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Modulação da expressão do gene de reparo de DNA xpa por meio de vetores genéticos em células humanas; XPA DNA repair gene modulation in human cell lines by genetic vectors

Muotri, Alysson Renato
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 19/04/2001 Português
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45.93%
A integridade do DNA é ameaçada pelos efeitos lesivos de inúmeros agentes físicos e químicos que podem vir a comprometer sua função. Um dos mais versáteis e estudados mecanismos de reparo de DNA é o reparo por excisão de nucleotídeos (NER). Este mecanismo remove lesões que causam distorções na dupla fita de DNA, incluindo dímeros de pirimidina ciclobutano (CPDs) e 6-4 fotoprodutos (6-4 PPs), provocados pela radiação de luz ultravioleta (UV). Em humanos, a síndrome genética xeroderma pigmentosum (XP) apresenta uma alta sensibilidade à luz solar, resultando em um grande aumento na incidência de tumores em regiões expostas da pele e degeneração neurológica progressiva. O gene xpa parece estar envolvido diretamente no reconhecimento de lesões produzidas pela luz UV, atuando tanto no reparo global (GGR) como no reparo acoplado à transcrição (TCR). A modulação da expressão deste gene deve alterar as taxas de reparo no genoma celular, fornecendo valiosa contribuição para o estudo do reparo de DNA no NER e em outras vias distintas. No entanto, não foi possível a atenuação ou inativação total do transcrito XPA, provavelmente devido ao baixo número de moléculas de mRNA nas células e da relativa estabilidade da proteína XPA. A expressão controlada do cDNA xpa em células deficientes XP12RO foi conseguida através da transfecção do vetor indutível por muristerona A...

Uso de vetores adenovirais na identificação de grupo de complementação gênica de pacientes com Xeroderma pigmentosum e em animais deficientes em reparo de DNA.; Use of adenoviral vectors in the identification of genetic complementation group of patients with Xeroderma pigmentosum um and animals deficient in DNA repair.

Leite, Ricardo Alexandre
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 30/09/2008 Português
Relevância na Pesquisa
36.15%
Um dos mais versáteis mecanismos de reparo de DNA é o reparo por excisão de nucleotídeos (nucleotide excision repair- NER). Defeitos genéticos associados a esta via podem gerar diferentes síndromes com deficiência de reparo. Dentre essas, Xeroderma pigmentosum (XP) é a que apresenta maior sensibilidade à luz solar, resultando em um grande aumento na incidência de tumores em regiões expostas da pele e, em alguns casos, degeneração neurológica progressiva e envelhecimento prematuro. Na primeira parte deste projeto é apresentado o uso de adenovírus recombinantes portando genes da via de NER para identificar a deficiência gênica de três pacientes portadores de XP. Na segunda parte do trabalho os estudos de reparo de DNA são estendidos a modelos animais, com deficiências nos mesmos genes carregados pelos vetores adenovirais. A expressão gênica do vetor foi avaliada pela detecção de proteína e por visualização da fluorescência de EGFP na pele dos animais infectados. Em resumo, este trabalho apresenta o uso eficiente de vetores adenovirais portando genes de reparo em ensaios in vitro e in vivo, e descreve duas mutações deletérias no gene XPC de pacientes XP brasileiros, incluindo uma mutação nova.; One of the most versatile mechanisms of DNA repair is the nucleotide excision repair (NER). Genetic defects in NER can generate different syndromes. Among these...

Sequenciamento de um código de barras como ferramenta para quantificação de alterações na dinâmica de populações celulares transduzidas com vetores lentivirais.; Sequencing of a barcode as a tool for the quantification of changes in the dynamics of cell populations transduced with lentiviral vectors.

