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Pereira, Tiago Campos. "Estudo de possiveis aplicações médicas da interferencia por RNA." [s.n.], 2005. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316861.
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Orientadores: Iscia Teresinha Lopes-Cendes, Ivan de Godoy MaiaTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Doutorado
Genetica Animal e Evolução
Doutor em Genetica e Biologia Molecular
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Abdel-Lateif, Khalid. "Flavonoids and actinorhizal symbiosis : Impact of RNA interference-mediated silencing of chalcone synthase gene on symbiosis between Casuarina glauca and Frankia." Thesis, Montpellier 2, 2012. http://www.theses.fr/2012MON20244/document.
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Les deux systèmes nodulaires symbiotiques les plus importants au niveau agronomique et environnemental sont, d'une part, les symbioses Rhizobium-légumineuses qui concernent environ 14 000 espèces, et d'autre part, les symbioses entre les plantes actinorhiziennes (environ 200 espèces) et l'actinomycète du sol Frankia. La plupart des plantes actinorhiziennes sont capables de fixer des quantités d'azote comparable à celles des Légumineuses ; ce sont généralement des plantes pionnières capables de coloniser des environnements pauvres en éléments minéraux. Elles représentent donc un atout écologique important. Si la symbiose Rhizobium-légumineuse est très étudiée, les mécanismes moléculaires à l'origine de la formation des nodules actinorhiziens restent actuellement peu connus. Ainsi, chez les Légumineuses, les flavonoïdes sont des molécules-clefs du processus de nodulation, alors que chez les plantes actinorhiziennes, l'implication des flavonoïdes dans la nodulation reste imprécise. L'objectif de cette thèse était de comprendre l'implication des flavonoïdes au cours de l'interaction symbiotique entre l'arbre actinorhizien tropical Casuarina glauca et son symbiote Frankia. L'analyse d'une base de données d'unigènes couplée à celle de données d'expression de puces à ADN a permis l'identification de huit genes de C. glauca impliqués dans la voie de biosynthèse des flavonoïdes. L'étude de leur expression dans les racines par PCR quantitative au cours d'une cinétique d'infection de C. glauca par Frankia a montré que les transcrits de la chalcone isomerase et de l'isoflavone reductase s'accumulaient très tôt après l'inoculation, suggérant ainsi une implication des isoflavonoïdes dans la symbiose actinorhizienne. Nous avons alors utilisé une stratégie d'ARN interférent pour réduire l'expression du gène de la chalcone synthase, la première enzyme de la voie de biosynthèse des flavonoïdes. La réduction de l'expression du gène de la chalcone synthase a provoqué une réduction significative du taux de flavonoïdes dans les racines ainsi qu'une très forte diminution du taux de nodulation chez les plantes transformées. Une restauration du taux de nodulation a pu être obtenu en présence de naringenin, une molécule centrale de la voie de biosynthèse des flavonoïdes.Nos résultats apportent donc, pour la première fois, une évidence directe de l'implication forte des flavonoïdes au cours de la nodulation des plantes actinorhiziennesNitrogen-fixing root nodulation, confined to four plant orders, encompasses more than 14,000 Leguminosae species, and approximately 200 actinorhizal species forming symbioses with rhizobia and Frankia bacterial species, respectively. Most actinorhizal plants are capable of high rates of nitrogen fixation comparable to the nitrogen fixing symbiosis between legumes and Rhizobium. As a consequence, these plants are able to grow in poor and disturbed soils and are important elements in plant community worldwide. The basic knowledge of the symbiotic interaction between Frankia and actinorhizal plants is still poorly understood, although it offers striking differences with the Rhizobium-legume symbiosis. In the symbiosis between legumes and Rhizobium, flavonoids are key molecules for nodulation. In actinorhizal plants, the involvement of flavonoids in symbiosis is poorly understood, but because of the similarities of the infection process between some actinorhizal plants and legumes, flavonoids were proposed to act as plant signals for the bacteria Frankia. The objective of this thesis was to investigate the involvement of flavonoids during the actinorhizal nodulation process resulting from the interaction between the tropical tree Casuarina glauca and the actinomycete Frankia.Eight C. glauca genes involved in flavonoid biosynthesis were identified from a unigene database and their expression patterns were monitored by quantitative real-time PCR during the nodulation time course. Our results showed that chalcone isomerase and isoflavone reductase transcripts accumulated preferentially early after inoculation with Frankia, suggesting thus for the first time that isoflavonoids are implicated in actinorhizal nodulation. To go deeper in the understanding of the role of these molecules in actinorhizal symbiosis, we used RNA interference strategy to silence chalcone synthase, the enzyme that catalyzes the first committed step of the flavonoid pathway. Knockdown of chalcone synthase expression led to a strong reduction of specific flavonoids levels and resulted in a severely impaired nodulation. Nodule formation could be rescued by supplementation of plants with naringenin, which is an upstream intermediate in flavonoid biosynthesis. Our results provide, for the first time, direct evidence of a strong implication of flavonoids during actinorhizal nodulation
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Shah, Samit Friedman Simon H. "Light activated RNA interference." Diss., UMK access, 2007.
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Thesis (Ph. D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2007."A dissertation in pharmaceutical science and chemistry." Advisor: Simon H. Friedman. Typescript. Vita. Description based on contents viewed July 16, 2008; title from "catalog record" of the print edition. Includes bibliographical references (leaves 206-220). Online version of the print edition.
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Fransecky, Lars. "Auswirkungen des LRRK2-Knockdown durch RNA-Interferenz auf die murine dopaminerge Zelllinie MN9D." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2009. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-21600.
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Mutationen im Protein LRRK2 wurden im Zusammenhang mit klinischen Symptomen beschrieben, die dem Idiopathischen Parkinsonsyndrom (IPS) nahezu gleichen. So findet sich neben vielen anderen Mutationen die häufigste pathogene Mutation für das IPS im LRRK2-Gen.
Die Aufklärung der molekularbiologischen Mechanismen, die zur Pathologie der spezifischen Neurodegeneration in der Substantia nigra Pars Compacta (SNpc) und somit zur Idiopathischen Parkinsonssyndrom führen, ist mit der Hoffnung auf kausale und kurative Therapieansätze verbunden.
In dieser medizinischen Doktorarbeit soll daher versucht werden, die biologische Funktion des LRRK2 in einem dopaminergen Mauszellmodell näher zu beschreiben. Hierfür soll die genetische Aktivität des LRRK2 in mesenzephalen, sogenannten MN9D-Zellen reduziert werden, indem der Mechanismus der RNA-Interferenz in vitro durch Transfektion von siRNA angestoßen wird. Durch die Reduktion der LRRK2-Aktivität sollen Veränderungen in den MN9D-Zellen induziert und diese objektiviert werden.
Die Darstellung der Beobachtungen konzentriert sich auf die transkriptionelle Expression von Genen des Zellzyklus sowie der neuralen und dopaminergen Differenzierung (Tyrosinhydroxylase, Nestin und β-Tubulin) durch PCR. Die Proliferation der Zellen vor und nach den RNA-Interferenzexperimenten soll global durch MTT- und BrdU-Test gemessen werden.
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Marr, Edward John. "RNA interference (RNAi) for selective gene silencing in Astigmatid mites." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25722.
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Psoroptic mange, caused by the Astigmatid mite Psoroptes ovis, is an ectoparasitic disease of significant economic importance to agriculture on a global scale and poses a serious welfare concern. With the current chemotherapeutic controls considered unsustainable, there is pressing need for novel control strategies. RNA interference has been proposed as a potential high throughput approach for the identification of novel therapeutic targets with high specificity, speed and at a relatively low cost compared to the existing methods. The presence of the components of the RNA interference (RNAi) pathway in P. ovis was first confirmed through in silico analyses of the P. ovis transcriptome and, following development of a non-invasive immersion method of double stranded RNA (dsRNA) delivery, gene silencing by RNAi was demonstrated in P. ovis. Statistically-significant reduction of transcript level was measured for the three genes targeted: P. ovis mite group 2 allergen (Pso o 2), P. ovis mu class glutathione S-transferase (PoGST-mu1) and P. ovis beta tubulin (Poβtub). This is the first demonstration of gene silencing by RNAi in P. ovis and provides a key mechanism for mining transcriptomic and genomic datasets in the future for novel targets of intervention against P. ovis. The first assessment of gene silencing was also performed in two related Astigmatid mites of high medical importance; the European house dust mite Dermatophagoides pteronyssinus and the scabies mite Sarcoptes scabiei. A statistically-significant reduction in expression of a D. pteronyssinus mu class glutathione S-transferase (DpGST-mu1) transcript was observed. No significant reduction in expression of a S. scabiei mu class glutathione S-transferase (SsGST-mu1) transcript was observed. Additionally, microRNAs (miRNAs) from the related miRNA pathway were identified in a P. ovis small RNA sample and were sequenced and annotated.
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Jagannath, Aarti. "Studies on the RNA interference pathway in mammalian cells." Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.711608.
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Knapinski, Sven. "Doppelstrang-RNA-vermittelte Gen-Interferenz (RNAi) im Nervensystem adulter Grillen (Gryllus bimaculatus)." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2010. http://dx.doi.org/10.18452/16157.
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Ziel der vorliegenden Dissertation war es, zum Verständnis der genetischen Grundlagen des akustischen Kommunikationssystems der Grille Gryllus bimaculatus beizutragen (s. auch 1.2). Dazu wurde die Expression eines Orthologs des no-on-transientA-Gens (nonA) mit Hilfe der RNA-Interferenz-Methode spezifisch herunterreguliert. Bei nonA handelt es sich um ein vielversprechendes Kandidatengen, da Punktmutationen in der codierenden Region des Gens die Eigenschaften des männlichen Balzgesangs bei Drosophila melanogaster beeinflussen. Zudem belegen Gentransfer-Experimente bei Drosophila, dass dieses Gen artspezifische Informationen des Balzgesangs enthält. Die Analyse der Gesangsdaten ergab, dass sich die Periodenlänge durch das Herunterregulieren von NONA nicht verändert. Außerdem konnte gezeigt werden, dass nonA-dsRNA-injizierte Tiere seltener 3-silbige Chirps produzieren, dafür aber mehr 4- und 5-silbige Chirps. Die Auswertung der tageszeitlichen Gesangsaktivität zeigte, dass alle Tiere signifikant am häufigsten im ersten Nachtquartal (nach Erlöschen der Beleuchtung) zirpten. Ein Effekt durch das Herunterregulieren von NONA konnte statistisch nicht belegt werden. Allerdings schien es einen Trend bei nonA-dsRNA-injizierten Tieren zu geben, gleichmäßiger über den Tag verteilt Gesangsaktivität zu zeigen. Transgene Drosophila melanogaster, deren arteigenes nonA durch das der Grille ersetzt bzw. ergänzt worden war, zeigten durchweg eine verbesserte Überlebensfähigkeit (Steigerungen zwischen 27 und 340%). Auch das positiv phototaktische Verhalten wurde durch das Grillen-NONA bei allen transformanten Fliegen verstärkt; allerdings fiel dieser Effekt eher marginal aus. Dennoch kann durchaus von einer zumindest teilweisen funktionellen Konservierung des nonA-Gens zwischen Gryllus bimaculatus und Drosophila melanogaster ausgegangen werden.The present thesis aims to widen our understanding of the genetic background of the acoustic communication system of the cricket Gryllus bimaculatus (see also 1.2). Therefore the expression of an ortholog of the no-on-transientA (nonA) gene was specifically inhibited via RNA-interference. The nonA gene is one of the most interesting candidate genes in this context, as point mutations in the coding region of the gene affect the characteristics of the male’s calling song. Furthermore, gene transfer experiments in Drosophila showed that this gene obviously carries species-specific song information. The analysis of the calling song of nonA-RNAi-treated crickets, revealed that the duration of the syllable period was not influenced by the “knock-down” of the gene, but that the inhibition had a certain impact on the maximum number of syllables per chirp, as nonA-dsRNA-injected crickets produced significantly less 3-syllable chirps and significantly more 4- and 5-syllable chirps. Differences in the daytime calling activity between nonA-dsRNA-injected crickets and control groups could not be verified. The calling activity of all groups reached its peak in the first quarter of the night and significantly differed from the low calling activity during the remaining quarters of the day. Although the activity of all animals reached its peak during the first quarter of the night, there seems to be a trend that this rhythmical behaviour was less pronounced in nonA-dsRNA-injected crickets. Drosophila melanogaster mutants, which had been transformed with the nonA ortholog of Gryllus bimaculatus, increased their survival by 27% to 340%. In addition, the positive phototactic behaviour was slightly increased in all tested animals - this effect, however, remained marginal. Nevertheless, the nonA gene seems to be at least partly functionally conserved between Gryllus bimaculatus and Drosophila melanogaster.
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Garbutt, Jennifer S. "RNA interference in insects : persistence and uptake of double-stranded RNA and activation of RNAi genes." Thesis, University of Bath, 2011. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.548101.
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RNA interference (RNAi) is a eukaryotic phenomenon where short double-stranded RNA molecules (dsRNAs) repress homologous sequences. In insects RNAi has been widely observed and has proved extremely useful as a reverse genetics tool to elucidate the function of newly identified genes, as well as showing potential as a novel insecticide. Unfortunately, however, not all insect species are equally susceptible to RNAi. This thesis explores whether persistence of dsRNA in insect hemolymph, uptake of dsRNA into insect tissue, or activation of RNAi genes could be limiting factors in RNAi experiments. Trials were conducted with the tobacco hornworm, Manduca sexta, a species in which experimental difficulty has been experienced with RNAi protocols and the German cockroach, Blattella germanica, which is known to be highly susceptible to experimental RNAi. In M. sexta larvae dsRNA disappeared rapidly from the hemolymph in vivo. By comparison, exogenous dsRNA persisted longer in the hemolymph of B. germanica adults. These findings lead me to propose that the rate of persistence of dsRNA in insect hemolymph may be a key factor in determining the susceptibility of insect species to RNAi. Despite such rapid breakdown of dsRNA in M. sexta larvae uptake of exogenous dsRNA into hemocytes, fat body and midgut could be detected by quantitative RT-PCR in vivo and was experimentally investigated in hemocytes in vivo and in vitro using fluorescently labelled dsRNA. Furthermore, quantitative-RT-PCR revealed that the expression of two M. sexta RNAi genes dicer-2 and argonaute-2 (partial sequences of which were isolated during this study) was specifically upregulated in response to injection with dsRNA.
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Zhang, Zhao. "piRNA Biogenesis and Transposon Silencing in Drosophila: A Dissertation." eScholarship@UMMS, 2013. http://escholarship.umassmed.edu/gsbs_diss/689.
