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Development of a Protein-Based Therapy for the Treatment of Spinal Muscular AtrophyBurns, Joseph 12 March 2014 (has links)
The autosomal recessive disorder spinal muscular atrophy (SMA) causes motor neuron degeneration and muscle wasting, progressing to paralysis and death in severe cases. The disease is caused by deficiency of survival motor neuron protein (SMN) due to deletion or mutation of the SMN1 gene. We seek to develop a protein-based therapy for SMA using an adenoviral vector which encodes a secretable form of SMN fused to a protein transduction domain (PTD) derived from the trans-acting activator of transcription (TAT) from HIV. We generated secretable GFP proteins using transient transfection in mammalian cells and determined that the secretory peptide was inefficient when paired with the native PTD. We generated TAT-GFP proteins in bacteria and observed that the variant TAT3 most reliably tranduced cells in vitro. We did not observe uptake of the therapeutic protein following infection with an adenoviral vector and subsequent secretion of the protein from infected cells.
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Rapid development of optimized recombinant adenoviral vaccines for biosafety level 4 virusesSahib, Mickey M. 10 September 2010 (has links)
This thesis describes the production of adenovirus-based vaccines containing codon-optimized genes from Nipah virus and Crimean-Congo Hemorrhagic Fever virus. Genes encoding envelope proteins from Crimean-Congo Hemorrhagic Fever Virus and Nipah Virus were codon-optimized for translation in human cells and constructed using a modified method of non-gapped gene synthesis, while the entire M segment encoding the glycoprotein precursor for Crimean-Congo Hemorrhagic Fever Virus was commercially synthesized. Genes were cloned into recombinant human adenovirus serotype 5 and the resulting viral particles were amplified, titred and analyzed for in vivo efficacy. Results show that a modified method of non-gapped gene synthesis is an effective and efficient method of producing antigen-encoded DNA and at a fraction of the cost and time required for commercial synthesis. Furthermore, adenovirus-based vaccines induce both cellular and humoral immune responses providing for a highly efficacious vaccine during potential disease outbreaks, where time to completion is of utmost importance. This study has shown that recombinant adenoviral vaccines for Crimean-Congo Hemorrhagic Fever virus and Nipah virus can be produced rapidly and efficiently from virtual DNA sequence to optimized recombinant vaccines in just eight months.
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Functional studies on the coxsackie and adenovirus receptor (CAR) in skeletal muscle cellsTai, Yunlin, 1962- January 2000 (has links)
CAR (for C&barbelow;oxsackievirus and A&barbelow;denovirus R&barbelow;eceptor) is a novel member of the Ig superfamily, which has recently been identified as a high affinity receptor for both Coxsackievirus and certain adenovirus (AV) serotypes. Virus bound by CAR is believed to be passed to integrins which bind an RGD (Arg-Gly-Asp) sequence in the viral penton base protein and act as secondary receptors responsible for virus internalization. / Recent studies have shown that, in integrin-expressing cells, CAR-mediated AV uptake does not require the cytoplasmic (CP) domain of CAR, presumably because virus bound to the CAR extracellular (EC) domain can be passed to integrins for subsequent internalization. It has however also been reported that CAR can directly mediate AV uptake in the absence of penton base RGD-alphav integrin interactions. I therefore attempted to determine whether the CP domain of CAR is required for CAR-mediated AV uptake in cells which do not express integrins, or in which integrin function has been blocked by RGD-containing peptide. / As CAR is the primary AV receptor and integrins are secondary AV receptors I investigated the possibility that these proteins associate in a functional complex in the cell membrane. (Abstract shortened by UMI.)
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Ganglioside Increases Metastatic Potential and Susceptibility of Prostate Cancer to Gene Therapy in vitroMiklavcic, John 11 1900 (has links)
Prostate cancer (CaP) is the 2nd most common cancer in North American men. Tumour management strategies are appropriate for early stage disease, but advanced disease has a poor prognosis and requires prompt treatment. Therefore, research into delay of tumour progression and efficacious treatment of aggressive cancer are of interest. Ganglioside was assessed for its role in altering markers of metastatic potential and susceptibility of CaP to adenovirus-mediated gene therapy. Healthy (RWPE-1) and malignant (DU-145, PC-3) prostate cells were cultured with or without mixed ganglioside. Differences in growth, ganglioside and integrin densities, and adenoviral infectivity were assessed between treatment and control groups. Ganglioside decreased (p<0.01) growth of PC-3 cells relative to untreated control. Ganglioside decreased (p<0.01) GD1a and increased (p<0.04) integrin densities in malignant prostate cells, suggesting ganglioside may increase metastatic potential of CaP. Ganglioside significantly increased adenovirus entry in PC-3 cells, thereby improving susceptibility of CaP to adenovirus-mediated gene therapy. / Nutrition and Metabolism
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The interaction of the adenovirus E1B-55K protein with a histone deacetylase complex : its importance in regulation of P53 protein functions /Punga, Tanel, January 2003 (has links)
Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 3 uppsatser.
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Effect of pre-existing adenovirus neutralizing antibody on vector infectivity and transgene expressionDahl, Noelle Parisi. January 2010 (has links)
Thesis (M.S.)--Villanova University, 2010. / Biology Dept. Includes bibliographical references.