Zanatta, Daniela Bertolini
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 28/06/2012 Português
Relevância na Pesquisa
36.02%
Os vetores retrovirais representam uma das melhores opções para transferência e terapia gênica, pois fornecem expressão do transgene em longo prazo. Entretanto, a inserção do provírus pode causar mutagênese insercional, induzindo proto-oncogenes. Eventos deste tipo têm sido descritos em protocolos clínicos para o tratamento de SCID-X1, doença granulomatosa crônica e talessemia beta, quando vetores retrovirais (oncorretrovirus) foram utilizados. Atualmente, existem poucos métodos simples e rápidos para revelar e quantificar a expansão clonal. Assim, descrevemos a construção uma biblioteca de vetores contendo uma marcação aleatória, denominada código de barras. O sequenciamento do código de barras permitirá revelar, caracterizar e até quantificar a expansão clonal de uma população de células transduzidas. Esta metodologia ajudará a testar novos arranjos de promotores e genes terapêuticos, para o desenvolvimento de vetores mais seguros contribuindo para a redução da probabilidade de um evento de proliferação clonal desencadeado pela mutagênese insercional.; Retroviral vectors represent one of the best options for gene transfer and therapy, where long-term transgene expression is required. However, insertion of the provirus can cause insertional mutagenesis...

A role for adeno-associated viral vectors in gene therapy

Coura, Renata dos Santos; Nardi, Nance Beyer
Fonte: Universidade Federal do Rio Grande do Sul Publicador: Universidade Federal do Rio Grande do Sul
Tipo: Artigo de Revista Científica Formato: application/pdf
Português
Relevância na Pesquisa
36.1%
Gene therapy constitutes a therapeutic intervention based on modification of the genetic material of living cells, by correcting genetic defects or overexpressing therapeutic proteins. The success of gene therapy protocols depends on the availability of therapeutically suitable genes, appropriate gene delivery systems and proof of safety and efficacy. Recent advances on the development of gene delivery systems, particularly on viral vectors engineering and improved gene regulatory systems, have led to marked progress in this field. Although the available vector systems can successfully transfer genes into cells, the ideal delivery vehicle has not been found. In this context, adeno-associated virus vectors (AAV) are arising as a promising tool for a wide range of applications, due to a combination of characteristics such as lack of pathogenicity and immunogenicity, wide range of cell tropism and long-term gene expression. Since its isolation, the biological properties of the adeno-associated virus have been increasingly understood, improving our ability to manipulate and use it as a safe and efficient gene therapy vector of wide spectrum. In this work, we review the bases of gene therapy, main types of gene transfer systems and basic properties and use of AAV vectors.

Molecular biological approaches to the study of vectors in relation to malaria control

Crampton,J. M.; Comley,I.; Eggleston,P.; Hill,S.; Hughes,M.; Knapp,T.; Lycett,G.; Urwin,R.; Warren,A.
Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/1992 Português
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36.02%
To a large extent, control of malaria vectors relies on the elimination of breeding sites and the application of chemical agents. There are increasing problems associated with the use of synthetic insecticides for vector control, including the evolution of resistance, the high cost of developing and registering new insecticides and an awareness of pollution from insecticide residues. These factors have stimulated interest in the application of molecular biology to the study of mosquito vectors of malaria; focussing primarily on two aspects. First, the improvement of existing control measures through the development of simplified DNA probe systems suitable for identification of vectors of malaria. The development of synthetic, non-radioactive DNA probes suitable for identification of species in the Anopheles gambiae complex is described with the aim of defining a simplified methodology wich is suitable for entomologist in the field. The second aspect to be considered is the development of completely novel strategies through the development of completely novel strategies through the genetic manipulation of insect vectors of malaria in order to alter their ability to transmit the disease. The major requirements for producing transgenic mosquitoes are outlined together with the progress wich has been made to date and discussed in relation to the prospects which this type of approach has for the future control of malaria.

