Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

No description available.

O Adenovírus (Ad) é um agente etiológico que causa infecções em diversas espécies e também pode ser utilizado na forma de vetor como ferramenta tecnológica para terapia gênica. O Controle sobre a replicação de Ad pode trazer beneficio para o combate de infecções e para as tecnologias de transferência genica. Porém, poucas ferramentas existem que podem inibir a replicação de Ad. Uma aplicação importante seria a inibição da replicação de Adenovírus helper utilizado na produção de Vírus Adenoassociado recombinante (rAAV), assim minimizando contaminação da produção de rAAV com o virus helper. Dessa maneira o objetivo desse trabalho foi investigar se há inibição da replicação do Ad mediada por RNA de interferência (RNAi) direcionada para alvos adenovirais chaves. Para isso foram construídos vetores lentivirais que codificam shRNAs para os genes hexon, IVa2 e pol. Em seguida foram criadas linhagens que expressam constitutivamente os shRNAs em 293T, células onde os vetores adenovirais conseguem se replicar. Os shRNAs específicos para hexon e IVa2 promoverem significantemente a redução dos níveis destes mRNAs conforme revelado utilizando RT-qPCR para quantificação dos transcritos adenovirais. Em seguida, knockdown do gene hexon se mostrou promissor em inibir a replicação do Ad, visto como redução de vírus produzido em células 293T anti-hexon. O knockdown do transcrito de hexon e a redução em replicação de Adenovírus foram mais acentuados após cell sorting e obtenção de clones celulares a partir da linhagem anti-hexon. O clone anti-hexon mostrou significante redução na quantidade de partículas adenovirais visualizadas por microscopia eletrônica e redução de 92% das partículas infecciosas em relação a 293T quando a produção foi realizada em larga escala. Esses resultados indicam que a tecnologia de shRNA para inibir a replicação do Ad é promissora e representa o primeiro passo de desenvolvimento de uma estratégia para a produção de rAAV livre de contaminação com Ad helper / Adenovirus (ad) is an etiologic agent that causes infections in diverse species and can also be used as a technologic resource, such as a vector applied in gene therapy. Control over Ad replication could be beneficial for the combat of infections and for the technology of gene transfer. However, few tools exist that may useful for the inhibition of Ad replication. One important application would be to impede replication of helper adenovirus utilized in the production of recombinant Adenoassociated Virus (rAAV), thus minimizing the contamination of the rAAV production with helper virus. The objective of the study was to investigate the use of RNA interference (RNAi) directed against key adenoviral targets as an inhibitor of Ad replication. For this, lentiviral vectors encoding shRNAs for hexon, IVa2 and pol were constructed. Next, constitutive expression of the shRNAs was established in 293T cells, the parental cell line that is permissive for adenovirus replication. The shRNAs specific for hexon or IVa2 significantly promoted reduction in the level of these mRNAs as revealed by RT-qPCR quantification of the adenoviral transcripts. Next, knockdown of hexon was shown to be promising as an inhibitor of Ad replication, seen as the reduction of Ad produced in the 293T anti-hexon cell line. Both the knockdown of the hexon transcript and reduction in adenovirus replication were accentuated after cell sorting and isolation of cellular clones from the anti-hexon cell line. The anti-hexon clone showed significant reduction in the quantity of adenovirus particles when visualized by electron microscopy and 92% fewer infectious particles as compared to the parental 293T cells when full scale production was made. These results indicate that the use of shRNA technology for the inhibition of Ad replication is promising and represents the first step for the development of a strategy for the production of rAAV free from helper virus contamination

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2009. / Härtill 4 uppsatser.

Thèse doctorat : Médecine. Virologie : Université de Nantes : 2005. / Bibliogr. 145-175 f. [587 réf.].

Thèse de doctorat : Médecine. Virologie : Nantes : 2007. / Bibliogr.

Thèse de doctorat : Médecine. Virologie : Nantes : 2006. / Bibliogr.

Reproduction de : Thèse de doctorat : Médecine. Virologie : Nantes : 2008. / Bibliogr.

Thesis (Ph. D.)--Case Western Reserve University, 2009. / [School of Medicine] Department of Physiology and Biophysics. Includes bibliographical references.

Since its identification in 1997 as the common receptor for Coxsackie and adenovirus (CAR) multiple lines of evidence argue in favor of CAR contributing to aspects of cell adhesion in addition to serving as a viral receptor. Nevertheless, a precise biological role for CAR remains to be identified suggesting the receptor may participate in a variety of cellular functions that reflect its tissue specific and developmentally regulated expression. This thesis elucidates aspects of CAR biology in mature striated muscle by providing studies that encompass (i) its physiological cellular/subcellular localization and expression in mature striated muscle (ii) its expression profile in human diseased skeletal muscle and (iii) the potential consequences of its sustained expression in mature striated muscle where its levels would otherwise be highly attenuated. / In non-diseased, mature striated muscle despite low and barely detectable levels of the CAR transcript (cardiac and skeletal muscle respectively), we identified CAR as a novel component of the neuromuscular junction and showed its expression to be isoform-specific in contrast to the intercalated discs, where both predominant CAR isoforms are detected. We then investigated the expression of CAR at the level of human skeletal muscle disease. From these studies we observed that in diseases characterized by active necrosis and regeneration, extrasynaptic CAR expression is detectable in regenerating fibers and co-expressed with other previously described markers of regeneration at a high degree of coincidence. Moreover, extrasynaptic CAR expression appears to be a highly reliable indicator of the regenerative process offering potential use at the diagnostic level. Following these investigations, our final studies involved assessing whether sustained CAR expression might affect the normal homeostasis in skeletal and cardiac muscle using a transgenic mouse model. We discovered that transgenic mice expressing sustained high levels of CAR (as seen in the CAR+/+ transgenics) develop a lethal necrotizing myopathy characterized by dual deficiencies in dystrophin and dysferlin, two proteins pivotal in maintaining plasmalemmal integrity, raising the possibility for a previously unrecognized cause of skeletal muscle dysfunction. / Collectively these findings argue that in non-diseased mature skeletal and cardiac muscle, CAR expression is restricted to the neuromuscular junction and cardiac intercalated discs but in diseases of skeletal muscle characterized by active necrosis and regeneration, extrasynaptic CAR expression is reexpressed at these sites of injury/repair. In addition they raise the possibility that sustained CAR expression in mature skeletal muscle may be associated with altered muscle homeostasis.