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Estudo comparativo de três diferentes procedimentos para extração de RNA a partir de amostras fixadas em formalina e embebidas em parafina; Comparative study of three different procedures for RNA extraction from formalin-fixed paraffin-embedded samples

RIBEIRO-SILVA, Alfredo; GARCIA, Sérgio Britto
Fonte: Sociedade Brasileira de Patologia ClínicaSociedade Brasileira de PatologiaSociedade Brasileira de Citopatologia Publicador: Sociedade Brasileira de Patologia ClínicaSociedade Brasileira de PatologiaSociedade Brasileira de Citopatologia
Tipo: Artigo de Revista Científica
Português
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INTRODUÇÃO: O desenvolvimento de métodos de extração de RNA a partir de amostras fixadas em formalina e embebidas em parafina (FFEP) possibilitou estudos retrospectivos de biologia molecular. Objetivos: Comparar a quantidade e a qualidade do RNA extraído de amostras FFEP a partir de três kits disponíveis comercialmente. MATERIAL E MÉTODO: Utilizando-se três diferentes procedimentos, o RNA total foi extraído de 14 blocos de parafina contendo fragmentos de carcinomas mamários, todos arquivados há 10 anos. A quantidade do RNA foi expressa em pg/µl; e a qualidade, pelo número de integridade do RNA (NIR), utilizando-se o Bioanalyzer da Agilent com o Pico LabChip. O RNA de maior NIR extraído de cada uma das 14 amostras foi amplificado por reação em cadeia da polimerase com transcrição reversa (RT-PCR) utilizando-se o gene G6PD, com primers designados para gerar fragmentos de 67, 151 e 242 pares de bases (pb). RESULTADOS: A média e a mediana da quantidade do RNA extraído para os três protocolos foram, respectivamente, 42,91 e 31,31 pg/µl. A média e a mediana do NIR foram, respectivamente, 1,8 e 2. Em todas as amostras, o gene G6PD foi amplificado para fragmentos de RNA de 67 e 151 pb. DISCUSSÃO: Como houve grande variação individual na quantidade e na qualidade do RNA extraído para cada amostra...

Problemas da biossíntese de RNA em eucariotos. O modelo glândulas salivares de Rhynchosciara angelae; Biosynthesis of RNA in eukaryotes. The model salivary glands of Rhynchosciara angelae

Armelin, Hugo Aguirre
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 11/12/1969 Português
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a. Do ponto de vista metodológico dois problemas foram enfrentados neste trabalho: i) extração dos RNA's e ii) fracionamento das células das glândulas salivares de R. angelae. i) O processo prático elaborado para resolver o primeiro problema se constituiu numa variação da técnica clássica de extração com fenol. Jogando com a presença de detergente e variações de pH e de temperatura, foi possível se chegar a um método que garante a extração e um fracionamento parcial de, praticamente, todo RNA celular. Com extrações a baixa temperatura obtém-se preferencialmente rRNA e tRNA. O RNA refratário a extração com fenol a frio tem propriedades do RNA nuclear, mas o citoplasma, também parece conter classes de RNA somente extraíveis a quente. A dificuldade de extração do RNA nuclear com fenol frio, foi especulativamente interpretada como uma propriedade das RNP's que encerram esta classe de RNA. A grande vantagem deste método de extração está no fato de ter permitido isolar, com as extrações a alta temperatura, frações de RNA enriquecidas em produtos de transcrição específicos de determinados períodos do desenvolvimento larval. ii) O fracionamento celular das glândulas salivares não é alcançado pela aplicação das técnicas tradicionais de fracionamento de tecido. Em vista disso foi necessário procurar uma alternativa experimental para êste caso. Combinando um enzima proteolítico com a ação de detergentes não iônicos foi conseguido um método útil para o fracionamento das células das glândulas salivares. Êste método foi aplicado nesta tese para um estudo inicial das RNP's e da transferência do rRNA do núcleo para o citoplasma nas células das glândulas salivares. b. Foi possível verificar com êste trabalho que o rRNA é sintetizado inicialmente na forma de um precursor primário de 37s...

