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1
Souza, Sandra Maria de. "Avaliação do silenciamento gênico em plantas utilizando estratégias baseadas em dsRNAs". reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2006. http://hdl.handle.net/10183/165640.
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O silenciamento gênico tem sido utilizado extensivamente para facilitar a investigação de eventos da biologia vegetal. Ele pode ser induzido de diferentes modos, sendo que o passo chave para sua ocorrência é a presença de RNA fita dupla (dsRNA). A fim de avaliar um método rápido de obtenção de silenciamento para análise funcional em larga escala, foi inoculado dsRNA ou siRNA diretamente em folhas de plantas de arroz e de Nicotiana benthamiana. Os dsRNAs de seqüências do gene pds e do gene codificante da ferritina foram obtidos através da síntese in vivo e in vitro e o siRNA de PDS de arroz, derivado somente de síntese in vitro. O gene pds codifica a enzima fitoeno desaturase (PDS), uma enzima chave na biossíntese de carotenóides, que protege a clorofila das plantas contra o foto-branqueamento. O silenciamento deste gene mostra um fenótipo visível, claramente demonstrado através do uso de um inibidor químico da síntese desta enzima. No presente estudo, plantas de arroz e de N. benthamiana inoculadas com dsRNAs derivados do gene pds não apresentaram mudanças fenotípicas. Para avaliação de um outro método foi adaptado um vetor de silenciamento estável, no qual foram inseridos fragmentos de cDNA nas orientações senso e antisenso do gene de ferritina de arroz. O papel da ferritina, segundo experimentação in vitro, pode estar ligada à tolerância de certos cultivares de arroz às altas concentrações de ferro. O aprimoramento das metodologias de silenciamento e de transformação poderá resultar em uma ferramenta mais útil para a análise funcional em larga escala de genes de arroz.Gene silencing has been used extensively to facilitate the investigation of different events in plant biology. It can be induced through different ways. The key step for its occurrence is the presence of double strand RNA (dsRNA). In order to evaluate a fast method to obtain silencing for functional analysis in a wide scale, either dsRNAs or siRNAs were directly inoculated in the leaves of rice and N. benthamiana plants. The dsRNAs the sequences of the pds gene and of the coding region of the ferritin gene were obtained through in vivo or in vitro synthesis, the siRNA PDS of rice were derived from synthesis in vitro. The pds gene codifies for the enzyme phytoene desaturase (PDS), a key enzyme in the biosynthesis of carotenoids, which protects the clorophil from the plants against photo-bleaching. The silencing of this gene shows a visible phenotype, clearly demonstrated through a chemical inhibition of PDS synthesis. However, no phenotypical changes were observed in either rice or N. benthamiana plants inoculated with dsRNA of the pds gene. In the present study, it was also adapted a vector for stable transformation, in which were inserted cDNA fragments of both, sense and antisense, orientations of the rice ferritin gene. In vitro experiments show that ferritin could be connected to the tolerance to high concentrations of iron of certain cultivars of rice. The improvement of silencing and transformation methodologies could result in a more useful tool for the functional analysis of rice genes in a wide scale.
2
Cole, Thomas Edwin. "Molecular studies of virus-like dsRNAs in a diseased isolate of Ophiostoma novo-ulmi". Thesis, Imperial College London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.327031.
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Vyas, Meenal, Amir Raza, Muhammad Yousaf Ali, Muhammad Aleem Ashraf, Shahid Mansoor, Ahmad Ali Shahid y Judith K. Brown. "Knock down of Whitefly Gut Gene Expression and Mortality by Orally Delivered Gut Gene-Specific dsRNAs". PUBLIC LIBRARY SCIENCE, 2017. http://hdl.handle.net/10150/622632.
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Control of the whitefly Bemisia tabaci (Genn.) agricultural pest and plant virus vector relies on the use of chemical insecticides. RNA-interference (RNAi) is a homology-dependent innate immune response in eukaryotes, including insects, which results in degradation of the corresponding transcript following its recognition by a double-stranded RNA (dsRNA) that shares 100% sequence homology. In this study, six whitefly `gut' genes were selected from an in silico-annotated transcriptome library constructed from the whitefly alimentary canal or 'gut' of the B biotype of B. tabaci, and tested for knock down efficacy, post-ingestion of dsRNAs that share 100% sequence homology to each respective gene target. Candidate genes were: Acetylcholine receptor subunit a, Alpha glucosidase 1, Aquaporin 1, Heat shock protein 70, Trehalasel, and Trehalose transported. The efficacy of RNAi knock down was further tested in a gene-specific functional bioassay, and mortality was recorded in 24 hr intervals, six days, post-treatment. Based on qPCR analysis, all six genes tested showed significantly reduced gene expression. Moderate-to-high whitefly mortality was associated with the down-regulation of osmoregulation, sugar metabolism and sugar transport -associated genes, demonstrating that whitefly survivability was linked with RNAi results. Silenced Acetylcholine receptor subunit a and Heat shock protein 70 genes showed an initial low whitefly mortality, however, following insecticide or high temperature treatments, respectively, significantly increased knockdown efficacy and death was observed, indicating enhanced post-knockdown sensitivity perhaps related to systemic silencing. The oral delivery of gut-specific dsRNAs, when combined with qPCR analysis of gene expression and a corresponding gene-specific bioassay that relates knockdown and mortality, offers a viable approach for functional genomics analysis and the discovery of prospective dsRNA biopesticide targets. The approach can be applied to functional genomics analyses to facilitate, species-specific dsRNA-mediated control of other non-model hemipterans.
4
Reddy, Vidita. "Role of dsRNA-induced DRAK1 in Apoptosis". University of Toledo / OhioLINK, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=toledo1513349817056948.
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Lu, Lenette L. "dsRNA Signaling in Innate Immunity and Viral Inhibition". Case Western Reserve University School of Graduate Studies / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1220030971.
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Muccioli, Maria. "Characterizing dsRNA-induced inflammation in ovarian cancer cells". Ohio University / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=ohiou1405707670.
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Kilic, Ozlem III. "Effect of dsRNA-containing and dsRNA-free hypovirulent isolates of Fusarium oxysporum on severity of Fusarium seedling disease of Essex soybean". Thesis, Virginia Tech, 1997. http://hdl.handle.net/10919/36965.
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Sixty-six isolates of F. oxysporum and F. solani were recovered from healthy and necrotic Essex soybean seedlings grown in naturally infested soil. These were tested for pathogenicity at 20 C and -0.01 MPa water potential in artificially infested, autoclaved field soil. Highly pathogenic, moderately pathogenic, and hypovirulent isolates of both species were identified. Fifty-seven F. oxysporum and nine F. solani isolates were tested for the presence of dsRNA. The presence of dsRNA was not associated with hypovirulence in F. oxysporum since some hypovirulent isolates contained dsRNA while other hypovirulent isolates did not. Furthermore, of six dsRNA-containing F. oxysporum isolates, three were hypovirulent, two were moderately pathogenic, and one isolate was highly pathogenic. Four segments of dsRNA, with sizes of 4.0, 3.1, 2.7, and 2.2 kb, were detected in extracts of all six F. oxysporum isolates. No morphological differences were found between dsRNA-containing and dsRNA-free F. oxysporum isolates. Attempts to cure dsRNA-containing hypovirulent F. oxysporum isolates, either by single-sporing of isolates or by using a range of concentrations of cycloheximide, were not successful. No dsRNA was found in any of the F. solani isolates tested.