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piRNAs guide PIWI proteins to silence transposons in animal germ cells. In Drosophila, the heterochromatic piRNA clusters transcribe piRNA precursors to be transported into nuage, a perinuclear structure for piRNA production and transposon silencing. At nuage, reciprocal cycles of piRNA-directed RNA cleavage—catalyzed by the PIWI proteins Aubergine (Aub) and Argonaute3 (Ago3) in Drosophila—destroy the sense transposon mRNA and expand the population of antisense piRNAs in response to transposon expression, a process called the Ping-Pong cycle. Heterotypic Ping-Pong between Aub and Ago3 ensures that antisense piRNAs predominate.
My thesis research mainly focuses on two fundamental questions about the piRNA production: How does the germ cell differentiate piRNA precursor from mRNAs for piRNA biogenesis? And what is the mechanism to impose Aub Ping-Pong with Ago3? For the first question, we show that the HP1 homolog protein Rhino marks the piRNA cluster regions in the genome for piRNA biogenesis. Rhino seems to anchor a nuclear complex that suppresses cluster transcript splicing, which may differentiate piRNA precursors from mature mRNAs. Moreover, LacI::Rhino fusion protein binding suppresses splicing of a reporter transgene and is sufficient to trigger de novo piRNA production from a trans combination of sense and antisense transgenes. For the second question, we show that Qin, a new piRNA pathway factor contains both E3 ligase and Tudor domains, colocalizes with Aub and Ago3 in nuage, enforces heterotypic Ping- Pong between Aub and Ago3. Loss of qinleads to less Ago3 binding to Aub, futile Aub:Aub homotypic Ping-Pong prevails, antisense piRNAs decrease, many families of mobile genetic elements are reactivated, DNA damage accumulates in the germ cells and flies are sterile.
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Hönemann, Mario. "RNA Interferenz unter Verwendung eines lentiviralen Vektosystems zur Modifikation einer persistierenden Masernvirusinfektion." Doctoral thesis, Universitätsbibliothek Leipzig, 2011. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-77084.
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Die Vorliegende Arbeit beschäftige sich mit der Etablierung eines lentiviralen Vektorsystems, mit dem es möglich ist die RNA-Interferenz experimentell zu nutzen. Dafür wurden SEC Sequenzen in den Vektor pGJ3-eGFP kloniert. Nach Optimierung der Transfektions und Transduktionsschritte wurden im Anschluss rekombinante virale Partikel hergestellt. Zur Überprüfung der Effektivität der Induzierten RNA-Interferenz erfolgte die Transduktion einer persistierend mit Masern infizierten Zelllinie (C6SSPE). Ziel der siRNA Sequenzen war dabei die mRNA des N-Proteins, welches eine zentrale Rolle im viralen Replikationszyklus spielt. Die Reduktion der mRNA wurde über quantitative real time RT-PCR nachgewiesen.
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Aitken, Amelia. "Blocking the RNA Interference Pathway Improves Oncolytic Virus Therapy." Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/36821.
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Oncolytic viruses are novel candidates for cancer therapy and their efficacy relies on their capacity to overcome the host’s anti-viral barriers. In mammalian cells, the anti-viral response involves a protein-signaling cascade known as the interferon pathway, which alerts the immune system and limits the propagation of infection. Given that most cancer cells have defects in this pathway, they are susceptible to viral infection and responsive to oncolytic virotherapy. For reasons that remain unknown, many cancers are still refractory to oncolytic viruses, which suggests the existence of additional antiviral mechanisms. In this study, we investigate the potential involvement of an alternative antiviral pathway in cancer cells. Given that insects and plants rely on the RNA silencing pathway for their anti-viral protection, we investigated the presence of a similar mechanism in cancer cells. We found viral genome-derived small RNAs in various cancer cell lines upon infection, which is indicative of an RNA-mediated antiviral response. Also, various viruses encode suppressors of the RNA interference pathway. To determine if an oncolytic virus could benefit from such a factor, we engineered an oncolytic virus variant to encode the Nodamura virus B2 protein, a known inhibitor of RNA silencing-mediated immune responses. Using this virus, we observed enhanced cytotoxicity in 33 out of the 38 human cancer cell lines tested. Furthermore, our results show inhibition of viral genome cleavage and altered microRNA processing by our B2-expressing oncolytic virus. Taken together, our data suggests the blockade of RNA silencing antiviral pathways and/or antiviral microRNA processing improves the efficacy of our B2-encoding virus in a cell-line specific manner. Overall, our results establish the improved potential of our novel virus therapy and demonstrate for the first time the involvement of RNA pathways in the antiviral defense of cancer cells.
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Schultes, Stephan. "Nanoparticles for RNA Interference." Diss., lmu, 2009. http://nbn-resolving.de/urn:nbn:de:bvb:19-113293.
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Micocci, Kelli Cristina. "Análise da expressão e do papel da ADAM9 humana na capacidade invasiva de células tumorais por silenciamento gênico." Universidade Federal de São Carlos, 2009. https://repositorio.ufscar.br/handle/ufscar/1319.
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ADAMs is a term used to describe the presence of disintegrin and metalloprotease domains(A Disintegrin And Metalloprotease) in a certain class of multi-functional membrane proteins,expressed in several animal species such as mammals and insects. They play important rolesin many physiological processes as in fertilization, myoblast fusion, migration, proliferationand cell survival, as well in diseases including breast, prostate, and pancreas cancer. ADAM9is involved in cellular processes such as adhesion, migration and signaling of tumor cells andit is involved in the metastatic spreading. Aims: To analyze the expression of ADAM9 intumor cell lines (MDA-MB-231 and DU-145), no tumors (FH and C2C12), and to generateknockout clones for ADAM9 expression using silencing RNA techniques for the study ofADAM9 effects on invasion, proliferation and gene expression of MDA-MB-231 cells.Methods: Cells were cultured and plated (2x106 cells/plate of 6cm) for 24 hours in DMEM(10% FBS) and lysed for western blotting and zymography. To check for inhibition ofadhesion to immobilized collagen I, MDA-MB-231 cells and FH (5x106 cells/ml) werelabeled with CMFDA, and then incubated with anti-ADAM9D and anti-RP3ADAM9 indifferent concentrations. For ADAM9 silencing, it was used a kit (Silencer ® siRNA StarterKit - Ambion), 2x105 cells (MDA-MB-231) and the transfection agent (Lipofectamine 2000 -Invitrogen). The cells were plated in 5ml of culture medium DMEM/plate. On the third daycells were treated with the transfection agent and RNA silencing primers. Total RNA wasisolated for RT-PCR, and proliferation and invasion in matrigel assays. Results: All the celllines studied expressed ADAM9 in the tested conditions. MMP-2 (Gelatinase-A) activity wasdetected in the cell extracts of all studied cell lines. Silencing of ADAM9 did not affect therate of MDA-MB-231 proliferation, at 4, 5, 6, 7, 8, 9 and 10 days after silencing (24 or 48hours of incubation in 96-well plates). Silencing of human ADAM9 inhibited tumor cellinvasion in matrigel (71,51±8,02%) when compared to control. Conclusion: The generation ofMDA-MB-231 knockout clones lacking ADAM9 expression using siRNA technique did notaffect the rate of cell proliferation but inhibited tumor cell matrigel invasion, suggesting thatADAM9 is an important molecule in the processes of invasion and metastasis.
ADAMs é um termo usado para descrever a presença de domínios desintegrina emetaloprotease (A Disintegrin And Metalloprotease) em uma determinada classe de proteínasde membrana, multifuncionais, expressas em diferentes espécies animais como mamíferos einsetos. Elas possuem funções importantes em muitos processos fisiológicos como nafertilização, fusão de mioblastos, migração, proliferação e sobrevivência celular, entre outros,bem como em processos patológicos tais como muitos tipos de tumores humanos, incluindomama, próstata, pâncreas, fígado, rins e pele. A ADAM9 está envolvida em diversosprocessos celulares, tais como adesão celular, migração e sinalização de células tumorais,contribuindo para o desenvolvimento de metástases. Objetivos: Analisar a expressão daADAM9 em linhagens de células tumorais (MDA-MB-231 e DU-145) e não tumorais (FH eC2C12), gerar clones com o gene que codifica para a ADAM9 silenciados e verificar o efeitoda ausência desta proteína na invasão, proliferação e expressão gênica das células MDA-MB-231. Métodos: As células foram cultivadas e posteriormente plaqueadas (2x106 células/placade 6cm) por 24 horas e em 5ml de meio de cultura DMEM (10% de FBS) e lisadas para oensaio de western blotting e zimografia. Para o ensaio de inibição de adesão as células MDAMB-231 e FH (5x106 células/ml) foram marcadas com CMFDA (clorometil diacetatofluoresceína) e posteriormente incubadas com os anticorpos anti-ADAM9D e anti-RP3ADAM9 em diferentes concentrações. Para a técnica de RNAi foi utilizado um kit(Silencer® siRNA Starter Kit Ambion), 2,0x105 de células (MDA-MB-231) e agente detransfecção (Lipofectamina 2000 - Invitrogen). As células foram plaqueadas em 5ml de meiode cultura DMEM/placa. No terceiro dia de plaqueamento as células foram tratadas comagente de transfecção e primer de silenciamento do RNA, para serem utilizadas na RT-PCR,ensaio de proliferação e invasão em matrigel. Resultados: Todas as linhagens estudadasexpressaram a ADAM9 nas condições de estudo. A atividade MMP-2 (gelatinase-A)intermediária estava presente em todos os tipos celulares testados. O silenciamento deADAM9 não afetou a taxa de proliferação das células MDA-MB-231 após 4, 5, 6, 7, 8, 9 e 10dias do silenciamento (24 ou 48 horas de incubação). O silenciamento da ADAM9 humanainibiu a invasão celular em células de câncer de mama (MDA-MB-231) em matrigel (71,51 ±8,02%) quando comparados com o controle. Conclusão: A geração de clones knockout sem aexpressão da ADAM9 utilizando a técnica de silenciamento de RNA em células de câncer demama (MDA-MB-231), não afetou a taxa de proliferação celular. No entanto, a invasão dascélulas tumorais em matrigel foi inibida em aproximadamente 70% quando comparada com ocontrole, demonstrando que ADAM9 é uma importante molécula envolvida no processo deinvasão e metástase.
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Kunze, Doreen. "Small interfering RNA-vermittelte Hemmung der Apoptoseinhibitoren BCL2, BCL-XL, XIAP und Survivin in Zellkultur- und Mausmodellen des humanen Harnblasenkarzinoms." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2012. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-81888.
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Das Harnblasenkarzinom (BCa) stellt in Deutschland die vierthäufigste Tumorneuerkrankung und die zehnthäufigste krebsbedingte Todesursache bei Männern dar. Nichtmuskelinvasive BCa werden organerhaltend aus der Blasenwand entfernt und zur Rezidiv- und Progressionsprophylaxe mittels intravesikaler Chemo- oder Immuntherapien behandelt. Trotz dieser adjuvanten Therapien, die mit starken Nebenwirkungen verbunden sein können, ist nur eine bedingte Minimierung des Rezidivrisikos möglich. Besonders im fortgeschrittenen Stadium weisen Harnblasenkarzinome eine schlechte Prognose auf. Obwohl das BCa eine chemosensitive Erkrankung darstellt, wird das Ansprechen auf lokale oder systemische Chemotherapien häufig durch auftretende Resistenzmechanismen limitiert. Daher stehen sowohl die Verbesserung konventioneller Chemotherapien als auch die Suche nach neuartigen Behandlungsstrategien im Fokus der experimentellen BCa-Forschung.
Die Apoptose, eine Form des programmierten Zelltodes, ist ein essenzieller, streng regulierter biologischer Prozess, welcher der Aufrechterhaltung der Gewebshomöostase und der gezielten, entzündungsfreien Eliminierung geschädigter Zellen dient. Fehlregulationen in den Apoptosesignalwegen stellen ein zentrales Ereignis in der Tumorgenese dar und tragen außerdem zur Entstehung von Chemo- und Radiotherapieresistenzen bei. Eine wichtige Rolle in der Apoptoseregulation spielen die Mitglieder der BCL2- und der Inhibitor of Apoptosis Protein (IAP)-Familien, deren wichtigste antiapoptotische Vertreter BCL2, BCL-XL, XIAP und Survivin häufig in Tumoren, einschließlich des BCa, überexprimiert sind.
Unter Verwendung von small interfering RNAs (siRNAs), synthetischen Nukleinsäurekonstrukten zur selektiven Geninhibition, wurde im Rahmen der Arbeit in vitro und in vivo untersucht, ob die Hemmung der Apoptoseinhibitoren BCL2, BCL-XL, XIAP und Survivin – allein und in Kombination mit Chemotherapie – eine Therapieoption zur Behandlung des BCa darstellen könnte. Da zur Tumorentstehung und -progression eine Vielzahl von genetischen Veränderungen beitragen, erscheint der Angriff eines einzelnen Zielgens unzureichend für eine effektive Tumortherapie. Aufgrund dessen wurde untersucht, ob durch simultane Reduktion der ausgewählten Apoptoseinhibitoren in BCa-Zellen stärkere wachstumsinhibitorische Effekte erzielt werden können.
In der vorliegenden Arbeit wurde gezeigt, dass insbesondere die siRNA-vermittelte Hemmung von BCL-XL und Survivin in den BCa-Zelllinien EJ28 und J82 antiproliferative Effekte hervorruft und diese Tumorzellen gegenüber einer nachgeschalteten Chemotherapie mit Mitomycin C oder Cisplatin sensitiviert. Hingegen bewirkte sowohl die transiente als auch die stabile RNAi-induzierte Hemmung von BCL2 und XIAP in den untersuchten BCa-Monolayerzellkulturen, möglicherweise infolge kontinuierlicher Versorgung der Tumorzellen mit Sauerstoff und Nährstoffen, keine Reduktion des Tumorwachstums.
Eine gegenüber den Einzelbehandlungen deutliche Verstärkung der antitumoralen und insbesondere der chemosensitivierenden Effekte in den BCa-Zelllinien wurde durch simultane Hemmung von BCL-XL und Survivin erzielt. Beispielsweise stieg der Anteil apoptotischer Zellen von 64 % nach Survivin-siRNA+Cisplatin-Behandlung auf 94 % nach gleichzeitiger BCL-XL+Survivin-Inhibition in Kombination mit Cisplatin. Folglich stellt die simultane Inhibition von BCL-XL und Survivin in Kombination mit Chemotherapeutika eine äußert viel versprechende BCa-Therapieoption dar. Tierexperimentelle Studien belegen die wachstumsinhibitorische Wirkung der Survivin-Reduktion und der kombinierten BCL-XL-siRNA+Chemotherapie-Behandlung, so wurde das Tumorendvolumen im Vergleich zur Kontrollbehandlung um 43 % bzw. um 48 % reduziert.
