24 results on '"Citoesqueleto de actina"'
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2. Supervivencia de células fibroblásticas humanas en ausencia de suplementación.
- Author
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Simancas-Escorcia, Víctor, Díaz-Caballero, Antonio, and Vergara Hernandez, Clara
- Abstract
Introduction. Gingival fibroblasts (GF) are cells of gingival connective tissue that have taken promising relevance in recent years due to their probable use in cell therapy, given their multipotencial and self-renewal capabilities. Objective. To know and to describe the impact of the absence of Fetal Bovine Serum (FBS supplementation on the survival of gingival fibroblasts in cultures. Materials and methods. Gingival fibroblasts were isolated from gingival tissue of healthy patients and cultured in DMEM (Dulbecco's Modified of Eagle Medium) culture media in absence and supplemented with 0.2% FBS at 37 ° C in a humid atmosphere with 5% CO2. A morphological evaluation, survival and proliferation of GF were carried out, as well as the identification by the immunofluorescence technique of cellular cytoskeleton markers such as actin and mitochondria. Results. The GF grown in the absence and with supplementation of 0.2% FBS showed a fusiform shape, with oval nuclei and numerous cytoplasmic extensions during the culture time. A slight increase in the proliferation of GF was observed in those cells in contact with the DMEM medium +0.2% FBS compared to the medium where the supplementation was absent. Immunostaining of actin and mitochondria showed that the absence and supplementation to 0.2% of FBS did not affect its location in the evaluated. Conclusion. Gingival fibroblasts survive and proliferate in the absence of FBS, preserving their cellular morphological characteristics. [ABSTRACT FROM AUTHOR]
- Published
- 2020
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3. Mecanismos moleculares asociados con el efecto inhibitorio de los anticuerpos anti-gangliósidos sobre la regeneración axonal en el sistema nervioso periférico
- Author
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Báez, Bárbara Beatriz, López, Pablo Héctor Horacio, Bisbal, Mariano, Kunda, Patricia, Lardone, Ricardo Dante, and Quinta, Héctor Ramiro
- Subjects
Anticuerpos ,Síndrome de Guillain Barré ,Enfermedades autoinmunes ,Enfermedades del sistema nervioso ,Neuropatía ,Citoesqueleto de actina ,Neurociencia ,Neuronas ,Gangliosidos - Abstract
Tesis (Doctora en Neurociencias) - - Universidad Nacional de Córdoba. Facultad de Ciencias Químicas, 2022 Resumen: El síndrome de Guillain Barré (SGB) es una polineuropatía aguda caracterizada por la presencia de parálisis muscular ascendente y arreflexia. Al presente los mecanismos patogénicos no han sido dilucidados en su totalidad, pero se asume que la parálisis en pacientes con SGB donde se ven afectados los axones estaría relacionada al desarrollo de una respuesta autoinmune humoral contra gangliósidos. Se ha descripto que la presencia de altos títulos de anticuerpos anti-gangliósidos se asocia con una recuperación clínica más lenta y una mala prognosis. Resultados previos de nuestro grupo de trabajo han demostrado en modelos in vitro usando neuronas del ganglio de la raíz dorsal (DRGn) que los anticuerpos anti-gangliósidos inhiben la extensión de neuritas y provocan colapso del cono de crecimiento a través de la activación de vías tanto dependientes de la GTPasa RhoA y su quinasa asociada ROCK (mediante la alteración de la dinámica de microtúbulos y reducción de la extensión de filopodias) como independientes de RhoA (colapso de la lamela de actina). Así los anticuerpos anti-gangliósidos ejercen un rol inhibitorio sobre la regeneración axonal mediante la activación de una cascada de señalización que involucra la interacción de gangliósidos con moléculas transductoras específicas, lo que se traduce en la activación de vías dependientes e independientes de RhoA, que modulan en forma negativa el citoesqueleto de actina y tubulina de los conos de crecimiento de las neuronas estudiadas. Los objetivos del presente trabajo fueron determinar la/las posibles moléculas compañeras de los gangliósidos en la membrana, capaces de transducir el efecto inhibitorio de los anticuerpos anti-gangliósidos sobre el crecimiento neurítico en modelos in vitro e in vivo; y estudiar las vías de señalización que conducen a la activación de la GTPasa pequeña RhoA y a la inhibición de la regeneración axonal. Comenzamos con una búsqueda bibliográfica e informática de las proteínas que se encontraron en un estudio de proteómica para determinar posibles interacciones con gangliósidos. Luego empleando diferentes técnicas experimentales pudimos encontrar que los efectos inhibitorios de los anticuerpos específicos contra el gangliósido GD1a (αGD1a) sobre el crecimiento neurítico de DRGn son dependientes del receptor del factor de necrosis tumoral alfa 1a (TNFR1a) y que éste actúa como molécula transductora y compañero molecular del gangliósido GD1a en forma específica. Complementariamente se realizó un análisis in sílico de la estructura 3D de la región del tallo del TNFR1a con la finalidad de identificar posibles sitios de interacción con el gangliósido GD1a. Esto derivó en la identificación de una mutante del TNFR1a caracterizada por la sustitución de ácido aspártico por alanina en la posición 165 la cual fue capaz de prevenir la transducción de la señal de los anticuerpos αGD1a específicos. Determinamos que los anticuerpos αGD1a (ya sean específicos o crosreactivos) activan a RhoA a través de TNFR1a por una vía alternativa al desplazamiento RhoGDI/RhoA por el dominio intracelular de éste receptor. Esto nos llevó a estudiar otra vía de señalización alternativa relacionada con la activación de RhoA. Otro candidato encontrado en la proteómica y confirmado experimentalmente por participar en la vía de señalización inhibitoria de los anticuerpos anti-gangliósidos fue la subunidad alfa de tipo inhibitoria 2 de la proteína G heterotrimérica (Gαi2). Esta proteína se relaciona con la disminución de los niveles de AMP cíclico intracelular y en consecuencia no se activa la proteína quinasa A que ejerce un efecto modulatorio negativo sobre RhoA. Confirmamos que Gαi2 en DRGn está involucrada en la inhibición del crecimiento axonal, en el colapso rápido de la lamela de actina y en la activación de RhoA mediada por los anticuerpos αGD1a específicos y crosreactivos. Por otra parte, identificamos a la GTPasa Rac1 como otro candidato encontrado en el estudio de proteómica y ya había sido relacionado con las alteraciones del citoesqueleto del cono de crecimiento. Determinamos que la Gαi2 está involucrada en el rápido descenso en la actividad de Rac1, mediado por el anticuerpo αGD1a/GT1b y esto se asocia al colapso de la lamela de actina (proceso RhoA independiente). En conclusión, nuestros resultados identifican un nuevo mecanismo molecular de señalización intracelular relacionado con el efecto inhibitorio de anticuerpos αGD1a específicos sobre el crecimiento neurítico y la reparación nerviosa, lo que abre la posibilidad al desarrollo de nuevas estrategias terapéuticas tendientes a mitigar el daño degenerativo que caracteriza a un porcentaje importante de pacientes con SGB. Abstract: Guillain-Barré Syndrome (GBS) is an acute polyneuropathy characterized by the presence of ascending muscle paralysis and arreflexia. The pathogenic mechanisms have not been fully elucidated, but it is assumed that the paralysis in patients with axonal forms of GBS is related to the development of a humoral autoimmune response against gangliosides. It has been described that the presence of high titers of anti-ganglioside antibodies is associated with a slower clinical recovery and poor prognosis. Previous results from our research group in in vitro paradigms of neurite outgrowth of dorsal root ganglion neurons (DRGn) have shown that anti-gangliosides antibodies inhibit neurite outgrowth and cause growth cone collapse through the activation of both dependent (alteration of microtubule dynamics and reduction of filopodia extension) and independent (collapse of lamella) RhoA pathways. Thus, anti-ganglioside antibodies exert an inhibitory role on axonal regeneration by activating a signalling cascade that involves the interaction of gangliosides with specific transducing molecules. This results in the negative modulation of actin and tubulin cytoskeleton on growth cones from the neurons in both RhoA dependent and independent manner. The goals of the present work were to determine the potential partner molecules of the gangliosides in the membrane, capable of transducing the inhibitory effect of the anti-gangliosides antibodies on neurite outgrowth and to study the signaling pathways that lead to the activation of the small GTPase RhoA resulting in inhibition of axonal regeneration. We first performed a bibliographic and bioinformatic search among proteins from a proteomic study to determine possible molecular partners for gangliosides. Then, using different experimental techniques, we were able to find that the inhibitory effects of anti-ganglioside GD1a (antibodies αGD1a) on neurite outgrowth of DRGn are dependent on the expression of Tumor Necrosis Factor alpha receptor 1a (TNFR1a), which acts as a specific transducing moleculepartner for GD1a ganglioside. In addition, we performed an in sílico analysis of the 3D structure of the TNFR1a stalk region in order to identify potential interaction sites with GD1a ganglioside. This led to the identification of a TNFR1a mutant characterized by an aspartic acid substitutionfor alanine at position 165 which was able to prevent the signal transduction of specific αGD1a antibodies. We also identified that αGD1a antibodies (either specific or cross-reactive) activate RhoA through TNFR1a by an alternative mechanism to RhoGDI/RhoA displacement via the intracellular domain of this receptor. This led us to study another alternative signalling pathway related to RhoA activation. Another candidate found in the proteomic study and experimentally confirmed to participate in the inhibitory signalling pathway of anti-ganglioside antibodies, was the inhibitory type 2 alpha subunit of the heterotrimeric G protein (Gαi2). This protein`srole is to decrease intracellular cyclic AMP levels and consequently, protein kinase A, which exerts a negative modulatory effect on RhoA. We confirmedin DRGn that Gαi2 is involved in the inhibition of axonal outgrowth, the rapid collapse of the actin lamella and RhoA activation triggered by specific and cross-reactive αGD1a antibodies. On the other hand, we identified that Gαi is involved in the early decrease of GTPase Rac1 activity mediated by the αGD1a/GT1b antibody, which precedes the collapse of the actin lamella (RhoA-independent process). In conclusion, our results identify a new molecular mechanism of intracellular signalling related to the inhibitory effect of specific anti-GD1a antibodies on neurite outgrowth and nerve repair, which opens the possibility of developing new therapeutic strategies aimed to mitigate the degenerative damage present in a significant percentage of GBS patients. 2024-03-31 Fil: Báez, Bárbara Beatriz Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina.