A role for adeno-associated viral vectors in gene therapy

Coura,Renata dos Santos; Nardi,Nance Beyer
Fonte: Sociedade Brasileira de Genética Publicador: Sociedade Brasileira de Genética
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2008 Português
Relevância na Pesquisa
36.1%
Gene therapy constitutes a therapeutic intervention based on modification of the genetic material of living cells, by correcting genetic defects or overexpressing therapeutic proteins. The success of gene therapy protocols depends on the availability of therapeutically suitable genes, appropriate gene delivery systems and proof of safety and efficacy. Recent advances on the development of gene delivery systems, particularly on viral vectors engineering and improved gene regulatory systems, have led to marked progress in this field. Although the available vector systems can successfully transfer genes into cells, the ideal delivery vehicle has not been found. In this context, adeno-associated virus vectors (AAV) are arising as a promising tool for a wide range of applications, due to a combination of characteristics such as lack of pathogenicity and immunogenicity, wide range of cell tropism and long-term gene expression. Since its isolation, the biological properties of the adeno-associated virus have been increasingly understood, improving our ability to manipulate and use it as a safe and efficient gene therapy vector of wide spectrum. In this work, we review the bases of gene therapy, main types of gene transfer systems and basic properties and use of AAV vectors.

Estimation of the Size of Genetic Bottlenecks in Cell-to-Cell Movement of Soil-Borne Wheat Mosaic Virus and the Possible Role of the Bottlenecks in Speeding Up Selection of Variations in trans-Acting Genes or Elements▿ §

Miyashita, Shuhei; Kishino, Hirohisa
Fonte: American Society for Microbiology (ASM) Publicador: American Society for Microbiology (ASM)
Tipo: Artigo de Revista Científica
Português
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36.06%
Genetic bottlenecks facilitate the fixation and extinction of variants in populations, and viral populations are no exception to this theory. To examine the existence of genetic bottlenecks in cell-to-cell movement of plant RNA viruses, we prepared constructs for Soil-borne wheat mosaic virus RNA2 vectors carrying two different fluorescent proteins, yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP). Coinoculation of host plant leaves with the two RNA2 vectors and the wild-type RNA1 showed separation of the two vector RNA2s, mostly within seven to nine cell-to-cell movements from individual initially coinfected cells. Our statistical analysis showed that the number of viral RNA genomes establishing infection in adjacent cells after the first cell-to-cell movement from an initially infected cell was 5.97 ± 0.22 on average and 5.02 ± 0.29 after the second cell-to-cell movement. These results indicate that plant RNA viruses may generally face narrow genetic bottlenecks in every cell-to-cell movement. Furthermore, our model suggests that, rather than suffering from fitness losses caused by the bottlenecks, the plant RNA viruses are utilizing the repeated genetic bottlenecks as an essential element of rapid selection of their adaptive variants in trans-acting genes or elements to respond to host shifting and changes in the growth conditions of the hosts.

Lymphomagenesis in SCID-X1 Mice Following Lentivirus-mediated Phenotype Correction Independent of Insertional Mutagenesis and gamma c Overexpression

Ginn, S.; Liao, S.; Dane, A.; Hu, M.; Hyman, J.; Finnie, J.; Zheng, M.; Cavazzana-Calvo, M.; Alexander, S.; Trasher, A.; Alexander, I.
Fonte: Academic Press Inc Elsevier Science Publicador: Academic Press Inc Elsevier Science
Tipo: Artigo de Revista Científica
Publicado em //2010 Português
Relevância na Pesquisa
36.07%
The development of leukemia as a consequence of vector-mediated genotoxicity in gene therapy trials for X-linked severe combined immunodeficiency (SCID-X1) has prompted substantial research effort into the design and safety testing of integrating vectors. An important element of vector design is the selection and evaluation of promoter-enhancer elements with sufficient strength to drive reliable immune reconstitution, but minimal propensity for enhancer-mediated insertional mutagenesis. In this study, we set out to explore the effect of promoter-enhancer selection on the efficacy and safety of human immunodeficiency virus-1-derived lentiviral vectors in γc-deficient mice. We observed incomplete or absent T- and B-cell development in mice transplanted with progenitors expressing γc from the phosphoglycerate kinase (PGK) and Wiscott–Aldrich syndrome (WAS) promoters, respectively. In contrast, functional T- and B-cell compartments were restored in mice receiving an equivalent vector containing the elongation factor-1-α (EF1α) promoter; however, 4 of 14 mice reconstituted with this vector subsequently developed lymphoma. Extensive analyses failed to implicate insertional mutagenesis or γc overexpression as the underlying mechanism. These findings highlight the need for detailed mechanistic analysis of tumor readouts in preclinical animal models assessing vector safety...