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Berenjian, Saideh. "Construction of Adenovirus Vectors for Studies of Protein Function and RNA Interference." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6328.
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Hall, Emma Andisi. "Screening for genes involved in cilia formation and function." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/9898.
Full textAbstract:
Cilia are small microtubule based structures found on the surface of almost all mammalian cells, enclosed in a highly specialised extension of the cell membrane. Components of several key developmental signalling pathways, in particular Hedgehog (Hh) signalling, are enriched in cilia and cells with mutations in cilia structure show aberrant signalling, suggesting cilia act as “antennae” to focus these signalling cascades. A spectrum of human diseases, termed ciliopathies, are caused by problems in cilia formation or cilia function, which display wide ranging phenotypes from embryonic lethality to retinal degeneration, polydactyly to cystic kidneys. Despite recent advances in the understanding of the essential roles cilia play in mammalian development, exactly how these complex structures are put together, how they carry out their diverse functions, and how they are regulated is not well understood. In this thesis, I describe a screen for genes involved in cilia formation and function. While optimising ciliogenesis and immunofluorescence protocols for the screen, the phenotypes of two ciliary mutant cell lines were analysed. Wdr35yet/yet and Dync2h1pol/pol mouse lines were identified in an ENU screen for genes involved in early development, and shown to have gross phenotypes similar to other ciliary mutants (Mill et al. 2011). Intraflagellar transport (IFT) is the active transport of proteins up and down the ciliary axoneme. Dync2h1 is a retrograde IFT motor component, whereas Wdr35 is part of the retrograde IFT-A complex. In this thesis, the cellular phenotypes of mouse embryonic fibroblasts derived from these mutants are described, showing that despite the fact both genes are thought to be involved in retrograde IFT, they show distinct ciliary phenotypes, suggesting novel roles for Wdr35 in mouse ciliogenesis. An siRNA screen was carried out in mouse fibroblasts to identify genes involved in (i) cilia formation, assayed by immunofluorescence for ciliary markers, and (ii) cilia function, assayed by activity of a Hh responsive luciferase transgene as an indirect readout of ciliary function. Although scalable, I initially screened a small test set of thirty-six putative cilia candidates, identified by cross species transcriptomic analysis. We identified several possible hits, many of which were in the ciliome database but also importantly, several genes with no known link to ciliogenesis. Repeats, correlation of phenotype to knockdown efficiencies and localisation studies validated two hits, Ccdc63 and Azi1. Ccdc63 is a novel coiled-coil gene with no previous link to ciliogenesis; the phenotype for this gene was analysed in real time using fluorescently tagged ciliary markers. A second hit, Azi1, was followed up in more detail. The reduction in ciliogenesis upon Azi1 knockdown was confirmed with separate siRNAs, and was rescued by overexpressing siRNA insensitive Azi1-GFP, confirming the phenotype is not due to off-target effects of the siRNAs. Azi1 gene trap mutant mice were generated and confirmed to be null mutations. Surprisingly, the mice survive, showing Azi1 is not essential for mammalian ciliogenesis. However, mutant males are infertile, with highly reduced sperm count and sperm abnormalities indicative of an arrest at Stage IX of spermiogenesis, when the flagellum, a highly specialised motile cilium, forms. The small number of sperm that do get to the epididymus are immotile. We suggest Azi1 is essential to in the formation of the sperm flagella and male fertility.
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Martin, Janine Nicole. "Developing RNAi therapy For DYT1 dystonia." Diss., University of Iowa, 2011. https://ir.uiowa.edu/etd/1019.
Full textAbstract:
DYT1 dystonia is an early onset central nervous system-based movement disorder characterized by uncontrolled sustained muscle contractions that can lead to debilitating abnormal postures. Though a genetic mutation in the gene TOR1A is responsible for most DYT1 cases, the low penetrance of the disease implicates additional genetic and environmental modifiers. Current therapeutic options for DYT1 dystonia are limited to symptomatic treatments with variable effectiveness. Currently, the underlying pathogenesis of this disease and the role of torsinA (torA), the protein product of TOR1A, in the development of this disease have yet to be established.
In the first part of this thesis we aimed to further understand the effects of the TOR1A mutation at the molecular, cellular and organismal level in order to identify disease associated biomarkers that can be later used to measure the effectiveness of novel therapies. We found that expression of mutant torsinA (torA(ÄE)) in a cellular and an animal model of DYT1 had no significant effect on global transcription, despite its interaction with nuclear envelope proteins. Recent research has unearthed a role for microRNAs (miRNAs) in neuronal development and maturation. Consequently we explored whether torA(ÄE) expression in murine neural tissue was associated with changes in miRNA expression in young DYT1 knockin (KI) mice. Since the primary sight of dysfunction is still being debated, we profiled miRNA expression of the two strongest candidates, the striatum and cerebellum, both of which have well established roles in the control and coordination of muscle movements. We have identified several microRNAs that were uniquely altered in either the striatum or cerebellum and further research will be conducted to determine their usability as disease biomarkers. Finally, we were unable to identify motor phenotypes in either a DYT1 (KI) mice or a novel DYT1 transgenic model in open field, rotarod or staircase forepaw reaching tests.
In the second part of this thesis we aimed to develop and evaluate the safety and efficacy of viral therapeutic RNAi constructs for in DYT1 murine models. DYT1 is an ideal candidate for this form of therapy due to its dominant inheritance, common mutation and potentially reversible phenotype. Virally delivered short-hairpin RNAs (shRNA) designed to knockdown torA(ÄE) in either an allele-specific or nonallele-specific manner were injected into the striatum of DYT1 transgenic or KI mice respectively. Unexpectedly, we found widespread lethal toxicity and behavioral abnormalities in mice injected with either therapeutic or control shRNAs that weren't observed in mice injected with no shRNAs. Further studies found that regions where toxic shRNAs were expressed corresponded with neuronal loss and glial activation. Finally, we found evidence that the severity of toxicity was influenced in part by the genetic background of the mice.
In summary, the studies completed in this thesis contribute important information to the fields of dystonia pathogenesis and therapeutics, and more broadly pertain to the development of therapeutic gene silencing for neurological disease.
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Wee, Liang Meng. "RNA Interference by the Numbers: Explaining Biology Through Enzymology: A Dissertation." eScholarship@UMMS, 2006. http://escholarship.umassmed.edu/gsbs_diss/661.
Full textAbstract:
Small silencing RNAs function in almost every aspect of cellular biology. Argonaute proteins bind small RNA and execute gene silencing. The number of Argonaute paralogs range from 5 in Drosophila melanogaster , 8 in Homo sapiens to an astounding 27 in Caenorhabditis elegans. This begs several questions: Do Argonaute proteins have different small RNA repertoires? Do Argonaute proteins behave differently? And if so, how are they functionally and mechanistically distinct?
To address these questions, we examined the thermodynamic, kinetic and functional properties of fly Argonaute1 (dAgo1), fly Argonaute2 (dAgo2) and mouse Argonaute2 (mAGO2). Our studies reveal that in fly, small RNA duplexes sort into Argonaute proteins based on their intrinsic structures: extensively paired siRNA duplex is preferentially sorted into dAgo2 while imperfectly paired miRNA duplex is channeled into dAgo1. The sorting of small RNA is uncoupled from its biogenesis. This is exemplified by mir-277, which is born a miRNA but its extensive duplex structure licenses its entry into dAgo2. In the Argonaute protein, the small RNA guide partitions into functional domains: anchor, seed, central, 3' supplementary and tail. Of these domains, the seed initiates binding to target.
Both dAgo2 and mAGO2 (more closely related to and a surrogate for dAgo1 in our studies) bind targets at astonishing diffusion-limited rates (~107–108 M−1s−1). The dissociation kinetics between dAgo2 and mAGO2 from their targets, however, are different. For a fully paired target, dAgo2 dissociates slowly (t½ ~2 hr) but for a seed-matched target, dAgo2 dissociates rapidly (t½ ~20 s). In comparison, mAGO2 does not discriminate between either targets and demonstrates an equivalent dissociation rate (t½ ~20 min). Regardless, both dAgo2 and mAGO2 demonstrate high binding affinity to perfect targets with equilibrium dissociation constants, KD ~4–20 pM. Functionally, we also showed that dAgo1 but not dAgo2 silence a centrally bulged target. By contrast, dAgo2 cleaved and destroyed perfectly paired targets 43-fold faster than dAgo1. In target cleavage, dAgo2 can tolerate mismatches, bulged and internal loop in the target but at the expense of reduced target binding affinities and cleavage rates.
Taken together, our studies indicate that small RNAs are actively sorted into different Argonaute proteins with distinct thermodynamic, kinetic and functional behaviors. Our quantitative biochemical analysis also allows us to model how Argonaute proteins find, bind and regulate their targets.
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Wee, Liang Meng. "RNA Interference by the Numbers: Explaining Biology Through Enzymology: A Dissertation." eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/661.
Full textAbstract:
Small silencing RNAs function in almost every aspect of cellular biology. Argonaute proteins bind small RNA and execute gene silencing. The number of Argonaute paralogs range from 5 in Drosophila melanogaster , 8 in Homo sapiens to an astounding 27 in Caenorhabditis elegans. This begs several questions: Do Argonaute proteins have different small RNA repertoires? Do Argonaute proteins behave differently? And if so, how are they functionally and mechanistically distinct?
To address these questions, we examined the thermodynamic, kinetic and functional properties of fly Argonaute1 (dAgo1), fly Argonaute2 (dAgo2) and mouse Argonaute2 (mAGO2). Our studies reveal that in fly, small RNA duplexes sort into Argonaute proteins based on their intrinsic structures: extensively paired siRNA duplex is preferentially sorted into dAgo2 while imperfectly paired miRNA duplex is channeled into dAgo1. The sorting of small RNA is uncoupled from its biogenesis. This is exemplified by mir-277, which is born a miRNA but its extensive duplex structure licenses its entry into dAgo2. In the Argonaute protein, the small RNA guide partitions into functional domains: anchor, seed, central, 3' supplementary and tail. Of these domains, the seed initiates binding to target.
Both dAgo2 and mAGO2 (more closely related to and a surrogate for dAgo1 in our studies) bind targets at astonishing diffusion-limited rates (~107–108 M−1s−1). The dissociation kinetics between dAgo2 and mAGO2 from their targets, however, are different. For a fully paired target, dAgo2 dissociates slowly (t½ ~2 hr) but for a seed-matched target, dAgo2 dissociates rapidly (t½ ~20 s). In comparison, mAGO2 does not discriminate between either targets and demonstrates an equivalent dissociation rate (t½ ~20 min). Regardless, both dAgo2 and mAGO2 demonstrate high binding affinity to perfect targets with equilibrium dissociation constants, KD ~4–20 pM. Functionally, we also showed that dAgo1 but not dAgo2 silence a centrally bulged target. By contrast, dAgo2 cleaved and destroyed perfectly paired targets 43-fold faster than dAgo1. In target cleavage, dAgo2 can tolerate mismatches, bulged and internal loop in the target but at the expense of reduced target binding affinities and cleavage rates.
Taken together, our studies indicate that small RNAs are actively sorted into different Argonaute proteins with distinct thermodynamic, kinetic and functional behaviors. Our quantitative biochemical analysis also allows us to model how Argonaute proteins find, bind and regulate their targets.
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Kittler, Ralf. "Functional genomic analysis of cell cycle progression in human tissue culture cells." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2006. http://nbn-resolving.de/urn:nbn:de:swb:14-1161253856455-48321.
Full textAbstract:
The eukaryotic cell cycle orchestrates the precise duplication and distribution of the genetic material, cytoplasm and membranes to daughter cells. In multicellular eukaryotes, cell cycle regulation also governs various organisatorial processes ranging from gametogenesis over multicellular development to tissue formation and repair. Consequently, defects in cell cycle regulation provoke a variety of human cancers. A global view of genes and pathways governing the human cell cycle would advance many research areas and may also deliver novel cancer targets. Therefore this work aimed on the genome-wide identification and systematic characterisation of genes required for cell cycle progression in human cells. I developed a highly specific and efficient RNA interference (RNAi) technology to realize the potential of RNAi for genome-wide screening of the genes essential for cell cycle progression in human tissue culture cells. This approach is based on the large-scale enzymatic digestion of long dsRNAs for the rapid and cost-efficient generation of libraries of highly complex pools of endoribonuclease-prepared siRNAs (esiRNAs). The analysis of the silencing efficiency and specificity of esiRNAs and siRNAs revealed that esiRNAs are as efficient for mRNA degradation as chemically synthesized siRNA designed with state-of-the-art design algorithms, while exhibiting a markedly reduced number of off-target effects. After demonstrating the effectiveness of this approach in a proof-of-concept study, I screened a genome-wide esiRNA library and used three assays to generate a quantitative and reproducible multi-parameter profile for the 1389 identified genes. The resulting phenotypic signatures were used to assign novel cell cycle functions to genes by combining hierarchical clustering, bioinformatics and proteomic data mining. This global perspective on gene functions in the human cell cycle presents a framework for the systematic documentation necessary for the understanding of cell cycle progression and its misregulation in diseases. The identification of novel genes with a role in human cell cycle progression is a starting point for an in-depth analysis of their specific functions, which requires the validation of the observed RNAi phenotype by genetic rescue, the study of the subcellular localisation and the identification of interaction partners of the expressed protein. One strategy to achieve these experimental goals is the expression of RNAi resistant and/or tagged transgenes. A major obstacle for transgenesis in mammalian tissue culture cells is the lack of efficient homologous recombination limiting the use of cultured mammalian cells as a real genetic system like yeast. I developed a technology circumventing this problem by expressing an orthologous gene from a closely related species including its regulatory sequences carried on a bacterial artificial chromosome (BAC). This technology allows physiological expression of the transgene, which cannot be achieved with conventional cDNA expression constructs. The use of the orthologous gene from a closely related species confers RNAi resistance to the transgene allowing the depletion of the endogenous gene by RNAi. Thus, this technology mimics homologous recombination by replacing an endogenous gene with a transgene while maintaining normal gene expression. In combination with recombineering strategies this technology is useful for RNAi rescue experiments, protein localisation and the identification of protein interaction partners in mammalian tissue culture cells. In summary, this thesis presents a major technical advance for large-scale functional genomic studies in mammalian tissue culture cells and provides novel insights into various aspects of cell cycle progression. (Die Druckexemplare enthalten jeweils eine CD-ROM als Anlagenteil: 217 MB: Movies, Rohdaten - Nutzung: Referat Informationsvermittlung der SLUB)
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Bhattacharjee, Puranjoy. "Correlation Between Computed Equilibrium Secondary Structure Free Energy and siRNA Efficiency." Thesis, Virginia Tech, 2009. http://hdl.handle.net/10919/34643.
