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Targeting polymer coated adenovirus to tumour-associated vasculatureBachtarzi, Houria January 2010 (has links)
Tumour-associated vasculature provides an accessible target for systemic gene therapy using targeted adenoviruses. The aim of this thesis is to develop strategies for targeting adenovirus infection to tumour-associated endothelium. Adenovirus expressing luciferase (Adluc) was coated with an amino-reactive polymer based on poly [N-(2-hydroxypropyl) methacrylamide] [pHPMA] to ablate normal infection pathways¬. This was a pre-requisite to redirecting virus tropism to infect endothelial cells via specific receptors. Direct attachment to the pHPMA-adenovirus (pcAdluc) of ligands including vascular endothelial growth factor (VEGF165) and a monoclonal antibody (RAFL) recognising VEGF receptor 2 (VEGFR-2) retargeted infectivity to VEGFR-2-positive endothelial cells and not to receptor-negative cells. Specificity of transduction in vitro was shown by competition with excess antibody. In vivo however, the VEGF165-retargeted virus failed to transduce tumour-associated endothelia following systemic administration. Similarly, direct linkage of a monoclonal antibody against E-Selectin (MHES) demonstrated E-Selectin-specific transduction of tumour necrosis factor-α (TNF-α)-activated endothelial cells, although overall levels of infection were not increased compared to unmodified Adluc. A two-component targeting system using protein A or protein G as ‘bridging’ agents was developed to ensure the required orientation of targeting antibodies. Using this system MHES mediated greater transduction of TNF-α-activated endothelial cells than Adluc. Conjugation using protein A also gave non-specific effects which were not seen with protein G. Whereas the unmodified Adluc virus failed to transduce TNF-α-activated endothelium in an umbilical vein model ex vivo, the MHES-protein G-pHPMA-adenovirus (MHES-StrepGpcAdluc) mediated good transduction. Similarly, StrepGpcAdluc retargeted with a chimeric P-Selectin Glycoprotein Ligand-1 (PSGL-1)-Fc fusion protein, showed good circulation kinetics and significant uptake into HepG2 xenografts following intravenous administration. Histological studies suggested selective targeting to tumour-associated endothelial cells. Overall these findings support the assertion that tumour-associated vasculature is an accessible target for systemic gene delivery, and the use of protein G as bridging agent facilitates rapid screening of Fc-bearing ligands for retargeting pcAd infection to tumour-associated endothelium.
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Construção e caracterização de vetor adenoviral com promotor responsivo ao seu próprio transgene p53 e sua comparação com um vetor de promoção constitutiva. / Construction and characterization of an adenoviral vector responsive to its own p53 transgene in a comparison with a constitutive promoter vector.Soares, Rafael Bento da Silva 22 March 2011 (has links)
Em sua maioria, estratégias de transferência gênica utilizam arranjos com promotores e transgenes funcionando de maneira independente entre si, como é o caso do amplamente utilizado promotor CMV. Nosso grupo foge dessa linha de promotores/transgenes independentes. Desenvolvemos uma nova estratégia de transferência gênica que combina estrategicamente a atividade do transgene e o do promotor de expressão gênica, onde o promotor foi modificado com a inserção do elemento PG, responsívo a p53. Neste trabalho construímos um novo vetor adenoviral (AdPGp53) contendo o gene da proteína supressora de tumor p53 cuja expressão é controlada por ela própria através do elemento PG. Em comparação com um vetor adenoviral que possui o gene da p53 sob ação do promotor tradicional CMV (AdCMVp53), o vetor AdPGp53 apresentou expressão superior de p53 em células humanas de carcinoma de próstata PC3, maior morte celular in vitro e parece ter diminuído o ritmo de crescimento tumoral in vivo em um modelo xenográfico de células PC3 em camundongos atímicos. / The majority of gene therapy strategies in use today are based on promoters and transgenes that work independently, and an example of this is the widely used CMV promoter. Our group breaks way from the use of independent promoter/transgene activity. We developed a new gene transfer strategy which combines the transgene activity and the promoter of gene expression. This was achieved by the insertion of the PG element, which is a p53-responsive enhancer, in the promoter. In the present work we built a new adenoviral vector (AdPGp53) containing the p53 tumor suppressor gene, whose expression is controlled by the p53 protein itself through the PG element. In comparative experiments, in which we used our AdPGp53 vector and another adenoviral vector, with a p53 gene and a traditional CMV promoter (AdCMVp53), our vector showed superior p53 expression in PC3 human prostate cancer cells, superior cell death in vitro and a tendency in diminishing tumor growth in an in vivo xenograft model in nude mice injected with PC3 cells.