Innate immune recognition of poxviral vaccine vectors

Lousberg, E.; Diener, K.; Brown, M.; Hayball, J.
Fonte: Future Drugs Ltd. Publicador: Future Drugs Ltd.
Tipo: Artigo de Revista Científica
Publicado em //2011 Português
Relevância na Pesquisa
45.91%
The study of poxviruses pioneered the field of vaccinology after Jenner’s remarkable discovery that ‘vaccination’ with the phylogenetically related cowpox virus conferred immunity to the devastating disease of smallpox. The study of poxviruses continues to enrich the field of virology because the global eradication of smallpox provides a unique example of the potency of effective immunization. Other poxviruses have since been developed as vaccine vectors for clinical and veterinary applications and include modified vaccinia virus strains such as modified vaccinia Ankara and NYVAC as well as the avipox viruses, fowlpox virus and canarypox virus. Despite the empirical development of poxvirus-based vectored vaccines, it is only now becoming apparent that we need to better understand how the innate arm of the immune system drives adaptive immunity to poxviruses, and how this information is relevant to vaccine design strategies, which are the topics addressed in this article.; Erin L. Lousberg, Kerrilyn R. Diener, Michael P. Brown and John D. Hayball

Heterologous prime-boost-boost immunisation of Chinese cynomolgus macaques using DNA and recombinant poxvirus vectors expressing HIV-1 virus-like particles

Bridge, S.H.; Sharpe, S.A.; Dennis, M.J.; Dowall, S.D.; Getty, B.; Anson, D.S.; Skinner, M.A.; Stewart, J.P.; Blanchard, T.J.
Fonte: BioMed Central Publicador: BioMed Central
Tipo: Artigo de Revista Científica
Publicado em //2011 Português
Relevância na Pesquisa
46%
BACKGROUND: There is renewed interest in the development of poxvirus vector-based HIV vaccines due to the protective effect observed with repeated recombinant canarypox priming with gp120 boosting in the recent Thai placebo-controlled trial. This study sought to investigate whether a heterologous prime-boost-boost vaccine regimen in Chinese cynomolgus macaques with a DNA vaccine and recombinant poxviral vectors expressing HIV virus-like particles bearing envelopes derived from the most prevalent clades circulating in sub-Saharan Africa, focused the antibody response to shared neutralising epitopes. METHODS: Three Chinese cynomolgus macaques were immunised via intramuscular injections using a regimen composed of a prime with two DNA vaccines expressing clade A Env/clade B Gag followed by boosting with recombinant fowlpox virus expressing HIV-1 clade D Gag, Env and cholera toxin B subunit followed by the final boost with recombinant modified vaccinia virus Ankara expressing HIV-1 clade C Env, Gag and human complement protein C3d. We measured the macaque serum antibody responses by ELISA, enumerated T cell responses by IFN-γ ELISpot and assessed seroneutralisation of HIV-1 using the TZM-bl β-galactosidase assay with primary isolates of HIV-1. RESULTS: This study shows that large and complex synthetic DNA sequences can be successfully cloned in a single step into two poxvirus vectors: MVA and FPV and the recombinant poxviruses could be grown to high titres. The vaccine candidates showed appropriate expression of recombinant proteins with the formation of authentic HIV virus-like particles seen on transmission electron microscopy. In addition the b12 epitope was shown to be held in common by the vaccine candidates using confocal immunofluorescent microscopy. The vaccine candidates were safely administered to Chinese cynomolgus macaques which elicited modest T cell responses at the end of the study but only one out of the three macaques elicited an HIV-specific antibody response. However...