Conservação do RNA leucocitário para detecção da expressão do gene MDR1 em equinos da Raça Crioulo; Preservation of leucocyte RNA for detection of MDR1 gene expression in crioulo horses

Lamberts, Marianne
Fonte: Universidade Federal do Rio Grande do Sul Publicador: Universidade Federal do Rio Grande do Sul
Tipo: Dissertação Formato: application/pdf
Português
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O estudo da expressão gênica é de grande aplicabilidade nas áreas de pesquisa científica e clínica. Por ser um método pouco invasivo e permitir coletas seriadas, a utilização de amostras sanguíneas facilita a análise da transcrição gênica. O maior desafio para a precisão nos ensaios moleculares é manter uma amostra com qualidade desde a coleta, armazenamento e transporte até o momento de sua análise. Este estudo utilizou um sistema de conservação de RNA sanguíneo que manteve amostras de qualidade após a coleta, transporte e armazenamento, permitindo extração de RNA e identificação da expressão do gene MDR1 em cavalos da raça Crioulo. Foram coletadas amostras sanguíneas de 27 cavalos a campo em tubos PAXgene® Blood RNA, que após o transporte e armazenamento a -20°C por 90 dias foram processadas em laboratório. A extração do RNA sanguíneo foi realizada com o kit Nucleo Spin® RNA II, a conversão em cDNA foi com o kit High-Capacity cDNA Reverse Transcription, utilizando a fluorimetria para as avaliações. A identificação da expressão do gene MDR1 em sangue conservado utilizou primer específico para cDNA e PCR em tempo real. Houve extração de RNA em todas as amostras coletadas, sendo que as leituras das concentrações de RNA das amostras com DNA contaminante não tiveram diferença estatística das amostras sem DNA contaminante. Ocorreu amplificação do gene MDR1 a partir do RNA das amostras...

Biorecognition by amino acid-based affinity chromatography for RNA purification

Martins, Ana Rita Nunes
Fonte: Universidade da Beira Interior Publicador: Universidade da Beira Interior
Tipo: Tese de Doutorado
Publicado em /10/2013 Português
Relevância na Pesquisa
36.31%
Following the decoding of the human genome, a new era was opened for developing new gene therapy strategies employing nucleic acids. Recently, RNA was renowned a central molecule in cellular processes with implications in many diseases as well as in understanding of evolution, becoming one of the most exciting research areas of molecular biology. From basic to applied research, many procedures employ pure and intact RNA molecules. On one hand, RNA purification is a first critical step of a number of molecular biology procedures and its quality is crucial to ensure reproducibility and biological relevance of an experiment. On the other hand, the promising and revolutionary RNA-based therapies of RNA vaccination, gene therapy or recombinant biopharmaceuticals involves RNA formulations which should fulfill rigorous quality criteria recommended by international regulatory agencies. However, the isolation and purification of RNA are critical steps because of the easy degradability of RNA, which can impair chemical stability and biological functionality essential for analysis. Many techniques have been development to overcome the challenges of purifying RNA molecules; nonetheless they still have several limitations in regard to time demanding and the requirement of toxic solvents and denaturing conditions. Therefore...

The bunyavirus nucleocapsid protein is an RNA chaperone: Possible roles in viral RNA panhandle formation and genome replication