Pythium ultimum, an associate in Essex seedling disease, was isolated from water-soaked lesions and interfered with evaluations of disease caused by the Fusarium spp. Metalaxyl was used to control P. ultimum and had no apparent effect on symptoms associated with F. oxysporum and F. solani in field soil. Prior inoculation of Essex soybean seeds with conidia of dsRNA-free hypovirulent F. oxysporum isolates, plus metalaxyl seed treatment, significantly (p<0.05) reduced disease severity on both cotyledons and hypocotyls and increased the rate of seedling emergence in field soil, compared to the control plants treated with metalaxyl alone or not treated with metalaxyl. No significant (p>0.05) differences were found between dsRNA-containing and dsRNA-free hypovirulent F. oxysporum isolates in their effects on the reduction of disease severity. A mixture of two hypovirulent F. oxysporum isolates was significantly (p<0.05) more effective than single hypovirulent F. oxysporum isolates in increasing the rate of seedling emergence. Symptoms associated with P. ultimum were not affected by the prior inoculation of seeds with individual hypovirulent F. oxysporum isolates.Master of Science
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Rodrigues, Paula. "Produção e caracterização de um antissoro policlonal para detecção de ds-RNA". Bachelor's thesis, UTAD, 1998. http://hdl.handle.net/10198/997.
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Os fitopatologistas têm feito um uso cada vez maior dos métodos serológicos na detecção e caracterização de fitopatogénios, por se tratar de técnicas rápidas, práticas e de elevada sensibilidade, que se podem adaptar às necessidades. De entre estes testes, as várias adaptações do ELISA (enzyme-linked immunosorbent assay) são, actualmente, os métodos mais divulgados, uma vez que permitem testar um elevado número de amostras num curto espaço de tempo e a preço moderado. A maioria dos vírus fitopatogénicos tem genoma de ss-RNA (ácido ribonucleico monocatenário) que, durante o processo replicativo, no interior das células do hospedeiro, dá origem a uma forma replicativa de ds-RNA (ácido ribonucleico bicatenário). Considerando que as plantas não infectadas não contêm quantidades detectáveis de ds-RNA, a sua presença em extractos vegetais é uma forte indicação de infecção viral. O presente trabalho desenvolveu-se no sentido de produzir um antissoro policlonal para um polinucleótido sintético bicatenário [poli(I):poli(C)] para detecção de ds-RNA. O antissoro foi caracterizado através de várias técnicas serológicas (ELISA-indirecto em placa de poliestireno, ELISA-indirecto em membrana de nitrocelulose e teste de difusão dupla em agar). O teste ELISA-indirecto em placa revelou ser mais sensível e prático do que o respectivo teste em membrana de nitrocelulose, tanto na detecção de poli(I):poli(C) como de ds-RNA purificado. Ambos se mostraram, no entanto, incapazes de detectar ds-RNA a partir de extractos aquosos de videira, o que dificulta o processo de detecção, uma vez que a extracção de ds-RNA de material vegetal é morosa e de baixo rendimento.
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Potgieter, Abraham Christiaan. "Cloning viral dsRNA genomes : analysis and application / A.C. Potgieter". Thesis, North-West University, 2004. http://hdl.handle.net/10394/334.
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Double-stranded RNA viruses occur in a large number of hosts in nature ranging from
bacteria to mammals. Molecular studies of the double-stranded RNA viruses have greatly
enhanced man's understanding of this large group of viruses as far as structure and function
of their genes and epidemiology is concerned. However, one of the major prerequisites of
obtaining this information is the ability to clone the genomes of these viruses for nucleotide
sequencing and recombinant protein expression studies. In the dsRNA field, cloning viral
genomes has historically been difficult and time consuming and created a bottleneck that
hampered molecular studies. The main aim of this investigation was to optimise a method for
cloning viral dsRNA genomes to the extent that it would be easy and fast as well as
applicable to most dsRNA viruses.
In this study a sequence-independent, oligo-ligation mediated dsRNA cloning procedure for
large genes (up to 6.8 kb) was perfected and tailored for routine use to amplify and clone
complete genome sets or individual genes. Complete genome sets could be amplified and
cloned from as little as 1 ng dsRNA. The method was shown to be simple and efficient
compared to other methods and is currently the only method that allows the amplification of
complete genomes in a single PCR reaction.
Complete gene sets of seven genomes from the Reovirus family, one from the Cystovirus
family and one mycovirus, have been amplified and cloned. The full-length VP2 genes of all
9 AHSV and 24 BTV serotypes were also cloned. Phylogenetic analysis of VP2-genes
revealed the same grouping of AHSVs and BTVs as serology. Several cloned genes of
AHSV, rotavirus and EEV have been utilised for recombinant protein production establishing
that the cloned cDNAs have full open reading frames. The nine AHSV VP2 genes have been
developed as serotype-specific probes which allowed serotyping of AHSV isolates within 4
days compared to 2-4 weeks needed with the traditional serological serotyping.
The new cloning procedure finally opens the bottleneck that hamstrung the development of
complete repertoires of recombinant vaccines, molecular diagnostics and epidemiology to
combat dsRNA viral diseases. It should now be possible to deliver on many of the
expectations that were envisaged for dsRNA virus research and biotechnology since the
advent of recombinant DNA technology.Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2004.
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Robinson, Helen Lynne. "Characterization of double-stranded RNA (dsRNA) from Rhizoctonia solani". Thesis, University of Edinburgh, 1999. http://webex.lib.ed.ac.uk/abstracts/robins01.pdf.
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Ho, Wing-tak y 何永德. "Glycyrrhizic acid potentiates dsRNA-induced nitric oxide generation inalveolar macrophages". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B31971799.
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Stricker, Ruth Lydia Olga [Verfasser], S. E. [Akademischer Betreuer] Behrens, E. [Akademischer Betreuer] Mundt y E. [Akademischer Betreuer] Vahlenkamp. "Influence of cellular dsRNA binding proteins in the replication process of a dsRNA virus / Ruth Lydia Olga Stricker. Betreuer: S.-E. Behrens ; E. Mundt ; E. Vahlenkamp". Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2012. http://d-nb.info/1025352467/34.
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Ho, Wing-tak. "Glycyrrhizic acid potentiates dsRNA-induced nitric oxide generation in alveolar macrophages". Click to view the E-thesis via HKUTO, 2004. http://sunzi.lib.hku.hk/hkuto/record/B31971799.
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Beyleveld, Mia. "Interaction of nonstructural protein NS3 of African horsesickness virus with viral and cellular proteins". Diss., Pretoria : [s.n.], 2007. http://upetd.up.ac.za/thesis/available/etd-12132007-115112.
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Brown, Joanne Louise. "The role of the dsRNA dependent protein kinase (PKR) in cell signalling". Thesis, St George's, University of London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.391775.
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Figueirêdo, Girlene Soares de. "Potencial Antagônico de Trichoderma spp. associados a dsRNA contra Colletotrichum guaranicola (Albuq.)". Universidade Federal do Amazonas, 2010. http://tede.ufam.edu.br/handle/tede/4491.
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CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico
Guaran´a is one of the most important crops in the state of Amazonas. Commercially, their use is as refrigerant, however, the industry of Guaran´a has been growing and diversifying its products, including the foreign market. The main disease that affects the culture of guarana is anthracnose, and its etiologic agent, is the pathogen Colletotrichum guaranicola. The main form of anthracnose control is the use of chemical fungicides, but in some regions, which have gained the recognition of organic agriculture, this control measure is not appropriate to the needs of production. Several species of genus Trichoderma, are antagonists used effectively in biological control of some plant pathogens, including the genus Colletotrichum. The presence of dsRNAs is common in fungi, being reported in the literature many studies associating them with various aspects of these hosts: hipovirulence morphological changes, the phenotype “ killer”in yeast, and others changes in these microorganisms. The main objective of this research was investigate the presence and influence of dsRNA on the antagonistic potential of Trichoderma spp. against C. guaranicola. We analyzed 100 isolates of Trichoderma spp., and only one presented dsRNA. The species was determined by sequencing the ITS region of rDNA and its micromorphological aspects (optical and electron microscopy) as Trichoderma asperellum. We proceeded to purify this material by column chromatography on cellulose and digestion with nucleases (DNase I and nuclease S1). To analyze the possible interference of these particles in the antagonistic potential of Trichoderma, the dsRNA was eliminated with sodium deoxycholate, added to PDA medium at a concentration of 200 mg / mL. Tests of antagonism in vitro (by the method of pairing in Petri dishes), showed a difference between inbred strains (with and without dsRNA) against the pathogen. In vivo tests, in plants of Mucuna aterrima, showed no statistical difference between the isolates with and without dsRNA. Morphological alterations were observed betweem isolates with and without dsRNA, the absence of dsRNA showed higher mycelial growth and higher production of spores. Based on these results it was concluded that dsRNA present in T. asperellum interfere in its potential antagonist in vitro tests, but not to its performance in vivo tests.