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Aplicação de técnicas moleculares no diagnóstico laboratorial complementar das infecções virais do sistema nervoso central no Hospital Universitário da USP. / Molecular techniques application for the complementary laboratory diagnosis of viral infections of the central nervous system, at the University Hospital of USP.Nunes, Rafaella Almeida Lima 22 August 2013 (has links)
Enterovírus (HEV), herpesvírus 1 e 2 (HHV-1 e HHV-2) e adenovírus (HAdV) são importantes agentes de infecções do SNC. Neste trabalho, técnicas moleculares foram aplicadas para a detecção destes vírus em quadros de infecção do SNC. Amostras de líquor foram colhidas de pacientes atendidos no HU-USP entre agosto e novembro/2010 e fevereiro/2012 a janeiro/2013. Através da Nested-PCR HEV foram detectados em 9,8% das amostras, HAdV em 2,5% e HHV-1 e 2 em 1,1%, além de 3 casos de coinfecção, 2 entre HEV e HHV, e 1 entre HEV e HAdV. O material genético viral foi extraído através dos métodos Qiaamp DNA Blood (Qiagen®) e MagMAXTM Viral RNA Isolation (Ambiom), e este último pareceu mais adequado à aplicação na rotina clínica. A análise quimiocitológica do líquor mostrou-se importante no direcionamento da conduta clínica, mas a detecção do vírus é fundamental para a conclusão do diagnóstico. A PCR em tempo real, cuja padronização foi iniciada neste trabalho, consiste em importante ferramenta para a utilização futura no diagnóstico complementar das infecções virais do SNC. / Enteroviruses (HEV), herpesviruses 1 and 2 (HHV-1 and HHV-2) and adenoviruses (HAdV) are important causative agents of infections of the CNS. In this study, molecular techniques were applied to the detection of these viruses. CSF samples were collected from patients treated at the University Hospital of USP, between August and November, 2010, and February 2012 and January 2013. By the Nested-PCR reaction, HEV were detected in 9.8% of the samples, HAdV in 2.5% and HHV-1 and 2 in 1.1%. There were 3 cases of coinfection: 2 with HEV and HHV and other with HEV and HAdV. The viral genetic materials were extracted by QIAamp DNA Blood kit (Qiagen®) and MagMAXTM Viral RNA Isolation (Ambiom), and the second one showed to be more suitable for the application in clinical diagnosis. The CSF chemocytologic analysis proved to be important in directing the clinical conduct, but the detection of viruses is essential for the diagnosis. The real time PCR, which standardization was initiated in this work, will be an important tool for complementary diagnosis of viral infections of the CNS.
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Utilização de shRNA anti-hexon, anti-IVa2 e anti-pol durante a produção de vírus adeno-associado como estratégia de eliminar Adenovírus helper: prova de princípio / Use of shRNAs directed against key adenoviral targets as an inhibitor of Helper Viruses: first stepLana, Marlous Vinicius Gomes 26 January 2016 (has links)
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
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Estabelecimento de métodos moleculares para aplicação no diagnóstico rápido de virus neurotrópicos. / The integration of molecular methods into the rapid laboratorial diagnostic of neurotropic viruses.Santos, Daniela Carvalho dos 04 September 2009 (has links)
Diversos agentes virais são causadores de meningites e meningoencefalites. Neste estudo, técnicas moleculares foram utilizadas para detecção de HEV, HHV e HAdV em amostras de líquor colhidas de janeiro de 2005 a março de 2007. Dos três métodos de extração de DNA e RNA testados, o kit DNA Qiablood Qiagen® se mostrou o mais sensível e específico. A nested PCR detectou HEV em 28% das amostras, HSV em 4%, HHV-3 em 1%; HHV-4 em 0,3%, HHV-5 em 0,3%, HHV-6 em 0,7% e HAdV em 13%. Através da PCR em tempo real os HEV foram detectados em 23,3% e HSV em 5,1%. Por neutralização, somente duas amostras foram sorotipadas (Echovirus 6 e