MIR, M. AYOUB; PANGANIBAN, ANTONITO T.
Fonte: Copyright 2006 by RNA Society Publicador: Copyright 2006 by RNA Society
Tipo: Artigo de Revista Científica
Publicado em /02/2006 Português
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Cellular RNA chaperones are crucial for the genesis of correctly folded functional RNAs. Using several complementary in vitro assays we find that the bunyavirus nucleocapsid protein (N) is an RNA chaperone. In the Bunyaviridae genomic RNA is in stable “panhandle” formation that arises through the hydrogen bonding of the terminal nucleotides of the RNA. The RNA chaperone function of N facilitates panhandle formation even though the termini are separated by >2 kb. RNA panhandle formation is likely driven by the exceptionally high base-pairing specificity of the terminal nucleotides as evidenced by P-num analysis. N protein can nonspecifically dissociate RNA duplexes. In addition, following panhandle formation, the RNA chaperone activity of N also appears to be involved in dissociation of the RNA panhandle and remains in association with the 5′ terminus of the viral RNA following dissociation. Thus, N likely functions in the initiation of genome replication to allow efficient initiation of RNA synthesis by the viral polymerase. The RNA chaperone activity of N may be facilitated by an intrinsically disordered domain that catalyzes RNA unfolding driven by reciprocal entropy transfer. These observations highlight the essential features that are probably common to all RNA chaperones in which the role of the chaperone is to nonspecifically dissociate higher order structure and formation of functional higher order structure may often be predicted by RNA P-num value. The data also highlight features of N that are probably specifically important during replication of bunyavirus RNA.

Identification of a structural element of the hepatitis C virus minus strand RNA involved in the initiation of RNA synthesis

Mahias, Kathleen; Ahmed-El-Sayed, Neveen; Masante, Cyril; Bitard, Juliette; Staedel, Cathy; Darfeuille, Fabien; Ventura, Michel; Astier-Gin, Thérèse
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
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The replication of the genomic RNA of the hepatitis C virus (HCV) of positive polarity involves the synthesis of a replication intermediate of negative polarity by the viral RNA-dependent RNA polymerase (NS5B). In vitro and likely in vivo, the NS5B initiates RNA synthesis without primers. This de novo mechanism needs specific interactions between the polymerase and viral RNA elements. Cis-acting elements involved in the initiation of (–) RNA synthesis have been identified in the 3′ non-coding region and in the NS5B coding region of the HCV RNA. However, the detailed contribution of sequences and/or structures of (–) RNA involved in the initiation of (+) RNA synthesis has been less studied. In this report, we identified an RNA element localized between nucleotides 177 and 222 from the 3′-end of the (–) RNA that is necessary for efficient initiation of RNA synthesis by the recombinant NS5B. By site-directed mutagenesis experiments, we demonstrate that the structure rather than the primary sequence of this domain is important for RNA synthesis. We also demonstrate that the intact structure of this RNA element is also needed for efficient RNA synthesis when the viral NS5B functions in association with other viral and cellular proteins in cultured hepatic cells.

RNA Sequences and Structures Required for the Recruitment and Activity of the Dengue Virus Polymerase*

Filomatori, Claudia V.; Iglesias, Nestor G.; Villordo, Sergio M.; Alvarez, Diego E.; Gamarnik, Andrea V.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Português
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Dengue virus RNA-dependent RNA polymerase specifically binds to the viral genome by interacting with a promoter element known as stem-loop A (SLA). Although a great deal has been learned in recent years about the function of this promoter in dengue virus-infected cells, the molecular details that explain how the SLA interacts with the polymerase to promote viral RNA synthesis remain poorly understood. Using RNA binding and polymerase activity assays, we defined two elements of the SLA that are involved in polymerase interaction and RNA synthesis. Mutations at the top of the SLA resulted in RNAs that retained the ability to bind the polymerase but impaired promoter-dependent RNA synthesis. These results indicate that protein binding to the SLA is not sufficient to induce polymerase activity and that specific nucleotides of the SLA are necessary to render an active polymerase-promoter complex for RNA synthesis. We also report that protein binding to the viral RNA induces conformational changes downstream of the promoter element. Furthermore, we found that structured RNA elements at the 3′ end of the template repress dengue virus polymerase activity in the context of a fully active SLA promoter. Using assays to evaluate initiation of RNA synthesis at the viral 3′-UTR...