O guaran´a (Paulinia cupana) ´e uma das mais importantes culturas do Estado do Amazonas. Comercialmente, sua utilizac¸ ˜ao ´e maior nos refrigerantes gaseificados, no entanto, a ind´ustria do guaran´a vem crescendo e diversificando seus produtos, inclusive no mercado internacional. A principal doenc¸a que afeta a cultura do guaran´a ´e a antracnose, sendo seu agente etiol´ogico o fitopat´ogeno Colletotrichum guaranicola. A principal forma de controle da antracnose ´e o emprego de fungicidas qu´ımicos, por´em, em algumas regi˜oes, que adquiriram o reconhecimento de cultura orgˆanica, esta medida de controle n˜ao se adequa `as necessidades da produc¸ ˜ao. As esp´ecies do gˆenero Trichoderma, s˜ao antagonistas utilizadas eficazmente no controle biol´ogico de algumas esp´ecies fitopatogˆenicas, incluindo o gˆenero Colletotrichum. A presenc¸a de dsRNAs ´e frequente em fungos, sendo relatada na literatura muitas pesquisas associando-os com v´arios aspectos nestes hospedeiros: fen´otipos hipovirulentos, alterac¸ ˜oes morfol´ogicas, o fen´otipo “killer” em leveduras, ou ainda, a nenhuma alterac¸ ˜ao nestes microrganismos. Esta pesquisa teve por objetivos investigar a presenc¸a e influˆencia de dsRNA sobre o potencial antagˆonico de Trichoderma spp. contra C. guaranicola. Foram analisados 100 isolados de Trichoderma spp., sendo que apenas um apresentou dsRNA. A esp´ecie foi determinada por sequenciamento da regi˜ao ITS do rDNA e seus aspectos micromorfol´ogicos (microscopia ´optica e eletrˆonica de varredura) como Trichoderma asperellum. Procedeu-se a purificac¸ ˜ao deste material por meio de cromatografia em coluna de celulose e a digest˜ao com nucleases (DNAse I e nuclease S1). Para analisar a poss´ıvel interferˆencia destas part´ıculas no potencial antagˆonico dos isolados de Trichoderma, eliminou-se o dsRNA do isolado infectado com a substˆancia desoxicolato de s´odio, adicionada ao meio BDA na concentrac¸ ˜ao de 200 mg/mL. Testes de antagonismo in vitro (pelo m´etodo de pareamento em placa), revelaram diferenc¸a entre as linhagens is´ogenas (com e sem dsRNA) contra o fitopat´ogeno. O teste in vivo, pelo m´etodo de adic¸ ˜ao de esporos de antagonista e fitopat´ogenos em plantas de Mucuna aterrima, n˜ao apresentou diferenc¸a estat´ıstica entre as li-nhagens. Morfologicamente houve alterac¸ ˜oes entre os isolados com e sem dsRNA, sendo que os sem dsRNA, apresentaram maior crescimento micelial e maior produc¸ ˜ao de esporos. Diante dos resultados obtidos concluiu-se que o dsRNA presente em T. asperellum interfere no seu potencial antagonista em testes in vitro, mas n˜ao no seu desempenho em testes in vivo.
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Moura, Vanessa Santos [UNESP]. "Caracterização bioquímica e funcional de toxina killer produzida por Saccharomyces cerevisiae". Universidade Estadual Paulista (UNESP), 2017. http://hdl.handle.net/11449/151466.
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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
O bolor verde e a podridão azeda destacam-se entre as doenças de pós-colheita em frutos cítricos, causados por Penicillium digitatum e Geotrichum citri-aurantii, diminuindo a qualidade e a quantidade dos frutos e, consequentemente, resultando em significativas perdas econômicas. Uma alternativa para controle destes fungos é através da toxinas killer produzidas por algumas espécies de levedura, capazes de matar fungos filamentosos. Saccharomyces cerevisiae produz toxinas killer proteicas que são letais para células sensíveis de levedura. Estas toxinas foram agrupadas em quatro tipos, K1, K2, K28 e Klus, codificado por elementos extra cromossomais associados a partículas virais na forma de dsRNA. Este trabalho tem como objetivo caracterizar a toxina killer de S. cerevisiae ACB-K1 e testar sua atividade antagônica em patógenos pós-colheita de citros. O isolado ACB-K1 apresentou atividade killer, sobre levedura sensível (S. cerevisae NCYC 1006) além do fitopatógeno P. digitatum, não apresentando porém inibição contra o patógeno G. citri-aurantii. A toxina apresentou máxima atividade em pH 4,1 a 22 °C, tanto para a levedura sensível quanto para o fitopatógeno P. digitatum. A toxina apresentou estabilidade em diferentes pH de 4,1 a 6,0, após a incubação de 24h a 22 °C sobre o fungo. O isolado ACB-K1 apresentou dsRNA, sendo detectadas duas formas (LA e M-dsRNA), sugerindo que a base genética para a produção da toxina é extra cromossomal, dado confirmado pela cura do fenótipo killer a 40 °C. As frações obtidas por cromatografia de exclusão molecular em gel de Sephadex G75 demonstraram características de biocontrole contra o fitopatógeno P. digitatum.
Green mold and sour rot are among post-harvest diseases in citrus fruits, caused by Penicillium digitatum and Geotrichum citri-aurantii, reducing a quality and quantity of fruits and, consequently, resulting in significant economic losses. An alternative for the control of fungi is using killer toxins produced by some species of yeasts, capable of killing filamentous fungi. Saccharomyces cerevisiae produces protein killer toxins that are lethal to yeast sensitive cells. These toxins were grouped into four types, K1, K2, K28 and Klus, encoded by extrachromosomal elements associated with viral particles in the form of dsRNA. This work aims to characterize a killer toxin of S. cerevisiae ACB-K1 and to test its antagonistic activity in post-harvest citrus pathogens. The isolate ACB-K1 showed activity killer on sensitive yeast (S. cerevisae NCYC 1006) besides the phytopathogenic P. digitatum, but did not present inhibition against the pathogen G. citri-aurantii. The killer toxin showed maximum activity at pH 4.1 at 22 ° C for both a sensitive yeast and the phytopathogenic P. digitatum. The toxin presented stability at pH range from 4.1 to 6.0, after a 24h incubation at 22 ° C on the fungus. The ACB-K1 isolate showed dsRNA and two forms were detected (LA and M-dsRNA), suggesting that a genetic basis for a toxin production is extrachromosomal, confirmed by curing the killer phenotype at 40 ° C. The fractions obtained by exclusion chromatography Sephadex G75 gel, demonstrated biocontrol characteristics against the phytopathogen P. digitatum.
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Noland, Jeffrey Edward. "RISK PARAMETERS AND ASSESSMENT OF DIETARY dsRNA EXPOSURE IN FOLSOMIA CANDIDA". UKnowledge, 2017. http://uknowledge.uky.edu/entomology_etds/37.