- Published
- 2022
4. Supervivencia de células fibroblásticas humanas en ausencia de suplementación
- Author
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Antonio Díaz-Caballero, Víctor Simancas-Escorcia, and Clara Inés Vergara Hernández
- Subjects
0301 basic medicine ,Proliferación celular ,Fibroblastos [(Decs Bireme)] ,Gingiva ,Fusiform shape ,Encía ,Culture Media Serum-Free ,Mitochondrion ,Immunofluorescence ,medio de cultivo libre de suero ,03 medical and health sciences ,medicine ,Cytoskeleton ,lcsh:QH301-705.5 ,Mitocondrias ,Actin ,Cell Proliferation ,Fibroblasts [(Mesh Database)] ,lcsh:R5-920 ,Fetus ,medicine.diagnostic_test ,Chemistry ,030111 toxicology ,Citoesqueleto de actina ,Molecular biology ,Mitochondria ,Actin Cytoskeleton ,030104 developmental biology ,lcsh:Biology (General) ,Cytoplasm ,lcsh:Medicine (General) - Abstract
Resumen Introducción. Los fibroblastos gingivales (FGs) son células del tejido conjuntivo gingival que han tomado en los últimos años una relevancia promisoria por su probable utilización en la terapia celular, dadas sus capacidades de multipotencialidad y de autorrenovación. Objetivo. Conocer y describir el impacto de la ausencia en la suplementación de Suero Fetal Bovino (SFB) en la supervivencia de fibroblastos gingivales en cultivos. Materiales y métodos. Fibroblastos gingivales fueron aislados de tejido gingival de pacientes sanos y cultivados en medios de cultivos DMEM (Dulbecco's Modified of Eagle Medium) en ausencia y suplementados con 0.2% de SFB a 37°C en una atmósfera húmeda con 5% de CO2. Se llevó a cabo una evaluación morfológica, de supervivencia y proliferación de los FGs, así como la identificación mediante la técnica de inmunofluorescencia de marcadores del citoesqueleto celular como la actina y mitocondrias. Resultados. Los FGs cultivados en ausencia y con suplementación de 0.2% de SFB evidenciaron una forma fusiforme, con núcleos ovalados y numerosas prolongaciones citoplasmáticas durante el tiempo de cultivo. Un leve aumento en la proliferación de FGs fue observado en aquellas células en contacto con el medio DMEM+0.2% de SFB comparadas con el medio donde estuvo ausente la suplementación. El inmunomarcaje de la actina y las mitocondrias dejó en evidencia que la ausencia y suplementación a 0.2% de SFB no afectó su localización en los FGs evaluados. Conclusión. Los fibroblastos gingivales sobreviven y proliferan en ausencia de SFB, conservando sus características morfológicas celulares. Abstract Introduction. Gingival fibroblasts (GF) are cells of gingival connective tissue that have taken promising relevance in recent years due to their probable use in cell therapy, given their multipotencial and self-renewal capabilities. Objective. To know and to describe the impact of the absence of Fetal Bovine Serum (FBS supplementation on the survival of gingival fibroblasts in cultures. Materials and methods. Gingival fibroblasts were isolated from gingival tissue of healthy patients and cultured in DMEM (Dulbecco's Modified of Eagle Medium) culture media in absence and supplemented with 0.2% FBS at 37 ° C in a humid atmosphere with 5% CO2. A morphological evaluation, survival and proliferation of GF were carried out, as well as the identification by the immunofluorescence technique of cellular cytoskeleton markers such as actin and mitochondria. Results. The GF grown in the absence and with supplementation of 0.2% FBS showed a fusiform shape, with oval nuclei and numerous cytoplasmic extensions during the culture time. A slight increase in the proliferation of GF was observed in those cells in contact with the DMEM medium +0.2% FBS compared to the medium where the supplementation was absent. Immunostaining of actin and mitochondria showed that the absence and supplementation to 0.2% of FBS did not affect its location in the evaluated. Conclusion. Gingival fibroblasts survive and proliferate in the absence of FBS, preserving their cellular morphological characteristics.
- Published
- 2020
5. Galectina-3, AFAP1-L1 e WASP na invasão e multiplicação de Trypanosoma cruzi e caracterização biológica da proteína P21-His6
- Author
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Fabrício Castro Machado, Silva, Claudio Vieira da, Ávila, Veridiana de Melo Rodrigues, and Mortara, Renato Arruda
- Subjects
CIENCIAS BIOLOGICAS::IMUNOLOGIA::IMUNOLOGIA APLICADA [CNPQ] ,Gene knockdown ,biology ,Angiogenesis ,Trypanosoma cruzi ,Angiogênese ,Imunologia ,Actin cytoskeleton ,Citoesqueleto de actina ,knockdown ,biology.organism_classification ,Molecular biology ,Bacteriophage ,Citoesqueleto ,Bacteriófagos - Abstract
The parasitism of cellular organisms is a topic of high relevance to human health and is under constant investigation. The process by which the flagellate protozoan Trypanosoma cruzi invades the host cells, thus causing the Chagas disease, is still not fully understood. In this work we study the role of AFAP-1L1, galectin-3 and WASP proteins on host cell invasion and characterize the biological functions of the P21 recombinant form, called P21-His6. The AFAP-1L1, galectin-3 and WASP proteins participate in numerous biological activities on the host cell, while the P21 protein, a recently characterized protein present in the membrane of T. cruzi, is presented in all developmental stages of the parasite. We assessed the involvement of galectin-3 in the intracellular traffic of T. cruzi by immunofluorescence tracking, and the role of the other host proteins by knockdown cell lines. The biological activities of P21-His6 were studied by selection of specific bacteriophages ligands with Biopanning technique, with implantation of sponges in Balb/c animals, treating or nor with P21-His6, and evaluating chemotaxis response when treating C57Bl/6 animals. We observed that galectin-3 is present in approximately 20% of cellular invasion events, forming structures containing polarized galectin-3 (G3CS) that fades over time. We also observed that AFAP-1L1 seems to have some importance in controlling early multiplications, and that reduction of WASP protein expression seems to facilitate parasite proliferation in parallel to the reduction of invasion. Thus, we conclude that these proteins are important for establishing T. cruzi infection. We observed that the P21-His6 has anti-angiogenic activity and capacity for cellular recruitment probably through interaction with the CXCR4 receptor especially of activated macrophages and neutrophils. We also obtained mimetic CXCR4 bacteriophages capable of specifically binding to P21-His6 and reducing its biological effects in vitro. Thus we conclude that P21-His6 has high chemotactic capacity that can be inhibited by specific ligands, and may have therapeutic anti-angiogenic activity. Together these results may open the door to novel therapeutic interventions. O parasitismo de organismos celulares é alvo de constantes estudos e o processo pelo qual o protozoário flagelado Trypanosoma cruzi invade a célula do hospedeiro ainda tem lacunas a serem preenchidas. As proteínas AFAP-1L1, galectina-3 e WASP são proteínas importantes para a célula do hospedeiro, participando de inúmeras atividades biológicas. Já a proteína P21, secretada pelo T. cruzi, foi recentemente caracterizada, se mostrando presente em todas as formas evolutivas do parasita. Nesse trabalho, estudamos a importância das proteínas AFAP-1L1, galectina-3 e WASP do hospedeiro durante a invasão e multiplicação do T. cruzi e também buscamos caracterizar funções biológicas da forma recombinante da P21, chamada P21-His6. A participação da galectina-3 durante o tráfego intracelular de T. cruzi foi acompanhada por imunofluorescência, e a importância das outras proteínas do hospedeiro foram analisadas através de linhagens celulares knockdown, com reduzida expressão, para as proteínas de interesse. Os estudos das atividades biológicas da P21-His6 se deram pela seleção de bacteriófagos ligantes específicos com a técnica de Biopanning, com o implante de esponjas interescapulares e a avaliação do processo inflamatório de corpo estranho e também pelo seu tratamento in vivo em animais Balb/c. Observamos que a proteína galectina-3 está presente em aproximadamente 20% dos eventos de invasão celular, formando estruturas contendo galectina-3 polarizada que vão desaparecendo ao longo do tempo, também observamos que a proteína AFAP-1L1 parece ter alguma importância no controle da multiplicação em tempos iniciais, assim como a redução da expressão proteína WASP parece facilitar a multiplicação do parasita, paralelamente ao fato de reduzir a invasão do mesmo. Observamos que a P21-His6 tem capacidade de recrutamento celular, provavelmente pela sua interação com o receptor CXCR4, especialmente de macrófagos e neutrófilos ativados, e que também possui atividade anti-angiogênica. Obtivemos também bacteriófagos miméticos do CXCR4, capaz de se ligar especificamente à P21-His6 e reduzir seus efeitos biológicos in vitro. Sendo assim concluímos a importância das proteínas do hospedeiro Galectina-3, AFAP-1L1 e WASP durante o estabelecimento da infecção por T. cruzi e concluímos que a P21-His6 pode ter função terapêutica pela sua atividade anti-angiogência, que possui alta capacidade quimiotática e que pode ser inibida por ligantes específicos. Mestre em Imunologia e Parasitologia Aplicadas
- Published
- 2021
6. Exploring the anticancer effect of new ruthenium-biotinylated conjugates in colorectal cancer
- Author
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Fernandes, Pedro José Pereira, Preto, Ana, and Universidade do Minho
- Subjects
Cancro colorretal ,Clonogenic ability ,Ciências Naturais::Ciências Biológicas ,Capacidade clonogénica ,Ruténio ,Actin cytoskeleton ,Citoesqueleto de actina ,Colorectal cancer ,Ruthenium - Abstract
Dissertação de mestrado em Genética Molecular, Colorectal cancer (CRC) is one of the most commonly diagnosed cancers and a leading cause of cancer death worldwide. The current chemotherapeutic agents available for CRC treatment are limited, accompanied by severe side effects and resistance acquisition. Ruthenium (Ru) compounds have been explored because they encompass several anticancer properties that make them promising therapeutic agents. Considering this, two new ruthenium-biotinylated conjugates (LCR134 and LCR220) were recently synthesized, in an attempt to bring more efficiency and selectivity to target cancer cells. LCR134 comprises a Ru cytotoxic fragment, with the addition of two molecules of biotin to the organometallic center (active targeting), while LCR220 has a polymeric chain, with two biotin molecules at the end (passive and active targeting). The aim of this work was to determine whether these Ru-biotinylated conjugates are promising candidates for CRC therapy. In this work, the effects of the newly Ru agents were assessed in CRC cells (SW480 and RKO) and in a “normal” colon-derived cell line (NCM460). In order to achieve our aim, we studied the effect of Ru compounds on cell viability, cell death, F-actin cytoskeleton structure, as well as in β-actin and glucose transporters 1 (GLUT1) expression levels. The results showed that the compounds were more cytotoxic for CRC cell lines in comparison with the normal colon cells. Moreover, all the half-maximum inhibitory concentration (IC50) values with the exception of LCR134 for SW480 cells, were lower than the values of the classical chemotherapeutic agent cisplatin, suggesting a higher efficacy of our ruthenium-biotinylated conjugates towards CRC cells. Ru compounds also interfere with the clonogenic ability of both CRC cell lines, without inducing cell death in a significant manner, suggesting that their mechanism of action affects proliferation. Additionally, these agents induced structural changes in the cytoskeleton, namely in F-actin filaments. We could not observe alterations on the expression levels of β-actin and GLUT1. Our results highlight the promising anticancer activities of these agents towards CRC cells., O cancro colorectal (CCR) é um dos cancros mais frequentemente diagnosticados e uma das principais causas de morte em todo o mundo. Os actuais agentes quimioterapêuticos disponíveis para o tratamento do CCR são limitados, acompanhados por graves efeitos secundários e aquisição de resistência. Os compostos de ruténio (Ru) têm sido explorados por abrangerem várias propriedades anticangerígenas que os tornam agentes terapêuticos promissores. Tendo isto em consideração, dois novos conjugados de ruténio-biotinilados (LCR134 e LCR220) foram sintetizados recentemente, numa tentativa de alcançar mais eficiência e selectividade para as células cancerígenas. O LCR134 é constituído por um fragmento citotóxico de Ru, com a adição de duas moléculas de biotina ao centro organometálico (alvo ativo), enquanto que o LCR220 têm uma cadeia polimérica, com duas moléculas de biotina no final (alvo passivo e ativo). O objetivo deste trabalho foi determinar se estes conjugados de Ru-biotinilados são candidatos promissores para a terapia do CCR. Neste trabalho, os efeitos dos novos agentes de Ru foram avaliados em células CCR (SW480 e RKO) e numa linha celular “normal” derivada do cólon (NCM460). Com esse propósito, estudámos os efeitos dos compostos de Ru na viabilidade celular, morte celular, na estrutura do citoesqueleto de F-actina, bem como nos níveis de expressão de β-actina e transportadores de glucose 1 (GLUT1). Os resultados mostraram que os compostos foram mais citotóxicos para as linhas celulares de CCR em comparação com a linha de células normais do cólon. Além disso, todos os valores de IC50, com a excepção do LCR134 para as células SW480 foram inferiores aos valores do agente quimioterapêutico clássico cisplatina, sugerindo uma maior eficácia dos nossos conjugados de ruténio-biotinilados, nas células de CCR. Os compostos de Ru também interferem com a capacidade clonogénica das linhas celulares de CCR, sem induzir a morte celular de maneira significativa, sugerindo um mecanismo de ação que interfere com a proliferação. Adicionalmente, os agentes induziram mudanças estruturais no citoesqueleto, mais precisamente nos filamentos de F-actina. Não observámos alterações nos níveis de expressão de β-actina e GLUT1. Estes resultados destacam actividades anticancerígenas promissoras destes agentes para as células de CCR., This work was financed by the Portuguese Foundation for Science and Technology (Fundação para a Ciência e a Tecnologia, FCT) within the scope of project PTDC/QUIQIN/28662/2017 and by the strategic programme UID/BIA/04050/2019 funded by national funds through the FCT I.P