Full textAbstract:
We have explored correlations between the measured efficiency of the RNAi process and
several computed signatures that characterize equilibrium secondary structure of the partic-
ipating mRNA, siRNA, and their complexes. A previously published data set of 609 exper-
imental points was used for the analysis. While virtually no correlation with the computed
structural signatures are observed for individual data points, several clear trends emerge
when the data is averaged over 10 bins of N â ¼ 60 data points per bin.
The strongest trend is a positive linear (r 2 = 0.87) correlation between ln(remaining mRNA)
and â Gms , the combined free energy cost of unraveling the siRNA and creating the break
in the mRNA secondary structure at the complementary target strand region. At the same
time, the free energy change â Gtotal of the entire process mRNA + siRNA â (mRNA â
siRNA)complex is not correlated with RNAi efficiency, even after averaging. These general
findings appear to be robust to details of the computational protocols. The correlation be-
tween computed â Gms and experimentally observed RNAi efficiency can be used to enhance
the ability of a machine learning algorithm based on a support vector machine (SVM) to
predict effective siRNA sequences for a given target mRNA. Specifically, we observe modest,
3 to 7%, but consistent improvement in the positive predictive value (PPV) when the SVM
training set is pre- or post-filtered according to a â Gms threshold.
Master of Science
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Stadler, Bradford Michael. "Interaction of a Mammalian Virus with Host RNA Silencing Pathways: A Dissertation." eScholarship@UMMS, 2007. https://escholarship.umassmed.edu/gsbs_diss/322.
Full textAbstract:
In the complex relationships of mammalian viruses with their hosts, it is currently unclear as to what role RNA silencing pathways play during the course of infection. RNA silencing-based immunity is the cornerstone of plant and invertebrate defense against viral pathogens, and examples of host defense mechanisms and numerous viral counterdefense mechanisms exist. Recent studies indicate that RNA silencing might also play an active role in the context of a mammalian virus infection. We show here that a mammalian virus, human adenovirus, interacts with RNA silencing pathways during infection, as the virus produces microRNAs (miRNAs) and regulates the expression of Dicer, a key component of RNA silencing mechanisms.
Our work demonstrates that adenovirus encodes two miRNAs within the loci of the virus-associated RNA I (VA RNA I). We find that one of these miRNAs, miR-VA “g”, enters into a functional, Argonaute-2 (Ago-2)-containing silencing complex during infection. Currently, the cellular or viral target genes for these miRNAs remain unidentified. Inhibition of the function of the miRNAs during infection did not affect viral growth in a highly cytopathic cell culture model. However, studies from other viruses implicate viral miRNAs in the establishment of latent or chronic infections.
Additionally, we find that adenovirus infection leads to the reduced expression of Dicer. This downregulation does not appear to be dependent on the presence of VA RNA or its associated miRNAs. Rather, Dicer levels appear to inversely correlate with the level of viral replication, indicating that another viral gene product is responsible for this activity. Misregulation of Dicer expression does not appear to influence viral growth in a cell culture model of infection, and also does not lead to gross changes in the pool of cellular miRNAs. Taken together, our results demonstrate that RNA silencing pathways are active participants in the process of infection with human adenovirus. The production of viral miRNAs and the regulation of cellular Dicer levels during infection implicate RNA silencing mechanisms in both viral fitness as well as potential host defense strategies.
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Werth, Stephanie [Verfasser], and Achim [Akademischer Betreuer] Aigner. "Neue Gentargeting-Strategien auf Basis der RNA interference (RNAi) / Stephanie Werth. Betreuer: Achim Aigner." Marburg : Philipps-Universität Marburg, 2013. http://d-nb.info/1038786754/34.
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Kandan-Kulangara, Febitha. "Poly(ADP-ribose) polymerase-1 (PARP-1) and RNA interference (RNAI) during cell death." Doctoral thesis, Université Laval, 2013. http://hdl.handle.net/20.500.11794/25972.
Full textAbstract:
L’activation de la poly(ADP-ribose) polymérase-1 (PARP-1) en réponse aux dommages à l’ADN est impliquée dans diverses réponses cellulaires, de la réparation de l’ADN à la mort cellulaire. Dans l'annexe I, nous avons décrit différentes techniques indispensables pour détecter le métabolisme de PARP-1 en réponse aux dommages à l’ADN in vitro et in vivo. Les travaux de cette thèse se concentrent sur le rôle de PARP-1 dans la mort cellulaire. PARP-1 est clivée et inactivée par des caspases pendant l’apoptose ; j’ai donc utilisé une PARP-1 non-clivable pour étudier le rôle de l’activation et de la fragmentation de PARP-1 dans la mort cellulaire induite par les UVB. Nous avons observé que, contrairement aux fibroblastes de peau humaine exprimant la PARP-1, les fibroblastes avec un "knockdown" de PARP-1 sont résistants à l’apoptose induite par les UVB, phénotype pouvant être totalement inversé par ré-expression de PARP-1 sauvage mais pas de PARP-1 non-clivable par les caspases, suggérant un rôle significatif du clivage de PARP-1 en réponse à la mort cellulaire induite par les UVB (chapitre 2). Dans ce contexte, nous avons récemment passé en revue comment les substrats non clivables par des caspases peuvent être utilisés comme outil important pour démystifier le rôle de ce clivage pour la mort comme pour la vie, avec l’exemple spécifique de PARP-1 non-clivable par les caspases (chapitre 3). Curieusement, en utilisant l’ARNi comme outil d’étude du rôle de PARP-1 dans la mort cellulaire, nous avons observé que l’ARNi stable (shRNA) de nombreux gènes, incluant PARP-1, échoue lors de l’apoptose, en raison de l’inactivation catalytique par clivage par une caspase de l’endoribonucléase Dicer-1, indispensable pour la régulation de l’ARNi et des miARN (chapitre 4). Cependant, nous avons découvert que l’ARNi transitoire persiste plusieurs jours même après induction de l’apoptose, soulignant des différences entre les ARNi stable et transitoire dans la dynamique de "knockdown" génétique et dans la dépendance de la fonction de Dicer-1 (chapitre 5). En résumé, mon travail a permis la découverte des avantages et des limites de l’ARNi durant l’apoptose et le rôle de PARP-1 dans la mort cellulaire induite par les UVB.
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Ellegast, Jana. "Interferon-Induktion durch Triphosphat-RNA." Diss., lmu, 2010. http://nbn-resolving.de/urn:nbn:de:bvb:19-113843.
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Seth, Meetu. "Functions of Argonaute Proteins in Self Versus Non-Self Recognition in the C. elegans Germline: A Dissertation." eScholarship@UMMS, 2008. http://escholarship.umassmed.edu/gsbs_diss/874.
Full textAbstract:
Organisms employ sophisticated mechanisms to silence foreign nucleic acid, such as viruses and transposons. Evidence exists for pathways that sense copy number, unpaired DNA, or aberrant RNA (e.g., dsRNA), but the mechanisms that distinguish “self” from “non-self” are not well understood. Our studies on transgene silencing in C. elegans have uncovered an RNA surveillance system in which the PIWI protein, PRG-1, uses a vast repertoire of piRNAs to recognize foreign transcripts and to initiate epigenetic silencing. Partial base pairing by piRNAs is sufficient to guide PRG-1 targeting. PRG-1 in turn recruits RdRP to synthesize perfectly matching antisense siRNAs (22G-RNAs) that are loaded onto worm-specific Argonaute (WAGO) proteins. WAGOs collaborate with chromatin factors to maintain epigenetic silencing (RNAe). Since mismatches are allowed during piRNA targeting, piRNAs could—in theory— target any transcript expressed in the germline, but germline genes are not subject to silencing by RNAe. Moreover, some foreign sequences are expressed and appear to be adopted as “self.” How are “self” transcripts distinguished from foreign transcripts? We have found that another Argonaute, CSR-1, and its siRNAs—also synthesized by RdRP—protect endogenous genes from silencing by RNAe. We refer to this pathway as RNA-mediated gene activation (RNAa). Reducing CSR-1 or PRG-1 or increasing piRNA targeting can shift the balance towards expression or silencing, indicating that PRG-1 and CSR-1 compete for control over their targets. Thus worms have evolved a remarkable nucleic acids immunity mechanism in which opposing Argonaute pathways generate and maintain epigenetic memories of self and non-self nucleotide sequences.
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Seth, Meetu. "Functions of Argonaute Proteins in Self Versus Non-Self Recognition in the C. elegans Germline: A Dissertation." eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/874.
Full textAbstract:
Organisms employ sophisticated mechanisms to silence foreign nucleic acid, such as viruses and transposons. Evidence exists for pathways that sense copy number, unpaired DNA, or aberrant RNA (e.g., dsRNA), but the mechanisms that distinguish “self” from “non-self” are not well understood. Our studies on transgene silencing in C. elegans have uncovered an RNA surveillance system in which the PIWI protein, PRG-1, uses a vast repertoire of piRNAs to recognize foreign transcripts and to initiate epigenetic silencing. Partial base pairing by piRNAs is sufficient to guide PRG-1 targeting. PRG-1 in turn recruits RdRP to synthesize perfectly matching antisense siRNAs (22G-RNAs) that are loaded onto worm-specific Argonaute (WAGO) proteins. WAGOs collaborate with chromatin factors to maintain epigenetic silencing (RNAe). Since mismatches are allowed during piRNA targeting, piRNAs could—in theory— target any transcript expressed in the germline, but germline genes are not subject to silencing by RNAe. Moreover, some foreign sequences are expressed and appear to be adopted as “self.” How are “self” transcripts distinguished from foreign transcripts? We have found that another Argonaute, CSR-1, and its siRNAs—also synthesized by RdRP—protect endogenous genes from silencing by RNAe. We refer to this pathway as RNA-mediated gene activation (RNAa). Reducing CSR-1 or PRG-1 or increasing piRNA targeting can shift the balance towards expression or silencing, indicating that PRG-1 and CSR-1 compete for control over their targets. Thus worms have evolved a remarkable nucleic acids immunity mechanism in which opposing Argonaute pathways generate and maintain epigenetic memories of self and non-self nucleotide sequences.
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Saadat, Angela P. "Identifying Novel Contributors to RNA Interference in Aedes aegypti." Thesis, Virginia Tech, 2015. http://hdl.handle.net/10919/75141.
Full textAbstract:
Aedes aegypti is an important vector of human pathogens including the viruses yellow fever, dengue and chikungunya. The small interfering RNA (siRNA) pathway is a critical immune response for controlling viral replication in Aedes aegypti. The goal of this research is to identify components of the Aedes aegypti genome that influence this pathway.
A transgenic mosquito strain that reports the status of the siRNA pathway via enhanced green fluorescent protein (EGFP) intensity was employed to differentiate silencing abilities among individuals. Extreme EGFP expression phenotypes, representing efficient and poor silencing abilities, were enriched over five generations.
Transcriptome sequencing and analyses were performed from pools of individuals from each enriched phenotype, revealing potential RNAi contributors. 1,120 transcripts were significantly different (FDR<0.0001) among the extreme phenotypes.
Four genes were chosen, amplified, sequenced for SNP analysis. These analyses were performed on samples obtained by crossing enriched, extreme phenotype F0 individuals, intercrossing their progeny, then selecting individuals representing the extreme phenotypes from the F2 population. Though further verification is needed, findings from these analyses imply the regions of Aedes aegypti, Liverpool strain (AAEL) gene identifiers AAEL005026, AAEL013438 and AAEL011704 amplified do not contribute to the two extreme, opposite RNAi silencing in the sensor strain used here. SNP analyses of AAEL000817 indicate this gene either influences extreme RNAi phenotypes or is closely linked to a gene(s) that contributes to RNAi in Aedes aegypti.
The 1,120 genes identified can be validated or eliminated as potential targets in the quest to mitigate the impact of Aedes aegypti.Master of Science in Life Sciences
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Araujo, Patricia Aline Oliveira Ribeiro de Aguiar. "Analise de expressão do gene Lgi1 durante o desenvolvimento do sistema nervoso central e seu silenciamento utilizando a tecnica de interferencia por RNA." [s.n.], 2008. http://repositorio.unicamp.br/jspui/handle/REPOSIP/309740.
