Rol de la proteína sparc en la biología adenoviral orientada al uso terapéutico

Today, great strides have been made in the field of gene therapy for cancer treatment, and in this field, the use of oncolytic viruses is presented with promising advances, since they not only have a preferential tropism for transformed cells but also they mediate cytotoxic processes in infected cel...

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Detalles Bibliográficos
Autor principal: Rivas Baquero, Elvia Carolina
Otros Autores: Salvatierra, Edgardo
Formato: Tesis de maestría acceptedVersion
Lenguaje:Español
Publicado: Facultad de Farmacia y Bioquímica 2015
Materias:
CRAds
Lisis
SPARC
Matriz extracelular
Adenovirus
Lysis
Ciencias de la vida
Acceso en línea:http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_1917
http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_1917.dir/1917.PDF
Aporte de:
Repositorio Digital de la Universidad de Buenos Aires (UBA) de Universidad de Buenos Aires
id I28-R145-HWA_1917
record_format dspace
institution Universidad de Buenos Aires
institution_str I-28
repository_str R-145
collection Repositorio Digital de la Universidad de Buenos Aires (UBA)
language Español
orig_language_str_mv spa
topic CRAds
Lisis
SPARC
Matriz extracelular
Adenovirus
CRAds
Lysis
SPARC
Ciencias de la vida
spellingShingle CRAds
Lisis
SPARC
Matriz extracelular
Adenovirus
CRAds
Lysis
SPARC
Ciencias de la vida
Rivas Baquero, Elvia Carolina
Rol de la proteína sparc en la biología adenoviral orientada al uso terapéutico
topic_facet CRAds
Lisis
SPARC
Matriz extracelular
Adenovirus
CRAds
Lysis
SPARC
Ciencias de la vida
description Today, great strides have been made in the field of gene therapy for cancer treatment, and in this field, the use of oncolytic viruses is presented with promising advances, since they not only have a preferential tropism for transformed cells but also they mediate cytotoxic processes in infected cells, without harming normal cells. Within oncolytic viruses are conditionally replicative adenovirus (CRAds) based in adenovirus that infect humans. In our laboratory was built a CRAd whose replication is driven by a chimeric promoter with a fragment of the SPARC promoter, hypoxia response elements and inflammation, besides having a chimera fiber 5/3. This virus was called adenovirus 5/3 NHS. In the search for new strategies for the delivery of CRAds to the tumor, mesenchymal cells were used (MSCs) pre-infected with Ad 5/3 NHS as a means of transport for the same. It was observed that by infecting these cells, both in the presence of conditioned medium of tumor cells that express the SPARC protein, as in the presence of purified protein SPARC, the infection turn out to be more efficient, demonstrating an increase in lysis at lower multiplicity of infection (MOI).\nStarting from this premise, this work aimed to assess whether the effect observed in MCSs reproduced in tumor cells. This three strategies were used: Infections in the presence of conditioned medium from cells secreting SPARC, infection in the presence of the purified protein SPARC and infections in cells expressing or not SPARC using 3 viruses: Ad5/3 Luciferase as a non-replicative control, Ad5/3 WT as positive control and the Ad5/3 NHS generated in our laboratory. The strategy that involve an infection in the presence of conditioned media do not represent an efficient test for evaluating the effect of SPARC, since it inhibited cell growth. SPARC expression and exogenously added protein promotes adenovirus mediated lysis. However, studies of expression of CAR, CD46 and Desmoglein-2 adenoviral receptors and virus entry by quantification of copies of E4 gene and immunofluorescence, suggest that the mechanism used by SPARC is unrelated to adenoviral cycle stages related to receptor binding and virus entry.
author2 Salvatierra, Edgardo
author_facet Salvatierra, Edgardo
Rivas Baquero, Elvia Carolina
format Tesis de maestría
Tesis de maestría
acceptedVersion
author Rivas Baquero, Elvia Carolina
author_sort Rivas Baquero, Elvia Carolina
title Rol de la proteína sparc en la biología adenoviral orientada al uso terapéutico
title_short Rol de la proteína sparc en la biología adenoviral orientada al uso terapéutico
title_full Rol de la proteína sparc en la biología adenoviral orientada al uso terapéutico
title_fullStr Rol de la proteína sparc en la biología adenoviral orientada al uso terapéutico
title_full_unstemmed Rol de la proteína sparc en la biología adenoviral orientada al uso terapéutico
title_sort rol de la proteína sparc en la biología adenoviral orientada al uso terapéutico
publisher Facultad de Farmacia y Bioquímica
publishDate 2015
url http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_1917
http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_1917.dir/1917.PDF
work_keys_str_mv AT rivasbaqueroelviacarolina roldelaproteinasparcenlabiologiaadenoviralorientadaalusoterapeutico