- Published
- 2020
7. Regulation of CFTR membrane stability - crosstalk between signalling pathways and the cytoskeleton
- Author
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Ferreira, João Frederico Santos Fernandes e Coelho and Farinha, Carlos,1973
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Ciências Naturais::Ciências Químicas [Domínio/Área Científica] ,Citoesqueleto de actina ,Fibrose Quística ,CFTR ,Sinalização de cAMP ,Teses de mestrado - 2020 - Abstract
Tese de mestrado em Bioquímica (Bioquímica Médica), Universidade de Lisboa, Faculdade de Ciências, 2020 Submitted by Cristina Manessiez (camanessiez@fc.ul.pt) on 2021-05-05T17:47:33Z No. of bitstreams: 1 ulfc126203_tm_João_Ferreira.pdf: 1726435 bytes, checksum: 79ab7d3a2329c7ef50b4e2e61ce55e43 (MD5) Made available in DSpace on 2021-05-05T17:47:47Z (GMT). No. of bitstreams: 1 ulfc126203_tm_João_Ferreira.pdf: 1726435 bytes, checksum: 79ab7d3a2329c7ef50b4e2e61ce55e43 (MD5) Previous issue date: 2020
- Published
- 2020
8. Role of action dynamics in the cooperative maintenance of synaptic plasticity
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Marcut, Cristina Flórica, Alvarez, Rosalina Maria Regada Carvalho Fonseca de, and Ferreira, Hugo Alexandre
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Citoesqueleto de actina ,Marcador sináptico ,Plasticidade sináptica ,Cdc42 ,Ciências Naturais::Ciências Biológicas [Domínio/Área Científica] ,Teses de mestrado - 2020 ,STC - Abstract
Tese de mestrado em Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2020 Submitted by Cristina Manessiez (camanessiez@fc.ul.pt) on 2020-12-16T17:17:48Z No. of bitstreams: 1 ulfc125980_tm_Cristina_Marcut.pdf: 1322720 bytes, checksum: b30ccecd85d96ea0646317f74bc05a9b (MD5) Made available in DSpace on 2020-12-16T17:17:59Z (GMT). No. of bitstreams: 1 ulfc125980_tm_Cristina_Marcut.pdf: 1322720 bytes, checksum: b30ccecd85d96ea0646317f74bc05a9b (MD5) Previous issue date: 2020
- Published
- 2020
9. Digital holographic imaging of dynamic cytoskeleton changes
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Schnekenburger, Jürgen, Bredebusch, Ilona, Domschke, Wolfram, Kemper, Björn, Langehanenberg, Patrik, and von Bally, Gert
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CANCER cells , *CELLS , *CELLULAR pathology , *ASCITES tumors - Abstract
Abstract: A variety of physical and biochemical cell properties depends on the function and integrity of the actin cytoskeleton. The actin cytoskeleton mediates crucial cellular functions as migration, intracellular transport, exocytosis, endocytosis, cell stiffness and force generation. Highly dynamic actin fibers are therefore targets for several drugs and toxins. However, the study of actin interfering processes by standard microscopy techniques fails in the detailed resolution of dynamic spatial alterations required for a deeper understanding of toxic or drug effects. Here we applied digital holographic microscopy in the online functional analysis of the actin cytoskeleton changes of a highly differentiated and a dedifferentiated pancreas tumor cell line induced by the marine toxin Latrunculin B. Scanning electron microscopy (SEM) and fluorescence microscopy showed rapid Latrunculin B induced alterations in cell morphology and actin fiber degradation in both pancreatic tumor cell lines. In contrast digital holographic in vivo analysis of the drug dependent dynamic cellular processes revealed unequal changes in cell morphology. While tumor cells with a low metastatic potential showed Latrunculin B induced cell collapse within 4h the metastatic tumor cells were resistant to Latrunculin B treatment. Spatial resolution of morphological alterations by digital holography detected so far unknown differences in the actin cytoskeleton stability of highly differentiated and dedifferentiated pancreas tumor cell lines. These data demonstrate that marker-free, non-destructive online analysis of cellular morphology and dynamic spatial processes in living cells by digital holography offers new insights in actin dependent cellular mechanisms. Digital holographic microscopy was shown to be a versatile tool in the screening of toxic drug effects and cancer cell biology. [Copyright &y& Elsevier]
- Published
- 2007
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- View/download PDF
10. Design and validation of advanced image analysis tools for the quantification of cancer cell migration in microfluidic microdevices
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Castilla-Ruiz, C.(Carlos) and Ortiz-de-Solorzano, C. (Carlos)
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Matrigel ,Confocal microscopy ,Citoesqueleto de actina ,Hydrogel ,Phase contrast microscopy ,Modelo de Chan-Vese ,Collagen ,Clahe ,Segmentación y tracking de células - Abstract
This thesis presents the design and validation of advanced image analysis and machine learning tools for the quantitative analysis of three-dimensional (3D) lung cancer cell migration. These tools were used to study the role that the composition and the morphological and mechanical properties of the microenvironment have in cancer cell migration. Cell migration experiments were performed using in vitro cellular models consisting of custom-made microfluidic devices, which provide a high level of control of the properties of the microenvironment, offer optimal optical properties for microscopic observation and ensure appropriate use of reagents due to their microscopic dimensions. Varying migration environmental conditions were simulated by embedding the cells in biomimetic hydrogels of different composition and, thus, with different morphological and mechanical properties. These hydrogels act as the 3D extracellular matrix (ECM) of the cells. Namely, we used hydrogels made of pure collagen type I to mimic normal connective tissue and hydrogels made of a mixed composition of collagen and Matrigel to simulate the disorganized basement membrane at the leading edge of cancer invasion. The migration experiments performed within the hydrogels were done with and without serum stimulation to determine the role of hydrogel extrinsic and intrinsic growth factors in cell migration. Furthermore, migration experiments were performed with and without blocking cellular integrins to determine the role of the cell-to-ECM interactions in cell migration, and with and without metalloproteinase (MMP) inhibitors to understand the role of ECM in cell migration. Migration experiments were performed at two resolution levels. First, low-resolution experiments were performed to determine globally the effect of the parameters analyzed in the dynamics of cancer cell migration. This required detecting and tracking a large number of cancer cells, imaged at low magnification (10×) using widefield phase-contrast and fluorescence microscopy. Second, high magnification (63×) migration experiments were used to study the morphology of migrating cancer cells, by measuring the number, length and life time of cell protrusions produced during the migration. This required the use of laser scanning confocal microscopy. The microscopy time-lapse sequences generated in both types of experiments were analyzed using novel image processing and machine learning techniques in order to obtain robust quantifiable metrics of cell migration dynamics and migrating phenotype in 3D environments. This thesis describes the experiments performed along with the image processing and computational algorithms used to analyze cell migration both in low- and high-magnification image data. We provide the source code of the developed analysis software, for its use and future algorithmic improvement by the research community. Esta tesis presenta el diseño y la validación de herramientas avanzadas de análisis de imagen y de aprendizaje automático para el análisis cuantitativo de la migración tridimensional (3D) de células de cáncer de pulmón. Estas herramientas se utilizaron para estudiar el papel que tiene la composición y las propiedades morfológicas y mecánicas del microambiente en la migración de células cancerígenas. Los experimentos de migración celular se llevaron a cabo usando modelos celulares in vitro consistentes en dispositivos microfluídicos hechos a medida, los cuales proporcionan un alto nivel de control de las propiedades del microambiente, ofrecen propiedades óptimas para la observación microscópica y aseguran el uso apropiado de reactivos debido a sus dimensiones microscópicas. Las diferentes condiciones ambientales de la migración se simularon embebiendo las células en hidrogeles biomiméticos de diferente composición y, por lo tanto, con diferentes propiedades morfológicas y mecánicas. Estos hidrogeles actúan como la matriz extracelular 3D (MEC) de las células. En concreto, se han utilizado hidrogeles hechos de colágeno puro tipo I para simular el tejido conectivo normal e hidrogeles de una composición mixta de colágeno y Matrigel para simular la membrana basal desorganizada en el frente de invasión del cáncer. Los experimentos de migración realizados dentro de los hidrogeles se realizaron con y sin estimulación con suero para determinar el papel de los factores de crecimiento extrínsecos e intrínsecos del hidrogel en la migración celular. Además, se realizaron experimentos de migración con y sin bloqueo de integrinas celulares para determinar el papel de las interacciones célula-MEC en la migración celular, y con y sin inhibidores de metaloproteinasas (MMP) para comprender el papel de la MEC en la migración celular. Los experimentos de migración se realizaron en dos niveles de resolución. Por una parte, se realizaron experimentos a baja resolución para determinar globalmente el efecto de los parámetros analizados en la dinámica de la migración de células cancerígenas. Esto requirió detectar y seguir un gran número de células cancerígenas, con imágenes de baja magnificación (10×) utilizando microscopía de contraste de fase y de fluorescencia de campo amplio. Por otra parte, se realizaron experimentos de migración a alta magnificación (63×) para estudiar la morfología de las células cancerígenas mientras migran, midiendo el número, longitud y tiempo de vida de las protrusiones celulares producidas durante la migración. Esto requirió el uso de microscopía confocal de barrido láser. Las secuencias de microscopía de lapso de tiempo generadas en ambos tipos de experimentos se analizaron utilizando novedosas técnicas de procesamiento de imagen y de aprendizaje automático para obtener métricas cuantificables robustas de la dinámica de la migración celular y del fenotipo de migración en entornos 3D. Esta tesis describe los experimentos realizados junto con los algoritmos computacionales y de procesamiento de imagen utilizados para analizar la migración celular en imágenes de baja y alta magnificación. Proporcionamos el código fuente del software de análisis desarrollado para su uso y futura mejora algorítmica por parte de la comunidad investigadora.
