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Universidade Estadual de Campinas

1. Toledo, Marcelo Augusto Szymanski de, 1987-. Trafego intracelular de vetores não-virais = desenvolvimento de proteínas de fusão para transporte de DNA plasmidial através da interação com proteínas motoras = Intracelullar traffic of non-viral vectors: development of recombinant fusion proteins to mediate plasmidial DNA transport by interaction with motor proteins: Intracelullar traffic of non-viral vectors : development of recombinant fusion proteins to mediate plasmidial DNA transport by interaction with motor proteins.

Degree: 2013, Universidade Estadual de Campinas

URL: http://repositorio.unicamp.br/jspui/handle/REPOSIP/316418 

Abstract: The use of non viral vectors such as plasmidial DNA (pDNA) in gene therapy and DNA vaccination protocols has been limited due to its low transfection efficiency when compared to viral vectors. This limitation occurs mainly due to the physical, enzymatic and diffusion barriers faced during the transport of the genetic material to the nucleus of target eukaryotic cells. Regarding this subject, the present work demonstrates the feasibility of using modified Dynein light chains (Lc8 and Rp3) as non viral vectors for gene delivery. The use of Dynein light chains relies on the possibility to exploit the Dynein based cellular retrograde transport in order to improve the exogenous genetic material transport across the citosol towards the nuclear periphery. By adding small peptide domains, based in positively charged aminoacids (arginine and lysine) to the N-terminal of Dynein light chains, the resulting recombinant proteins were able to interact and condense genetic material into delivery particles. Transfection assays demonstrated that these particles are highly efficient to delivery plasmidial DNA to nucleus of HeLa cells when compared to the transfection efficiency presented by protamine, a well characterized non viral vector peptide. Ternary complexes formed by modified Dynein light chains, pDNA and a cationic lipid showed even higher transfection efficiency. Additionally, the light chain based non viral delivery vectors presented low citotoxic effect to HeLa cells, a valuable feature as toxicity is regarded as one of the main concerns on delivery vectors development. The mechanism by which the modified Dynein light chain based vectors mediates gene delivery was also investigated and we could observe that (1) the internalization process deeply relies on endocytosis, (2) it depends on the microtubule network and (3) a significant fraction of the delivery complexes are trapped and degraded in the endocytic pathway. The non viral vectors developed in the present study combine high transfection efficiency, low toxicity and relative low production cost, as all modified proteins were produced in Escherichia coli prokaryotic host. Its noteworthy that additional peptide domains can be further associated to the delivery vectors described providing it with new abilities such as higher internalization or endosomal escape capacity. The approach to use the cellular retrograde transport in order to develop non viral vectors is poorly exploited by the scientific community and the present study stands among few in the field hopefully contributing to the development of more efficient and safer non viral vectors for gene delivery Advisors/Committee Members: UNIVERSIDADE ESTADUAL DE CAMPINAS (CRUESP), Azzoni, Adriano Rodrigues, 1971- (advisor), Souza, Anete Pereira de, 1962- (coadvisor), Universidade Estadual de Campinas. Instituto de Biologia (institution), Programa de Pós-Graduação em Genética e Biologia Molecular (nameofprogram), Torre, Lucimara Gaziola de la (committee member), Massirer, Katlin Brauer (committee member), Peroni, Luis Antonio (committee member), Santos, Sandra Aparecida Cororato dos (committee member).

Subjects/Keywords: Entrega gênica; Dineínas; Transfecção; Terapia genética; Plasmideos; Gene delivery; Dyneins; Transfection; Gene therapy; Plasmids

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Toledo, Marcelo Augusto Szymanski de, 1. (2013). Trafego intracelular de vetores não-virais = desenvolvimento de proteínas de fusão para transporte de DNA plasmidial através da interação com proteínas motoras = Intracelullar traffic of non-viral vectors: development of recombinant fusion proteins to mediate plasmidial DNA transport by interaction with motor proteins: Intracelullar traffic of non-viral vectors : development of recombinant fusion proteins to mediate plasmidial DNA transport by interaction with motor proteins. (Thesis). Universidade Estadual de Campinas. Retrieved from http://repositorio.unicamp.br/jspui/handle/REPOSIP/316418