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spelling I28-R145-HWA_19172019-09-27 Today, great strides have been made in the field of gene therapy for cancer treatment, and in this field, the use of oncolytic viruses is presented with promising advances, since they not only have a preferential tropism for transformed cells but also they mediate cytotoxic processes in infected cells, without harming normal cells. Within oncolytic viruses are conditionally replicative adenovirus (CRAds) based in adenovirus that infect humans. In our laboratory was built a CRAd whose replication is driven by a chimeric promoter with a fragment of the SPARC promoter, hypoxia response elements and inflammation, besides having a chimera fiber 5/3. This virus was called adenovirus 5/3 NHS. In the search for new strategies for the delivery of CRAds to the tumor, mesenchymal cells were used (MSCs) pre-infected with Ad 5/3 NHS as a means of transport for the same. It was observed that by infecting these cells, both in the presence of conditioned medium of tumor cells that express the SPARC protein, as in the presence of purified protein SPARC, the infection turn out to be more efficient, demonstrating an increase in lysis at lower multiplicity of infection (MOI).\nStarting from this premise, this work aimed to assess whether the effect observed in MCSs reproduced in tumor cells. This three strategies were used: Infections in the presence of conditioned medium from cells secreting SPARC, infection in the presence of the purified protein SPARC and infections in cells expressing or not SPARC using 3 viruses: Ad5/3 Luciferase as a non-replicative control, Ad5/3 WT as positive control and the Ad5/3 NHS generated in our laboratory. The strategy that involve an infection in the presence of conditioned media do not represent an efficient test for evaluating the effect of SPARC, since it inhibited cell growth. SPARC expression and exogenously added protein promotes adenovirus mediated lysis. However, studies of expression of CAR, CD46 and Desmoglein-2 adenoviral receptors and virus entry by quantification of copies of E4 gene and immunofluorescence, suggest that the mechanism used by SPARC is unrelated to adenoviral cycle stages related to receptor binding and virus entry. Fil: Rivas Baquero, Elvia Carolina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Buenos Aires, Argentina Salvatierra, Edgardo Facultad de Farmacia y Bioquímica Podhajcer, Osvaldo Rivas Baquero, Elvia Carolina 2015-12-18 En la actualidad, grandes avances se han realizado en el campo de la terapia génica para el tratamiento del cáncer, y dentro de este campo, el uso de virus oncolíticos se presenta con adelantos prometedores, ya que no solo presentan un tropismo preferencial por células transformadas, sino que también median procesos citotóxicos en las células infectadas, sin dañar a las células normales. Dentro de los virus oncolíticos encontramos los adenovirus de replicación condicional basados en adenovirus que infectan a humanos (?CRAds? en sus siglas en inglés). En nuestro laboratorio se construyó un CRAd cuya replicación es dirigida por un promotor quimera con: un fragmento del promotor de SPARC, elementos respondedores a hipoxia y a inflamación; además posee una fibra quimera 5/3. Este virus se denominó Adenovirus 5/3 NHS. En la búsqueda de nuevas estrategias para el ?delivery? de CRAds al tumor, se utilizaron células mesenquimales (?MSCs? en sus siglas en inglés) pre infectadas con el Ad 5/3 NHS como medio de transporte para el mismo. Se observó que al infectar estas células tanto en presencia de medio condicionado de células tumorales que expresan la proteína SPARC, como en presencia de la proteína purificada SPARC, la infección fue más eficiente, evidenciándose en un aumento en la lisis a menor multiplicidad de infección (?MOI? en sus siglas en inglés).\nPartiendo de esta premisa, en este trabajo se planteó evaluar si el efecto observado en MSC se reproducía en células tumorales. Para ello se utilizaron 3 estrategias: infecciones en presencia de medio condicionado proveniente de células que secretan SPARC, infección en presencia de la proteína purificada SPARC e infecciones en células que expresan o no SPARC, empleando 3 virus: Ad5/3 Luciferasa como control no replicativo, Ad5/3 WT como control positivo y el Ad5/3 NHS generado en nuestro laboratorio. La estrategia en la que infectamos en presencia del medio condicionado no represento un ensayo eficiente para la evaluación del efecto de SPARC, ya que el mismo inhibió el crecimiento celular. La expresión de SPARC y el agregado exógeno de la proteína favorece la lisis mediada por los adenovirus utilizados. Sin embargo, estudios de expresión de los receptores adenovirales CAR, CD46 y Desmogleína-2, y de entrada de virus por cuantificación de copias del gen E4, nos sugieren que el mecanismo utilizado por SPARC no está relacionado con las etapas del ciclo adenoviral que abarcan la unión a receptores y la entrada del virus. application/pdf Cavallaro, Lucía Flichman, Diego Eijan, Ana María CRAds Lisis SPARC Matriz extracelular Adenovirus CRAds Lysis SPARC spa Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica info:eu-repo/semantics/openAccess http://creativecommons.org/licenses/by-nc-nd/2.5/ar/ Ciencias de la vida Rol de la proteína sparc en la biología adenoviral orientada al uso terapéutico info:eu-repo/semantics/masterThesis info:ar-repo/semantics/tesis de maestría info:eu-repo/semantics/acceptedVersion http://repositoriouba.sisbi.uba.ar/gsdl/cgi-bin/library.cgi?a=d&c=afamaster&cl=CL1&d=HWA_1917 http://repositoriouba.sisbi.uba.ar/gsdl/collect/afamaster/index/assoc/HWA_1917.dir/1917.PDF

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