- Published
- 2019
11. Caracterización de los mecanismos que modifican el citoesqueleto de actina en degeneración axonal de neuronas sensoriales y su impacto en el proceso degenerativo
- Author
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Martínez, Gaby Fabiana, Unsain, Nicolás, Cáceres, Alfredo Oscar, Hereñú, Claudia Beatriz, Quiroga, Santiago, and Cáceres, Alfredo
- Subjects
Ciencias Médicas y de la Salud ,Medicina Básica ,Microscopía confocal ,Sistema nervioso ,Citoesqueleto de Actina ,Neurociencias ,Microscopia de super-resolución ,Actinas ,Lesión Axonal difusa ,Degeneración Walleriana ,Enfermedades del Sistema Nervioso ,Neuronas ,Citoesqueleto, MPS, Actina, degeneración - Abstract
Tesis (Doctora en Neurociencias) - - Universidad Nacional de Córdoba. Facultad de Ciencias Químicas, 2019 La Degeneración Axonal (DA) es un proceso normal del desarrollo del sistema nervioso. La DA participa junto con otros procesos (como el guiado axonal, el colapso de conos de crecimiento o la retracción axonal) en la maduración de los circuitos neuronales para que lleguen a ser funcionales. Interesantemente, muchos mecanismos moleculares que controlan la DA del desarrollo, también participan de la DA en condiciones patológicas, como en el caso de una lesión axonal (como ocurre en la Degeneración Walleriana) o en enfermedades neurodegenerativas (como en Alzheirmer o Parkinson). Es por todo esto que ha resultado de gran interés en los últimos años conocer en detalle los mecanismos involucrados en la destrucción del axón en diversos contextos. El citoesqueleto axonal participa directamente en la mantención estructural y funcional de los axones. Evidencias previas sugieren un rol de vías de señalización que controlan la dinámica del citoesqueleto de actina durante el desarrollo, lesiones axonales y algunas patologías. Sin embargo, hasta la realización del presente trabajo no existían evidencias de los cambios directos provocados por la degeneración en la estructura del citoesqueleto de actina y sus componentes asociados, como las estructuras periódicas de citoesqueleto asociado a membrana de actina/espectrina (MPS, por sus siglas en inglés). En este marco, el objetivo general de la tesis fue evidenciar cambios estructurales en el citoesqueleto de actina y el MPS de neuronas sensoriales en un modelo de DA del desarrollo (Privación de NGF, PNGF) y en un modelo de Degeneración Walleriana (DW, corte del axón), mediante métodos de microscopía de super-resolusión (STED y Microscopía de Expansión) y microscopía confocal. En primer lugar evidenciamos que la privación de NGF provoca cambios tempranos en el citoesqueleto de F-actina (axones y conos de crecimientos) y por nanoscopía STED demostramos que el remodelado de MPS es temprano y que su posterior pérdida precede la fragmentación axonal. Identificamos su rol clave en la mantención axonal cuando evidenciamos que la protección farmacológica del MPS retarda la fragmentación de los axones en vías de degeneración. En el segundo objetivo, validamos la utilización de Microscopía de Expansión cuantitativa para evidenciar el MPS y evaluar cambios en su abundancia y organización. En el tercer objetivo de la tesis utilizamos esta técnia para evaluar cambios en el citoesqueleto de actina y en la organización del MPS axonales durante la Degeneración Walleriana. Pudimos evidenciar cambios tempranos de F-actina en conos de crecimiento de axones distales. La organización y abundancia del MPS, también fue afectada significativamente a tiempos tempranos de la Degeneración Walleriana. Esta pérdida del MPS precedió la fragmentación axonal y la disrupción farmacológica de los mismos aceleró el proceso de pérdidas de axones. En su conjunto, los resultados del presente trabajo de tesis, aportan por primera vez datos claves sobre el rol estructural del citoesqueleto de F-actina y componentes asociados (MPS) en la mantención axonal en dos modelos de degeneración. Sumado a esto, también se logró poner a punto la técnica de Microscopía de Expansión como método cuantitativo de súperresolución para la observación del MPS utilizando microscopios de fluorescencia convencionales. 2021-12-31 Martínez, Gaby Fabiana. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina. Unsain, Nicolás. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina. Cáceres, Alfredo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina. Hereñú, Claudia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Farmacología Experimental de Córdoba; Argentina. Quiroga, Santiago. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina.
- Published
- 2019
12. Análisis de la expresión génica diferencial de células eritroleucémicas resistentes a la diferenciación: relevancia de las proteínas del citoesqueleto de actina
- Author
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Krimer, Dora B., Ministerio de Economía, Industria y Competitividad (España), Fernández-Calleja, Vanessa, Krimer, Dora B., Ministerio de Economía, Industria y Competitividad (España), and Fernández-Calleja, Vanessa
- Abstract
Friend murine erythroleukemia cells (MEL) derive from proerythroblasts transformed with the Spleen Focus Forming Virus (SFFV) (Friend et al. 1966; Ruscetti (1999) where integration occurred several kilobases upstream the PU.1 locus (Fernandez-Nestosa et al. 2013). These cells remain in a proliferative state and do not differentiate in the presence of erythropoietin. MEL-DS19 cell line may overcome the blockage, however, and reinitiate differentiation when exposed to a number of different chemical agents, such as hexamethylene-bis-acetamide (HMBA). This feature makes MEL-DS19 cells an extremely useful model to study reprogramming of tumor cells to a non-malignant phenotype and to analyze the mode of action of different chemotheraupetics compounds. We previously reported the establishment of HMBA-resistant cell lines (MEL-R) that are unable to differentiate, even in the presence of the inducer (Fernandez-Nestosa et al. 2008). On the other hand, the network of actin filaments provides mechanical support to the cell cytoskeleton, but it is increasingly acknowledged that it also contributes to other critical cellular processes. Emerging evidence points to a role for the actin cytoskeleton in controlling and regulating receptor signaling (Mattila et al. (2016). In this line, changes in the regulation of actin cytoskeleton could be implicated in the blockade of the erythroid differentiation program. The aims of this thesis were: -Identify genes whose differential expression is higher in MEL-DS19 in relation to MEL-R, and viceversa by using RNA-seq., Analyze specific hematopoietic genes whose functions are related to the organization and polymerization of the actin cytoskeleton, i.e. Was (Wiskott-Aldrich syndrome), Btk (Bruton’s tyrosine kinase) and Plek (pleckstrin). - Generate genomic deletions of Was, Btk and Plek in MEL-DS19 cells via CRISPR/Cas9. - Established MEL-R transfectants that overexpressed those same genes. -Analyze the possible implication of increased expression of histone-encoding genes in the resistant line MEL-R. Our results can be summarize as follows: Resistant cells (MEL-R) showed phenotypic differences in relation to the parental cell line. We corroborated a significant increase in the size of MEL-R cells that parallel a prolonging doubling time. The distribution of MEL-R cells along the cell cycle follows the same pattern observed in uninduced MEL-DS19 cells. However, we found that the DNA content in the resistant lines has doubled, generating tetraploid cells. This data supports the idea that tetraploid cells enhance tumorigenic capacity relative to diploid cells. In an attempt to identify genes related to the resistant phenotype we used high-throughput RNA sequencing (RNA-seq) to compare the transcriptomes of the erythroleukemia progenitor and the resistance cell lines. RNA-seq revealed a total of 596 genes with a p-value adjusted less than 0.05 that were differentially expressed by more than two-fold, of which 486 genes were up-regulated in MELDS19 cells and 110 in MEL-R cells, enlightening that the number of genes expressed in the parental cell line decreased as the cells acquired the resistant phenotype. The progressive gene silencing observed in MEL-R cells involves several mechanisms. We proved that heterochromatinization, a marker for transcription down-regulation, is enhanced in MEL-R cells relative to undifferentiated MEL-DS19 cells, but is nevertheless lower than in HMBA-differentiated cells. We also examined the methylation status of Was, Btk and Plek genes and found that in, The expression pattern of enzymes that catalyze DNA methylation and demethylation revealed an up-regulation of Dnmt1 and Tet3 expression in MEL-R cells. These results showed that the increase in DNA methylation by Dnmt1 in MEL-R cells overlaps with a decrease in demethylation by Tet3, which presumably results in the silencing of gene promoters. RNA-seq analysis showed highest differential expression allowed identification of a group among genes up-regulated in MEL cells. These genes are related with the organization of the actin cytoskeleton network, the majority of these genes are preferentially expressed in the erythroid lineage. Among this group stand out Bruton's tyrosine kinase (Btk), Wiskott Aldrich syndrome (Was) and pleckstrin (Plek). In order to study the potential implications of these genes on resistance to cell differentiation we performed genome deletions of Was, Btk or Plek in MEL-DS19 based on CRISPR/Cas9 system. In the first two cases, deficiencies were observed in the organization and polymerization of the actin cytoskeleton as opposed to the results obtained when Plek was deleted. The ability of the resistant line to reverse the wild phenotype was also determined. We established stable transfectants expressing each of the proteins in MEL-R cells. We observed a recovery in the organization of the actin cytoskeleton in Was and Btk. Moreover, we demonstrated that Was and Plek regulate the transcription expression of Btk, function that could be independent of the organization of the actin cytoskeleton. Altogether, these results suggest that disturbances of genes involved in the regulation and polymerization of actin cytoskeleton are coupled to a resistant differentiation phenotype.