Full textAbstract:
Orientador: Iscia Teresinha Lopes-CendesTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: Introdução/Objetivo: Mutações no gene LGI1 foram descritas como causa da Epilepsia Parcial Autossômica Dominante com Sintomas Auditivos em algumas famílias. Alguns estudos apontam para um possível envolvimento do gene LGI1 com migração e/ou proliferação neuronal, porém a função exata desse gene permanece desconhecida. O objetivo deste trabalho foi determinar o perfil de expressão do gene Lgi1 em cérebro de camundongos durante o desenvolvimento do sistema nervoso central (SNC) e na fase adulta e, ainda, silenciar este gene em cérebros de camundongos utilizando a técnica de interferência por RNA (RNAi). Métodos: Acasalamentos programados foram realizados, utilizando camundongos Balb/c, para a obtenção de fetos de diferentes idades. Os cérebros de três animais, nas seguintes idades, foram retirados e os hemisférios direito e esquerdo separados: E15, E17, E18 dias (E:dias embrionários), P1, P7, P14 dias (P: dias pós-natal), 4, 6, 8 e 24 semanas. Também utilizamos cabeças inteiras de animais E13. Além disso, foram utilizados três animais adultos para a análise do gene Lgi1 em neocórtex, hipocampo e cerebelo. O perfil de expressão gênico foi determinado pela PCR em tempo real utilizando o sistema TaqMan® e por western blot. A técnica de RNAi foi realizada utilizando diferentes métodos de introdução de pequenas moléculas interferentes no cérebro de animais neonatos e adultos. Utilizamos também vários parâmetros diferentes no que se refere ao desenho das moléculas interferentes, suas concentrações, o local e o número de injeções. Além disso, experimentos de RNAi in vitro foram realizados, utilizando uma linhagem celular de glioblastoma humano, a U138MG, e uma linhagem de neuroblastoma murino, a Neuro2a. A confirmação do silenciamento gênico foi feita por PCR em tempo real e, em alguns experimentos, também por western blot. Resultados: A expressão do gene Lgi1 se apresentou baixa durante as idades intra-uterinas, aumentando progressivamente. Os animais em idade adulta apresentaram um aumento de expressão de 35 vezes quando comparadas às amostras E13, utilizando Gapdh como normalizador e de aproximadamente 28 vezes, utilizando ?-actina. Embora o teste estatístico não tenha encontrado diferença na expressão do gene Lgi1 entre os hemisférios cerebrais, ele revelou uma diferença significativa entre as idades estudadas. Os experimentos de western blot confirmaram o perfil de expressão determinado pelos estudos de PCR em tempo real, encontrando-se, a proteína Lgi1, em maior quantidade nas idades mais avançadas analisadas. O estudo de expressão das três regiões do cérebro não resultou em diferença estatisticamente significativa. O silenciamento do gene Lgi1 foi realizado com sucesso pela técnica de RNAi, em cérebro de animais adultos, sendo que os resultados mais consistentes, redução da expressão de aproximadamente 50%, foram observados com o método de eletroporação local e confirmação do silenciamento por PCR em tempo real. Além disso, nós conseguimos demonstrar silenciamento de até 99% do gene Lgi1 em cultura de células. Conclusões/Discussão: O padrão de expressão baixo do gene Lgi1 durante o desenvolvimento, com aumento progressivo e alta expressão na idade adulta aponta para uma potencial função inibitória da proliferação celular. Tal suposição encontra apoio em achados de neuroimagem de alguns pacientes com mutação em LGI1. O silenciamento do gene Lgi1 em cérebro de camundongos, utilizando a técnica de RNAi, foi alcançado, porém com grande dificuldade técnica. Esses obstáculos encontrados apontam para a existência de possíveis características moleculares próprias do gene LGI1 que poderiam dificultar seu silenciamento pela técnica da RNAi, tais como: um RNA mensageiro rico em proteínas associadas, impedindo o acesso da maquinaria de RNAi ou, ainda, LGI1 poderia ser um gene essencial, onde a diminuição de sua expressão ativaria processos celulares compensatórios
Abstract: Introduction/Objectives: Mutations in the LGI1 gene were described in some patients with autosomal dominant partial epilepsy with auditory features and preliminary functional studies point to a possible involvement of LGI1 with migration and/or neuronal proliferation. However, the precise function of LGI1 remains unknown. The objective of the present study was to determine the expression pattern of the Lgi1 gene in mice brain during central nervous system (CNS) development and in adult animals. In addition, we aimed to silence Lgi1 in mouse brain using RNA interference (RNAi). Methods: Programmed mating was carried with Balb/c mice in order to obtain embryos of different ages. The brains of three animals at the following ages were removed and the right and left hemispheres were separated: E15, E17, E18 days (E: embryo days), P1, P7, P14 (P: post-natal days), 4, 6, 8 and 24 weeks old. We also studied E13 whole head animals. In addition we studied three different regions from 5 weeks-old animal brains: neocortex, hippocampus and cerebellum. Gene expression assays were carried out using real time PCR with the TaqMan® system and western blot experiments. RNAi was performed using different methods for injection of interfering molecules into the neonate and adult brains. We also used different molecule designs and concentrations, as well as the number and the local of injections was varied. Furthermore, we performed in vitro RNAi experiments using a glioblastoma cell line, U138MG, and a murine cell line, Neuro2a. Gene silencing confirmation was carried out by real time PCR and western blot assays. Results: Lgi1 gene expression was significantly low during the intrauterine ages increasing progressively until the adult stages. Samples from adult animals presented a 35 fold increase in expression as compared to E13 samples, using Gapdh as endogenous control, and when we use ?-actin, adult samples presented approximately 28 fold increase in Lgi1 expression. There were no statistical differences between Lgi1 gene expression test between right and left hemispheres. However, a significant difference in expression was found among the different ages studied. The western blot showed higher expression of the Lgi1 protein in the most advanced ages analyzed, confirming the expression profile observed in the real time PCR studies. However, we did not find any statistic difference between the three regions of the brain studied. In addition, we achieved significant gene silencing of Lgi1, reduction of expression of approximately 50%, in brain of adult animals using RNAi and the local electroporation method. In addition, we demonstrated up to 99% silencing of LGI1 in cell culture. Conclusions/Discussion: The Lgi1 expression profile, which is characterized by low expression in the initial stages of development with progressive increase as the animal developed, could be explained by a possible inhibitory functional role in neuronal proliferation during CNS development. Lgi1 gene silencing in adult brain using RNAi technique was achieved after several attempts. These difficulties in gene silencing, point to the presence of intrinsic molecular characteristics of LGI1 which could be preventing silencing by RNAi, such as a message RNA too rich of associated proteins that may impairing the action of RNAi machinery; or LGI1 could be an essential gene, with very strong and stringent compensatory mechanisms; therefore, when attempting to decreased its expression one would activate the compensatory processes
Doutorado
Neurociencias
Doutor em Fisiopatologia Medica
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Zhuang, Jimmy Jiajia. "Phenotypes and genetic mechanisms of C. elegans enhanced RNAi." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:10758.
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RNA interference (RNAi) potently and specifically induces gene knockdown, and its potential for reverse genetics in Caenorhabditis elegans is enormous. However, even in these nematodes, RNAi can be induced more effectively via enhanced RNAi (Eri) mutant backgrounds. With advances in small RNA sequencing, evidence has suggested that the eri pathway plays an endogenous gene regulatory role, which competes with experimentally introduced RNAi triggers for limiting resources. However, the nature, cellular location, and physiological consequences of this small RNA pathways competition remain unclear. To answer these questions, I first fully characterized the genetic phenotypes of all known Eri mutants. I discovered that different components of the eri pathway have subtle differences upon mutation, which affects more than exogenous RNAi. I then attempted to screen for novel enhanced RNAi mutants, guided by hypothetical mechanisms or tissues of expression not associated with known mutants. After these attempts, I fully characterized the genetic mechanisms that account for enhanced RNAi. Surprisingly, I discovered that the nuclear Argonaute nrde-3 and the peri-nuclear P-granule component pgl-1 are necessary and sufficient for an Eri response. Finally, I examined the impact of the competition among microRNA, endogenous siRNA, and exogenous RNAi pathways. I discovered that C. elegans develops slower upon perturbations to its normal flux of small RNA pathways. Insights from these phenotypes and genetic mechanisms shed light on the importance of small RNA biology and offer a novel suite of tools for sensitizing RNAi in broader contexts, especially given the deep evolutionary conservation of most eri-associated genes.
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Ono, Ekaterina Alexandrovna Durymanova. "Silenciamento gênico pós-transicional por interferência por RNA (RNAi) com terapia antiviral para a raiva." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-18082015-153913/.
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A raiva é uma zoonose que afeta todos os mamíferos e causa cerca de 55.000 mortes humanas por ano, causada pelo vírus da raiva. O vírus da raiva pertence à Ordem Mononegavirales, Família Rhabdoviridae e o Gênero Lyssavirus. Ultimamente, o Protocolo de Milwaukee é a base do tratamento humano, com indução do paciente ao coma e uso de massiva terapia antiviral. O protocolo, embora tenha sido utilizado duas vezes com sucesso, inclusive em um caso brasileiro, ainda requer aperfeiçoamentos. Neste sentido, a interferência por RNA (RNAi) é uma nova abordagem para terapia de doenças virais. O objetivo deste trabalho foi avaliar a inibição da replicação do vírus da raiva in vitro e in vivo utilizando RNAi. Para este fim, foram utilizados três siRNAs (siRNA 360, siRNA 652, siRNA 649) com a fita antisenso complementar ao mRNA da fosfoproteína (P) e três siRNAs (Le 1, Le 2, Le 3) contra o RNA líder do vírus da raiva. Para o ensaio in vitro foram utilizadas as amostras PV e 4005 (AgV3) do vírus da raiva e as células de BHK-21 (Baby hamster kidney). As monocamadas celulares foram infectadas com as amostras PV ou 4005 e depois de 2 horas de incubação transfectadas com cada um dos siRNAs em combinação com Lipofectamine 2000TM. Depois de 24 e 48 horas as placas teste e controle foram submetidas à imunofluorescência direta (IFD) com conjugado globulina de coelho anti-ribonucleocapsídeo do vírus da raiva/isotiocianato de fluoresceína (Instituto Pasteur de São Paulo). Os resultados revelaram que os siRNAs contra o RNA líder do vírus não foram capazes de inibir a replicação do vírus. A utilização dos siRNAs contra mRNA P resultaram em títulos de 3,625logTCID50/ml, 3,875logTCID50/ml e 4,125logTCID50/ml para os siRNAs 360, 649 e 652, respectivamente, enquanto que, para a placa controle, o título foi 4,0logTCID50/ml nas placas infectadas com PV e período de incubação de 24h. Nas placas infectadas com a amostra 4005 e tratadas com siRNAs, a maior queda de título viral foi na placa tratada com siRNA 360, de 1,0 log, comparando-se com a placa controle de incubação de 24 para a amostra 4005. Nas placas tratadas com siRNA 649 e siRNA 652, também houve a diminuição de título viral, mas em uma escala menor (0,25log e 0,125log, respectivamente) comparando-se com o controle. Nas placas infectadas com PV e incubadas durante 48h, os títulos apresentados foram de 5,625logTCID50/ml, 4,625logTCID50/ml e 4,75logTCID50%/ml para os siRNAs 360, 649 e 652, respectivamente, enquanto que na placa controle o título foi 6,0logTCID50C%/ml. A placa com período de incubação de 48h com a amostra 4005 e tratada com siRNA 360 apresentou a maior queda de título viral entre os três siRNAs, o que resultou em 1,125log de diferença. Nas monocamadas onde foram administrados siRNA 649 e siRNA 652, observou-se também uma pequena queda de título viral igual a 0,875log e 0,295log, respectivamente, comparando-se com a placa não tratada. Para o ensaio in vivo, foram usados camundongos albino suíços de 21 dias com peso entre 11 e 14g, infectados com a cepa PV e AgV3 em 10DL50% via intracerebral. Duas horas depois da infecção, foi inoculada por via intracerebral uma solução do siRNA 360 com Lipofectamine 2000TM. Os animais com paralisia foram eutanasiados e aqueles sobreviventes foram observados até completar 30 dias de observação quando foram, então, eutanasiados. O sistema nervoso central de todos os animas foi recolhido e submetido a IFD. A utilização do siRNA 360 em camundongos resultou em 30% de animais sobreviventes frente amostra 4005, enquanto que a mortalidade nos animais não tratados foi de 90%. Nos animais inoculados com a amostra PV e tratados com este siRNA, a sobrevivência foi de 40%, enquanto que no grupo controle a mortalidade foi de 100%. O resultado do ensaio in vitro demonstra que os siRNAs utilizados são capazes de inibir a replicação do vírus da raiva, com eficiência mais pronunciada para o siRNA 360. In vivo, este siRNA foi capaz de induzir a proteção parcial dos animais inoculados com as duas variantes virais. Estes resultados, ainda que indiquem a necessidade de mais estudos, permitem concluir que a RNAi é uma tecnologia promissora como antiviral contra a raivaRabies is a zoonotic disease that affects all mammals and causes more than 55.000 human deaths every year, caused by rabies virus (RABV) a virus of the Mononegavirales order, Family Rhabdoviridae and the Lyssavirus genus. After the onset of the symptoms, the illness has a fast progression and the patients feel intense physical suffering. Currently, human rabies treatment has been based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. Despite this protocol has been successful in two cases, including a Brazilian one, more studies on antivirals for human rabies treatment are required. RNA interference is a new antiviral approach, which gives hope to the possibility of rabies antiviral treatment. The aim of this study was to assess the decrease in titres of rabies virus in vitro and in vivo using short-interfering RNAs. To this end, three siRNAs (siRNA 360, siRNA 652, and siRNA 649) were used with antisense strands complementary to rabies virus phosphoprotein (P) mRNA and three other (Le 1, Le 2, Le 3) to the leader RNA. Pasteur virus strain (PV) and strain 4005 (AgV3) of rabies virus and BHK-21 cells were used, and the monolayers were transfected with each of the RNAs with Lipofectamine-2000 TM. After 22 hours, the siRNA-treated and the control plates were tested by direct fluorescent antibody test (DFAT) with anti-rabies virus nucleocapsid antibody conjugate with fluorescein isothiocianate (Pasteur Insitutte, Brazil). The plates transfected with siRNA against phosphoprotein mRNA were also incubated for 48 hours and subjected to IFD assay. Virus titres were calculated by the Spearman-Karber method. The results showed that siRNAs against virus leader RNA were not able to inhibit the replication of the virus. The use of siRNAs against P mRNA resulting titres of 3.625logTCID50/ml 3.875logTCID50/ml and 4.125logTCID50/ml for siRNAs 360, 649 and 652, respectively, while, for the control plate, the titre was 4.0logTCID50/ml in plates with PV and 24h incubation period. In plates with strain 4005 and treated with siRNAs, the highest viral titre decrease was obtained with siRNA 360, with a 1.0 log difference compared to the control plate of strain 4005 incubated for 24h. The plates treated with siRNA 649 and siRNA 652 have was also shown a decrease in viral titres, but on a smaller scale (0.25log and 0.125log, respectively) compared to the control. The plates infected with PV and incubated for 48 hours showed titre of 5.625logTCID50/ml, 4.625logTCID50/ml and 4.75logTCID50%/ml for siRNAs 360, 649 and 652, respectively, while for the control plate the titre was 6.0logTCID50C%/ml. The plate with strain 4005 and then treated with siRNA360 and incubated for a total of 48h had the highest viral titre decrease among the three siRNAs, which resulted in a 1.125log difference compared to the control plate. In monolayers treated with siRNA649 and siRNA652 there was also a discrete drop in viral titres (0.875log and 0.295log, respectively) compared to the control plate. For the in vivo assay, 21-day old Swiss albino mice weighing between 11 and 14g were intracerebrally inoculated with PV or 4005 strains (10DL50%). Two hours after inoculation, a solution of siRNA360 with Lipofectamine 2000 TM was also intracerebrally injected. Mice presenting paralysis and those that survived the 30 days of observation were euthanized. The central nervous system of all animals was collected and submitted to IFD. The use of siRNA360 in mice resulted in survival of 30% of animals in the group inoculated with strain 4005, whereas 90% mortality was observed in the control group. In animals inoculated with the PV strain, and treated with siRNA360, the survival rate was 40% and in the control group the mortality was 100%. The results of the in vitro assay demonstrate that the siRNAs used are effective in inhibiting the replication of rabies virus with a more intense inhibition regarding siRNA 360. In vivo, this siRNA was able to induce partial protection of animals infected with both viral variants. These results also indicate that, despite the need for further studies, RNAi is a promising technology as antiviral against rabies
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Sharma, Nidhi. "Developmental expression analysis and RNA interference (RNAi) screen of putative neuromuscular receptors of «Schistosoma mansoni»." Thesis, McGill University, 2014. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=123300.