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16th Edition):

Toledo, Marcelo Augusto Szymanski de, 1987-. “Trafego intracelular de vetores não-virais = desenvolvimento de proteínas de fusão para transporte de DNA plasmidial através da interação com proteínas motoras = Intracelullar traffic of non-viral vectors: development of recombinant fusion proteins to mediate plasmidial DNA transport by interaction with motor proteins: Intracelullar traffic of non-viral vectors : development of recombinant fusion proteins to mediate plasmidial DNA transport by interaction with motor proteins.” 2013. Thesis, Universidade Estadual de Campinas. Accessed March 03, 2021. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316418.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7th Edition):

Toledo, Marcelo Augusto Szymanski de, 1987-. “Trafego intracelular de vetores não-virais = desenvolvimento de proteínas de fusão para transporte de DNA plasmidial através da interação com proteínas motoras = Intracelullar traffic of non-viral vectors: development of recombinant fusion proteins to mediate plasmidial DNA transport by interaction with motor proteins: Intracelullar traffic of non-viral vectors : development of recombinant fusion proteins to mediate plasmidial DNA transport by interaction with motor proteins.” 2013. Web. 03 Mar 2021.

Vancouver:

Toledo, Marcelo Augusto Szymanski de 1. Trafego intracelular de vetores não-virais = desenvolvimento de proteínas de fusão para transporte de DNA plasmidial através da interação com proteínas motoras = Intracelullar traffic of non-viral vectors: development of recombinant fusion proteins to mediate plasmidial DNA transport by interaction with motor proteins: Intracelullar traffic of non-viral vectors : development of recombinant fusion proteins to mediate plasmidial DNA transport by interaction with motor proteins. [Internet] [Thesis]. Universidade Estadual de Campinas; 2013. [cited 2021 Mar 03]. Available from: http://repositorio.unicamp.br/jspui/handle/REPOSIP/316418.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Toledo, Marcelo Augusto Szymanski de 1. Trafego intracelular de vetores não-virais = desenvolvimento de proteínas de fusão para transporte de DNA plasmidial através da interação com proteínas motoras = Intracelullar traffic of non-viral vectors: development of recombinant fusion proteins to mediate plasmidial DNA transport by interaction with motor proteins: Intracelullar traffic of non-viral vectors : development of recombinant fusion proteins to mediate plasmidial DNA transport by interaction with motor proteins. [Thesis]. Universidade Estadual de Campinas; 2013. Available from: http://repositorio.unicamp.br/jspui/handle/REPOSIP/316418

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

2. Vanessa Bastos Pereira. Construção e avaliação funcional de um plasmídeo vacinal para a expressão do antígeno ESAT-6 de Mycobacterium Tuberculosis em células mamíferas, utilizando uma bactéria láctica invasiva como veículo carreador.