β-Catenin recognizes a specific RNA motif in the cyclooxygenase-2 mRNA 3′-UTR and interacts with HuR in colon cancer cells

Kim, Inae; Kwak, Hoyun; Lee, Hee Kyu; Hyun, Soonsil; Jeong, Sunjoo
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
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36.27%
RNA-binding proteins regulate multiple steps of RNA metabolism through both dynamic and combined binding. In addition to its crucial roles in cell adhesion and Wnt-activated transcription in cancer cells, β-catenin regulates RNA alternative splicing and stability possibly by binding to target RNA in cells. An RNA aptamer was selected for specific binding to β-catenin to address RNA recognition by β-catenin more specifically. Here, we characterized the structural properties of the RNA aptamer as a model and identified a β-catenin RNA motif. Similar RNA motif was found in cellular RNA, Cyclooxygenase-2 (COX-2) mRNA 3′-untranslated region (3′-UTR). More significantly, the C-terminal domain of β-catenin interacted with HuR and the Armadillo repeat domain associated with RNA to form the RNA–β-catenin–HuR complex in vitro and in cells. Furthermore, the tertiary RNA–protein complex was predominantly found in the cytoplasm of colon cancer cells; thus, it might be related to COX-2 protein level and cancer progression. Taken together, the β-catenin RNA aptamer was valuable for deducing the cellular RNA aptamer and identifying novel and oncogenic RNA–protein networks in colon cancer cells.

Recognition of two distinct elements in the RNA substrate by the RNA-binding domain of the T. thermophilus DEAD box helicase Hera

Steimer, Lenz; Wurm, Jan Philip; Linden, Martin H.; Rudolph, Markus G.; Wöhnert, Jens; Klostermeier, Dagmar
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
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DEAD box helicases catalyze the ATP-dependent destabilization of RNA duplexes. Whereas duplex separation is mediated by the helicase core shared by all members of the family, flanking domains often contribute to binding of the RNA substrate. The Thermus thermophilus DEAD-box helicase Hera (for “heat-resistant RNA-binding ATPase”) contains a C-terminal RNA-binding domain (RBD). We have analyzed RNA binding to the Hera RBD by a combination of mutational analyses, nuclear magnetic resonance and X-ray crystallography, and identify residues on helix α1 and the C-terminus as the main determinants for high-affinity RNA binding. A crystal structure of the RBD in complex with a single-stranded RNA resolves the RNA–protein interactions in the RBD core region around helix α1. Differences in RNA binding to the Hera RBD and to the structurally similar RBD of the Bacillus subtilis DEAD box helicase YxiN illustrate the versatility of RNA recognition motifs as RNA-binding platforms. Comparison of chemical shift perturbation patterns elicited by different RNAs, and the effect of sequence changes in the RNA on binding and unwinding show that the RBD binds a single-stranded RNA region at the core and simultaneously contacts double-stranded RNA through its C-terminal tail. The helicase core then unwinds an adjacent RNA duplex. Overall...

Initiating nucleotide identity determines efficiency of RNA synthesis from 6S RNA templates in Bacillus subtilis but not Escherichia coli

Cabrera-Ostertag, Ignacio J.; Cavanagh, Amy T.; Wassarman, Karen M.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Português
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36.27%
The 6S RNA is a non-coding small RNA that binds within the active site of housekeeping forms of RNA polymerases (e.g. Eσ70 in Escherichia coli, EσA in Bacillus subtilis) and regulates transcription. Efficient release of RNA polymerase from 6S RNA regulation during outgrowth from stationary phase is dependent on use of 6S RNA as a template to generate a product RNA (pRNA). Interestingly, B. subtilis has two 6S RNAs, 6S-1 and 6S-2, but only 6S-1 RNA appears to be used efficiently as a template for pRNA synthesis during outgrowth. Here, we demonstrate that the identity of the initiating nucleotide is particularly important for the B. subtilis RNA polymerase to use RNA templates. Specifically, initiation with guanosine triphosphate (GTP) is required for efficient pRNA synthesis, providing mechanistic insight into why 6S-2 RNA does not support robust pRNA synthesis as it initiates with adenosine triphosphate (ATP). Intriguingly, E. coli RNA polymerase does not have a strong preference for initiating nucleotide identity. These observations highlight an important difference in biochemical properties of B. subtilis and E. coli RNA polymerases, specifically in their ability to use RNA templates efficiently, which also may reflect the differences in GTP and ATP metabolism in these two organisms.