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Assessing the risk of transgenic crop products is essential when determining the safety of a crop for deregulation and commercialization. The Organization of Economic and Cooperative Development (OECD), International Standards Organization (ISO), and governmental regulatory agencies require a battery of tests to demonstrate the safety of a GM product against several surrogate species of organisms that perform various ecosystem services. Assays are performed using toxicology methods established for pesticides. These methods have been applied to testing the safety, specificity and fate of Bacillus thuringiensis (Bt) Cry protein toxins engineered into crop plants and information exists on the effects on non-target organisms (NTOs). Toxicology assays are typically evaluated through a tier-based approach, where, if no or negligible risk via oral toxicity or phenotypic changes then a risk decision can be made. Long term exposure studies are often performed after commercial release of the crop occurs and provide a more in depth understanding of environmental impacts. Risk analyses are currently being performed on the product of the next generation of GM crops that express dsRNAs against Western Corn Rootworm. This thesis provides another such study, primarily focused on Folsomia candida, a microarthropod that is the subject of numerous toxicological studies. I describe the development of dsRNA stability assays, which indicate stability of dsRNA across assay time, both with and without F. candida. When exposed to dsRNA levels several orders of magnitude higher that what would be encountered in the environment, F. candida is not negatively impacted as seen through gene expression and life history trait analysis.
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Ostertag, Derek Glenn. "Novel dsRNA-dependent activation of a cellular antiviral response to vesicular stomatitis virus /". Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2005. http://wwwlib.umi.com/cr/ucsd/fullcit?p3167840.
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DAINO, GIANLUCA. "Characterization of Ebola virus VP35-dsRNA binding for drug development against Ebola virus disease". Doctoral thesis, Università degli Studi di Cagliari, 2017. http://hdl.handle.net/11584/248693.
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The Ebola virus (EBOV) VP35 protein plays an important role in the inhibition of the initial innate immune responses to EBOV infection leading to Ebola virus disease development. In fact, VP35 interaction with the RIG-I like receptors (RLR) cascade components inhibits the interferon (IFN) production, impeding proper host immune response. Hence, it has been shown that VP35 is a validate drug target. Full-length His-tagged recombinant VP35 (rVP35) has been previously expressed in prokaryotic system and used to validate a biochemical pull-down assay for the screening of small molecules targeted to the VP35-double strand (ds)-RNA interactions. However, low rVP35 amount of purified protein and the use of radioactive substrate for binding evaluation, strongly limited the screening system. In the present study, a new method for high-yield rVP35 expression and purification, based on denaturation and subsequent protein refolding was established. Subsequently, a novel assay based on the use of Nickel-coated plates using a fluorescent labeled 30mer dsRNA was validated, showing a VP35 Kd value for dsRNA binding around 4 nM, comparable to the one previously reported, and a Z'-factor equal to 0.69, that indicate a good assay.
The use of this biochemical assay to screen the ability of plant extracts and derived compounds to inhibit the rVP35 binding to dsRNA allowed to identified a few small molecules able to inhibit the VP35-dsRNA binding with IC50 values in the low micromolar range. Active plant extracts and derived compounds were also tested in a cellular assay to evaluate their ability to counteract the inhibitory activity of VP35 on the IFN response. We identified a number of compounds able to inhibit the VP35 function in biochemical assay but ineffective in the cellular system. Conversely, some other compound, unable to inhibit rVP35 binding to dsRNA in biochemical assay, subverted VP35 inhibition of IFN production in cellular assay. These results suggested that the VP35 binding to dsRNA may not be the driving force of the VP35 inhibition of the IFN cascade and that an alternative mechanisms of action could be hypothesized for those compounds not able to inhibit VP35-dsRNA binding but effective against the VP35 IFN inhibitory effect. An interesting compound showed activity in both biochemical and cellular assays, suggesting the possibility that some small molecules interact with VP35 in such way to disrupt its interaction with multiple targets.
In order to understand the relative role of VP35-dsRNA binding in inhibiting the RLR cascade, we studied the effects of the lack of VP35 dimerization, considered essential for VP35 binding to dsRNA. For this purpose, three single point mutations, proposed to be essential for VP35 dimerization, were introduced into the coiled-coil VP35 domain. We confirmed by in silico studies that introduction of these three mutations disrupted coiled-coil dimerization. The importance of VP35 dimerization for VP35-dsRNA binding was confirmed by biochemical assay studies, where the mutant rVP35 showed a reduce ability to bind dsRNA. However, the mutant VP35 expressed in a cellular system showed a limited reduction of the ability of IFN production inhibition compared to the wild-type VP35. These results seem to confirm the hypothesis that VP35 binding to dsRNA is not the main interaction needed for VP35 inhibition of the IFN production and suggested that the VP35 interactions with cellular components do not require VP35 dimerization.
In conclusion, we demonstrated that small molecules interacting with VP35 can subvert its inhibition of the IFN production, possibly inhibiting its interactions with cellular components of RLR pathway and suggested that VP35 binding to dsRNA is not the driving for VP35 inhibition RLR cascade activation.
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Schwartz, Thomas. "Structural basis for left handed Z-DNA binding by human dsRNA specific adenosine deaminase ADAR1". [S.l.] : [s.n.], 1999. http://www.diss.fu-berlin.de/2000/34/index.html.
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Elco, Christopher. "Regulation of dsRNA-induced transcription by NFêB and IRF-3 through TLR3 and RIG-1". Connect to text online, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=case1182005526.
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Lowe, Katie. "An investigation into the induction of tumour cell apoptosis by dsRNA : a pro-inflammatory event?" Thesis, St George's, University of London, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.568723.
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Dendritic cells (DCs) exhibit the capacity to recognise and phagocytose dying cells and invading pathogens and to present antigens from this internalised material on their surface. The recognition of these antigens by T cells can induce specific cytotoxic responses against other cells expressing the same antigen. These characteristics suggest the potential for DCs to be used a Cancer vaccine, due to their ability to stimulate an immune response against tumour cells. To ensure that cytotoxicity, rather than tolerance to the presented antigens is induced, co- stimulatory molecule expression on a DC is necessary. These molecules provide additional signals to T cells to ensure that only antigens presented in the appropriate environment are capable of inducing cytotoxic responses. This thesis investigates the hypothesis that poly (I:C), a synthetic dsRNA analogue, may induce an inflammatory form of apoptosis, releasing tumour antigens and inflammatory mediators capable of maturing DCs, for use as a Cancer vaccine. It has previously been shown that the administration of poly (I:C) induces apoptosis and the release of inflammatory cytokines in intestinal epithelial cells'. Results presented here demonstrate apoptosis, cytokine release and the up regulation of mRNA encoding several inflammatory factors in poly (I:C) treated tumour cells. Internalisation of apoptotic cells by DCs is shown and DCs cultured in supernatants from poly (I:C) treated tumour cells express co-stimulatory and MHC molecules, indicating their potential to stimulate T cells. Results indicate that poly (I:C) treated tumour cells may provide a more efficient method for loading DCs with antigens and inducing DC maturation than the loading of DCs with irradiated tumour material
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Özkan, Selin. "Investigations of Aspergillus fumigatus RNA silencing mechanisms and pathogenicity in the presence of dsRNA mycoviruses". Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/44122.
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Mycoviruses are a specific group of viruses that naturally infect and replicate in fungi. Aspergillus fumigatus, an opportunistic pathogen causing fungal lung diseases in humans and animals, is recently shown to harbour at least three different types of mycoviruses. RNA silencing mechanisms exist in fungi against mycoviruses and it is also known that A. fumigatus encodes all the proteins involved in RNA silencing, including Dicer and Argonaute homologues. Therefore, it is anticipated that small interfering RNAs (siRNAs) are generated and silencing of viral dsRNAs occurs as seen with other mycoviruses. This study compares the gene expression levels in isogenic lines of virus-free and virus-infected A. fumigatus. Eight different genes that play a role in RNA silencing were selected and analysed by quantitative PCR in order to determine whether the expression levels of these genes correlate with virus infection. To further evaluate the potential existence of siRNAs, small RNA profiles of virus-free and virus-infected isolates were compared using next generation sequencing. Virus-derived siRNAs were detected in the presence of three A. fumigatus virus infections. Moreover first miRNA-like candidates in A. fumigatus were identified. Additionally, the effects of mycoviruses on the pathogenicity of A. fumigatus were assessed using larvae of the greater wax moth Galleria mellonella. A. fumigatus uncharacterised virus was found to cause mild hypervirulence in A. fumigatus. To our knowledge this is the first study reporting the small RNA profiles of A. fumigatus isolates and also the interaction between mycoviruses and virulence of A. fumigatus.