Effect of deletion and the site of insertion in double copy anti-tat retroviral vectors: viral titres and production of anti-tat mRNA

Carr, J.; Calvert, J.; Kumar, R.; Burrell, C.; Li, P.
Fonte: Springer Wien Publicador: Springer Wien
Tipo: Artigo de Revista Científica
Publicado em //2001 Português
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46.15%
Summary. In attempts to further develop murine leukemia virus (MLV) based retroviral vectors for gene therapy, we investigated vector production and anti-sense expression from retroviral constructs with U3 deletions or insertions. Promoter elements in the U3 region of the 3' LTR of the vector pLXSN were deleted and replaced with DNA encoding the HIV anti-tat gene under control of the tRNAmet promoter to produce a double copy self inactivating vector (DC-SIN). DC-SIN constructs were compared to vectors containing the anti-tat cassette inserted at 5 different sites of the U3 region (DC-insertions). Titres of DC-SIN and DC-insertion vectors were similar but approximately 10 fold lower than parental pLXSN. Cells transduced with DC-SIN and DC-insertion vectors all expressed anti-tat mRNA. Transcripts from the MLV-LTR were detected in cells transduced with DC-insertion but not DC-SIN vectors or a vector with the anti-tat cassette between CAAT and TATA boxes of the promoter, indicating inactivation of the viral promoter in the latter vectors. Cells transduced with constructs of either design showed comparable efficacy of protection against HIV challenge. Thus, no U3 insertion site was preferred for virus production. Insertion of a tRNA promoter between CAAT and TATA boxes and the DC-SIN design which would not introduce an active RNA pol II promoter into the genome are attractive for further development of safe gene therapy agents.; The original publication is available at www.springerlink.com

Refinement of lentiviral vector for improved RNA processing and reduced rates of self inactivation repair

Koldej, R.; Anson, D.
Fonte: BioMed Central Ltd. Publicador: BioMed Central Ltd.
Tipo: Artigo de Revista Científica
Publicado em //2009 Português
Relevância na Pesquisa
36.06%
Background Lentiviral gene therapy vectors are now finding clinical application. In order to fully exploit their potential it is important that vectors are made as efficient and as safe as possible. Accordingly, we have modified a previously reported vector to improve RNA processing, minimise Human Immunodeficiency Virus Type-1 (HIV-1) sequence content and reduce repair of the self inactivating (SIN) deletion. Results HIV-1 sequence in the vector was reduced by substituting the polyadenylation signal with a heterologous signal. Mutation of splice donor sites was undertaken to prevent the majority of splicing within the vector genomic RNA. In addition, a number of other sequences within the vector were deleted. The combination of these modifications was able to significantly reduce the rates of both vector mobilisation and repair of the self inactivating deletion after two rounds of marker rescue. Conclusion RNA processing can be improved by mutation of the major and minor HIV-1 splice donor sites in the vector. In addition the rate of vector mobilisation and repair of SIN vectors can be successfully reduced by careful vector design that reduces homology between the 5' and 3' long terminal repeats (LTRs) to a minimum.; Rachel M Koldej and Donald S Anson

Interference between effector RNAs expressed from conventional dual-function anti-HIV retroviral vectors can be circumvented using dual-effector-cassette retroviral vectors