- Published
- 2017
13. Wiskott-Aldrich syndrome protein regulates autophagy and inflammasome activity in innate immune cells
- Author
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Pamela P. Lee, Damián Lobato-Márquez, Nayani Pramanik, Andrea Sirianni, Vanessa Daza-Cajigal, Elizabeth Rivers, Alessia Cavazza, Gerben Bouma, Dale Moulding, Kjell Hultenby, Lisa S. Westerberg, Michael Hollinshead, Yu-Lung Lau, Siobhan O. Burns, Serge Mostowy, Mona Bajaj-Elliott, and Adrian J. Thrasher
- Subjects
MECHANISM ,THP-1 Cells ,Inflammasomes ,humanos ,citoesqueleto de actina ,Escherichia coli enteropatógena ,Monocytes ,Shigella flexneri ,ARP2/3 COMPLEX ,ACTIVATION ,Enteropathogenic Escherichia coli ,Mice ,hemic and lymphatic diseases ,inflamasomas ,lcsh:Science ,Mice, Knockout ,inmunidad ,línea celular ,Wiskott-Aldrich Syndrome ,interferón de tipo I ,Multidisciplinary Sciences ,DEFICIENCY ,Actin Cytoskeleton ,ESCHERICHIA-COLI ,Interferon Type I ,Science & Technology - Other Topics ,nigericina ,Wiskott-Aldrich Syndrome Protein ,autofagia ,proteína del síndrome de Wiskott-Aldrich ,Science ,macromolecular substances ,DENDRITIC CELLS ,Article ,Cell Line ,síndrome de Wiskott-Aldrich ,ACTIN NUCLEATION ,Cell Line, Tumor ,NLR Family, Pyrin Domain-Containing 3 Protein ,septinas ,MD Multidisciplinary ,Autophagy ,Humans ,Animals ,I INTERFERON-PRODUCTION ,Science & Technology ,CUTTING EDGE ,carga bacteriana ,fungi ,Immunity ,RECOGNITION ,células dendríticas ,receptor 4 similar a toll ,Immunity, Innate ,Bacterial Load ,Mice, Inbred C57BL ,Toll-Like Receptor 4 ,monocitos ,Nigericin ,animales ,lcsh:Q ,ratones ,Septins - Abstract
Dysregulation of autophagy and inflammasome activity contributes to the development of auto-inflammatory diseases. Emerging evidence highlights the importance of the actin cytoskeleton in modulating inflammatory responses. Here we show that deficiency of Wiskott–Aldrich syndrome protein (WASp), which signals to the actin cytoskeleton, modulates autophagy and inflammasome function. In a model of sterile inflammation utilizing TLR4 ligation followed by ATP or nigericin treatment, inflammasome activation is enhanced in monocytes from WAS patients and in WAS-knockout mouse dendritic cells. In ex vivo models of enteropathogenic Escherichia coli and Shigella flexneri infection, WASp deficiency causes defective bacterial clearance, excessive inflammasome activation and host cell death that are associated with dysregulated septin cage-like formation, impaired autophagic p62/LC3 recruitment and defective formation of canonical autophagosomes. Taken together, we propose that dysregulation of autophagy and inflammasome activities contribute to the autoinflammatory manifestations of WAS, thereby identifying potential targets for therapeutic intervention., Wiskott-Aldrich syndrome protein (WASp) is essential for controlling the cytoskeleton, but its function in innate immunity is unclear. Here the authors show that WASp deficiency is associated with dysregulated septin cage formation, excessive inflammasome activation, elevated immune cell death and reduced bacterial clearance.
- Published
- 2017
14. Role of actin regulatory proteins in Tunneling Nanotube formation and intercellular transfer in HeLa cells
- Author
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Lemos, Miguel Jorge Macedo, Zurzolo, Chiara, and Girão, Henrique Manuel Paixão dos Santos
- Subjects
Filopodia ,Túneis de Nanotubos ,Transferência Intercelular ,Intercellular transfer ,Actin cytoskeleton ,Citoesqueleto de actina ,Proteínas reguladoras do citoesquleto de actina ,Tunneling Nanotubes ,Actin regulatory proteins - Published
- 2017
15. Involvement of actin cytoskeleton and intracellular calcium in the control of sperm acrosomal exocytosis
- Author
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Romarowski, Ana and Buffone, Mariano G.
- Subjects
PEQUEÑAS GTPASAS ,COFILIN ,CALCIO ,ACTIN CYTOSKELETON ,PROGESTERONE ,CITOESQUELETO DE ACTINA ,PROGESTERONA ,CALCIUM ,ACROSOMAL EXOCYTOSIS ,LIMK1 ,SMALL GTPASES ,ESPERMATOZOIDE ,EXOCITOSIS ACROSOMAL ,SPERM - Abstract
Los espermatozoides de mamífero adquieren su capacidad fecundante después de una serie de modificaciones bioquímicas en el tracto reproductor femenino, colectivamente llamadas capacitación. Estos cambios son esenciales para que los espermatozoides puedan realizarla exocitosis acrosomal (EA), un proceso que es fundamental para la fecundación. En este trabajo se estudiaron los cambios en el citoesqueleto de actina en la preparación para la ocurrencia de la EA así como también, los cambios en los niveles de calcio intracelular como evento fundamental para las etapas finales de la fusión de membranas. La dinámica del citoesqueleto de actina juega un rol central en controlar el proceso de exocitosis en células somáticas así como en los espermatozoides de varias especies demamíferos. A pesar de que en células somáticas, laspequeñas GTPasas de la familia Rho son ampliamente conocidas como reguladores principales de la dinámica dela actina, su función en espermatozoides es desconocida. En el presente trabajo se caracterizó la participación de las pequeñas GTPasas de la familia Rho en la vía de señalización que conduce a la polimerización de actina durante la capacitación del espermatozoide de ratón. Se observó que la mayoría de las proteínas de esta cascada de señalización y sus proteínas efectoras se expresan en el espermatozoide de ratón. La activación de las vías de señalización de AMPc/PKA, RhoA/C y Rac1 es esencial para la activación por fosforilación de LIMK1 en Treonina 508. Cofilin es fosforilada en Serina 3porLIMK1 durante la capacitación de manera transitoria. La inhibición de LIMK1 por un inhibidor específico (BMS-3) resultó en menores niveles de polimerización de actina durante la capacitación y una marcada disminución en el porcentaje de espermatozoides que realizan EA. Asimismo se sabe que es necesario un aumento en el Ca2+ intracelular ([Ca2+]i) para que la EA se produzca. La progesterona producida por las células del cúmulus ha sido asociada con diversos procesos fisiológicos en los espermatozoides, incluyendo la estimulación de la EA. En este trabajo, investigamos la correlación espacio temporal entre los cambios en el [Ca2+]iy la EA en espermatozoidesindividuales de ratón en respuesta a la progesterona. Se encontró que la progesterona estimula un incremento en el [Ca2+]i encinco patrones diferentes: gradual,oscilatorio, transitorio tardío, transitorioinmediato, y sostenido.Tambiénse observó que el aumento en el [Ca2+]ipromovido por la progesterona puedecomenzar tanto en el flagelo como en la cabeza del espermatozoide. Se validó la utilización de FM4-64 como un indicador de la ocurrencia de la EA mediante la detección simultánea del aumento de su fluorescencia y la pérdida del EGFP en espermatozoides transgénicos EGFPAcr. Por primera vez, se logró visualizar simultáneamente el aumento en el [Ca2+]i y el proceso de exocitosis en respuesta a la progesterona, observándose que sólo un aumento transitorio específico en el [Ca2+]i originado en la cabeza del espermatozoide promueve la iniciación de la EA. En conclusión, en esta tesis se logró evidenciar la importanciade las pequeñas GTPasas de la familia Rho y sus efectores principales LIMK1 y Cofilin en la regulación de la dinámica del citoesqueleto de actina en la preparación del espermatozoide para poder realizar la EA. A su vez, se identificó el tipo de aumento de [Ca2+]i específico que es necesario para iniciar los eventos finales de la EA estimulada por progesterona. Mammalia sperm must acquire their fertilizing ability after a series of biochemical modifications in the female reproductive tract collectively called capacitation to undergo acrosomal exocytosis (AE), a process that is essential for fertilization. In this work, the changes in the actin cytoskeleton in preparation for the occurrence of the AE as well as the changes in intracellular calcium levels as a crucial event to the final stages of membrane fusion, were studied. Actin dynamics play a central role in controlling the process of exocytosis in somatic cells as well as in sperm from several mammalian species. In somatic cells, small GTPases of the Rho family are widely known as master regulators of actin dynamics. However, the role of these proteins in sperm has not been studied in detail. In the present work the role of the small GTPases of the Rho family in the signaling pathway that leads to actin polymerization during mouse sperm capacitation was studied. It was observed that most of the proteins of this signaling cascade and their downstream effector proteins are expressed in mouse sperm. The activation of the cAMP/PKA, RhoA/C and Rac1 signaling pathways are essential for LIMK1 activation by phosphorylation on Threonine 508. Serine 3 of Cofilin is phosphorylated by LIMK1 during capacitation in a transiently manner. Inhibition of LIMK1 by specific inhibitors (BMS-3) resulted in lower levels of actin polymerization during capacitation and a dramatic decrease in the percentage of sperm that undergo acrosomal exocytosis. In addition, it is well known that an increase in intracellular Ca2+ ([Ca2+]i) is necessary for AE to occur. Progesterone produced by cumulus cells has been associated with various physiological processes in sperm, including stimulation of AE. In this study, the spatio-temporal correlation between the changes in [Ca2+]i and AEin single mouse spermatozoa in response to progesterone was investigated. Progesterone stimulated an [Ca2+]i increase in five different patterns: gradual, oscillatory, late transitory, immediate transitory and sustained. It was also observed that the [Ca2+]i increase promoted by progesterone started at either the flagellum or the head. The use of FM4-64 as an indicator for the occurrence of the AE was validated by simultaneously detecting its fluorescence increase and the loss of EGFP in transgenic EGFPAcr sperm. For the first time, it was simultaneously visualized the rise in [Ca2+]i at the onset of AE in response to progesterone. Only a specific transitory increase in [Ca2+]i originated in the sperm head promoted the initiation of AE. In conclusion, in this thesis it was demonstrated the importance of the small GTPases of the Rho family and its main effector proteins LIMK1 and Cofilin in the regulation of the actin cytoskeleton in preparation for AE. Moreover, it was identified the type of [Ca2 +]i increase necessary to trigger the final events of AE induced by progesterone. Fil: Romarowski, Ana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentina.