Coxsackievirus B). Os HEV detectados foram então seqüenciados para a determinação do sorotipo. Os sorotipos Echovirus 18 (53%) e Coxsackievirus B5 (26%) foram os mais freqüentes. As técnicas de biologia molecular aplicadas na detecção de HEV, HHV e HAdV no líquor trazem grandes vantagens ao diagnóstico de doenças do SNC graças à rapidez no diagnóstico, alta sensibilidade e especificidade. / Several viruses are etiological agents of meningitis and meningoencephalitis. In this study, molecular techniques were used for the detection of HEV, HHV and HAdV in liquor samples, collected from January 2005 to March 2007. Among the three methods tested for the extraction of DNA and RNA, the DNA Qiablood kit - Qiagen® was the most sensible and specific. HEV (28%), HSV (4%), HHV-3 (1%), HHV-4 (0.3%), HHV-5 (0.3%), HHV-6 (0.7%) and HAdV (13%) were detected by Nested PCR in the samples. By real time PCR, HEV were detected in 23.3% and HSV in 5.1%. Only two HEV could be serotyped by neutralization (Echovirus 6 and Coxsackievirus B). All detected HEV were then sequenced to determine the serotype. The serotypes echovirus 18 (53%) and Coxsackievirus B5 (26%) were the most frequent. Molecular biology techniques applied in the detection of HEV, HAdV and HHV in CSF bring major benefits to the diagnosis of meningitis thanks to the rapid diagnosis, high sensibility and specificity.
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Aplicação de técnicas moleculares no diagnóstico laboratorial complementar das infecções virais do sistema nervoso central no Hospital Universitário da USP. / Molecular techniques application for the complementary laboratory diagnosis of viral infections of the central nervous system, at the University Hospital of USP.Rafaella Almeida Lima Nunes 22 August 2013 (has links)
Enterovírus (HEV), herpesvírus 1 e 2 (HHV-1 e HHV-2) e adenovírus (HAdV) são importantes agentes de infecções do SNC. Neste trabalho, técnicas moleculares foram aplicadas para a detecção destes vírus em quadros de infecção do SNC. Amostras de líquor foram colhidas de pacientes atendidos no HU-USP entre agosto e novembro/2010 e fevereiro/2012 a janeiro/2013. Através da Nested-PCR HEV foram detectados em 9,8% das amostras, HAdV em 2,5% e HHV-1 e 2 em 1,1%, além de 3 casos de coinfecção, 2 entre HEV e HHV, e 1 entre HEV e HAdV. O material genético viral foi extraído através dos métodos Qiaamp DNA Blood (Qiagen®) e MagMAXTM Viral RNA Isolation (Ambiom), e este último pareceu mais adequado à aplicação na rotina clínica. A análise quimiocitológica do líquor mostrou-se importante no direcionamento da conduta clínica, mas a detecção do vírus é fundamental para a conclusão do diagnóstico. A PCR em tempo real, cuja padronização foi iniciada neste trabalho, consiste em importante ferramenta para a utilização futura no diagnóstico complementar das infecções virais do SNC. / Enteroviruses (HEV), herpesviruses 1 and 2 (HHV-1 and HHV-2) and adenoviruses (HAdV) are important causative agents of infections of the CNS. In this study, molecular techniques were applied to the detection of these viruses. CSF samples were collected from patients treated at the University Hospital of USP, between August and November, 2010, and February 2012 and January 2013. By the Nested-PCR reaction, HEV were detected in 9.8% of the samples, HAdV in 2.5% and HHV-1 and 2 in 1.1%. There were 3 cases of coinfection: 2 with HEV and HHV and other with HEV and HAdV. The viral genetic materials were extracted by QIAamp DNA Blood kit (Qiagen®) and MagMAXTM Viral RNA Isolation (Ambiom), and the second one showed to be more suitable for the application in clinical diagnosis. The CSF chemocytologic analysis proved to be important in directing the clinical conduct, but the detection of viruses is essential for the diagnosis. The real time PCR, which standardization was initiated in this work, will be an important tool for complementary diagnosis of viral infections of the CNS.