Peng, H.; Callison, D.; Li, P.; Burrell, C.
Fonte: MARY ANN LIEBERT INC PUBL Publicador: MARY ANN LIEBERT INC PUBL
Tipo: Artigo de Revista Científica
Publicado em //1999 Português
Relevância na Pesquisa
46.15%
Coexpression of different effector molecules from a single vector (a dual-function vector) may provide enhanced efficacy. Thus far most of the reported anti-HIV dual-function vectors express different effector RNAs as a chimeric molecule. In our study involving retroviral vectors coexpressing a U5 ribozyme and either an anti-tat or anti-rev antisense RNA, chimeric vectors exhibit poor potency in several important functional aspects, including inhibition of HIV replication, protection against cytopathic effects, and suppression of target gene function. Surprisingly, such a poor efficacy of chimeric vector function was not associated with a lower level of effector RNA expression. These results indicate that expression of two effector RNAs as a chimeric molecule can lead to interference, reducing their global biological effects. More importantly, we have demonstrated that such interference can be avoided by coexpressing these effector RNAs as separate molecules through a new dual-function vector, called a dual-effector cassette (Dec) vector, developed in this study. We also define some of the design alterations that might affect the efficacy of the Dec vector and demonstrate that forward-designed Dec vectors are more efficacious than reverse-designed Dec vectors...

The Elucidation of the involvement of endonuclease DNase y in reducing DNA transfection efficiency in mammalian cells

Fonte: Brock University Publicador: Brock University
Tipo: Electronic Thesis or Dissertation
Português
Relevância na Pesquisa
36.1%
Gene therapy is predicated upon efficient gene transfer. While viral vectors are the method of choice for transformation efficiency, the immunogenicity and safety concerns remain problematic. Non-viral vectors, on the other hand, have shown high degrees of safety and are mostly non-immunogenic in nature. However, non-viral vectors usually suffer from low levels oftransformation efficiency and transgene expression. Thus, increasing transformation efficiency ofnon-viral vectors, in particular by calcium phosphate co-precipitation technique, is a way of generating a suitable vector for gene therapy and is the aim of this study. It is a long known fact that different cell lines have different transfection efficiencies regardless oftransfection methodology (Lin et a!., 1994). Using commonly available cell lines Madine-Darby Bovine Kidney (MDBK), HeLa and Human Embryonic Kidney (HEK-293), we have shown a decreasing trend ofDNase activity based on a plasmid digestion assay. From densitometry studies, as much as a 40% reduction in DNase activity was observed when comparing HEK-293 (least active) to MDBK (most active). Using various biochemical assays, it was determined that DNase y, in particular, was expressed more highly in MDBK cells than both HeLa and HEK-293. Upon cloning of the bovine DNase y gene...

Development of packaging cell lines for rescuing BAV2 viral vectors

Salamé, Mohamad S.
Fonte: Brock University Publicador: Brock University
Tipo: Electronic Thesis or Dissertation
Português
Relevância na Pesquisa
45.91%
The construction of adenovirus vectors for cloning and foreign gene expression requires packaging cell lines that can complement missing viral functions caused by sequence deletions and/or replacement with foreign DNA sequences. In this study, packaging cell lines were designed to provide in trans the missing bovine adenovirus functions, so that recombinant viruses could be generated. Fetal bovine kidney and lUng cells, acquired at the trimester term from a pregnant cow, were tranfected with both digested wild type BAV2 genomic DNA and pCMV-EI. The plasmid pCMV-EI was specifically constructed to express El of BAV2 under the control of the cytomegalovirus enhancer/promoter (CMV). Selection for "true" transformants by continuous passaging showed no success in isolating immortalised cells, since the cells underwent crisis resulting in complete cell death. Moreover, selection for G418 resistance, using the same cells, also did not result in the isolation of an immortalised cell line and the same culture-collapse event was observed. The lack of success in establishing an immortalised cell line from fetal tissue prompted us to transfect a pre-established cell line. We began by transfecting MDBK (Mardin-Dardy bovine kidney) cells with pCMV-El-neo...