- Published
- 2017
16. Análisis de la expresión génica diferencial de células eritroleucémicas resistentes a la diferenciación: relevancia de las proteínas del citoesqueleto de actina
- Author
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Fernández-Calleja, Vanessa, Krimer., Dora B., Krimer, Dora B., and Ministerio de Economía, Industria y Competitividad (España)
- Subjects
Células enteroendocrinas ,hemic and lymphatic diseases ,Biología ,Citoesqueleto de actina ,Diferenciación celular ,RNAseq ,CRISPR/Cas9 - Abstract
131 p.-49 fig.-6 tab.-anexo, Friend murine erythroleukemia cells (MEL) derive from proerythroblasts transformed with the Spleen Focus Forming Virus (SFFV) (Friend et al. 1966; Ruscetti (1999) where integration occurred several kilobases upstream the PU.1 locus (Fernandez-Nestosa et al. 2013). These cells remain in a proliferative state and do not differentiate in the presence of erythropoietin. MEL-DS19 cell line may overcome the blockage, however, and reinitiate differentiation when exposed to a number of different chemical agents, such as hexamethylene-bis-acetamide (HMBA). This feature makes MEL-DS19 cells an extremely useful model to study reprogramming of tumor cells to a non-malignant phenotype and to analyze the mode of action of different chemotheraupetics compounds. We previously reported the establishment of HMBA-resistant cell lines (MEL-R) that are unable to differentiate, even in the presence of the inducer (Fernandez-Nestosa et al. 2008). On the other hand, the network of actin filaments provides mechanical support to the cell cytoskeleton, but it is increasingly acknowledged that it also contributes to other critical cellular processes. Emerging evidence points to a role for the actin cytoskeleton in controlling and regulating receptor signaling (Mattila et al. (2016). In this line, changes in the regulation of actin cytoskeleton could be implicated in the blockade of the erythroid differentiation program. The aims of this thesis were: -Identify genes whose differential expression is higher in MEL-DS19 in relation to MEL-R, and viceversa by using RNA-seq., Analyze specific hematopoietic genes whose functions are related to the organization and polymerization of the actin cytoskeleton, i.e. Was (Wiskott-Aldrich syndrome), Btk (Bruton’s tyrosine kinase) and Plek (pleckstrin). - Generate genomic deletions of Was, Btk and Plek in MEL-DS19 cells via CRISPR/Cas9. - Established MEL-R transfectants that overexpressed those same genes. -Analyze the possible implication of increased expression of histone-encoding genes in the resistant line MEL-R. Our results can be summarize as follows: Resistant cells (MEL-R) showed phenotypic differences in relation to the parental cell line. We corroborated a significant increase in the size of MEL-R cells that parallel a prolonging doubling time. The distribution of MEL-R cells along the cell cycle follows the same pattern observed in uninduced MEL-DS19 cells. However, we found that the DNA content in the resistant lines has doubled, generating tetraploid cells. This data supports the idea that tetraploid cells enhance tumorigenic capacity relative to diploid cells. In an attempt to identify genes related to the resistant phenotype we used high-throughput RNA sequencing (RNA-seq) to compare the transcriptomes of the erythroleukemia progenitor and the resistance cell lines. RNA-seq revealed a total of 596 genes with a p-value adjusted less than 0.05 that were differentially expressed by more than two-fold, of which 486 genes were up-regulated in MELDS19 cells and 110 in MEL-R cells, enlightening that the number of genes expressed in the parental cell line decreased as the cells acquired the resistant phenotype. The progressive gene silencing observed in MEL-R cells involves several mechanisms. We proved that heterochromatinization, a marker for transcription down-regulation, is enhanced in MEL-R cells relative to undifferentiated MEL-DS19 cells, but is nevertheless lower than in HMBA-differentiated cells. We also examined the methylation status of Was, Btk and Plek genes and found that in MEL-R cells the CpG islands remained methylated in contrast to their non-methylated status in MEL-DS19 and HMBA-differentiated cells., The expression pattern of enzymes that catalyze DNA methylation and demethylation revealed an up-regulation of Dnmt1 and Tet3 expression in MEL-R cells. These results showed that the increase in DNA methylation by Dnmt1 in MEL-R cells overlaps with a decrease in demethylation by Tet3, which presumably results in the silencing of gene promoters. RNA-seq analysis showed highest differential expression allowed identification of a group among genes up-regulated in MEL cells. These genes are related with the organization of the actin cytoskeleton network, the majority of these genes are preferentially expressed in the erythroid lineage. Among this group stand out Bruton's tyrosine kinase (Btk), Wiskott Aldrich syndrome (Was) and pleckstrin (Plek). In order to study the potential implications of these genes on resistance to cell differentiation we performed genome deletions of Was, Btk or Plek in MEL-DS19 based on CRISPR/Cas9 system. In the first two cases, deficiencies were observed in the organization and polymerization of the actin cytoskeleton as opposed to the results obtained when Plek was deleted. The ability of the resistant line to reverse the wild phenotype was also determined. We established stable transfectants expressing each of the proteins in MEL-R cells. We observed a recovery in the organization of the actin cytoskeleton in Was and Btk. Moreover, we demonstrated that Was and Plek regulate the transcription expression of Btk, function that could be independent of the organization of the actin cytoskeleton. Altogether, these results suggest that disturbances of genes involved in the regulation and polymerization of actin cytoskeleton are coupled to a resistant differentiation phenotype.
- Published
- 2017
17. Ghrelin receptor activation regulates hippocampal spine dynamics
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Serrenho, Débora Vanessa Lourenço, Carvalho, Ana Luísa, and Santos, Sandra
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Hippocampo ,Plasticidade estrutural ,Grelina ,Citoesqueleto de actina ,Receptor da grelina - Published
- 2016
18. Citocinas, citoesqueleto de actina e a P21 de Trypanosoma cruzi na multiplicação intracelular do parasito in vitro
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Martins, Flávia Alves, Silva, Marcelo José Barbosa, Silva, Claudio Vieira da, Fonseca, Belchiolina Beatriz, and Ferreira Júnior, Álvaro
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Intracellular multiplication ,CIENCIAS BIOLOGICAS::MORFOLOGIA::CITOLOGIA E BIOLOGIA CELULAR [CNPQ] ,Citoesqueleto ,Trypanosoma cruzi ,Citoesqueleto de actina ,Multiplicação intracelular ,Citologia ,Actin polymerization ,Chagas, Doença de - Abstract
Chagas disease, caused by the protozoan Trypanosoma cruzi, affects about 8 million people worldwide. The disease is manifested in two phases, acute, characterized by high parasitaemia, and chronic by control of infection by the host. An adequate immune response is required to control infection. The response profile to infection by T. cruzi is the T helper type 1 (Th1). In recent studies, it was shown that the cytokines IL-3, IL-7 and IL-10 are highly expressed during the chronic phase of experimental infection by T. cruzi. The IFN-γ and IL-10 cytokines have been extensively studied to infection by T. cruzi, and IFN-γ plays a fundamental role in controlling parasitemia in the acute phase. The rP21 protein based on the P21 of T. cruzi induces nonspecific phagocytosis, polymerize actin cytoskeleton and seems to be matter in the passage to the chronic phase of the disease. Thus, in this study we analyzed the role of IFN-γ, IL-3, IL-7, IL-10 cytokines and the rP21 protein on in vitro infection by T. cruzi, and analyzed the role of these cytocines in the polymerization of actin. It was observed on peritoneal macrophages and myoblasts C2C12, that the cytokines IFN-γ, IL-7 and rP21 protein, induce the polymerization of actin cytoskeleton. Moreover, in macrophages and myoblasts infected with G and Y strain was seen differential effect of cytokines and rP21 in the parasite multiplication. However, in all analyzed groups IFN-γ and rP21 decreased parasite multiplication. This effect may be due to the induction of actin polymerization, along with induction of nitric oxide (NO) and reactive oxygen species (ROS) for the groups treated with IFN-γ. Meanwhile, the observed effect of rP21 seems to be linked by only with the actin cytoskeleton where the polymerization thereof, act in forming a mechanical barrier for parasite multiplication. Therefore, it is important to investigate the potential role of the actin cytoskeleton during intracellular multiplication of the parasite and its interaction with the host\'s immune response once an understanding of the modulation of such properties, would provide a basis for better understanding of the biology of the parasite. A Doença de Chagas, causada pelo protozoário Trypanosoma cruzi, afeta em torno de oito milhões de pessoas em todo o mundo. A doença se manifesta em duas fases, sendo a aguda caracterizada por alta parasitemia, e a crônica por controle da infecção por parte do hospedeiro. Uma resposta imune adequada é necessária para controle da infecção. O perfil de resposta frente à infecção por T. cruzi é do tipo T helper 1 (Th1). Estudos recentes demonstraram que as citocinas IL-3, IL-7 e IL-10 são altamente expressas na fase crônica da infecção experimental por T. cruzi. As citocinas IFN-γ e IL-10 têm sido amplamente estudadas frente à infecção por T. cruzi, sendo que IFN-γ tem papel fundamental no controle da parasitemia na fase aguda. A proteína rP21 baseada da P21 de T. cruzi, induz a fagocitose inespecífica, polimeriza o citoesqueleto de actina e parece ter importância na passagem para a fase crônica da doença. Assim, nesse trabalho, avaliamos o papel das citocinas IFN-γ, IL-3, IL-7, IL-10 e da proteína rP21 na infecção in vitro por T. cruzi, e analisamos o papel das mesmas na polimerização de actina. Observou-se que em macrófagos peritoneais e em mioblastos C2C12, as citocinas IFN-γ, IL-7 e a proteína rP21, induzem a polimerização do citoesqueleto de actina. Além disso, em macrófagos e mioblastos infectados com a cepa G e Y, foi visto efeito diferencial das citocinas e da rP21 sobre a multiplicação parasitária. Porém, em todos grupos analisados, IFN-γ e rP21 diminuíram a multiplicação parasitária. Tal efeito pode ser devido à indução na polimerização de actina, juntamente com a indução da produção de óxido nítrico e espécies reativas de oxigênio pelos grupos tratados com IFN-γ. Enquanto isso, o efeito observado pela rP21 aparenta ter ligação apenas com o citoesqueleto de actina, onde a polimerização deste, atuaria na formação de uma barreira mecânica para a multiplicação parasitária. Sendo assim, é importante investigar os potenciais papéis do citoesqueleto de actina durante a multiplicação intracelular do parasita e sua interação com a resposta imune do hospedeiro, uma vez que o entendimento da modulação de tais propriedades forneceria base para melhor entendimento da biologia do parasita. Mestre em Biologia Celular e Estrutural Aplicadas
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- 2015
19. Estudos da interação Trypanosoma cruzi hospedeiro mamífero enfatizando a resposta imunológica e caracterização biológica da proteína P21 do parasito
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Rodrigues, Adele Aud, Silva, Claudio Vieira da, Silva, Marcelo José Barbosa, Silber, Ariel Mariano, Véliz, Mauro Javier Cortez, and Vieira, Leda Quércia
- Subjects
CIENCIAS BIOLOGICAS::IMUNOLOGIA::IMUNOLOGIA APLICADA [CNPQ] ,P21 ,PI3-Kinase ,KC ,Trypanosoma cruzi ,Fagocitose ,Actin cytoskeleton ,Resposta imune ,Citoesqueleto de actina ,Chagas, Doença de ,Tropismo ,Tropism ,Phagocytosis ,PI3-quinase ,Immune response ,IFN-γ - Abstract