Degree: 2011, Universidade Federal de Minas Gerais

URL: http://hdl.handle.net/1843/BUOS-8EMR7C 

O uso de bactérias como veículos para a entrega de plasmídeos vacinais pela rota oral constitui uma estratégia de vacinação promissora contra diversas doenças infecciosas. Para isto, bactérias patogênicas atenuadas como Shigella, Listeria e Salmonella vêm sendo utilizadas, ainda que as mesmas apresentem risco de reversão ao seu fenótipo selvagem. Sendo assim, a utilização de bactérias não patogênicas poderia constituir uma alternativa mais segura para este propósito. Lactococcus lactis é uma bactéria láctica modelo considerada GRAS (Generally Recognized As Safe) que vem sendo extensivamente utilizada para a produção e entrega de antígenos e citocinas ao nível de mucosas. Assim, L. lactis pode representar uma alternativa para a entrega de plasmídeos vacinais em relação aos patógenos atenuados, ainda que o mesmo não tenha capacidade invasiva. Neste contexto, foram construídas linhagens de L. lactis invasivas (L. lactis FnBPA, Innocentin et al., 2009) e também um plasmídeo replicativo em L. lactis, contendo um cassete de expressão eucariótica (pValac, Guimarães et al., 2009). Assim, a utilização de uma linhagem invasiva de L. lactis contendo o vetor pValac para a expressão eucariótica do antígeno ESAT-6 (6-kDa Early Secreted Antigenic Target) de Mycobacterium tuberculosis, pode vir a ser uma nova estratégia para o controle da tuberculose; uma doença infecto-contagiosa que atinge 1/3 da população mundial na forma latente. Desta forma, este trabalho teve como objetivo a construção do plasmídeo vacinal pValac:ESAT-6 e verificação de sua funcionalidade, in vitro, assim como a clonagem na linhagem invasiva de L. lactis para sua utilização como uma via de entrega oral de vacinas gênicas. A seqüência codificadora de ESAT-6 foi isolada por PCR a partir do DNA genômico de M. tuberculosis linhagem H37Rv para clonagem no vetor Zero Blunt® TOPO® e posteriormente no vetor pValac. A construção final, pValac:ESAT-6, foi primeiramente obtida em Escherichia coli TG1. Para a avaliação da funcionalidade do plasmídeo, células da linhagem CHO (Chinese Hamster Ovary) foram transfectadas com o plasmídeo pValac:ESAT-6 e a expressão de ESAT-6 foi avaliada por RT-PCR, Western blotting e Imunocitoquímica, sendo, a funcionalidade do plasmídeo pValac:ESAT-6, assim confirmada. Por fim, o plasmídeo pValac:ESAT-6 foi transformado na linhagem invasiva de L. lactis, gerando a linhagem L. lactis FnBPA(pValac:ESAT-6). Enfim, este trabalho constitui um primeiro passo rumo à validação da eficácia e efetividade de novas vacinas gênicas baseadas em bactérias lácticas geneticamente modificadas, por via de administração oral em mucosas; o que também poderá fornecer informações valiosas para a pesquisa e o desenvolvimento de vacinas contra outros patógenos.

The use of bacteria as vehicles for the delivery of vaccine plasmids by oral route constitutes a promising strategy for vaccination against various infectious diseases. Attenuated pathogenic bacteria such as Shigella, Listeria and Salmonella have been widely used for such purposes, although presenting…

Advisors/Committee Members: Anderson Miyoshi, Anderson Miyoshi, Alvaro Cantini Nunes, Dawidson Assis Gomes, Sophie Yvette Leclercq.

Subjects/Keywords: Genética Teses.; Lactococcus lactis Teses.; Vacinas de DNA Teses.; Tuberculose Teses.; Plasmideos Teses.; Antígenos Teses.; Vacinas Teses.

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Pereira, V. B. (2011). Construção e avaliação funcional de um plasmídeo vacinal para a expressão do antígeno ESAT-6 de Mycobacterium Tuberculosis em células mamíferas, utilizando uma bactéria láctica invasiva como veículo carreador. (Thesis). Universidade Federal de Minas Gerais. Retrieved from http://hdl.handle.net/1843/BUOS-8EMR7C

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16th Edition):

Pereira, Vanessa Bastos. “Construção e avaliação funcional de um plasmídeo vacinal para a expressão do antígeno ESAT-6 de Mycobacterium Tuberculosis em células mamíferas, utilizando uma bactéria láctica invasiva como veículo carreador.” 2011. Thesis, Universidade Federal de Minas Gerais. Accessed March 03, 2021. http://hdl.handle.net/1843/BUOS-8EMR7C.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7th Edition):

Pereira, Vanessa Bastos. “Construção e avaliação funcional de um plasmídeo vacinal para a expressão do antígeno ESAT-6 de Mycobacterium Tuberculosis em células mamíferas, utilizando uma bactéria láctica invasiva como veículo carreador.” 2011. Web. 03 Mar 2021.