Obten????o de RNA odontobl??stico de alta qualidade ap??s o armazenamento de dentes em diferentes condi????es de temperatura; Gene expression of odontoblast markers of human teeth using different RNA extraction protocols 2010

CONDE, Marcus Cristian Muniz
Fonte: Universidade Federal de Pelotas; Odontologia; Programa de P??s-Gradua????o em Odontologia; UFPel; BR Publicador: Universidade Federal de Pelotas; Odontologia; Programa de P??s-Gradua????o em Odontologia; UFPel; BR
Tipo: Dissertação Formato: application/pdf
Português
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Isolate high quality RNA form dental tissues is a most critical step to perform gene expression analysis. In some situations it is impossible to achieve the RNA isolation after tooth extraction, which leads to tooth discarding. Since, the aim of this experiment was to verify the effect of different teeth storage methods in the quality of RNA obtained from freshly extracted third molars. The teeth were randomly divided in five groups according to the temperature and storage time conditions. In control group RNA was isolated immediately after tooth extraction in room temperature. Experimental storage conditions evaluated were: liquid nitrogen, -80??C, -20??C (24h) and 4??C (6h). To RNA isolation, teeth were longitudinally sectioned and then pulp and pre-dentin were submerged in TRIzol??. Semi-quantitative RT-PCR was used to analyze the expression of odontoblast makers (DSPP, DMP1, and MEPE), which were normalized against the GAPDH gene. DSPP, DMP1 and MEPE were amplified in all storage conditions evaluated, regardless of storage method or tissue analyzed. Was possible to obtaining high quality RNA from pulp and dentin in all storage conditions appraised, increasing the RNA available to be used as positive control in cell differentiation studies; Extrair RNA de qualidade dos tecidos dentais ?? um passo cr??tico para a realiza????o da an??lise de express??o g??nica. Em algumas situa????es n??o ?? poss??vel realizar o isolamento do material gen??tico dos tecidos dent??rios logo ap??s a exodontia...

CLIP: cross-linking and immunoprecipitation of in vivo RNA targets of RNA binding proteins

Jensen, K.; Darnell, R.
Fonte: Humana Press; USA Publicador: Humana Press; USA
Tipo: Parte de Livro
Publicado em //2008 Português
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We present a newly developed method for fixing RNA-protein complexes in situ in living cells and the subsequent purification of the RNA targets. Using this approach, complex tissue such as mouse brain can be ultraviolet (UV) irradiated to covalently crosslink RNA-protein complexes. Once covalently bound, RNA-protein complexes can be purified under stringent conditions, allowing a highly specific purification scheme to be employed. After UV irradiation, the tissue is solubilized and the RNA partially digested, allowing a small fragment to remain attached to protein. RNA-protein complexes of interest are partially purified by immunoprecipitation and noncovalently associated RNA removed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). These purified RNA-protein complexes are isolated and treated with proteinase K, which removes protein but leaves intact RNA. This RNA is abundant enough, and competent for, RNA linker ligation, reverse transcriptase polymerase chain reaction (RT-PCR) amplification, and sequencing. Database matching of these short 70- to 100-nt RNA CLIP (crosslinking and immunoprecipitation of RNA-protein complexes) "tags," which mark the native binding sites of RNA binding proteins, potentially allows the entire target repertoire of an RNA binding protein to be determined.; Kirk B. Jensen...

Induktion von CLL-spezifischen CD4- und CD8-positiven T-Lymphozyten mit Hilfe von RNA-transfizierten dendritischen Zellen; Induction of chronic lymphocytic leukaemia (CLL) - specific CD4- and CD8-mediated T-cell responses using RNA transfected dendritic cells