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Trypanosoma cruzi is the etiologic agent of Chagas disease. Due to its genetic diversity, species have been divided into six groups denominated Discrete Typing Units (DTU I to VI). The G strain belongs to T. cruzi I group and CL to T. cruzi VI. Extracellular amastigotes forms (EA) are considered important in maintaining the vertebrate host cycle. Studies concerning the difference of infectiveness and the characterization of immunologic susceptibility in parasites belonging to different groups are relevant so that therapies based upon immunological response may be efficient for all distinct phylogenetic groups. T. cruzi´s P21 protein has recently been characterized and its probable action in parasites internalization process into host cell was observed. To give continuity to the functional characterization of T. cruzi´s P21 employing its recombinant form (P21-His6) is of great relevance taking into account that these could in the future take part into the potential therapeutic goals. The objectives of this study were to analyze the immunological profile by T. cruzi infection, seeking to identify the differentiated performance of this response against G and CL strains and different forms of the parasite (metacyclic trypomastigotes or extracellular amastigotes); to analyze the differential tropism of CL and G strains during the acute and chronic infection; characterize the biological activity of P21 T. cruzi protein, trying to analyze its function to the parasite and performance in the host cell. We observed that extracellular amastigotes from G strain does not induce patent infection in vivo due to their high susceptibility to IFN-γ production early in infection; There were differences in tropism between the different strains, since during the kinetics infection, the CL strain was maintained preferably in the stomach, both in the acute and chronic phase and G strain remained in the stomach throughout the entire infection, but was also able to migrate into the heart tissue, particularly in chronic infection; The chemokine KC seems to play an important role in controlling the infection and may influence the course of infection, as well as the possible migration of parasites; the P21-His6 enhances phagocytosis and the remodeling of the actin cytoskeleton by binding to CXCR4 receptor and activate via PI3-k. Trypanosoma cruzi é o agente etiológico da doença de Chagas. Devido à ampla diversidade genética e fenotípica existente em T. cruzi, a espécie foi dividida em seis discretas unidades de tipagem (DTU), denominadas T.cruzi I a VI. A cepa G pertence ao grupo T.cruzi I e a CL a T.cruzi VI. Formas amastigotas extracelulares (AE) são consideradas importantes na manutenção do ciclo no hospedeiro vertebrado. Estudos quanto à diferença de infectividade e caracterização da suscetibilidade imunológica de parasitas pertencentes aos diferentes grupos mostram-se relevantes para que terapias baseadas na resposta imunológica sejam eficientes para todos os grupos filogeneticamente distintos. A proteína P21 de T. cruzi foi recentemente caracterizada e sua possível atuação no processo de internalização do parasito na célula hospedeira foi observada. Dar continuidade à caracterização funcional da P21 de T. cruzi por meio do emprego de sua forma recombinante (P21-His6) é de grande relevância, pois esta poderá futuramente figurar entre os potenciais alvos terapêuticos para o tratamento da doença de Chagas. Os objetivos desse trabalho foram analisar o perfil imunológico mediante a infecção por T. cruzi, buscando identificar a atuação diferenciada dessa resposta frente a duas cepas (G e CL) e formas distintas do parasito (tripomastigotas metacíclios ou amastigotas extracelulares); analisar o tropismo diferencial das cepas G e CL de T. cruzi durante a infecção aguda e crônica em camundongos; caracterizar a atividade biológica da proteína P21 de T. cruzi, buscando analisar sua função para o parasito e atuação na célula hospedeira. No presente trabalho mostra-se que amastigotas extracelulares da cepa G não induzem infecção patente in vivo devido a sua alta susceptibilidade à produção de IFN-γ no início da infecção. Houve diferença no tropismo entre as distintas cepas, durante a cinética de infecção, sendo que a cepa CL manteve-se preferencialmente no estômago, tanto na fase aguda quanto crônica, e a cepa G manteve-se no estômago no decorrer de toda a infecção, mas também foi capaz de migrar para o tecido cardíaco, principalmente no processo de cronificação. A quimiocina KC parece exercer um importante papel no controle da infecção, podendo interferir no curso da mesma, assim como na possível migração dos parasitos; a P21- His6 aumenta a fagocitose e a remodelação do citoesqueleto de actina, ao se ligar no receptor CXCR4 e ativar a via de PI3-k. Doutor em Imunologia e Parasitologia Aplicadas
- Published
- 2015
20. Hipusinación del factor de traducción eIF5A dependiente de poliaminas
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Ferrando Monleón, Alejandro Ramón, Serrano Salom, Ramón, Universitat Politècnica de València. Departamento de Biotecnología - Departament de Biotecnologia, Belda Palazón, Borja, Ferrando Monleón, Alejandro Ramón, Serrano Salom, Ramón, Universitat Politècnica de València. Departamento de Biotecnología - Departament de Biotecnologia, and Belda Palazón, Borja
- Abstract
RESUMEN La hipusinación es una modificación post-traduccional dependiente de espermidina que activa al factor de traducción eIF5A, y que es esencial en todos los eucariotas. En los últimos años se ha sugerido un importante papel para eIF5A en los procesos de senescencia y respuesta a estrés ambiental en plantas, en el establecimiento de la polaridad celular en levadura y su implicación en enfermedades tales como diabetes, VIH-1 o cáncer en humanos. Con el objetivo de caracterizar a nivel molecular la actividad biológica de eIF5A en plantas, hemos establecido una metodología basada en técnicas bioquímicas e inmunológicas para determinar el patrón de hipusinación de eIF5A en A. thaliana. La puesta a punto de esta metodología nos ha permitido demostrar que el tratamiento con ácido abscísico inhibe la activación por hipusinación de la isoforma eIF5A1. Además, para tratar de estudiar la función de eIF5A durante el desarrollo de A. thaliana, hemos realizado estudios funcionales basados en la caracterización de plantas transgénicas capaces de desactivar genéticamente la ruta dependiente de eIF5A mediante ARN de interferencia, condicionado a la aplicación de dexametasona. La desactivación condicional de la enzima de hipusinación desoxihipusina sintasa, produjo alteraciones durante el desarrollo y en respuesta a condiciones adversas de crecimiento, tales como floración temprana, inhibición del crecimiento de la raíz, alteraciones en los pelos radiculares, ramificación exacerbada del tallo, presencia de elementos traqueales completamente lignificados en hipocotilos, niveles reducidos de óxido nítrico e hipersensibilidad al ácido abscísico, sal y glucosa. Recientemente se ha demostrado que eIF5A es necesario para la traducción de proteínas con más de 3 prolinas sucesivas en su secuencia. El análisis de ontología realizado reveló un enriquecimiento de proteínas con poli-prolinas entre las implicadas en la organización del citoesqueleto de actina. La alteración de la actividad d
- Published
- 2015
21. Acción antitumoral del ácido carnósico, sobre células humanas de cáncer colorrectal
- Author
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Baroni, María Verónica, Moreno, Silvia, Wall, Luis, Casas, Adriana Gabriela, Ripoll, Giselle Vanina, and Ranuncolo, Stella Maris
- Subjects
Plantas medicinales ,Organic chemicals ,Metaloproteases ,Ensaios de seleção de medicamentos antitumorais ,Plantas medicinais ,Apoptosis ,Ensayos de selección de medicamentos antitumorales ,Medicinal plants ,Productos biológicos ,Ciclooxigenasa 2 ,Produtos biológicos ,Metaloproteasas ,Biological products ,Química farmacêutica ,Productos químicos orgánicos ,Cancer ,Activador de plasminógeno de tipo uroquinasa ,Produtos químicos orgânicos ,Produtos farmacêuticos ,Apoptose ,Células (biologia) ,Actin cytoskeleton ,Citoesqueleto de actina ,Drugs ,Productos farmacéuticos ,Cáncer ,Células (biología) ,Drug screening assays, antitumor ,Química farmacéutica ,Ativador de plasminogênio tipo uroquinase ,Cyclooxygenase 2 ,Metalloproteases ,Pharmaceutical chemestry ,Urokinase-type plasminogen activator ,Câncer ,Ciclo-oxigenase 2 ,Cells (biology) - Abstract
Fil: Baroni, María Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Ciencia y Tecnología de Alimentos Córdoba; Argentina. Fil: Baroni, María Verónica. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico-Córdoba; Argentina. Baroni, M. V. (2014). Acción antitumoral del ácido carnósico, sobre células humanas de cáncer colorrectal. (Tesis de doctorado). Universidad Nacional de Quilmes, Bernal, Argentina. El cáncer de colorrectal (CCR) es una de las enfermedades de mayor incidencia a nivel mundial y la segunda causa de muerte en Argentina. El presente estudio pretende contribuir al conocimiento de los efectos antitumorales del ácido carnósico, principal bioactivo de la planta de Rosmarinus Officinalis L. sobre líneas celulares de CCR de origen humano. El ácido carnósico (AC) es el principal diterpeno y uno de los compuestos bioactivos mayoritario que hemos identificado de un extracto de R. Officinalis. En este trabajo, se estudió su acción antitumoral sobre diferentes líneas celulares de cáncer colorrectal (CCR) humanas. Se determinó que el AC posee acción antiproliferativa, de modo dosis dependiente, sobre tres líneas de CCR: Caco-2, HT29 y LoVo. En la línea celular Caco-2, se determinó que la exposición al AC provocó la inducción de la muerte celular mediante procesos asociados a la apoptosis. Se llevaron a cabo investigaciones a nivel celular para dilucidar el efecto del AC sobre diversos procesos involucrados en la progresión tumoral del CCR como adhesión, migración e invasión celular. Se estableció que el AC inhibió la migración y la invasión celular en la línea Caco-2. Se observó además que el diterpeno inhibe la adhesión celular a sustratos de la matriz extracelular (MEC) como colágeno tipo I y fibronectina. En cuanto a la búsqueda de potenciales blancos moleculares de acción del AC involucrados en los procesos anteriormente mencionados, se encontró que el AC fue capaz de reducir la expresión y la actividad proteolítica de proteasas de la MEC como ser la metaloproteasa 9 (MMP-9) y el activador del plasminógeno del tipo uroquinasa (uPA). Además, dada la eficacia del AC para inhibir la adhesión celular sobre distintos sustratos de la MEC, se estudió su efecto sobre el camino de señalización de la quinasa de adhesión focal (del inglés “focal adhesión kinase”, FAK), que desempeña un papel fundamental en la migración celular, invasión y en la organización del citoesqueleto celular. Los resultados revelaron que el AC fue capaz de alterar la fosforilación de proteínas que median los contactos focales, así como de regular la organización del citoesqueleto de actina y también de disminuir significativamente la capacidad invasiva de las células Caco-2 sobre una membrana de matrigel, todos estos, eventos claves en la progresión tumoral. Por otro lado, se observó un efecto inhibitorio del AC sobre la expresión a nivel de ARNm y proteíco de la ciclooxigenasa-2 (COX-2), proteína asociada a procesos inflamatorios que se sobreexpresa en células de CCR. Por último, se evaluó in vivo la toxicidad y los efectos antitumorales del AC en animales de experimentación (ratones). El AC administrado a 5mg/kg durante 15 días, no evidenció nefrotoxicidad o hepatotoxicidad y no alteró significativamente parámetros bioquímicos y fisiológicos en los ratones. Se obtuvieron indicios auspiciosos sobre la acción antitumoral de AC en tumores producidos a partir de la línea celular HT29. En resúmen, los resultados de este trabajo de tesis doctoral indicarían el potencial uso del AC como nueva alternativa terapéutica contra el cáncer colorrectal humano ya sea como un posible agente preventivo o terapéutico.