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In parasitic platyhelminths, including Schistosoma mansoni the coordination of neuromuscular system is critical for continued propagation, development and successful completion of the lifecycle. Neuromuscular signaling in these parasites is mediated by a variety of neurotransmitters, both small molecules ("classical") transmitters and neuropeptides. Biogenic amines (BA) constitute the largest subset of classical neurotransmitters and play several key roles in the control of schistosome muscle function and movement. There are several putative BA receptors identified in the S. mansoni genome, the majority of which are Class A G protein coupled receptors (GPCRs). Here we report the functional role of these putative BA receptors in parasite development and motility by developmental expression analysis and RNAi screening. We performed an expression analysis of several putative BA receptors at the RNA level in different developmental stages of the parasite, using reverse-transcription coupled to quantitative PCR (RT-qPCR). One of these proteins is a previously described serotonin receptor of S. mansoni (named Sm5HTR) and the others are novel "orphan" BA-like receptors. The analysis showed that the BA receptors tested are expressed in all developmental stages, however the majority are preferentially expressed in cercaria and schistosomula, suggesting these receptors play particularly important roles in parasite larvae. Next we performed RNA interference (RNAi) targeting the same BA receptors by transfecting S.mansoni larvae with small interfering RNA (siRNA) and analyzed for effects on motor activity by comparing with the control groups. Given that BAs are known modulators of schistosome movement, we hypothesized that the RNAi would produce a motor phenotype in the larvae and this was confirmed by the data. The results identified strongly hypoactive phenotypes for three out of four receptors tested, including Sm5HTR (Smp_126730), Smp_150180 and Smp_120620), all showing significant reduction in movement compared to control larvae transfected with irrelevant (scrambled) siRNAs. The RNAi phenotype correlated with a significant and specific knockdown in transcript levels as determined by RT-qPCR. To elucidate the mode of action of Sm5HTR we also performed confocal immunolocalization analysis using a specific peptide antibody. The expression pattern suggests Sm5HTR is highly abundant in the central and peripheral nervous system of the parasite, including the peripheral innervation of the body wall muscles responsible for movement. Together the results suggest that Sm5HTR and other BA receptors play an important role in the control of schistosome motility, particularly the larvae, and could be potential targets for new drug discovery.En plathelminthes parasites, y compris Schistosoma mansoni la coordination de système neuromusculaire est essentiel pour continuer à se propager, le développement et la réussite du cycle de vie. Signalisation neuromusculaire chez ces parasites est médiée par une variété de neurotransmetteurs, les petites molécules (classique) des émetteurs et des neuropeptides. Les amines biogènes (BA) constituent le plus grand sous-ensemble de neurotransmetteurs classiques et jouent plusieurs rôles clés dans le contrôle de la fonction musculaire schistosome et le mouvement. Il RNA ya plusieurs récepteurs BA putatifs identifiés dans le génome de S. mansoni, dont la majorité sont des récepteurs couplés aux protéines de classe AG (GPCR). Nous rapportons ici le rôle fonctionnel de ces récepteurs putatifs de BA dans le développement du parasite et de la motilité par analyse de l'expression du développement et de dépistage RNAi. Nous avons effectué une analyse de l'expression de plusieurs récepteurs de BA putatifs au niveau de l' dans les différents stades de développement du parasite, en utilisant la transcription inverse couplée à une PCR quantitative (RT- qPCR). Une de ces protéines est un récepteur de la sérotonine décrit précédemment de S. mansoni (nommé Sm5HTR) et les autres sont de nouveaux récepteurs "orphelins" BA -like . L'analyse a montré que les récepteurs de la BA testés sont exprimés dans tous les stades de développement mais la majorité sont préférentiellement exprimé dans les cercaires et schistosomule, suggérant que ces récepteurs jouent un rôle particulièrement important dans les larves de parasite. Suivant nous avons effectué l'interférence RNA (RNAi) de cibler les mêmes récepteurs de BA par transfection larves S.mansoni avec petits RNA interférents (siRNA) et analysé les effets sur l'activité motrice par comparaison avec les groupes témoins. Étant donné que le BAs sont des modulateurs du mouvement schistosome connu, nous avons supposé que l' RNAi serait de produire un phénotype de moteur dans les larves et cela a été confirmé par les données. Les résultats identifiés phénotypes fortement hypoactif pour trois des quatre récepteurs testés, y compris Sm5HTR (Smp_126730), Smp_150180 et Smp_120620), tous montrant une réduction significative de mouvement par rapport à lutter contre les larves transfectées avec non pertinentes (brouillés) siRNA. Le phénotype RNAi corrélée avec un effet de choc important et spécifique dans les niveaux de transcription tel que déterminé par RT- qPCR. Pour élucider le mode d'action de Sm5HTR nous avons également effectué une analyse de immunolocalisation confocale en utilisant un anticorps anti- peptide spécifique. Le profil d'expression suggère Sm5HTR est très abondant dans le système nerveux central et périphérique du parasite, y compris l'innervation périphérique des muscles de la paroi du corps chargés de mouvement. Ensemble, les résultats suggèrent que Sm5HTR et d'autres récepteurs de la BA jouent un rôle important dans le contrôle de la motilité schistosome, en particulier les larves, et pourraient être des cibles potentielles pour la découverte de nouveaux médicaments.
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Shoji, Masanobu. "RNA interference during spermatogenesis in mice." Kyoto University, 2006. http://hdl.handle.net/2433/143821.
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Kim, NaJung. "Rationale design of polymeric siRNA delivery systems." Diss., University of Iowa, 2011. https://ir.uiowa.edu/etd/1237.
Full textAbstract:
Regulation of gene expression using small interfering RNA (siRNA) is a promising strategy for research and treatment of numerous diseases. However, siRNA cannot easily cross the cell membrane due to its inherent instability, large molecular weight and anionic nature. For this reason, a carrier that protects, delivers and unloads siRNA is required for successful gene silencing. The goal of this research was to develop a potential siRNA delivery system for in vitro and in vivo applications using cationic polymers, chitosan and polyethylenimine (PEI), poly(ethylene glycol) (PEG), mannose, and poly(D,L-lactic-co-glycolic acid) (PLGA). Furthermore, the delivery system was constructed in two different ways to explore the effect of mannose location in the structure. In the first approach, mannose and PEG were directly conjugated to the chitosan/PEI backbone, while mannose was connected to the chitosan/PEI backbone through PEG spacer in the second approach. First, the ability of modified chitosan polymers to complex and deliver siRNA for gene silencing was investigated. Despite the modified chitosan polymers successfully formed nanoplexes with siRNA, entered target cells and reduced cytotoxicity of unmodified chitosan, they showed limited gene silencing efficiency. For this reason, modified PEIs were examined to improve in vitro gene knockdown. The modified PEI polymers also complexed with siRNA and facilitated endocytosis of the nanoplexes. In addition, the modifications reduced inherent cytotoxicity of unmodified PEI without compromising the gene silencing efficiency on both mRNA and protein levels. Interestingly, we found that complexation of siRNA with PEI-PEG-mannose resulted in higher cell uptake and gene silencing than complexes made with mannose-PEI-PEG. Finally, the effect of sustained release of the mannosylated pegylated PEI/siRNA nanoplexes on gene silencing was tested by encapsulating the nanoplexes within PLGA microparticles. The modified PEIs enhanced the entrapment efficiency of siRNA into the particles and resulted in reduced initial burst followed by sustained release. Incorporating the modified PEIs increased cellular uptake of siRNA, whereas it did not enhance in vitro gene knockdown efficiency due to the sustained release properties. The modified PEIs reduced the in vitro cytotoxicity and in vivo hepatotoxicity of the PLGA microparticles. In addition, encapsulating the nanoplexes into PLGA microparticles further reduced the cytotoxicity of PEI. Throughout the study, the second structure was proven more efficacious than the first structure in cellular uptake, gene silencing, siRNA encapsulation, and sustained release. We have developed novel polymeric siRNA delivery systems that enhance delivery efficiency and cellular uptake of siRNA. They have great potential for utility as a long-acting siRNA delivery system in biomedical research.
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Soler, Calvo Nuria. "Transgenic resistance against Citrus tristeza virus (CTV) and analysis of the viral p23 protein as pathogenicity determinant in citrus." Doctoral thesis, Universitat Politècnica de València, 2013. http://hdl.handle.net/10251/31631.
Full textAbstract:
El virus de la tristeza de los cítricos (Citrus tristeza virus; CTV) es el agente causal de unas de
las enfermedades virales de los árboles cítricos más devastadoras en el mundo. CTV está restringido al
floema en su huésped cítrico natural, y ha desarrollado tres proteínas supresoras de silenciamiento que
actúan a nivel intra-(p23 y p20) e intercelular (p20 y p25) para superar la fuerte defensa antiviral del
huésped. La interferencia de RNA, una aproximación basada en el uso de dsRNA para desencadenar el
silenciamiento de RNA, ha sido utilizada ampliamente para generar plantas transgénicas resistentes a
virus. Considerando el importante papel de p23, p20 y p25 en la patogénesis de CTV, hemos
transformado plantas de lima Mexicana con un vector intrón-horquilla que porta la secuencia completa en
versión no traducible de los genes p25, p20, p23 y el extremo 3¿-UTR de la cepa T36 de CTV, para
intentar silenciar su expresión en células infectadas.
Se ha observado resistencia completa a la infección viral en tres líneas transgénicas,
manteniéndose todas sus propagaciones asintomáticas y libres de virus tras ser inoculadas mediante
injerto con CTV-T36, tanto en el portainjertos no transgénico como directamente sobre la variedad
transgénica. La acumulación de siRNA derivados del transgén fue necesaria pero no suficiente para lograr
resistencia frente a CTV en las plantas. Al inocular propagaciones de las líneas transgénicas inmunes con
una cepa de CTV divergente, la resistencia fue parcialmente superada, destacando la importancia de la
identidad de secuencia en el mecanismo subyacente a la interferencia de RNA. Este trabajo es el primero
en que se consigue resistencia completa a CTV en un huésped cítrico muy sensible, actuando
simultáneamente sobre los tres supresores virales de silenciamiento mediante interferencia de RNA. La
proteína p23 codificada por el virus es además un importante factor de patogenicidad. La expresión
ectópica de p23 en plantas de cítricos induce aberraciones fenológicas semejantes a síntomas de CTV.
Para estudiar en más detalle el papel de p23 en la patogénesis de CTV, se ha sobre-expresado en lima
Mexicana el gen p23 de CTV T36 y tres versiones truncadas del mismo bajo el control del promotor 35S
del virus del mosaico de la coliflor (Cauliflower mosaic virus). Solo la versión truncada, que expresa los
aminoácidos del 1 al 157 (p23-¿157) indujo síntomas similares a los producidos por CTV, aunque más
suaves que los inducidos por la expresión de la proteína p23 entera (209 aminoácidos), permitiendo
delimitar la región responsable de la patogénesis de p23 en cítricos a un fragmento de 157 aminoácidos
que incluye el dedo de zinc y los motivos básicos flanqueantes de la proteína. La actividad de p23 como
supresor de silenciamiento de RNA en N. benthamiana se perdía en todos los mutantes de p23 probados,
lo cual indica que la supresión de silenciamiento implica a la mayoría de las regiones de la proteína. Para
profundizar más en el papel de p23 en la patogénesis, en un siguiente paso hemos restringido la expresión
de transgenes derivados de p23 a células asociadas al floema de lima Mexicana mediante el uso del
promotor especifico de floema del virus del moteado amarillo de la comelina (Commelina yellow mottle
virus, CoYMV). Se transformó lima Mexicana con construcciones que portaban el gen p23 completo, ya
sea de la cepa agresiva de CTV T36 o de la suave T317, o con un fragmento que comprende el dedo de
zinc y los motivos básicos flanqueantes de la primera, todas ellas bajo el control bien del promotor de
CoYMV o bien del promotor constitutivo 35S. La expresión de estas construcciones en el floema dio
lugar a aberraciones semejantes a los síntomas específicos de CTV, pero no a los síntomas inespecíficos
observados cuando se expresaba p23 de forma constitutiva. Por otra parte, la apariencia e intensidad de
las aberraciones fenotípicas más notorias similares a síntomas inducidos por CTV generadas por la
expresión específica en floema del gen p23 se relacionó positivamente con la agresividad de la cepa
origen utilizada. Además, la expresión en tejidos floemáticos del fragmento de p23 que comprende el
dominio de dedo de zinc y los motivos básicos flanqueantes fue suficiente para inducir síntomas
semejantes a los producidos por la infección con CTV, confirmando así que la región N-terminal
delimitada por los aminoácidos 1 y 157 podría determinar, al menos en parte, la patogénesis de CTV en
lima Mexicana.Citrus tristeza virus (CTV) is the causal agent of one of the most devastating viral diseases of citrus trees in the world. CTV is phloem-restricted in natural citrus hosts, and has evolved three silencing suppressor proteins acting at intra- (p23 and p20) and inter-cellular level (p20 and p25) to overcome strong host antiviral defense in citrus. RNA interference (RNAi), an approach based on using dsRNA to trigger RNA silencing, has been widely used for generating transgenic plants resistant against viruses. Considering the important role of p23, p20 and p25 in CTV pathogenesis, we have transformed Mexican lime plants with an intron-hairpin vector carrying full untranslatable versions of genes p25, p20, p23 and the 3¿-UTR from the CTV strain T36, to attempt silencing their expression in CTV-infected cells. Complete resistance to viral infection was observed in three transgenic lines, with all their propagations remaining symptomless and virus-free after graft-inoculation with CTV-T36, either in the non-transgenic rootstock or directly in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. Challenging immune transformants with a divergent CTV strain resulted in partial breakage of the resistance, stressing the importance of sequence identity in the underlying RNAi mechanism. This is the first evidence that it is possible to achieve full resistance to CTV in a highly sensitive citrus host by targeting simultaneously its three viral silencing suppressors through RNAi. The p23 protein encoded by the virus is additionally an important pathogenicity factor. Ectopic expression of p23 in transgenic citrus plants induces developmental aberrations resembling CTV symptoms. To explore in more detail the role of p23 in CTV pathogenesis, the p23 gene from CTV T36 and three truncated versions thereof under the control of the Cauliflower mosaic virus 35S promoter were used to transform Mexican lime. Only the truncated version expressing amino acids 1 to 157 (p23¿158-209) elicited CTV-like symptoms, similar to, albeit milder than, those incited by expressing the whole p23 protein (209 amino acids), thus delimiting the region responsible for p23 pathogenesis in citrus to a 157 amino acid fragment including the Zn finger and flanking basic motifs of the protein. RNA silencing suppressor activity of p23 in N. benthamiana was abolished by all mutants tested, indicating that silencing suppression involves most p23 regions. To better define the role of p23 in CTV pathogenesis, we next restricted the expression of p23-derived transgenes to phloem-associated cells in Mexican lime plants by means of using the phloem-specific promoter from Commelina yellow mottle virus (CoYMV). Constructions carrying the complete gene p23 from either the severe T36 or the mild T317 CTV strains, or a fragment comprising the zinc-finger and flanking basic motifs from the former, either under the control of the CoYMV promoter or the constitutive 35S promoter were used for genetic transformation of Mexican lime. Expression of these constructs in the phloem incited aberrations resembling CTV-specific symptoms, but not the unspecific symptoms observed when p23 was constitutively expressed. Moreover, appearance and intensity of the most notorious CTV-like phenotypic aberrations induced by the phloem-specific expression of the p23 gene were positively related with the aggressiveness of the source CTV strain used. Additionally, expression in phloem-tissues of the p23 fragment comprising the zinc-finger domain and flanking basic motifs was sufficient to induce CTV-like symptoms, corroborating that the N-terminal region (delimited by amino acids 1 and 157) determines, at least in part, CTV pathogenesis in Mexican lime.