Tsakou, Garyfalia
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
Português
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36.27%
In der vorliegenden Arbeit wurde die Effizienz RNA-transfizierter DC, in vitro Leukämie-spezifische autologe T-Zellantworten zu generieren, analysiert und es konnte gezeigt werden, dass aus Monozyten stammende DC, transfiziert mit Gesamttumor-RNA oder PCR-amplifizierter RNA aus CLL-Zellen, die Möglichkeit haben potente autologe zytotoxische und proliferative T-Zellantworten hervorzurufen. Diese Ergebnisse weisen darauf hin, dass sowohl funktionelle DC als auch funktionelle T-Zellen aus dem peripheren Blut der CLL-Patienten generiert werden konnten. Die DC der CLL-Patienten waren in der Lage CD8- und CD4-gerichtete Immunantworten zu induzieren. Die induzierten CTL und T-Helferzellen erkannten autologe unstimulierte CLL-Zellen und DC, die mit aus autologen malignen Zellen isolierter RNA transfiziert wurden, aber nicht die autologen nicht-malignen B-Lymphozyten oder mit irrelevanter RNA transfizierte DC. Darüber hinaus erkannten diese CTL autologe mit RNA aus verschiedenen CLL-Patienten transfizierte DC, was darauf hindeutet, dass die zytotoxische Aktivität gegen bestimmte Antigene gerichtet ist, die in vielen malignen leukämischen Zellen exprimiert werden. Zusammenfassend konnte mit den durchgeführten Experimenten gezeigt werden...

Untersuchungen zum Nachweis der Hepatitis A Virus RNA in menschlichen Seren; Investigations to detect Hepatitis A virus RNA in human blood samples

Heege,Ulrike
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
Português
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Einleitung: Ziel dieser Arbeit war es, ein einfaches Verfahren zu entwickeln, um Hepatitis A Viren (HAV) in Patientenseren anzureichern. Eine weitere Aufgabe war es, in diesen Seren mittels RT/PCR und zwei verschiedenen Primerpaaren HAV-RNA nachzuweisen. Nach Nachweis und Anreicherung von HAV sollten MRC-5 Zellen infiziert werden, um das Virus aus den Seren anzuzüchten. Ergebnisse: Es wurden 15 verschiedene Seren von Patienten mittels RT/PCR untersucht, die mit Hepatitis A infiziert waren. Von den untersuchten 15 Seren war ohne Anreicherungsverfahren durch Zentrifugation nur ein Serum in der nested PCR positiv. Nach Zentrifugation von je 200 µl Serum bei 4 Grad C und 12000rpm für drei Stunden zeigte sich ein Serum bereits in der ersten Runde positiv und sieben der untersuchten Patientenseren waren in der nested PCR positiv. In dieser Arbeit wurden zudem zwei Primerpaare, die beide Abschnitte in der 5’ Region der Hepatitis A Virus RNA amplifizieren, miteinander verglichen. Nicht nur Patientenseren wurden mit diesen verschiedenen Primerpaaren untersucht, sondern auch verschiedene Chargen inaktivierter Hepatitis A Virus Pools. Die aus den 4 inaktivierten Hepatitis A Virus Pools extrahierte HAV-RNA ließ sich mittels RT/PCR sicher mit den Primern N537/P452 amplifizieren...

RNA-Transfektion von dendritischen Zellen zur Induktion von antigenspezifischen zytotoxischen T-Lymphozyten; RNA-transfection of dendritic cells for the induction of antigen-specific cytotoxic T cells

Müller, Martin Rudolf
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
Português
Relevância na Pesquisa
36.28%
Dendritische Zellen (DC) sind professionelle antigenpräsentierende Zellen (APC), die bei der Induktion einer antigenspezifischen Immunantwort eine entscheidende Rolle spielen. Kürzlich konnte gezeigt werden, daß mit aus Tumoren isolierter RNA transfizierte DC effektive T-Zell-Immunantworten in vitro und in vivo induzieren können. Der entscheidende Vorteil dieser Vakzinierungsmethode besteht darin, daß zu ihrer Anwendung weder ein charakterisiertes Tumorantigen noch ein bestimmter HLA-Typ des Patienten notwendig ist. Mögliche Nachteile dieser Strategie könnten in der Induktion von Immunantworten gegen körpereigene Gewebsstrukturen (Autoimmunrekationen) bestehen. Im ersten Teil der vorliegenden Arbeit evaluierten wir drei verschiedene Methoden zur RNA-Transfektion von DC (Elektroporation, Lipofektion mit dem kationischen Lipid Unifectin M, Transferrinrezeptor-(CD71)-vermittelte Endozytose) hinsichtlich ihrer Effizienz. Bei Experimenten mit in vitro transkribierter EGFP mRNA zeigte sich eine deutliche Überlegenheit der Elektroporation mit einer erzielten Transfektionseffizienz von annähernd 30%, während die beiden anderen Methoden deutlich niedrigere Transfektionseffizienzen lieferten. Die Lipofektion mit Unifectin M ergab eine Transfektionseffizienz von ca. 17.5%...