- Published
- 2014
22. Hipusinación del factor de traducción eIF5A dependiente de poliaminas
- Author
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Borja Belda Palazón, Ferrando Monleón, Alejandro Ramón, Serrano Salom, Ramón, and Universitat Politècnica de València. Departamento de Biotecnología - Departament de Biotecnologia
- Subjects
Hipusina ,Electroforesis-2D ,Traducción ,Espermidina ,Citoesqueleto de actina ,Xilogénesis ,ARN de interferencia ,Forminas ,GC7 ,Estrés abiótico ,BIOQUIMICA Y BIOLOGIA MOLECULAR ,EIF5A ,Floración ,Ácido abscísico - Abstract
RESUMEN La hipusinación es una modificación post-traduccional dependiente de espermidina que activa al factor de traducción eIF5A, y que es esencial en todos los eucariotas. En los últimos años se ha sugerido un importante papel para eIF5A en los procesos de senescencia y respuesta a estrés ambiental en plantas, en el establecimiento de la polaridad celular en levadura y su implicación en enfermedades tales como diabetes, VIH-1 o cáncer en humanos. Con el objetivo de caracterizar a nivel molecular la actividad biológica de eIF5A en plantas, hemos establecido una metodología basada en técnicas bioquímicas e inmunológicas para determinar el patrón de hipusinación de eIF5A en A. thaliana. La puesta a punto de esta metodología nos ha permitido demostrar que el tratamiento con ácido abscísico inhibe la activación por hipusinación de la isoforma eIF5A1. Además, para tratar de estudiar la función de eIF5A durante el desarrollo de A. thaliana, hemos realizado estudios funcionales basados en la caracterización de plantas transgénicas capaces de desactivar genéticamente la ruta dependiente de eIF5A mediante ARN de interferencia, condicionado a la aplicación de dexametasona. La desactivación condicional de la enzima de hipusinación desoxihipusina sintasa, produjo alteraciones durante el desarrollo y en respuesta a condiciones adversas de crecimiento, tales como floración temprana, inhibición del crecimiento de la raíz, alteraciones en los pelos radiculares, ramificación exacerbada del tallo, presencia de elementos traqueales completamente lignificados en hipocotilos, niveles reducidos de óxido nítrico e hipersensibilidad al ácido abscísico, sal y glucosa. Recientemente se ha demostrado que eIF5A es necesario para la traducción de proteínas con más de 3 prolinas sucesivas en su secuencia. El análisis de ontología realizado reveló un enriquecimiento de proteínas con poli-prolinas entre las implicadas en la organización del citoesqueleto de actina. La alteración de la actividad de eIF5A provocó defectos en la estructuración de los filamentos de actina en A. thaliana, S. cerevisiae y H. sapiens. El estudio de mutantes termosensibles de levadura demostró el requerimiento de eIF5A durante el proceso de reproducción sexual a través de la traducción de la formina Bni1. Los experimentos de regulación traduccional en células HeLa demostraron que el silenciamiento vía ARN de interferencia de eIF5A1 provocaba un defecto en la tasa de traducción de la formina FMNL1 y la proteína ezrina. Estos resultados confirman que la actividad esencial de eIF5A en el ribosoma parece conservada en organismos eucariotas, y afecta fundamentalmente a proteínas con poli-prolinas implicadas en la organización del citoesqueleto de actina., Belda Palazón, B. (2014). Hipusinación del factor de traducción eIF5A dependiente de poliaminas [Tesis doctoral no publicada]. Universitat Politècnica de València. doi:10.4995/Thesis/10251/48474., TESIS
- Published
- 2014
23. Estudo da suscetibilidade de amastigotas extracelulares das cepas G e CL de Trypanosoma cruzi a componentes do sistema imune inato e adaptativo. Avaliação da atividade pró-fagocítica da P21-His6
- Author
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Rodrigues, Adele Aud, Ferro, Eloisa Amália Vieira, Silva, Claudio Vieira da, Mineo, Tiago Wilson Patriarca, and Padrón, Thais Cristina Baeta S. Souto
- Subjects
CIENCIAS BIOLOGICAS::IMUNOLOGIA::IMUNOLOGIA APLICADA [CNPQ] ,P21 ,PI3-Kinase ,Trypanosoma cruzi ,Fagocitose ,Actin cytoskeleton ,Citoesqueleto de actina ,Resposta imune ,Chagas, Doença de ,Phagocytosis ,parasitic diseases ,PI3-quinase ,Immune response ,IFN-γ - Abstract
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior Trypanosoma cruzi is the ethiologic agent of Human Chagas disease. Due to T. cruzi wide genetic diversity, the species has been divided in six Discrete Typing Units (DTU), named T. cruzi I to VI. The G strain belongs to T. cruzi I group and CL to T. cruzi VI. Extracellular amastigotes forms (EA) are considered important in maintaining the vertebrate host cycle. Studies concerning the difference of infectiveness and the characterization of immunologic susceptibility in parasites belonging to different groups are relevant so that therapies based upon immunological response may be efficient for all distinct phylogenetic groups. T. cruzi´s P21 protein has recently been characterized and its probable action in parasites internalization process into host cell was observed. To give continuity to the functional characterization of T. cruzi´s P21 employing its recombinant form (P21-His6) is of great relevance taking into account that these could in the future take part into the potential therapeutic goals. This work intended to compare the in vivo and in vitro infection of EA from G and CL strains, as well as to assess P21-His6pro-phagocytic potential. So that experiments in vitro and in vivo have been made. in vitro assays showed a greater infectiveness and multiplication of EA from G strain than of CL. Nevertheless EA from G strain showed more susceptible as to inflammatory macrophages action in ex vivo experiments as to those differenciated and in vitro stimulated with IFN-γ. Among the G strain infected animals, only Calomys callosus showed sub-patent parasitemia and the IL-12 and IFN-γ knockouts showed patent parasitemia and high mortality. All the CL strain infected animals showed parasitemia, but the TNF-α, iNOS, Myd-88 and IL-12 knockouts showed greater parasitemia and mortality in comparision to the wild ones. In the assays of immunossupressed mice, it has been observed high parasitemia for CL and G strains. We, then conclude that EA from G strain probably don t show in vivo parasitemia due to its greater susceptibility to IFN-γ activated macrophages. To study P21 protein it has been made in vitro invasion and phagocytosis assayes in that C. callosus peritoneal macrophages were infected with T. cruzi´s EA, Leishmania amazonensis promastigotes, Toxoplasma gondii taquizoitas or otherwise invadid with zymozan particle, and then treated or not with P21-His6. It has also been made immunofluorescence of actin filaments in macrophages treated or not with P21-His6. Finaly, another immunofluorescence reaction was made in order to mark actin filaments in macrophage submitted to P21-His6, to wortmanin treatment, or both. Results showed that P21-His6 hightened all parasites and the inactive particle internalization. Besides macrophages treated with the protein showed increase in cortical actin polymerization. Nevertheless, the treatment with wortmanin together with P21-His6 inhibited its action in actin cellular polymerization. We conclude that P21-His6 is capable of inducing inespecific phagocytic process probably to its action into the cortical actin cytoskeletal, by means of signalization process probably depending on PI3-kinase activation. Trypanosoma cruzi é o agente etiológico da doença de Chagas. Devido à ampla diversidade genética existente em T. cruzi, a espécie foi dividida em seis discretas unidades de tipagem (DTU), denominadas T.cruzi I a VI. A cepa G pertence ao grupo T.cruzi I e a CL a T.cruzi VI. Formas amastigotas extracelulares (AE) são consideradas importantes na manutenção do ciclo no hospedeiro vertebrado. Estudos quanto à diferença de infectividade e caracterização da suscetibilidade imunológica de parasitas pertencentes aos diferentes grupos mostram-se relevantes para que terapias baseadas na resposta imunológica sejam eficientes para todos os grupos filogeneticamente distintos. A proteína P21 de T. cruzi foi recentemente caracterizada e sua possível atuação no processo de internalização do parasito na célula hospedeira foi observada. Dar continuidade à caracterização funcional da P21 de T. cruzi por meio do emprego de sua forma recombinante (P21-His6) é de grande relevância, pois esta poderá futuramente figurar entre os potenciais alvos terapêuticos para o tratamento da doença de Chagas. O objetivo deste trabalho foi comparar a infecção in vitro e in vivo por amastigotas extracelulares das cepas G e CL, bem como avaliar o potencial pró-fagocítico da P21- His6. Para isto, foram realizadas experimentações in vitro e in vivo. Nos experimentos in vitro foi observado maior infectividade e multiplicação de AE da cepa G do que de CL. Contudo, AE da cepa G mostraram-se mais suscetíveis, tanto a atuação de macrófagos inflamatórios, em experimentos ex vivo, quanto daqueles diferenciados e estimulados com IFN-γ in vitro. Dentre os animais infectados com a cepa G, somente C. callosus apresentou parasitemia sub-patente e os nocautes para IL-12 e IFN-γ apresentaram parasitemia patente e alta mortalidade. Todos os animais infectados com a cepa CL apresentaram parasitemia, contudo os nocautes para TNF-α, iNOS, Myd-88 e IL-12 apresentaram maior parasitemia e mortalidade em relação aos selvagens. Em experimentos onde camundongos foram imunossuprimidos foi observada elevada parasitemia para a cepa CL e para a G. Conclui-se, portanto, que AE da cepa G provavelmente não apresentam parasitemia in vivo devido a sua maior suscetibilidade a atuação de macrófagos ativados com IFN-γ. Para o estudo da proteína P21, realizaramse ensaios de invasão e de fagocitose in vitro, no qual macrófagos peritoneais de C. callosus foram infectados com AE de T. cruzi, promastigotas de Leishmania amazonensis, taquizoitas de Toxoplasma gondii, ou ainda incubados com partículas de zimozan, e foram tratados ou não com P21-His6. Também realizou-se imunofluorescência de filamentos de actina em macrófagos tratados ou não com P21- His6. Finalmente, realizou-se outra reação de imunofluorescência para marcação de filamentos de actina em macrófagos que se submeteram ao tratamento de P21-His6, ao tratamento com wortmanina, ou ambos. Os resultados demonstraram que P21-His6 aumentou a internalização de todos os parasitos e da partícula inerte. Além disso, macrófagos tratados com a proteína apresentaram aumento na polimerização de actina cortical. No entanto, o tratamento de wortmanina conjuntamente a P21-His6 inibiu sua atuação na polimerização da actina celular. Conclui-se, portanto, que P21-His6 é capaz de induzir processo fagocítico inespecificamente, sendo devido a atuação da proteína no citoesqueleto de actina cortical, por um processo de sinalização provavelmente dependente da ativação de PI3-quinase. Mestre em Imunologia e Parasitologia Aplicadas
- Published
- 2011
24. Role of Tropomyosin2 in cell proliferation and survival of Drosophila melanogaster
- Author
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Viegas, Filipe Paulos e Cruz Oliveira, Janody, Florence, and Rodrigues, Maria Gabriela,1965
- Subjects
Tropomiosina ,Teses de mestrado - 2016 ,Drosophila melanogaster ,Via de sinalização Hippo ,Citoesqueleto de actina ,Controlo de crescimento ,Ciências Naturais::Ciências Biológicas [Domínio/Área Científica] - Abstract
Tese de mestrado em Biologia Evolutiva e do Desenvolvimento, apresentada à Universidade de Lisboa, através da Faculdade de Ciências, 2016 Submitted by Cristina Manessiez (camanessiez@fc.ul.pt) on 2017-02-10T16:07:22Z No. of bitstreams: 1 ulfc118905_tm_Filipe_Viegas.pdf: 4056710 bytes, checksum: aced3cd2e4e1b5a5b5b69b92fe8cd850 (MD5) Made available in DSpace on 2017-02-10T16:07:42Z (GMT). No. of bitstreams: 1 ulfc118905_tm_Filipe_Viegas.pdf: 4056710 bytes, checksum: aced3cd2e4e1b5a5b5b69b92fe8cd850 (MD5) Previous issue date: 2016
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