Soler Calvo, N. (2013). Transgenic resistance against Citrus tristeza virus (CTV) and analysis of the viral p23 protein as pathogenicity determinant in citrus [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/31631
TESIS
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Chu, Chia-Ying. "Molecular Mechanism of RNA-Mediated Gene Silencing in Human Cells: A Dissertation." eScholarship@UMMS, 2008. https://escholarship.umassmed.edu/gsbs_diss/388.
Full textAbstract:
Small non-coding RNAs regulate gene expression at posttranscriptional level in eukaryotic cells. Two classes of such small (~21-25 nt) RNAs that have been extensively studied in gene silencing are short interfering RNAs (siRNAs) and microRNAs (miRNAs). RNA interference (RNAi) is process whereby double-stranded RNA induces the sequence-specific degradation of homologous mRNA. The RNAi machinery can also be programmed in human cells by introducing 21-nt siRNA duplexes that are assembled into RNA-induced silencing complexes (RISC). In this dissertation, systematic analysis of siRNAs with deletions at the passenger and/or guide strand reveals that a short RNAi trigger, 16-nt siRNA, induces potent RNAi in human cells. The 16-nt siRNA more effectively knocked down mRNA and protein levels than 19-nt siRNA when targeting the endogenous CDK9 gene. In vitro kinetic analysis of human RISC indicates that 16-nt siRNA has a higher RISC-loading capacity than 19-nt siRNA. These results suggest that 16-nt duplexes can be designed as potent triggers for RNAi.
RISC can be programmed by small interfering RNAs (siRISC) to cleave a perfectly complementary target mRNA, or endogenous microRNAs (miRISC) to inhibit translation by binding imperfectly matched sequences in the 3’-untranslated region (3’-UTR) of target mRNA. Both RISCs contain Argonaute2 (Ago2), which localizes to cytoplasmic mRNA processing P-bodies. This dissertation shows that RCK/p54, a DEAD box helicase, interacts with Ago2, in affinity-purified active siRISC or miRISC, facilitates formation of P-bodies. Depletion of RCK/p54 disrupted P-bodies and dispersed Ago2 throughout the cytoplasm, but did not significantly affect siRNA-mediated RNAi. Depleting RCK/p54 releases general and miRNA-induced translational repression. These findings imply that miRISC-mediated translation repression requires RCK/p54, also suggest that location of miRISC to P-bodies is not required for miRNA function, but is the consequence of translation repression.
To elucidate the function of RCK/p54 in miRNA-mediated gene silencing, analysis of a series of YFP-tagged RCK/p54 mutants reveals the motif required for P-body localization, interaction with Ago2, and/or facilitating the miRNA-mediated translation repression. Additionally, rabbit reticulocyte lysate system was used to recapitulate the miRISC function in a cell-free system and confirmed the requirement of RCK/p54 for miRNA function in vitro. Analysis of Ago2 distribution in the polysome profiling in RCK/p54-depleted cells, compared to that in normal cells, revealed that RCK/p54 facilitates miRISC by trapping it at translation initiation complex. These data suggest that interaction of RCK/p54 with Ago2 is involved in the repression of translation initiation of miRNA function.
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Vasale, Jessica J. "Roles of Cellular RNA-Dependent RNA Polymerases in Endogenous Small RNA Pathways in Caenorhabditis elegans: A Dissertation." eScholarship@UMMS, 2010. https://escholarship.umassmed.edu/gsbs_diss/481.
Full textAbstract:
The RNA interference (RNAi) pathway in Caenorhabditis elegans is a two-step, small RNA-mediated silencing pathway. Unlike in other organisms, Dicer processing of double-stranded RNA into small interfering (si) RNAs is not sufficient in worms to induce gene silencing. The activity of cellular RNA-dependent RNA polymerase (RdRP) is necessary to synthesize a secondary pool of siRNAs, which interact with a unique class of Argonaute proteins to form the functional effector complexes that mediate silencing. The aims of this thesis were to: 1) characterize the role of RdRP family members in endogenous small RNA biogenesis; 2) identify the Argonaute proteins that interact with RdRP-dependent small RNAs; and 3) investigate the biological function of RdRP-dependent small RNA pathways in C. elegans.
In this thesis, I describe genetic, deep sequencing, and molecular studies, which identify 22G-RNAs as the most abundant class of endogenous small RNA in C. elegans. The 22G-RNAs resemble RdRP-dependent secondary siRNAs produced during exogenous RNAi, in that they possess a triphosphorylated 5’ guanine residue and exhibit a remarkable strand bias at target loci. Indeed, I show that 22G-RNAs are dependent on the activity of the RdRPs RRF-1 and EGO-1 and function in multiple distinct endogenous small RNA pathways. Interestingly, I have found that RRF-1 and EGO-1 function redundantly in the germline to generate 22G-RNAs that are dependent on and interact with members of an expanded family of worm-specific Argonaute (WAGO) proteins. The WAGO/22G-RNA pathway appears to be a transcriptome surveillance pathway that silences coding genes, pseudogenes, transposons, and non-annotated, or cryptic, transcripts. In contrast, I have found that EGO-1 alone is required for the biogenesis of a distinct class of 22G-RNAs that interact with the Argonaute CSR-1. Surprisingly, the CSR-1/22G-RNA pathway does not appear to silence its targets transcripts. Instead, the CSR-1/22G-RNA pathway is essential for the proper assembly of holocentric kinetochores and chromosome segregation.
Lastly, I show that a third endogenous small RNA pathway, the ERI pathway, is a two-step silencing pathway that requires the sequential activity of distinct RdRPs and Argonautes. In the first step of this pathway, the RdRP, RRF- 3, is required for the biogenesis of 26G-RNAs that associate with the Argonaute, ERGO-1. In the second step, RRF-1 and EGO-1 generate 22G-RNAs that associate with the WAGO Argonautes.
This work demonstrates how several C. elegans small RNAs pathways utilize RdRPs to generate abundant populations of small RNAs. These distinct categories of small RNAs function together with specific Argonaute proteins to affect gene expression, to play essential roles in development, and in the maintenance of genome and transcriptome integrity.
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Hinrichsen, Lars. "Analyse der Clathrin-vermittelten Endocytose mittels RNA-Interferenz." [S.l.] : [s.n.], 2005. http://deposit.ddb.de/cgi-bin/dokserv?idn=976698862.
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Mankin, Danielle N. "MC3R and MC4R Knockdown via RNA Interference." Digital Archive @ GSU, 2012. http://digitalarchive.gsu.edu/biology_theses/37.
Full textAbstract:
Melanocortins (MCs) play an important role in feeding, metabolism, and energy expenditure. While melanocortin receptor (MCR) mRNA has been found in the mesolimbic dopamine (DA) pathway, the ability of melanocortins to regulate feeding and other behaviors through actions on the mesolimbic DA system have not been examined. Short-hairpin RNAs (shRNAs) were created targeting MC3R and MC4R and were tested via in vitro studies for their ability to knockdown their target receptor. A total of three shRNAs were created targeting each receptor, and each shRNA caused successful knockdown. These shRNAs are tools that can be used for future in vivo studies to examine the various behavioral effects of melanocortins on the mesolimbic DA pathway.
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Lee, Hung-chiu. "Synthetic RNA interference against influenza A virus." Click to view the E-thesis via HKUTO, 2005. http://sunzi.lib.hku.hk/hkuto/record/B35537814.
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Lee, Hung-chiu, and 李洪釗. "Synthetic RNA interference against influenza A virus." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B35537814.
Full text
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Neofytou, Giannis. "Mathematical models of RNA interference in plants." Thesis, University of Sussex, 2017. http://sro.sussex.ac.uk/id/eprint/67207/.
Full textAbstract:
RNA interference (RNAi), or Post-Transcriptional Gene Silencing (PTGS), is a biological process which uses small RNAs to regulate gene expression on a cellular level, typically by causing the destruction of specfic mRNA molecules. This biological pathway is found in both plants and animals, and can be used as an effective strategy in defending cells against parasitic nucleotide sequences, viruses and transposons. In the case of plants, it also constitutes a major component of the adaptive immune system. RNAi is characterised by the ability to induce sequence-specific degradation of target messenger RNA (mRNAs) and methylation of target gene sequences. The small interfering RNA produced within the initiated cell is not only used locally but can also be transported into neighbouring cells, thus acting as a mobile warning signal. In the first part of the thesis I develop and analyse a new mathematical model of the plant immune response to a viral infection, with particular emphasis on the role of RNA interference. The model explicitly includes two different time delays, one to represent the maturation period of undifferentiated cells, and another to account for the time required for the RNAi propagating signal to reach other parts of the plant, resulting in either recovery or warning of susceptible cells. Analytical and numerical bifurcation theory is used to identify parameter regions associated with recovery and resistant plant phenotypes, as well as possible chronic infections. The analysis shows that the maturation time plays an important role in determining the dynamics, and that long-term host recovery does not depend on the speed of the warning signal but rather on the strength of local recovery. At best, the warning signal can amplify and hasten recovery, but by itself it is not competent at eradicating the infection. In the second part of the thesis I derive and analyse a new mathematical model of plant viral co-infection with particular account for RNA-mediated cross-protection in a single plant host. The model exhibits four non-trivial steady states, i.e. a disease-free steady state, two one-virus endemic equilibria, and a co-infected steady state. I obtained the basic reproduction number for each of the two viral strains and performed extensive numerical bifurcation analysis to investigate the stability of all steady states and identified parameter regions where the system exhibits synergistic or antagonistic interactions between viral strains, as well as different types of host recovery. The results indicate that the propagating component of RNA interference plays a significant role in determining whether both viruses can persist simultaneously, and as such, it controls whether the plant is able to support some constant level of both infections. If the two viruses are sufficiently immunologically related, the least harmful of the two viruses becomes dominant, and the plant experiences cross-protection. In the third part of the thesis I investigate the properties of intracellular dynamics of RNA interference and its capacity as a gene regulator by extending a well known model of RNA interference with time delays. For each of the two amplification pathways of the model, I consider the cumulative effects of delay in dsRNA-primed synthesis associated with the non-instantaneous nature of chemical signals and component transportation delay. An extensive bifurcation analysis is performed to demonstrate the significance of different parameters, and to investigate how time delays can affect the bi-stable regime in the model.
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Yoon, June-Sun. "THE MECHANISM OF RNA INTERFERENCE IN ARTHROPODS." UKnowledge, 2018. https://uknowledge.uky.edu/entomology_etds/45.
Full textAbstract:
RNA interference (RNAi) is a useful reverse genetics tool for investigation of gene function as well as for practical applications in many fields including medicine and agriculture. Due to the variability in RNAi efficiency, RNAi-based methods are currently being developed for controlling only coleopteran insects which are known to be amenable to RNAi. The first chapter of my thesis includes findings from research to investigate what are the factors that make coleopteran insects relatively more efficient in RNAi. I used Colorado potato beetle (CPB), Leptinotarsa decemlineata and its cell line (Lepd-SL1) as study models to identify genes that play key roles in RNAi pathway. Five genes including Argonaute-1 (microRNA Argonaute) and Aubergine (PiwiRNA Argonaute) were identified as those required for siRNA (short interfering RNA) RNAi pathway. I also found that RNAi is completely blocked in StaufenC knockdown cells. StaufenC belongs to dsRNA binding protein family and binds to dsRNA as shown by gel mobility shift and the pull-down assays. Interestingly, I also found that StaufenC is downregulated in RNAi resistant cells and StaufenC homologous sequences are present in only coleopteran insects where RNAi works efficiently. These data suggest that StaufenC is a major contributor to efficient RNAi in coleopteran insects and is a potential target for RNAi resistance. The second part of my research is to understand the mechanisms of RNAi in those insects refractory to RNAi. The barriers for successful RNAi include the presence of double-stranded ribonucleases (dsRNase) in the lumen and hemolymph that could potentially digest double-stranded RNA (dsRNA) and the variability in the transport of dsRNA into and within the cells. Recent work in our laboratory showed that the dsRNAs are transported into lepidopteran cells, but they are not processed into siRNAs because they are trapped in acidic bodies. I focused on identification of these acidic bodies in which dsRNAs accumulate in Spodoptera frugiperda Sf9 cells. These studies showed that entrapment of internalized dsRNA in endosomes is one of the major factors contributing to inefficient RNAi. Overall, my research revealed important players involved in successful and unsuccessful RNAi in insects.
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Ramachandran, Pavitra Shyam. "RNA interference therapy for the Spinocerebellar ataxias." Diss., University of Iowa, 2014. https://ir.uiowa.edu/etd/4730.
Full textAbstract:
The spinocerebellar ataxias are a group of diseases characterized by loss of motor coordination. Spinocerebellar ataxia types 2 and 7 are monogenic, autosomal dominant, late-onset neurodegenerative diseases characterized by ataxia with no effective treatments in the clinic. The most striking feature of these diseases is the degeneration of Purkinje neurons of the cerebellum. Spinocerebellar ataxia type 7 is also characterized by vision loss due to degeneration of the retinal photoreceptors. In this work, we tested the hypothesis that reducing mutant gene expression by RNAi would alleviate disease phenotypes in these two spinocerebellar ataxias.
For spinocerebellar ataxia type 7 (SCA7), we designed and tested RNAi sequences that could reduce the expression of both wildtype and mutant ataxin-7, an approach that would be applicable to all SCA7 patients. We found that AAV1-mediated delivery of a candidate RNAi sequence to the Purkinje neurons of SCA7 mice resulted in long-term sustained reduction of both wildtype and mutant ataxin-7 and resulted in significant improvements in ataxic and neuropathological phenotypes. We also delivered the RNAi sequence (AAV1-mediated) to reduce the expression of both mutant and wildtype ataxin-7 in the SCA7 mouse retina and evaluated retinal function long-term. We observed a preservation of normal retinal function and no adverse toxicity with reduction of wildtype and mutant ataxin-7 alleles. These studies address an important safety concern regarding non-allele specific silencing of ataxin-7 for SCA7 therapy.