Caracterização das proteinas humanas Mov34 e PACT e analise da sua interação com o RNA do virus da dengue; Characterization of the human Mov34 and PACT proteins and analyses of their interaction with dengue virus RNA

Beatriz Santos Capela Alves
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 21/08/2008 Português
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O combate à dengue atualmente está limitado praticamente aos esforços de eliminação do mosquito transmissor, o Aedes aegypti, porém esta estratégia não tem se mostrado eficiente. O desenvolvimento de novos instrumentos de combate à dengue requer, portanto, maior conhecimento sobre a biologia do vírus com relação à sua interação com seus hospedeiros. O genoma do vírus é constituído por um RNA simples-fita de polaridade positiva e possui duas regiões não traduzidas (5’ e 3’ UTR). A região 5’UTR viral possui organização similar à dos mRNAs eucarióticos, diferentemente da região 3’UTR que é longa e não possui cauda de poli(A). Em vez disso, na região 3’UTR encontram-se estruturas conservadas entre os diferentes Flavivirus, dentre elas a estrutura 3’ stem-loop (3’SL) que é indispensável para a replicação do RNA viral. O objetivo do nosso estudo foi identificar novas proteínas humanas capazes de interagir com a estrutura 3’SL do RNA do vírus da dengue. Dados da literatura descrevem que a proteína Mov34 de camundongo interage com 3’SL do vírus da encefalite japonesa. Devido à alta similaridade entre as proteínas ortólogas humana e de camundongo, bem como das respectivas estruturas 3’SL dos vírus da dengue e da encefalite japonesa...

Biorecognition by amino acid-based affinity chromatography for RNA purification

Martins, Ana Rita Nunes
Fonte: Universidade da Beira Interior Publicador: Universidade da Beira Interior
Tipo: Tese de Doutorado
Publicado em /10/2013 Português
Relevância na Pesquisa
36.31%
Following the decoding of the human genome, a new era was opened for developing new gene therapy strategies employing nucleic acids. Recently, RNA was renowned a central molecule in cellular processes with implications in many diseases as well as in understanding of evolution, becoming one of the most exciting research areas of molecular biology. From basic to applied research, many procedures employ pure and intact RNA molecules. On one hand, RNA purification is a first critical step of a number of molecular biology procedures and its quality is crucial to ensure reproducibility and biological relevance of an experiment. On the other hand, the promising and revolutionary RNA-based therapies of RNA vaccination, gene therapy or recombinant biopharmaceuticals involves RNA formulations which should fulfill rigorous quality criteria recommended by international regulatory agencies. However, the isolation and purification of RNA are critical steps because of the easy degradability of RNA, which can impair chemical stability and biological functionality essential for analysis. Many techniques have been development to overcome the challenges of purifying RNA molecules; nonetheless they still have several limitations in regard to time demanding and the requirement of toxic solvents and denaturing conditions. Therefore...