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An investigation of Coxsackie and Adenovirus receptor in the human pancreatic beta cellsIfie, Eseoghene January 2018 (has links)
Human pancreatic beta cells are susceptible to infection by enteroviruses, especially Coxsackie B viruses, and such infections could contribute to the development of Type 1 diabetes. Enteroviruses gain entry via cell surface receptors, one of which, the Coxsackie and Adenovirus receptor (CAR), is a transmembrane cell adhesion protein which serves as a key entry receptor for Coxsackie B viruses and is thought to be localised mainly within regions where contacts are formed between adjacent cells. CAR exists as at least 5 isoforms and this study has examined their expression profile and distribution in the human pancreas utilising; formalin-fixed paraffin-embedded pancreatic sections from non-diabetic individuals, type 1 diabetes patients and a human tissue microarray. Isolated human islets, human pancreatic beta and ductal cell lines were also studied. Immunological and molecular approaches were employed to examine the expression and cellular localisation of the known CAR isoforms in human pancreas. One specific isoform of CAR (CAR-SIV) with a unique C terminal PDZ binding domain, was highly expressed in human beta cells at the protein level. Surprisingly, it was distributed in a punctate manner mainly within the cytoplasm of the cells, rather than at the cell surface. In human beta cells, within the cytoplasm CAR-SIV co-localised with ZnT8, PC1/3 and insulin but less so with proinsulin suggesting that CAR-SIV is associated with insulin secretory granules. Immunogold labelling and electron microscopic analysis revealed that CAR-SIV is localised both to maturing insulin secretory granules and to fully mature, dense-core (insulin) secretory granules. Intriguingly, CAR-SIV colocalises and interacts with a cytosolic protein, PICK1, which plays a role in the budding, maturation and trafficking of insulin secretory granules. On this basis, a model is proposed whereby CAR-SIV and PICK1 interact to regulate the maturation and trafficking of insulin secretory granules. Overall, this study suggests that the specialised role and subcellular localisation of CAR-SIV in human beta cells may contribute to their sensitivity to enteroviral infection following externalisation of the protein at the cell surface, during insulin exocytosis.
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The Modular Domain Structure of ASF/SF2: Significance for its Function as a Regulator of RNA SplicingDauksaite, Vita January 2003 (has links)
<p>ASF/SF2 is an essential splicing factor, required for constitutive splicing, and functioning as a regulator of alternative splicing. ASF/SF2 is modular in structure and contains two amino-terminal RNA binding domains (RBD1 and RBD2), and a carboxy-terminal RS domain. The results from my studies show that the different activities of ASF/SF2 as a regulator of alternative 5’ and 3’ splice site selection can be attributed to distinct domains of ASF/SF2.</p><p>I show that ASF/SF2-RBD2 is both necessary and sufficient to reproduce the splicing repressor function of ASF/SF2. A SWQDLKD motif was shown to be essential for the splicing repressor activity of ASF/SF2. In conclusion, this study demonstrated that ASF/SF2 encodes for distinct domains responsible for its function as a splicing enhancer (the RS domain) or a splicing repressor (the RBD2) protein. Using a model transcript containing two competing 3’ splice sites it was further demonstrated that the activity of ASF/SF2 as a regulator of alternative 3’ splice site selection was directional: i.e. resulting in RS or RBD1 mediated activation of upstream 3’ splice site selection while simultaneously causing an RBD2 mediated repression of downstream 3’ splice site usage.</p><p>In alternative 5’ splice site selection, the RBD2 alone was sufficient to reproduce the activity of the full-length protein as an inducer of proximal 5’ splice site usage, while RBD1 had the opposite effect and induced distal 5’ splice site selection. The conserved SWQDLKD motif and the RNP-1 type RNA recognition motif in ASF/SF2-RBD2 were both essential for this induction. The activity of the ASF/SF2-RBD2 domain as a regulator of alternative 5’ splice site was shown to correlate with the RNA binding capacity of the domain.</p><p>Collectively, my results suggest that the RBD2 domain in ASF/SF2 plays the most decisive role in the alternative 5’ and 3’ splice site regulatory activities of ASF/SF2.</p>
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The Modular Domain Structure of ASF/SF2: Significance for its Function as a Regulator of RNA SplicingDauksaite, Vita January 2003 (has links)