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Elco, Christopher. "REGULATION OF dsRNA-INDUCED TRANSCRIPTION BY NFêB AND IRF-3 THROUGH TLR3 AND RIG-I". Case Western Reserve University School of Graduate Studies / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=case1182005526.
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Obadia, Benjamin. "Antiviral RNAi is initiated by dsRNA internalization into midgut cells in the insect model Drosophila". Paris 6, 2013. http://www.theses.fr/2013PA066227.
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Les insectes se sont adaptés à une variété admirable d'environnements où ils rencontrent divers microorganismes. Par conséquent, au cours de leur évolution, leurs mécanismes de défense ont été modelés pour générer un systme immunitaire flexible, leur permettant de survivre aux infections, mais favorisant, par ailleurs, une meilleure diffusion des microbes aux autres animaux. Historiquement, et de faon récurrente, les maladies transmises par les insectes ont constitué une grave menace pour les humains et ont concentré les efforts de recherche sur les interactions insecte-bactérie pluôt que sur les interactions insecte-virus. Récemment, l'implication du mécanisme d'ARN interférence (ARNi) dans la défense antivirale a donné des perspectives quant à la compréhension de l'immunité des insectes et à un éventuel contrôle des virus dont ils sont vecteurs. L'ARNi est activée par la présence d'ARN double-brin (ARNdb) généré au cours de la réplication virale, qui confre à l'insecte un moyen spécifique afin de contenir cette réplication. Chez l'insecte, la réponse antivirale supportée par l'ARNi peut être déclenchée par une immunisation artificielle d'ARNdb, établissant une protection antivirale au sein de l'organisme. La présente étude utilise l'insecte modèle Drosophila afin d'élucider in vivo la capacité de l'ARNdb à générer une réponse antivirale systémique contre des virus à ARN. L'étude fournit des évidences quant à l'implication majeure de l'épithélium intestinal dans l'internalisation de l'ARNdb présent dans l'environnement ou dans le systme circulatoir. Les rôles et propriétés de l'intestin dans la réponse immunitaire antivirale supportée par l'ARNi sont aussi discutésInsects have successfully adapted to an incredible variety of environments where they co-exist with diverse microorganisms (e. G. , fungi, bacteria, viruses). Consequently, insect defense mechanisms have been shaped over their evolution to generate a versatile immune system, allowing them to better survive infection, but also to better spread infection to others. Although insect-borne diseases have caused severe threats to humans since recorded history, studies had mainly focused on the dissection and understanding of insect-bacterium interactions while insect-virus interactions have remained poorly characterized. A few years ago, the discovery of RNA interference (RNAi) as an antiviral immune mechanism opened new perspectives on understanding insect immunity, which may potentially lead to the control of insect-borne viruses. RNAi is naturally triggered by virus-derived double-stranded (ds) RNA molecules and gives the insect a sequence-specific way of controlling viral replication. In insects, the antiviral RNAi response may also be mounted after artificial immunization with dsRNA, and evidence shows that a systemic protective state is established against viruses. Using the insect model Drosophila melanogaster, the present work inquires on the capacity of dsRNA to generate such a systemic silencing response against RNA viruses and focuses on the fate of dsRNA once in the insect organism. We provide evidence that the intestinal epithelium is the principal tissue involved in dsRNA uptake from both environmental and systemic media. The antiviral immune property of the midgut mediated by RNA interference is also debated
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Cramer, Tamlyn Jill. "Monitoring the African horsesickness virus life cycle by real-time RT-PCR of viral dsRNA". Diss., University of Pretoria, 2010. http://hdl.handle.net/2263/29034.
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African horsesickness (AHS), caused by African horsesickness virus (AHSV), is an infectious, non-contagious, insect-borne viral disease that affects members of the Equidae family. AHSV is a non-enveloped virus, consisting of 10 segments of double stranded RNA (dsRNA) encoding seven structural and four non-structural proteins. Infection of mammalian cell cultures with AHSV leads to severe cellular pathogenesis effects (CPE), whereas insect cells show no noticeable CPE. Differences are also apparent between different serotypes of AHSV with regards to viral production, viral release, membrane permeabilisation and CPE. In this study we investigated different aspects of the AHSV life cycle in cell culture. The first aim of this study was the development of a real-time RT-PCR assay to quantify and monitor dsRNA from AHSV-infected cells. The dsRNA was used to quantify viral production, as dsRNA (one copy of each segment) is found only within viral particles and is not free within the cytoplasm of infected cells, thus giving a true representation of the amount of virus. This was achieved by cloning genome segment 5, optimising the extraction and purification of dsRNA, optimising the cDNA synthesis reaction, as well as the establishment and standardisation of the real-time PCR reaction. The second part of the study investigated and compared viral production and viral release between three different serotypes of AHSV in either mammalian or insect cell lines. The amount of dsRNA, which represented cell associated virus from AHSV-3- and AHSV-4-infected BHK cells over a 48 hr time period, was monitored by real-time RT-PCR and revealed a second wave of dsRNA production. These findings possibly suggest that a second round of infection of released viruses is re-entering previously uninfected or infected cells to replicate further. AHSV production was monitored in KC cells and indicated no production of progeny virions. However, an improvement was obtained when AHSV was first passaged on KC cells before being used for infections. The results from this study are in agreement with the fact that for a particular virus to replicate efficiently in a specific cell line, it should first be adapted to those cells. The dsRNA was quantified from samples representing equivalent amounts of infectious virus (i.e. same titre values) of AHSV serotypes 2, 3 or 4. The amount of dsRNA was approximately four-fold higher from serotype 2 than from serotypes 3 and 4. When the percentage of viral entry into cells was analysed, the majority (approximately 90%) of virus from serotypes 3 and 4 entered the cells, whereas serotype 2 showed viral entry of only about 50%. These findings suggested that a large amount of virus from serotype 2 was non-infectious, while the majority of virus from serotypes 3 and 4 was infectious. However, serotype 2 was a great deal more cytotoxic to cells (e.g. earlier onset and severity of CPE) when compared to cells infected with either serotypes 3 or 4.Dissertation (MSc)--University of Pretoria, 2010.
Genetics
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Chukwudi, Chinwe Uzoma. "dsRNA as a target for tetracyclines and berberine, and a stress response regulator in Escherichia coli". Thesis, Royal Veterinary College (University of London), 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.572453.
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Ghazal, Ghada. "Biochemical and genetic analysis of RNA processing and decay". Thèse, Université de Sherbrooke, 2009. http://savoirs.usherbrooke.ca/handle/11143/4287.