Avaliação da factibilidade da terapia gênica com vetores não virais na sepse experimental murina; Evaluation of the feasibility of gene transfer with non-viral vectors in a murine model of sepsis

Vanessa Boury Faiotto
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 19/12/2014 Português
Relevância na Pesquisa
46.12%
A sepse representa uma condição potencialmente fatal em que a resposta do organismo a uma infecção resulta em lesão em seus próprios tecidos. A terapia gênica (TG) consiste na modificação do repertório de células somáticas com fins terapêuticos, substituindo genes defeituosos que causam doenças. Na sepse, vetores não virais podem representar uma estratégia excelente para a transferência do gene terapêutico, uma vez que não provoca resposta imune significativa e são expressos apenas transitoriamente. Além disso, a sua produção é mais simples. Métodos: O estudo foi dividido em três etapas. Em primeiro lugar, dois genes repórter (lacZ e F9) foram testados em dois modelos experimentais de sepse (endotoxemia e ligadura e punção cecal, CLP) para confirmar a viabilidade da transferência gênica no contexto da sepse. A expressão foi avaliada por métodos qualitativos (histoquímica) e quantitativos (avaliação funcional; métodos coagulométricos). Em seguida avaliou-se a eficácia terapêutica da transferência do gene de sFlt-1, um antagonista de VEGF natural, no modelo de endotoxemia. A expressão foi avaliada por ELISA, e a eficácia foi avaliada através de uma curva de sobrevida. Os camundongos foram tratados com o plasmídeo DNA (pDNA) contendo o cDNA do gene Flt1 (tratamento) lacZ (controle)...

Plasmid Vectors and Molecular Building Blocks for the Development of Genetic Manipulation Tools for Trypanosoma cruzi

Bouvier, Leon Alberto; Camara, Maria de Los Milagros; Canepa, Gaspar Exequiel; Miranda, Mariana Reneé; Pereira, Claudio Alejandro
Fonte: Public Library Science Publicador: Public Library Science
Tipo: info:eu-repo/semantics/publishedVersion; info:eu-repo/semantics/article; info:ar-repo/semantics/artículo Formato: application/pdf
Português
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36.04%
The post genomic era revealed the need for developing better performing, easier to use and more sophisticated genetic manipulation tools for the study of Trypanosoma cruzi, the etiological agent of Chagas disease. In this work a series of plasmids that allow genetic manipulation of this protozoan parasite were developed. First of all we focused on useful tools to establish selection strategies for different strains and which can be employed as expression vectors. On the other hand molecular building blocks in the form of diverse selectable markers, modifiable fluorescent protein and epitope-tag coding sequences were produced. Both types of modules were harboured in backbone molecules conceived to offer multiple construction and sub-cloning strategies. These can be used to confer new properties to already available genetic manipulation tools or as starting points for whole novel design. The performance of each plasmid and building block was determined independently. For illustration purposes, some simple direct practical applications were conducted.

Genetic Diversity in Introduced Golden Mussel Populations Corresponds to Vector Activity

Ghabooli, Sara; Zhan, Aibin; Sardiña, Paula; Paolucci, Esteban; Sylvester, Francisco; Perepelizin, Pablo Victor; Briski, Elizabeta; Cristescu, Melania; Maclsaac, Hugh
Fonte: Public Library Science Publicador: Public Library Science
Tipo: info:eu-repo/semantics/article; info:ar-repo/semantics/artículo; info:eu-repo/semantics/publishedVersion Formato: application/pdf
Português
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36.09%
We explored possible links between vector activity and genetic diversity in introduced populations of Limnoperna fortunei by characterizing the genetic structure in native and introduced ranges in Asia and South America. We surveyed 24 populations: ten in Asia and 14 in South America using the mitochondrial cytochrome c oxidase subunit I (COI) gene, as well as eight polymorphic microsatellite markers. We performed population genetics and phylogenetic analyses to investigate population genetic structure across native and introduced regions. Introduced populations in Asia exhibit higher genetic diversity (HE = 0.667–0.746) than those in South America (HE = 0.519–0.575), suggesting higher introduction effort for the former populations. We observed pronounced geographical structuring in introduced regions, as indicated by both mitochondrial and nuclear markers based on multiple genetic analyses including pairwise FST, FST, Bayesian clustering method, and three-dimensional factorial correspondence analyses. Pairwise FST values within both Asia (FST = 0.017–0.126, P = 0.000–0.009) and South America (FST = 0.004–0.107, P = 0.000–0.721) were lower than those between continents (FST = 0.180–0.319, P = 0.000). Fine-scale genetic structuring was also apparent among introduced populations in both Asia and South America...