ASF/SF2 is an essential splicing factor, required for constitutive splicing, and functioning as a regulator of alternative splicing. ASF/SF2 is modular in structure and contains two amino-terminal RNA binding domains (RBD1 and RBD2), and a carboxy-terminal RS domain. The results from my studies show that the different activities of ASF/SF2 as a regulator of alternative 5’ and 3’ splice site selection can be attributed to distinct domains of ASF/SF2. I show that ASF/SF2-RBD2 is both necessary and sufficient to reproduce the splicing repressor function of ASF/SF2. A SWQDLKD motif was shown to be essential for the splicing repressor activity of ASF/SF2. In conclusion, this study demonstrated that ASF/SF2 encodes for distinct domains responsible for its function as a splicing enhancer (the RS domain) or a splicing repressor (the RBD2) protein. Using a model transcript containing two competing 3’ splice sites it was further demonstrated that the activity of ASF/SF2 as a regulator of alternative 3’ splice site selection was directional: i.e. resulting in RS or RBD1 mediated activation of upstream 3’ splice site selection while simultaneously causing an RBD2 mediated repression of downstream 3’ splice site usage. In alternative 5’ splice site selection, the RBD2 alone was sufficient to reproduce the activity of the full-length protein as an inducer of proximal 5’ splice site usage, while RBD1 had the opposite effect and induced distal 5’ splice site selection. The conserved SWQDLKD motif and the RNP-1 type RNA recognition motif in ASF/SF2-RBD2 were both essential for this induction. The activity of the ASF/SF2-RBD2 domain as a regulator of alternative 5’ splice site was shown to correlate with the RNA binding capacity of the domain. Collectively, my results suggest that the RBD2 domain in ASF/SF2 plays the most decisive role in the alternative 5’ and 3’ splice site regulatory activities of ASF/SF2.
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Estrategias para la diferenciación in vitro de células ES de ratón a células acinares pancreáticasRovira Clusellas, Meritxell 31 January 2007 (has links)
Las patologías más importantes del páncreas exocrino, como la pancreatitis crónica (PC) o el cáncer de páncreas, representan un gran problema de salud pública en Europa. En la PC, el tejido acinar es substituido por complejos ductales. Además, es difícil mantener el fenotipo diferenciado de las células acinares en cultivo ya que sufren una transdiferenciación acinar-ductal.Las células madre embrionarias (ES) de ratón han sido utilizadas en la última década para generar in vitro células completamente diferenciadas de varios linajes celulares. No obstante, la capacidad de las células ES a diferenciarse a tipos celulares de origen endodérmico es muy limitada. El objetivo principal de este proyecto ha consistido en desarrollar estrategias para diferenciar células ES de ratón a células pancreáticas acinares con una elevada eficiencia mediante 1) la optimización de las condiciones de cultivo con tal de activar vías de señalización implicadas en el desarrollo/diferenciación pancreáticas; 2) la sobreexpresión de factores transcripcionales maestros utilizando vectores virales con el fin de recapitular específicamente un programa de diferenciación acinar; 3) la selección genética de las células comprometidas al linaje acinar con el objetivo de purificar las células acinares diferenciadas.Mediante la integración de estos abordajes, hemos conseguido aislar células que comparten características fenotípicas con células acinares inmaduras según la expresión de marcadores de diferenciación y la respuesta funcional a secretagogos. / Exocrine pancreatic diseases such as chronic pancreatitis (PC) or pancreatic cancer are major health issues in Europe. In CP, the acinar tissue is substituted by ductal complexes. In addition, it is difficult to maintain the differentiated phenotype of the acinar cells in culture as within few days an acinar-ductal transdifferentiation takes place.In the last decade, mouse embryonic stem cells (mES) have been used to generate differentiated cells of a variety of cellular lineages in vitro. However, the ability of ES cells to differentiate into endodermal lineages is limited. The main objective of this project has focused on the development of strategies to differentiate mES to pancreatic acinar cells with high efficiency by means of: 1) Optimization of cell culture conditions to activate signalling pathways involved in pancreatic differentiation/development; 2) the overexpression of master transcription factors involved in pancreas development using viral vectors in order to recapitulate specific acinar differentiation program; 3) the genetic selection of cells committed to the acinar linage in order to purify the differentiated cells.The integration of these different strategies allowed us to isolate cells that share phenotypic features with immature acinar cells according to the expression of differentiation markers and the functional response to acinar secretegogues.
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