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Gene expression is the conduit by which genetic information is connected into cellular phenotypes. Recently, it was shown that gene expression in mammalian cells is governed, at least in part, by the expression of short double stranded RNA (dsRNA). This mode of gene regulation is influenced by a large group of dsRNA binding proteins that could either stabilize or trigger the degradation of dsRNA. Indeed, double stranded RNA (dsRNA) specific ribonucleases (RNases) play an important role in regulating gene expression. In most eukaryotes, members of the dsRNA specific RNase III family trigger RNA degradation and initiate cellular immune response. Disruption of human . RNase III (Dicer) deregulates fetal gene expression and promotes the development of cancer. However, very little is known about the housekeeping function of eukaryotic RNase III and the mechanism by which they distinguish between exogenous and endogenous cellular RNA species. This thesis elucidates how dsRNAs are selected for cleavage and demonstrates their contribution to RNA metabolism in yeast as model eukaryote. Initially, the reactivity determinants of yeast RNase III (Rnt1p) were identified in vitro and used to study the global impact of Rnt1p on the processing of non-coding RNA. The results indicate that Rnt1p is required for the processing of all small nucleolar RNAs (snoRNAs) involved in rRNA methylation and identify a new role of Rnt1p in the processing of intronic snoRNAs. It was shown that Rnt1p cleavage helps to coordinate the expression of some ribosomal protein genes hosting intronic snoRNAs. Direct snoRNA processing from the pre-mRNA blocks the expression of the host gene, while delayed snoRNA processing from the excised intron allows the expression of both genes. In this way, the cell can carefully calibrate the amount of snoRNA and ribosomal proteins required for ribosome biogenesis. In addition, a global analysis of snoRNA processing identified new forms of Rnt1p cleavage signals that do not exhibit a conserved sequence motif but instead use a new RNA fold to recruit the enzyme to the cleavage site. This finding led to the conclusion that Rnt1p may use a wide combination of structural motifs to identify its substrates and thus increases the theoretical number of potential degradation targets in vivo . To evaluate this possibility, a new search for snoRNA independent Rnt1p cleavage targets was performed. Interestingly, many Rnt1p cleavage signals were identified in intergenic regions devoid of known RNA transcripts. In vivo , it was shown that Rnt1p induce the termination of non-polyadenylated transcripts and functions as a surveillance mechanism for transcription read-through. This finding directly links Rnt1p to the transcription machinery and provides a new mechanism for polyadenylation independent transcription termination. Together the work described in this thesis presents an example of how eukaryotic RNase III may identify its substrates and present a case study where transcription, RNA processing and stability are linked.
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Abraham, Ninan. "Consequences of loss of the murine dsRNA-dependent protein kinase, PKR, by natural mutation or genetic ablation". Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1998. http://www.collectionscanada.ca/obj/s4/f2/dsk2/ftp03/NQ36761.pdf.
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Blanc, Antony. "Capture of eukaryotic mRNA cap structures by the coat protein of the yeast L-A dsRNA virus". Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=41540.
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The eukaryotic mRNA 5$ sp prime$ cap structure m$ sp7$GpppX (where X is any nucleotide) is of crucial importance in translation initiation and protects messenger RNAs (mRNAs) against exonucleolytic degradation. L-A and L-BC are double-stranded RNA viruses which are persistently maintained in the cytoplasm of many Saccharomyces cerevisiae strains. Unexpectedly, their coat proteins exhibited a strong cap-binding activity. Unlike any other cap-binding protein, the coat protein (gag) of the L-A virus was shown to attach covalently to the cap structure of mRNAs, resulting in the cleavage of the cap structure and in the capture of m$ sp7$Gp. The linkage was determined to be a phosphoroimidazole bond between the $ alpha$ phosphate of the cap structure and a nitrogen ($N sp1$ or $N sp3$) in the gag His154 imidazole side-chain. Mutation of His154 abrogated the ability of gag to covalently link to the cap structure, without affecting cap recognition, viral particle formation from an L-A cDNA clone in vivo, or specific binding and replication of (+) single-stranded RNA in vitro. However, genetic analysis demonstrated that His154 was essential for the expression of viral proteins, possibly because efficient translation of the viral mRNA requires capping of the initially ppG-terminated viral transcripts by transfer of the m$ sp7$Gp captured by gag.
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Sehki, Hayat. "Rôle d’un suppresseur endogène de RNAi dans le développement de la plante et ses interactions avec les pathogènes". Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASB034.
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Le Post-Transcriptional Gene Silencing (PTGS) est un mécanisme dirigé contre les acides nucléiques invasifs endogènes (transposons) et exogènes (pathogènes, transgènes). Dans le cas des virus, le PTGS peut s’attaquer aux ARNs double-brin (dsRNAs) intermédiaires de la réplication virale et aux ARNs simple-brin viraux, mais il est souvent inhibé par des protéines virales appelées Viral Suppressor of RNAi (VSR). Chez la plante modèle Arabidopsis thaliana, une enzyme appelée RNase THREE-LIKE 1 (RTL1) est induite en réponse à l'infection virale et détruit les dsRNAs de manière non sélective. Cette enzyme devrait assurer à la plante une seconde ligne de défense en clivant les dsRNAs viraux, mais les VSR qui inhibent le PTGS inhibent généralement RTL1, indiquant que les virus ont mis en place des outils capables de combattre simultanément ces deux mécanismes de défense. Toutefois, un virus, le Turnip yellow mosaic virus (TYMV), est incapable d’inhiber RTL1 et semble même tirer profit de RTL1 pour réussir à infecter A. thaliana (Shamandi et al., 2015).Au cours de cette thèse, nous avons approfondi l’étude de l’interaction Arabidopsis-TYMV. Nous avons montré que le TYMV est incapable d’inhiber l’exécution du PTGS, mais est toutefois capable d’inhiber l’étape d’amplification du PTGS. Cette action est due à la protéine virale P69, et nous avons montré que P69 est retrouvée dans des corpuscules cytoplasmiques appelés siRNA-bodies qui sont le siège de l’amplification du PTGS. Par ailleurs, nous avons généré des mutants rtl1 et montré que l’absence de RTL1 retarde l’infection par le TYMV et augmente la quantité de siRNAs dirigés contre le virus, tandis que la surexpression de RTL1 favorise l’infection et inhibe la production des siRNAs anti-viraux. Nous avons observé que RTL1 était retrouvée dans les siRNA-bodies, et nous avons montré que RTL1 était capable de détruire non seulement les dsRNAs mais également les siRNAs. Ces résultats indiquent donc que le TYMV réussit à infecter A. thaliana en : i) se répliquant dans des invaginations de la membrane chloroplastique (Prod’homme et al., 2003) qui mettent vraisemblablement les dsRNAs intermédiaires de la réplication à l’abri du PTGS et de RTL1, ii) en induisant l’expression de RTL1 qui s’attaque aux dsRNAs et siRNAs induits par le PTGS dans les siRNA-bodies en réponse à l’infection, et iii) en exprimant la protéine P69 qui complète l’action de RTL1 en inhibant l’amplification du PTGS résiduel.Malgré un effet neutre ou négatif pour la plante vis-à-vis des virus, RTL1 est conservé dans toutes les accessions d’Arabidopsis, et l’étude du ratio des mutations synonymes et non synonymes dans les gènes RTL1 de 42 Eucotylédones suggère que RTL1 subit une pression de sélection de conservation, suggérant un rôle essentiel. Chez A. thaliana, RTL1 est exprimé faiblement dans la racine, les tissus en sénescence, et au cours du développement de la graine. Le phénotypage des plantes sauvages et des mutants rtl1 n’a pas révélé de différences morphologiques notables, mais nous avons observé que le poids des graines était supérieur chez les mutants rtl1. Par ailleurs, nous avons observé une senescence accrue chez le mutant rtl1, en particulier dans l’accession Ler. Cette différence entre Ler et Col nous a poussé à examiner si RTL1 pouvait contribuer à la variabilité naturelle