilizadas 13 amostras de tecido de glândula perianal normal, como controle. De cada animal foram obtidas informações como: sexo, idade, raça, presença de outras neoplasias, se castrado ou não, se em caso de fêmea havia o uso de anticoncepcional, tempo de evolução, recidiva e tempo de sobrevida. Dos 42 casos de neoplasias de glândulas perianais, 34 (80,95%) foram provenientes de cães machos e 8 (19,05%) de fêmeas. A média de idade dos machos com a neoplasia foi de 9,8 anos, e das fêmeas 7,4 anos, sendo que para os dois sexos a idade variou de 5 a 15 anos. Em relação às raças, 11 (26,19%) eram S.R.D. (sem raça definida), 9 (21,42%) Poodle, 4 (9,52%) Cocker Spaniel e Husky Siberiano. O histórico de diagnóstico de outras neoplasias, foi constatado em 23,80% dos animais deste estudo. Em relação à castração, 32 (94,11%) dos machos não eram castrados e apenas 8 (19,05%) fêmeas eram. O tempo de evolução da neoplasia apresentou média de 1,2 anos. Histórico de recidiva da neoplasia foram relatados em 14 animais, sendo maior... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The cause of perianal gland tumors is unknown, though they may be modulated by the influence of gonadal hormones, primarily testosterone, since there is a high prevalence (up to 95%) of regression of the benign tumor after males castration. Protein Ki-67 was first described in 1983, and it is a nonhistone protein, which is not expressed in G0 phase cells, but can be detected in the active phases of the cell cycle, G1, S, G2 and mitosis. It was found that Ki-67 is associated with cell proliferation, and the population of tumor cells positive for Ki-67 correlated with clinical outcome. In this present work we studied 42 cases of perineal gland neoplasms, being 15 adenomas, 15 epitheliomas and 12 carcinomas. We used 13 tissue samples of normal perianal gland as control. From each animal we obtained information such as gender, age, race, presence of other malignancies, neutering status, whether if there were female contraceptive use, duration, recurrence and survival time. Of the 42 cases of perianal gland neoplasms, 34 (80.95%) were from males and eight dogs (19.05%) females. The males average age with cancer was 9.8 years and 7.4 years for females and for both sexes aged between 5 and 15 years. In regard to race, 11 (26.19%) were no breed, 9 (21.42%) were Poodle, and 4 (9.52%) were Siberian Husky and Cocker Spaniels. The history of diagnosis of other cancers, was found in 23.80% of the animals in this study. In relation to castration, 32 (94.11%) of males were not castrated and only 8 (19.05%) were females. The progression of the tumor showed an average of 1.2 years. History of recurrence of cancer were reported in 14 animals, and was higher (66.66%) for carcinomas. Ki-67 staining revealed that the carcinomas showed higher proliferation rate (9.87%) compared to groups of epitheliomas... (Complete abstract click electronic access below) / Mestre
3

Avaliação imuno-histoquímica do marcado de proliferação celular KI-67 em glandula perianal normal e em neoplástica de cães

Pereira, Rodrigo Storti [UNESP] 18 March 2011 (has links) (PDF)
Made available in DSpace on 2014-06-11T19:27:18Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-03-18Bitstream added on 2014-06-13T19:35:13Z : No. of bitstreams: 1 pereira_rs_me_araca.pdf: 790587 bytes, checksum: 194f37c505d12a98d7b75720d14a4e5f (MD5) / A causa dos tumores da glândula perianal é desconhecida, entretanto eles podem ser modulados por influência de hormônios gonadais, basicamente a testosterona, uma vez que há uma alta prevalência (até 95%) de regressão da forma benigna dessa neoplasia após a castração dos machos. A proteína Ki-67 foi descrita pela primeira vez em 1983 é uma proteína nãohistona, que não é expressa em células na fase G0, mas que pode ser detectada nas fases ativas do ciclo celular, G1, S, G2 e mitose Verificou-se que o Ki-67 está associado com a proliferação celular, estando a população de células tumorais positivas ao Ki-67 correlacionada com a evolução clínica da doença. Foram estudados 42 casos de neoplasias da glândula perineal, sendo 15 adenomas, 15 epiteliomas, 12 carcinomas. Foram utilizadas 13 amostras de tecido de glândula perianal normal, como controle. De cada animal foram obtidas informações como: sexo, idade, raça, presença de outras neoplasias, se castrado ou não, se em caso de fêmea havia o uso de anticoncepcional, tempo de evolução, recidiva e tempo de sobrevida. Dos 42 casos de neoplasias de glândulas perianais, 34 (80,95%) foram provenientes de cães machos e 8 (19,05%) de fêmeas. A média de idade dos machos com a neoplasia foi de 9,8 anos, e das fêmeas 7,4 anos, sendo que para os dois sexos a idade variou de 5 a 15 anos. Em relação às raças, 11 (26,19%) eram S.R.D. (sem raça definida), 9 (21,42%) Poodle, 4 (9,52%) Cocker Spaniel e Husky Siberiano. O histórico de diagnóstico de outras neoplasias, foi constatado em 23,80% dos animais deste estudo. Em relação à castração, 32 (94,11%) dos machos não eram castrados e apenas 8 (19,05%) fêmeas eram. O tempo de evolução da neoplasia apresentou média de 1,2 anos. Histórico de recidiva da neoplasia foram relatados em 14 animais, sendo maior... / The cause of perianal gland tumors is unknown, though they may be modulated by the influence of gonadal hormones, primarily testosterone, since there is a high prevalence (up to 95%) of regression of the benign tumor after males castration. Protein Ki-67 was first described in 1983, and it is a nonhistone protein, which is not expressed in G0 phase cells, but can be detected in the active phases of the cell cycle, G1, S, G2 and mitosis. It was found that Ki-67 is associated with cell proliferation, and the population of tumor cells positive for Ki-67 correlated with clinical outcome. In this present work we studied 42 cases of perineal gland neoplasms, being 15 adenomas, 15 epitheliomas and 12 carcinomas. We used 13 tissue samples of normal perianal gland as control. From each animal we obtained information such as gender, age, race, presence of other malignancies, neutering status, whether if there were female contraceptive use, duration, recurrence and survival time. Of the 42 cases of perianal gland neoplasms, 34 (80.95%) were from males and eight dogs (19.05%) females. The males average age with cancer was 9.8 years and 7.4 years for females and for both sexes aged between 5 and 15 years. In regard to race, 11 (26.19%) were no breed, 9 (21.42%) were Poodle, and 4 (9.52%) were Siberian Husky and Cocker Spaniels. The history of diagnosis of other cancers, was found in 23.80% of the animals in this study. In relation to castration, 32 (94.11%) of males were not castrated and only 8 (19.05%) were females. The progression of the tumor showed an average of 1.2 years. History of recurrence of cancer were reported in 14 animals, and was higher (66.66%) for carcinomas. Ki-67 staining revealed that the carcinomas showed higher proliferation rate (9.87%) compared to groups of epitheliomas... (Complete abstract click electronic access below)

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