Genetic engineering of the skeletal muscle to counteract insulin resistance and obesity

Roca Lecha, Carles
Fonte: [Barcelona] : Universitat Autònoma de Barcelona, Publicador: [Barcelona] : Universitat Autònoma de Barcelona,
Tipo: Tesis i dissertacions electròniques; info:eu-repo/semantics/doctoralThesis; info:eu-repo/semantics/publishedVersion
Publicado em //2014 Português
Relevância na Pesquisa
36.12%
La diabetis tipus 2 és la malaltia metabòlica més freqüent a tot el món. Malgrat que els tractaments farmacològics són útils en les primeres etapes de la malaltia, no han sigut capaços prevenir la pèrdua de control de la glucèmia a llarg termini. A més, aquests tractaments presenten efectes secundaris indesitjables. Per tant, el desenvolupament de nous tractaments per a la diabetis tipus 2 és avui en dia un gran repte per a la investigació científica. El desenvolupament de la teràpia gènica ha proporcionat una nova eina per al tractament de malalties humanes. No obstant això, no s'han desenvolupat fins ara tractaments de teràpia gènica per a la diabetis tipus 2. Un 90% de la diabetis tipus 2 és conseqüència d'un excés de pes. L'acumulació de triglicèrids en els teixits perifèrics està vinculada a l'aparició de resistència a la insulina i la reducció de la captació de glucosa, conduint a un disfunció de la cèl·lula β i a la diabetis tipus 2. Per tant, promoure la captació de glucosa o l'oxidació d'àcids grassos podria prevenir el desenvolupament de la diabetis tipus 2. El múscul esquelètic juga un paper clau en l'homeòstasi de la glucosa i posseeix una gran capacitat d'utilitzar els àcids grassos per a la producció d'energia. És també un teixit ideal per a la transferència de gens...

A minimally invasive, lentiviral based method for the rapid and sustained genetic manipulation of renal tubules.

Espana-Augusti, Judit; Tuveson, David A.; Adams, David J.; Matakidou, Athena
Fonte: NPG Publicador: NPG
Tipo: Article; published version
Português
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This is the final version. It was first published by NPG at http://www.nature.com/srep/2015/150605/srep11061/full/srep11061.html.; The accelerated discovery of disease-related genes emerging from genomic studies has strained the capacity of traditional genetically engineered mouse models (GEMMs) to provide in-vivo validation. Direct, somatic, genetic engineering approaches allow for accelerated and flexible genetic manipulation and represent an attractive alternative to GEMMs. In this study we investigated the feasibility, safety and efficiency of a minimally invasive, lentiviral based approach for the sustained in-vivo modification of renal tubular epithelial cells. Using ultrasound guidance, reporter vectors were directly injected into the mouse renal parenchyma. We observed transgene expression confined to the renal cortex (specifically proximal and distal tubules) and sustained beyond 2 months post injection. Furthermore, we demonstrate the ability of this methodology to induce long-term, in-vivo knockdown of candidate genes either through somatic recombination of floxed alleles or by direct delivery of specific shRNA sequences. This study demonstrates that ultrasound-guided injection of lentiviral vectors provides a safe and efficient method for the genetic manipulation of renal tubules...