du PTGS des transgènes entre les accessions Ler (PTGS peu efficace) et Col (PTGS très efficace). Nous avons observé que la mutation rtl1 n’affectait pas sensiblement l’efficacité du PTGS chez Col mais augmentait celle de Ler au niveau de Col, ce qui pourrait s’expliquer par une plus forte expression de RTL1 chez Ler que chez Col. L’effet de l’absence de RTL1 devra donc être précisé en condition normale et en condition d’infection en privilégiant Ler plutôt que ColPost-Transcriptional Gene Silencing (PTGS) is a defense mechanism that targets invading nucleic acids of endogenous (transposons) or exogenous (pathogens, transgenes) origins. During virus infection, PTGS theoretically targets double-stranded (ds)RNA intermediates of viral replication and viral single-stranded RNAs; however, most viruses encode proteins, referred to as viral suppressor of RNAi (VSR), which inhibit PTGS. In the model plant Arabidopsis thaliana, an enzyme referred to as RNase THREE-LIKE 1 (RTL1) is induced in response to viral infection and cleaves dsRNAs in a non-specific manner. This enzyme should provide a second line of defense by cleaving viral dsRNAs, but VSR that inhibit PTGS generally inhibit RTL1, indicating that viruses had put in place tools that simultaneously counteract these two defense mechanisms. Nevertheless, at least one virus, Turnip yellow mosaic virus (TYMV), is not able to inhibit RTL1 and in fact seems to take advantage of RTL1 to successfully infect A. thaliana (Shamandi et al., 2015).In this thesis, we deepened the study of Arabidopsis-TYMV interaction. We show that TYMV is not able to inhibit PTGS execution but is able to inhibit PTGS amplification. This effect is due to the viral protein P69, and we show that P69 localizes in cytoplasmic foci called siRNA-bodies, where PTGS amplification takes place. Furthermore, using in house-generated rtl1 mutants, we show that the lack of RTL1 delays TYMV infection and promotes the production of siRNAs directed against the virus, whereas RTL1 overexpression enhances viral symptoms and suppresses the production of anti-viral siRNAs. We show that RTL1 is found in siRNA-bodies, and we show that RTL1 attacks not only dsRNAs but also siRNAs. These results indicate that, TYMV successfully infect A. thaliana by : i) replicating in chloroplast membrane invaginations (Prod’homme et al., 2003), which likely shelter dsRNAs intermediates of replication from PTGS and RTL1, ii) inducing RTL1 expression, which promotes the destruction of dsRNAs and siRNAs produced by PTGS in siRNA-bodies in response to TYMV infection, and iii) expressing the P69 protein to inhibit residual PTGS amplification.Despite a neutral or detrimental effect on plant anti-viral PTGS, RTL1 is conserved in all Arabidopsis accessions, and the study of synonymous and non-synonymous substitutions ratios in RTL1 genes from 42 dicotyledonous plant reveals that RTL1 is under the control of a conservative selection, suggesting an essential role. In A. thaliana, RTL1 is weakly expressed in roots, in senescent tissues and during seed development. Phenotyping wild-type plants and rtl1 mutants did not revealed any significant morphological differences, but we observed that seeds weight is enhanced in rtl1 mutants. Moreover, we observed an increased senescence in rtl1 mutants, in particular in the Ler accession. This difference between Ler and Col prompted us to determine if RTL1 could participate in the natural variability of transgene PTGS efficiency between Ler (weak PTGS) and Col (strong PTGS). We observed that rtl1 mutations have no significant effect on PTGS efficiency in Col, but enhances PTGS efficiency in Ler, up to the level of Col, which could be explained by a strongest RTL1 expression in Ler compared to Col. These results indicate that the effect of RTL1 impairment should be further examined in normal and infectious contexts by focusing on Ler rather than Col
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DeWitte-Orr, Stephanie. "A study of innate antiviral mechanisms using fish cell lines". Thesis, University of Waterloo, 2006. http://hdl.handle.net/10012/1272.
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Understanding basic antiviral mechanisms in vertebrates is essential for developing methods to enhance antiviral responses and promote human and animal health. In fish these antiviral mechanisms are poorly understood, but are important to understand because of the devastating impact of viral diseases on aquaculture. Therefore, the antiviral responses of a rainbow trout macrophage-like cell line, RTS11, and two non-immune cell lines, the rainbow trout fibroblast RTG-2 and Chinook salmon embryo CHSE-214 were studied. Three antiviral responses were first characterized using the viral mimic, synthetic double-stranded RNA (poly IC), and then their induction was investigated using Chum salmon reovirus (CSV). The responses were: 1) apoptosis, which is programmed cell death and a primitive antiviral defense; 2) homotypic aggregation (HA), which is clustering of like immune cells; and 3) expression of Mxs, which are antiviral proteins belonging to GTPase super-family. Some of these antiviral mechanisms were investigated using a novel continuous cell line, PBLE, developed from a peripheral blood leukocyte preparation of the American eel, Anguilla rostrata. RTS11 was exceptionally susceptible to apoptosis. The cells died at lower concentrations of poly IC and other agents, including the translation inhibitor, cycloheximide (CHX), and fungal metabolite, gliotoxin. Death was predominantly by apoptosis, as judged by DNA ladders, nuclear fragmentation, and protection by caspase inhibitors. By contrast, the other two cell lines died most commonly by necrosis, when death did occur. Co-treating RTS11 with CHX greatly sensitized the cells to poly IC. Based on the protection afforded by inhibitors of dsRNA-dependent protein kinase (PKR), RTS11 apoptosis induced by poly IC with CHX co-treatment but not gliotoxin was mediated by PKR. As macrophages are likely among the first cells to contact viruses during an infection in vivo and are mobile, the sensitivity of RTS11 to dsRNA killing could reflect a protective mechanism by which virus spread is limited by the early death of these first responders.
HA of RTS11 was induced by poly IC. HA required divalent cations and was blocked by CHX and by PKR inhibitors. This suggested that HA induction was PKR-mediated and involved the synthesis of new cell surface molecule(s), possibly galectins. As an antiviral mechanism, HA induction by dsRNA could be interpreted as an initial protective response, allowing cell localization at the site of infection, but once translation becomes inhibited, apoptosis ensues.
Mx was induced by poly IC in RTS11 and RTG-2 as judged by RT-PCR. Western blotting revealed constitutive Mx expression more consistantly in RTS11, but induction by poly IC in both cell lines. Medium conditioned by cells previously exposed to poly IC and assumed to contain interferon also induced Mx transcripts in RTS11 but not RTG-2. In RTS11, poly IC activated PKR activity, and PKR inhibitors blocked Mx induction, which is the first demonstration of PKR mediating Mx expression.
The dsRNA virus, CSV, also induced apoptosis, HA, and Mx expression, but in some cases contrasting with poly IC experiments. CSV induced apoptosis in RTG-2 and CHSE-214 but not in RTS11, and HA induction by CSV in RTS11 was not dependent on PKR. Mx induction was sustained in RTG-2 and transitory in RTS11; however, both cell lines supported CSV replication.
The novel cell line, PBLE, was also characterized in this study. PBLE was derived from an adherent culture of peripheral blood leukocytes from the American eel, Anguilla rostrata. PBLE were found to grow over a wide range of temperatures and fetal bovine serum (FBS) concentrations. This cell line was able to undergo apoptosis in response to gliotoxin. PBLE was also susceptible to a number of viruses, including CSV; however, CSV infection did not lead to apoptosis.
This study suggests that antiviral responses are likely numerous and overlapping and depend on cell type and virus. Understanding them should lead to novel methods for protecting fish from viral diseases. More specifically, using cell lines such as PBLE may aid in the understanding of species specific and perhaps even cell type specific antiviral mechanisms.
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Rocha, Guilherme Zweig 1983. "A atuação da proteína quinase dependente de dsRNA (PKR) no desenvolvimento de tumor de cólon em camundongos obesos". [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/312750.