Vancouver:

Pereira VB. Construção e avaliação funcional de um plasmídeo vacinal para a expressão do antígeno ESAT-6 de Mycobacterium Tuberculosis em células mamíferas, utilizando uma bactéria láctica invasiva como veículo carreador. [Internet] [Thesis]. Universidade Federal de Minas Gerais; 2011. [cited 2021 Mar 03]. Available from: http://hdl.handle.net/1843/BUOS-8EMR7C.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Pereira VB. Construção e avaliação funcional de um plasmídeo vacinal para a expressão do antígeno ESAT-6 de Mycobacterium Tuberculosis em células mamíferas, utilizando uma bactéria láctica invasiva como veículo carreador. [Thesis]. Universidade Federal de Minas Gerais; 2011. Available from: http://hdl.handle.net/1843/BUOS-8EMR7C

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation


Universidade Estadual de Campinas

3. Kuboyama, Rogerio Hakio. Detecção de cepas de Klebsiella pneumoniea produtoras de beta-lactamases de espectro estendido em pacientes assistidos em hospitais terciarios na cidade de Campinas : epidemiologia molecular e fatores de risco: Detection of extended-spectrum-beta-lactamase-producing strains of Klebsiella pneumoniea isolated from patients hospitalized in tertiary-care hospitals in Campinas : molecular epidemiology and risk factors.

Degree: 2009, Universidade Estadual de Campinas

URL: http://repositorio.unicamp.br/jspui/handle/REPOSIP/311054 

Abstract: The objectives of this study conducted restrospectively using strains isolated from clinical specimens obtained from patients hospitalized in two Brazilian hospitals between February 2001 to June 2004 were to describe the presence of extended-spectrum b-lactamase (ESBL)-producing Klebsiella pneumoniae strains, the best method and the preferred substrate for screening and confirming ESBL production and the epidemiological relatedness of ESBL-producing strains and analyse the risk factors for infection due to ESBL-producing K. pneumoniae. To investigate the genetic relatedness of the strains, plasmid analysis and chromosomal DNA analysis by pulsed-field gel electrophoresis were used. A total of 89 K. pneumoniae were collected from diverse body sites. The most commom specimens yielding ESBL-producing strains were urine (12.4%) and blood (10.1%). Using CLSI criteria (ESBL screening breakpoints), 35 K. pneumoniae (39.3%) had presumptive ESBL phenotype, while using the double-disk approximation test (DDAT) characteristic clavulanate-induced distortions of inhibition zones were found in 96, 62.5, 50, 18.8 and 12.5% of the strains around the disks containing aztreonam, cefotaxime, ceftazidime, ceftriaxone and cefpodoxime, respectively. Of 89 isolates, 32 (36%) produced ESBL based on the confirmatory Oxoid combination disk method. The disk containing cefotaxime was able to confirm 100% of the ESBL producers while the disk containing ceftazidime was not able to confirm 2 ESBL-positive strains. Ten and 32 different plasmid profiles were observed among the ESBL-producing K. pneumoniae and non ESBL producers, respectively. Pulsed-field gel electrophoresis showed the best discriminatory power giving 15 and 55 different chromosomal DNA profiles among the ESBL-positive and ESBL-negative K. pneumoniae, respectively. From univariate analysis, variables significantly associated with infection by an ESBL-producing strain of K. pneumoniae included the following: use of 4st-generation cephalosporins, lincosamide, carbapenems, glycopeptides, recent surgery, tracheostomy, age, days in using of lincosamide, days in using of glycopeptides, total number of antibiotics and duration of the antimicrobial therapy. The only variable that remained independent risk factor for acquiring infection due to ESBL-producing K. pneumoniae after multivariable analysis using a logistic regression model, which included the variables associated with acquiring infection by ESBL-producing K. pneumoniae by univariate analysis (P < 0.05), was total number of antibiotics (OR, 1.60; 95%CI, 1.194-2.145; P = 0.0017). In summary, these data indicate that ESBL-producing K. pneumoniae occur at a relatively high incidence at our institutions and the plasmid analysis is not sufficient to identify relationships between ESBL-producing strains of K. pneumoniae. The ESBL screening breakpoints and DDAT with aztreonam appear to be good indicators in presumptive detection of ESBL-producing strains and the cefotaxime used in the Oxoid combination disk method constituted… Advisors/Committee Members: UNIVERSIDADE ESTADUAL DE CAMPINAS (CRUESP), Moretti, Maria Luiza, 1953- (advisor), Universidade Estadual de Campinas. Faculdade de Ciências Médicas (institution), Programa de Pós-Graduação em Clínica Médica (nameofprogram), Yasuda, Maria Aparecida Shikanai (committee member), Pereira, Graziela Hanna (committee member), Aoki, Francisco Hideo (committee member), Yano, Tomomasa (committee member).