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In-vitro- und In-vivo-Untersuchungen funktioneller Bereiche des Adenovirus-Typ-5-E1B-55kDa-ProteinsZeller, Thomas. January 1900 (has links) (PDF)
Regensburg, Univ., Diss., 2004. / Erscheinungsjahr an der Haupttitelstelle: 2003
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Identifizierung regulatorischer Bereiche im E1B-55kDa-Protein von Adenovirus Serotyp 5 und ihre Rolle bei der TransformationEndter, Christian. January 2002 (has links) (PDF)
Regensburg, Univ., Diss., 2002.
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Triagem da potencial atividade antiviral de produtos marinhosSilva, Alexandre Cordeiro da January 2005 (has links)
Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pós-Graduação em Farmácia. / Made available in DSpace on 2013-07-16T01:22:19Z (GMT). No. of bitstreams: 1
214187.pdf: 538408 bytes, checksum: bb6dfd55649ed893ed0487c38357ff01 (MD5)
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Avaliação da citotoxicidade, da genotoxicidade e da potencial atividade antiviral da violaceína, produzida pela Chromobacterium violaceumFröhner, Carla Regina Andrighetti January 2003 (has links)
Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências da Saúde. Programa de Pós-Graduação em Farmácia. / Made available in DSpace on 2012-10-21T03:20:29Z (GMT). No. of bitstreams: 1
191935.pdf: 429500 bytes, checksum: 0d6ca425ea42609de18160f27941049e (MD5) / Os produtos naturais constituem uma fonte inesgotável de compostos com atividades farmacológicas promissoras, incluindo ação antiviral. A Chromobacterium violaceum é uma bactéria Gram (-) encontrada em amostras de solo e água de regiões tropicais e subtropicais de diversos continentes. No Brasil, esta bactéria é amplamente distribuída no Rio Negro e no solo de bancos dos rios da região da Amazônia. A violaceína, o pigmento majoritário produzido por esta bactéria, apresenta várias atividades biológicas, tais como antibiótica, antitumoral, tripanossomicida e antifúngica. Este trabalho teve o objetivo de avaliar a citotoxicidade, a genotoxicidade e a potencial atividade antiviral da violaceína. A citotoxicidade da violaceína, frente às células VERO, MA104, HEp-2 e FRhK-4, foi avaliada por três metodologias e as concentrações citotóxicas a 50% (CC50) obtidas foram diferentes de acordo com o método empregado: (I) avaliação microscópica das alterações morfológicas celulares: 2,29 ± 0,23 mM (VERO); 2,69 ±0,12 mM (MA104); 2,42 ± 0,22 mM (FRhK-4) e 2,78 ± 0,13 mM (HEp-2); (II) viabilidade celular - método de exclusão do corante azul de Trypan: 2,23 ± 0,06 mM (VERO); 2,54 ± 0,10 mM (MA104); 2,07 ± 0,05 mM (FRhK-4) e 2,70 ± 0,12 mM (HEp-2). e (III) viabilidade celular - ensaio colorimétrico do MTT: 2,96 ± 0,12 mM (VERO); 3,55± 0,22 mM (MA104); 3,14 ± 0,32 mM (FRhK-4) e 3,42 ± 0,05 mM (HEp-2). A violaceína não causou danos significativos ao DNA das células HEp-2 e MA104, nas concentrações testadas (0,19 - 1,5 mM), através do Ensaio do Cometa, quando comparados ao controle negativo (teste t-Student p>0,05); entretanto, apresentou ação genotóxica para as células VERO e FRhK-4, na concentração de 1,5 mM, quando comparada com o controle negativo (teste t-Student p<0,05). As demais concentrações (0,75; 0,37 e 0,19 mM) não causaram danos ao DNA celular e são estatisticamente semelhantes entre si (teste SNK p>0,05) e ao controle negativo (teste t-Student p>0,05). A potencial atividade antiviral da violaceína foi avaliada pelo método de inibição do efeito citopático viral e pelo ensaio do MTT. A violaceína não inibiu o efeito citopático do herpes vírus simplex humano tipo 1 (HSV-1): cepas 29-R, KOS e ATCC/VR-733, do poliovírus humano tipo 2 (PV-2), do rotavírus símio SA11 (RV), do vírus da hepatite A (HAV) cepas: HM-175 e HAF-203, e do adenovírus tipo 5 (AdV-5). As porcentagens de inibição da replicação dos diferentes vírus pela violaceína, obtidas através do ensaio do MTT, foram: 1,42 ± 0,68%; 14,48 ± 5,06% e 21,47 ± 3,74% para o HSV-1 cepa KOS; 5,96 ± 2,51%; 8,75 ± 3,08% e 17,75 ± 5,19%, para o HSV-1 cepa ATCC/VR-733; 5,13 ± 2,38 %; 8,18 ± 1,11% e 8,51 ± 1,94% para o PV-2 e 8,30 ± 4,24%; 13,33 ± 4,66% e 24,27 ± 2,18% para o RV, nas concentrações de 0,312; 0,625 e 1,250 mM, respectivamente. A violaceína não demonstrou ação inibitória da replicação do HSV-1 cepa 29-R, do HAV cepas HM-175 e HAF-203 e do AdV-5, através do ensaio do MTT.
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