Exploring the Immunogenicity and Therapeutic Applications of Boranophosphate-modified RNA: siRNA and RNA Aptamers

Sharaf, Mariam Lucila
Fonte: Universidade Duke Publicador: Universidade Duke
Tipo: Dissertação
Publicado em //2011 Português
Relevância na Pesquisa
36.29%

Borane (BH3) chemistry offers unique chemical characteristics that enable boranophosphate (BP) oligonucleotides with potential to enhance RNA therapeutic applications such as RNA interference (RNAi) and RNA aptamers. Further, BP nucleotides are substrates for RNA polymerases which allow the enzymatic synthesis of stereoregular boranophosphate (BP)-RNA molecules of different lengths and properties. We expect that these BP-RNAs will interact in a novel way with the desired target molecules because they can coordinate with a diverse array of ligand sites in proteins or other RNA molecules. This is due to the distinct hydrophobicity, sterospecificity, and polarity properties imparted by the phosphorus-boron (P-B) chemical bond compared to the natural phosphorus-oxygen (P-O) bond.

The object of this dissertation is to explore the therapeutic applications of the BP-RNA such as siRNA, RNA aptamers, and in addition investigate the immunogenicity of this modification. We used mouse cells to determine if BP-RNA would activate toll-like receptor (TLR 7), which is involved in innate immune response to foreign single stranded RNA (ssRNA). This response is undesired when applied to oligonucleotide therapeutics such as siRNA and RNA aptamers. In terms of RNAi...

RNA Backbone Validation, Correction, and Implications for RNA-Protein Interfaces

Kapral, Gary Joseph
Fonte: Universidade Duke Publicador: Universidade Duke
Tipo: Dissertação
Publicado em //2013 Português
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RNA is the molecular workhorse of nature, capable of doing many cellular tasks, from genetic data storage and regulation, to enzymatic synthesis--even to the point of self-catalyzing its own replication. While RNA can act as a catalyst on its own, as in the hammerhead ribozyme, the added efficiency of proteins is often a necessity; the ribosome--the large ribozyme responsible for peptide chain formation, is aided by proteins which ensure correct assembly and structural stability. These complexes of RNA and proteins feature in many essential cellular processes, including the RISC silencing complex and in the spliceosome. Despite its enormous utility, structural determination of RNA is notoriously difficult--particularly in the backbone, since a nucleotide standardly has 12 torsion angles (including χ) and 12 non-hydrogen atoms, compared to 4 torsions (including χ1) and 4 non-H atoms in a typical amino acid. The abundance of backbone atoms, their conformational flexibility, and experimental resolution limitations often result in systematic errors that can have a significant impact on the interpretation. False trails due to structural errors can lead to significant loss of time and effort, especially with such high-profile complexes as the ribosome and the RISC complex.

My research has focused on harnessing the recently discovered ribosome structures and the Richardsons' RNA dataset to find trends in RNA backbone conformations and motifs that were then used to develop structural validation techniques and provide improved diagnosis and correction techniques for RNA backbone. Methods for fixing RNA structure have been developed for both NMR and X-ray crystallography. For NMR structures...

RNA 3D Structure Analysis and Validation, and Design Algorithms for Proteins and RNA

Jain, Swati
Fonte: Universidade Duke Publicador: Universidade Duke
Tipo: Dissertação
Publicado em //2015 Português
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RNA, or ribonucleic acid, is one of the three biological macromolecule types essential for all known life forms, and is a critical part of a variety of cellular processes. The well known functions of RNA molecules include acting as carriers of genetic information in the form of mRNAs, and then assisting in translation of that information to protein molecules as tRNAs and rRNAs. In recent years, many other kinds of non-coding RNAs have been found, like miRNAs and siRNAs, that are important for gene regulation. Some RNA molecules, called ribozymes, are also known to catalyze biochemical reactions. Functions carried out by these recently discovered RNAs, coupled with the traditionally known functions of tRNAs, mRNAs, and rRNAs make RNA molecules even more crucial and essential components in biology.

Most of the functions mentioned above are carried out by RNA molecules associ- ating themselves with proteins to form Ribonucleoprotein (RNP) complexes, e.g. the ribosome or the splicesosome. RNA molecules also bind a variety of small molecules, such as metabolites, and their binding can turn on or off gene expression. These RNP complexes and small molecule binding RNAs are increasingly being recognized as potential therapeutic targets for drug design. The technique of computational structure-based rational design has been successfully used for designing drugs and inhibitors for protein function...