Subjects/Keywords: Klebsiella pneumoniae; Eletroforese em gel de campo pulsado; Plasmideos; Fatores de risco; Epidemiologia molecular; Infecção hospitalar; Beta-lactamas; Klebsiella pneumoniae; Electrophoresis, gel, pulsed-field; Plasmids; Risk factors; Molecular epidemiology; Nosocomial infections; Beta-lactamases

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Kuboyama, R. H. (2009). Detecção de cepas de Klebsiella pneumoniea produtoras de beta-lactamases de espectro estendido em pacientes assistidos em hospitais terciarios na cidade de Campinas : epidemiologia molecular e fatores de risco: Detection of extended-spectrum-beta-lactamase-producing strains of Klebsiella pneumoniea isolated from patients hospitalized in tertiary-care hospitals in Campinas : molecular epidemiology and risk factors. (Thesis). Universidade Estadual de Campinas. Retrieved from http://repositorio.unicamp.br/jspui/handle/REPOSIP/311054

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16th Edition):

Kuboyama, Rogerio Hakio. “Detecção de cepas de Klebsiella pneumoniea produtoras de beta-lactamases de espectro estendido em pacientes assistidos em hospitais terciarios na cidade de Campinas : epidemiologia molecular e fatores de risco: Detection of extended-spectrum-beta-lactamase-producing strains of Klebsiella pneumoniea isolated from patients hospitalized in tertiary-care hospitals in Campinas : molecular epidemiology and risk factors.” 2009. Thesis, Universidade Estadual de Campinas. Accessed March 03, 2021. http://repositorio.unicamp.br/jspui/handle/REPOSIP/311054.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7th Edition):

Kuboyama, Rogerio Hakio. “Detecção de cepas de Klebsiella pneumoniea produtoras de beta-lactamases de espectro estendido em pacientes assistidos em hospitais terciarios na cidade de Campinas : epidemiologia molecular e fatores de risco: Detection of extended-spectrum-beta-lactamase-producing strains of Klebsiella pneumoniea isolated from patients hospitalized in tertiary-care hospitals in Campinas : molecular epidemiology and risk factors.” 2009. Web. 03 Mar 2021.

Vancouver:

Kuboyama RH. Detecção de cepas de Klebsiella pneumoniea produtoras de beta-lactamases de espectro estendido em pacientes assistidos em hospitais terciarios na cidade de Campinas : epidemiologia molecular e fatores de risco: Detection of extended-spectrum-beta-lactamase-producing strains of Klebsiella pneumoniea isolated from patients hospitalized in tertiary-care hospitals in Campinas : molecular epidemiology and risk factors. [Internet] [Thesis]. Universidade Estadual de Campinas; 2009. [cited 2021 Mar 03]. Available from: http://repositorio.unicamp.br/jspui/handle/REPOSIP/311054.

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

Kuboyama RH. Detecção de cepas de Klebsiella pneumoniea produtoras de beta-lactamases de espectro estendido em pacientes assistidos em hospitais terciarios na cidade de Campinas : epidemiologia molecular e fatores de risco: Detection of extended-spectrum-beta-lactamase-producing strains of Klebsiella pneumoniea isolated from patients hospitalized in tertiary-care hospitals in Campinas : molecular epidemiology and risk factors. [Thesis]. Universidade Estadual de Campinas; 2009. Available from: http://repositorio.unicamp.br/jspui/handle/REPOSIP/311054

Note: this citation may be lacking information needed for this citation format:
Not specified: Masters Thesis or Doctoral Dissertation

.