To identify therapies for spinocerebellar ataxia type 2 (SCA2), we designed and tested several RNAi sequences to reduce the expression of both wildtype and mutant ataxin-2 in vitro and in vivo. We found that reduction of wildtype ataxin-2 expression in the mouse cerebellum was tolerated 4 months post injection without inducing behavioral deficits or cerebellar pathology. Additionally, we tested other sequences for improved silencing efficacy, and identified a potent RNAi sequence that significantly reduced the expression of both mutant and wildtype ataxin-2 in the cerebellum of a SCA2 mouse model. Ongoing work will establish if long-term reduction of both mutant and wildtype ataxin-2 will provide therapeutic benefit in the SCA2 mouse setting, and the safety of this sequence in normal cerebella.
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Huang, Ching-Cheng. "Development of RNA interference in parasitic nematodes." Thesis, Imperial College London, 2010. http://hdl.handle.net/10044/1/5745.
Full textAbstract:
Exploitation of RNA interference (RNAi) has revolutionised work on Caenorhabditis elegans, and considerable success has been recently reported with plant-parasitic nematodes. It has proven difficult to transfer this technology to animal parasitic species, and previous attempts in this laboratory by feeding Nippostrongylus brasiliensis larvae with Escherichia coli expressing double-stranded RNA (dsRNA) gave no consistent reductions in levels of target transcripts. The aim of this study was to develop methods for RNAi in N. brasiliensis, a rodent strongylid nematode which is closely related to gastrointestinal nematodes of humans and livestock, in order to explore the biological functions of parasite secreted proteins. In order to promote uptake of exogenous macromolecules such as dsRNA and small interfering RNA (siRNA) by infective larvae, the process of activation, whereby larvae are induced to resume feeding and development, was studied in vitro. Activation could be induced solely by exposure of larvae to elevated temperature (37oC), whereas host serum or glutathione had no effect. Neither a membrane permeant analogue of cyclic GMP nor muscarinic receptor agonists promoted activation, suggesting that a cholinergic neural pathway is not involved in the process. Activation at 37oC could be blocked by inhibitors of phosphatidylinositol 3-kinase, Akt protein kinase & cytochrome P450. These data indicate that the early signalling events for larval activation in N. brasiliensis differ substantially from the hookworm Ancylostoma caninum, most probably acting through thermosensory rather than chemosensory neurons, but that they may converge downstream of a step dependent on phosphatidylinositol 3-kinase. Stimulation of protein secretion paralleled resumption of feeding, suggesting that these processes were tightly linked and regulated by similar pathways during activation. Temperature-activated larvae were exposed to dsRNA and siRNA via electroporation or soaking in the presence of a variety of transfection reagents, but RNAi was unsuccessful. Serotonin was demonstrated to increase the rate of uptake of macromolecules, yet larvae exposed to exogenous dsRNA in the presence of serotonin still failed to show RNAi-mediated gene silencing. In addition, RNAi was also observed to be irreproducible in adult N. brasiliensis using the same methods of delivery. The use of mRNA encoding firefly luciferase identified uptake into larvae or adult worms as a major impediment in the process. Venom Allergen Homologue/ASP-Like (VAL) molecules have been identified as major components of secreted proteins from many species of parasitic nematodes. In this study, eight N. brasiliensis VALs were identified and sequenced, and all were shown to be present in products secreted by adult parasites. NbVAL-7 and NbVAL-8 were also detected in secreted products of infective larvae. Structural similarities to other members of the Pathogenesis- Related protein superfamily are discussed, as are possible functions for these proteins in N. brasiliensis.
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Stege, Alexandra Eva. "Überwindung der P-Glykoprotein (MDR1)-abhängigen Multidrugresistenz mittels RNA-Interferenz." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2007. http://dx.doi.org/10.18452/15578.
Full textAbstract:
P-Glykoprotein als Produkt des MDR1-Gens stellt einen gut untersuchten Mediator der Multidrugresistenz (MDR) in humanen Malignomen dar. Die Überexpression dieses ABC-Transporters steht in Korrelation zu einer erniedrigten Tumorremission und einer kürzeren Überlebensrate der Patienten. Bisherige Versuche, das Protein über niedermolekulare Substanzen (MDR-Modulatoren) zu inhibieren, vermochten in allen bisherigen klinischen Studien nicht zu überzeugen, so daß diese bis heute keinen Eingang in Standardtherapieschemata gefunden haben. Ziel dieser Arbeit war es, mittels RNA-Interferenz Strategien die Expression von MDR1 zu hemmen und eine Reversion der zellulären Chemoresistenz sowohl im Zellkultur- als auch im Tiermodell zu erreichen. Für die in vitro Untersuchungen an drei humanen multidrug-resistenten Karzinomzellinien wurden verschiedene siRNA (short interfering) Duplexe und shRNA (short hairpin)-exprimierende Vektoren gegen die MDR1 mRNA entwickelt. Die Behandlung der Zellen mit siRNAs führte zu einer bis zu 91 %igen Inhibition der MDR1 mRNA-Expression und zu einer Sensitivierung der Zellen gegenüber dem Anthrazyklin um 89 %. Diese Effekte konnte über einen Zeitraum von drei bis fünf Tagen aufrechterhalten werden. Die stabile Expression von anti-MDR1 shRNAs führte in zwei der untersuchten Zellmodelle zu einer dauerhaften und kompletten Überwindung des MDR1-abhängigen Resistenzphänotyps. Im Mausmodell konnte durch intratumorale Applikation des anti-MDR1 shRNA-kodierenden Vektors mittels low-volume Jet-Injektion eine komplette Reversion der MDR1-Überexpression sowie eine Wiederherstellung der Chemosensitivität gegenüber Doxorubicin in dem resistenten Tumormodell erreicht werden. Die Effizienz der kombinierten Gen- und Chemotherapie wird durch die Verminderung des in vivo Tumorwachstums auf das Volumen des von der sensiblen Zellinien-abgeleiteten Tumors reflektiert.Multidrug resistance (MDR) is the major cause of failure of effective chemotherapeutic treatment of disseminated neoplasms. The "classical" MDR phenotype of human malignancies is mediated by drug extrusion by the adenosine triphosphate binding cassette (ABC)-transporter P-glycoprotein (MDR1/P-gp). For stable reversal of "classical" MDR in three human cancer cell lines by RNA interference (RNAi) technology, two small interfering RNA (siRNA) constructs and four H1-RNA gene promoter-driven expression vectors encoding anti-MDR1/P-gp short hairpin RNA (shRNA) molecules were constructed. In all cellular systems, siRNAs could specifically inhibit MDR1 expression up to 91% at the mRNA and protein levels. Resistance against daunorubicin was decreased to a maximum of 89%. The introduction of anti-MDR1/P-gp shRNA expression vectors leads in two of the three human cancer cell lines to a complete reversion of the MDR phenotype. The reversal of MDR was accompanied by a complete suppression of MDR1/P-gp expression on mRNA and protein level, and by a considerable increased intracellular anthracyline accumulation in the anti-MDR1/P-gp shRNA-treated cells. In a mouse xenograft model a complete in vivo restoration of MDR1 overexpression and chemosensitivity to doxorubicin could be obtained by intratumorally jet-injected anti-MDR1 shRNA in a multidrug resistant human cancer tumor model.
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Cheng, Chi-Ping. "VIRAL RNA ELEMENTS AND HOST GENES AFFECTING RNA RECOMBINATION IN TOMBUSVIRUSES." UKnowledge, 2005. http://uknowledge.uky.edu/gradschool_diss/436.
Full textAbstract:
RNA recombination is a major factor driving viral evolution and contributing to new disease outbreaks. Therefore, understanding the mechanism of RNA recombination can help scientists to develop longer lasting antiviral strategies. Tombusviruses are one of the best model RNA viruses to study RNA virus recombination. My goals were to dissect the mechanism of tombusviral RNA recombination. To do so, in my thesis, I describe my results on the roles of (i) the viral replicase and the viral RNA templates; and (ii) the effect of host factors on tombusvirus recombination events. To study the mechanism of RNA recombination without the influence of selection pressure on the emerging recombinants, we developed an in vitro RNA recombination assay based on viral RNA templates and purified viral replicase preparations. Using this in vitro assay, we demonstrated that replicase driven template switching is the mechanism of recombination, whereas RNA ligation seems less likely to be a major mechanism. In addition, we also studied the role of RNA substrates, in more detail. Our results showed that viral replicase preferred to use functional RNA domains in the acceptor RNAs over random switching events. Host factors may also play important roles in RNA recombination. Using yeast as a model system for studying replication and recombination of a tombusvirus replicon, we identified 9 host genes affecting tombusvirus RNA recombination. Separate deletion of five of these genes enhanced generation of novel viral RNA recombinants. Further studies on one of these genes, XRN1, a 5-3 exoribonuclease, indicated that it might be involved in degradation of tombusvirus RNAs. Lack of Xrn1p resulted in accumulation of truncated (partially degraded) replicon RNAs, which became good templates for RNA recombination. To further study Xrn1p, we overexpressed Xrn4p of Arabidopsis thaliana, a functional analogue of the yeast Xrn1p, in Nicotiana benthamiana plants. After superinfecting the Xrn4p-overexpressing N. benthamiana with tombusvirus, truncated tombusvirus genomic and subgenomic RNA1 were observed. Some of the identified tombusvirus variants were infectious in protoplasts and could systemically infected N. benthamiana plants. Overall, this is the first report that a single host gene can affect rapid viral evolution and RNA recombination.
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Aldabbagh, Souhaib. "Modulation der Masernvirusinfektion durch RNA-Interferenz mittels miRNA Expressionskassetten." Doctoral thesis, Universitätsbibliothek Leipzig, 2011. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-73836.
Full textAbstract:
Die subakute sklerosierende Panenzephalitis (SSPE), eine durch das Masernvirus (MV) verursachte sogenannte „ slow virus “ Infektion des zentralen Nervensystems, ist eine progrediente chronische Erkrankung, die zum Tod führt und bisher medikamentös nicht heilbar ist. Da die RNAi-Strategien grundsätzlich zur Inhibition von Viren in Säugetierzellen
geeignet sind, stellt die RNAi eine Möglichkeit dar, die Infektion auf molekularer Ebene anzugreifen. Dafür wurden verschiedene miRNA-Expressionskassetten, welche gegen zwei Sequenzen im MV- Hämagglutinin-Gen (H) und sechs Sequenzen im MV-Nukleokapsid-Gen
(N) gerichtet sind, konstruiert und in MV infizierte Zellen eingebracht. Diese miRNAExpressionskassetten wurden auf zwei verschiedenen Wegen in die Zelle eingebracht: Zum einen wurden sie über ein miRNA-Expressionsplasmid (pmiR), welches in die Zellen transfiziert wird, transient exprimiert; zum anderen wurden sie durch virale Vektoren (HIV, SIV und MoMLV) stabil in die Zellen transduziert. Dies ermöglicht die Integration der miRNAExpressionskassette in das Genom der Zelle und dadurch die Expression der miRNAs für einige Wochen.
In erster Linie zielt die Wirkung der RNA-Interferenz auf die Degradierung der spezifischen MV-mRNAs. Diese Degradierung konnte mit Hilfe quantitative Reverse Transkription real time-PCR (qRT-PCR) nachgewiesen werden. Die transiente Expression der verschiedenen
miRNAs gegen das MV-N-Gen bzw. MV-H-Gen führte in jedem Fall zu einer Reduktion der viralen genspezifischen mRNAs. Die Reduktion der MV spezifischen mRNA betrug 99,8% für das MV-N-Gen und 91,2% für das MV-H-Gen.
Die Wirkung der RNA-Interferenz zielt am Ende auf die Reduktion der neu gebildeten infektiösen Viruspartikel und ihrer Verbreitung in der Zellkultur, welche die spezifische miRNA gegen MV-N oder MV-H exprimiert. Dieser Effekt konnte nur durch Plaque-Assay überprüft werden. Die Plaque-Assays, die mit den Überständen der miRNA-behandelten Zelllinien durchgeführt wurden, zeigten ebenfalls eine Reduktion der neu gebildeten infektiösen MV-Partikeln von 97,6% für die miRNA gegen das MV-H-Gen und 99,0 % für die miRNA gegen das MV-N-Gen.
Die intrazelluläre Expression der miRNAs führte zu einer Hemmung der Virus-Ausbreitung in MV-infizierten Zellen. Die Reduktion betrug hier durch die Expression der miRNA-N10 98,8% und durch die Expression der miRNA-H2 80,0%. Hier zeigte sich, dass die Inhibition der viralen Proteinsynthese durch den RNAi-Mechanismus auch die Verbreitung der MVInfektion durch Zell-Zell-Fusion behindern kann. Dies zeigte sich durch die verringerte Bildung von Plaques bzw. Synzytien in miRNA-behandelten Zelllinien.
Die vorliegende Arbeit zeigte, dass RNAi effektive gegen MV-Infektion in Zellkultur eingesetzt werden kann. Als nächster Schritt sollte daher dieser RNAi-Effekt im etablierten Tiermodell ausgetestet werden.
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Wohlbold, Lara. "Expressionshemmung des humanen Onkogens Bcr-Abl durch RNA-Interferenz." [S.l. : s.n.], 2004. http://www.bsz-bw.de/cgi-bin/xvms.cgi?SWB11730032.
Full text
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Warnock, N. D. "Investigations on RNA interference susceptibility in selected nematodes." Thesis, Queen's University Belfast, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.557851.
Full textAbstract:
Parasitic nematodes continue to be the causative agents of some of the most prevalent diseases of man and cause significant economic losses to the worldwide agri-food industry. With anthelmintic resistance on the rise and environmental concerns over the use of many nematicides the effectiveness of current control options is diminishing. The only viable alternative is the creation of new, environmentally safe drugs/control options. The reverse genetic technique of RNA interference (RNAi) facilitates the examination of gene function by the application of double stranded (ds)RNA complementary to a gene of interest which triggers endogenous cellular mechanisms that destroy the target gene transcript. Of particular interest here is the application of RNAi to nematode species and how this technology could be harnessed to improve our understanding of nematode biology and, ultimately, exploited to inform drug target selection. This study demonstrates that RNAi remains a potentially useful tool for the validation of gene function despite the pressing need to improve experimental rigor and statistical analysis when validating RNAi induced gene knockdown. Although RNAi failed to silence gene expression in the free-living nematode Panagrellus redivivus and was limited in its efficacy in l3 stage worms of the pig parasite Ascaris suum, it did trigger consistent knockdown of selected genes in A. suum adults. A bioinformatic analysis of the RNAi pathway components in the genomes/transcriptomes of selected nematodes indicated the occurrence of all functional groupings within diverse nematodes, suggesting that RNAi mechanisms are in place broadly in the phylum. There were no RNAi effector deficiencies that specifically mapped to RNAi incompetency. As such there remains hope that with further optimisation, and as culture and delivery techniques continue to improve, RNAi could be an important tool for screening gene function and validating drug targets in nematode parasites.