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Orientador: José Barreto Campello CarvalheiraTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas
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Resumo: Embora a obesidade seja reconhecida como importante causa de diabetes e doença cardiovascular, a associação entre obesidade e diferentes tipos de câncer tem recebido muito menos atenção. A associação entre obesidade e o desenvolvimento de câncer de cólon representa um dos principais avanços conceituais na patogênese do câncer de cólon da última década. Recentemente a atuação da inflamação subclínica da obesidade na carcinogênese ganhou destaque. Mecanisticamente acredita-se que a obesidade atue como promotor tumoral, e seus efeitos pró-tumorigênicos dependam principalmente da resposta inflamatória de baixo grau ocasionada pela obesidade que envolve a produção de citocinas inflamatórias e pró-tumorigênicas (TNF e IL-6). Uma das principais características da inflamação induzida por obesidade é a infiltração de macrófagos no tecido adiposo, produzindo citocinas inflamatórias e outros mediadores que interferem na sinalização insulínica. Inflamação e estresse de retículo que são conectadas em diversos níveis, são sistemas adaptativos de curto período de expressão necessárias para a função e sobrevivência do organismo, e ambas são prejudiciais quando ativadas cronicamente. Neste sentido, a ativação da PKR durante a inflamação e posterior ativação de JNK pela PKR, também interfere e prejudica a via de sinalização da insulina. A relação entre o câncer de cólon e obesidade pode ser devido a ação, em nível molecular, da inflamação subclínica de baixo grau e ao estresse celular causado por essa sinalização inflamatória. Sendo a PKR responsiva à sinalização inflamatória e também à via insulínica em outros tecidos, e relacionada à carcinogênese e à progressão em diversos tipos de câncer, a investigação de sua participação é relevante a medida que propicia o entendimento da fisiopatologia molecular de tumores de cólon. Assim, o objetivo principal do estudo foi avaliar o papel da PKR no desenvolvimento de tumores de cólon em camundongos submetido a dieta hiperlipídica. A ausência de PKR previne a formação de tumores. Além disso, aparentemente a ausência de PKR em células mielóides também confere proteção contra a resistência à insulina induzida por dieta hiperlipídica, reduzindo a inflamação induzida pela obesidade. Essas observações demostram que a PKR pode ser um ponto principal durante a carcinogênese associada à inflamação e pode representar um promissor alvo para a intervenção terapêutica
Abstract: Although obesity is recognized as a major cause of diabetes and cardiovascular disease, the association between obesity and different types of cancer has received much less attention. The association between obesity and the development of colon cancer is one of the major conceptual advances in the pathogenesis of colon cancer in the last decade. Recently the role of subclinical inflammation in obesity and in carcinogenesis gained prominence. Mechanistically it is believed that obesity acts as a tumor promoter, and their pro-tumorigenic effects depend mainly on low-grade inflammatory response caused by obesity, involving the production of inflammatory and pro-tumorigenic cytokines (TNF and IL-6). A key feature of obesity-induced inflammation is the infiltration of macrophages in adipose tissue, producing inflammatory cytokines and other mediators that interfere with insulin signaling. Reticulum stress and inflammation are connected on many levels and work as short period adaptive systems required for the function and survival of the organism, and both are detrimental when chronically activated. In this regard, the activation of PKR during inflammation and subsequent activation of JNK by PKR also interferes and impairs insulin signaling pathway. Thus, PKR can form a metabolically active inflammatory complex which then becomes part of the of insulin pathway and of the pathogens response pathway and control of translation sensible to nutrients. The relationship between colon cancer and obesity may be due to action at the molecular level, subclinical low-grade inflammation and cellular stress caused by this inflammatory signaling. PKR is responsive to inflammatory signaling and also to the insulin pathway in other tissues, and related to carcinogenesis and progression in several types of cancer. Thus, investigation of it's participation is relevant as it provides the understanding of the molecular pathophysiology of colon tumors. Thus, the main objective of the study was to evaluate the role of PKR in the development of colon tumors in mice subjected to a high-fat diet. The absence of PKR prevents the formation of tumors. Moreover, apparently the absence of PKR in myeloid cells also confers protection against resistance to insulin induced by a high-fat diet, reducing inflammation induced by obesity. These observations demonstrate that PKR can be a primary point during carcinogenesis associated with inflammation and may represent a promising target for therapeutic intervention
Doutorado
Fisiopatologia Médica
Doutor em Ciências
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Lima, Swiany Silveira. "Incidência e transmissão de dsRNA em Pseudocercospora griseola, agente causal da mancha-angular do feijoeiro comum (Phaseolus vulgaris)". Universidade Federal de Viçosa, 2008. http://locus.ufv.br/handle/123456789/5300.
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Previous issue date: 2008-07-29Conselho Nacional de Desenvolvimento Científico e Tecnológico
The common bean Phaseolus vulgaris shows great importance under feeding and economical aspects for the Brazilian people. However, its productivity has been low due to the occurrence of diseases and other factors. The angular leaf spot is distinguished among those diseases. Its causal agent is the Pseudocercospora griseola (Sacc.) Crous & U. Braun. Some mycovirus or virus-like particles were already described in several phytopathogenic fungus. Those viruses are unable to penetrating and lysing the host cells, and the intracytoplasmic transmission is accomplished by anastomosis among hyphae and the sporogenesis. Most mycovirus are found as multiple dsRNA fragments. In general, the mycovirus are cryptic (latent) concerning to the effects caused into phenotype of the host fungus, but they can affect the biology of their host by provoking morphological changes, hyper or hypovirulence. Because they are associated to the hypovirulence phenomenon, the mycoviruses show a potential use in the biocontroll of the phytopathogenic fungus. The general objective of this work was to characterize the mycovirus in the isolates of P. griseola, since they were recently detected in this fungus species for the first time. To reach this objective, the following were performed: the characterization of the dsRNA in different isolates; the vertical transmission analysis; and the obtainment of isogenic lines by the virus cure. The dsRNAs were detected in 31 from those 49 isolates of P. griseola under analysis. In the present study, most isolates showed multiple dsRNA fragments varying from zero to 10, as being the sizes estimated between 0.8 and 4.8 kb. The dsRNA fragment of 4.8 kb from the isolate was efficiently transmitted to the asexual spores. However, not all dsRNA fragments (between 1 and 6) found in the isolate Ig848 were transmitted to monosporic colonies. The cycloheximide was used at concentration of 20 µg/mL in order to obtain the mycovirus cure. This treatment was ineffective for the isolate 29-3, since those three colonies transplanted to cyclohexymide during four generations showed the same profile as the total nucleic acids found in the wild isolate. In the case of the isolate Ig848, this same chemical treatment eliminated the fragments 2.2; 2.0; 1.8; 1.2 and 1.0 kb of the colonies Ch2 and Ch4 after seven successive transplantings in medium containing cycloheximide. Several phytopathogenic fungus are attacked by viral infections, and this variation in the profile of the nucleic acid found in the P. griseola isolates is also observed in other plant pathogens. The presence of multiple fragments in only one isolate may be due to the infection by virus with segmented genome, RNA satellite, defective RNA or mixed infections. The efficiency of the transmission by conidia is variable, as depending on the fungus species under consideration, but it is usually near 100% as occurred for the isolate 29-3 of P. griseola. Either cure of some dsRNA fragments and the spontaneous loss during conidiogenesis observed for the isolate Ig848 rather indicate the infection by independent replicons. In those isogenic lines with and without some dsRNA fragments, the effect of the viral infection will be evaluated under some aspects such as the sporulation rate, growth and pathogenicity. The characterization of those viruses found in P. griseola will allow for further studies concerning to their use in the biological control of the angular leaf spot, which will turn possible to reduce the economical losses caused by this disease in agriculture.
O feijoeiro comum Phaseolus vulgaris apresenta grande importância alimentar e econômica para o brasileiro. No entanto, sua produtividade é baixa devido, em parte, à ocorrência de doenças. Entre essas doenças, destaca-se a mancha-angular, cujo agente causal é o fungo Pseudocercospora griseola (Sacc.) Crous & U. Braun. Micovírus ou partículas semelhantes a vírus já foram descritas em diversos fungos fitopatogênicos. Esses vírus são incapazes de penetrar e lisar as células hospedeiras, sendo a transmissão intracitoplasmática por meio da anastomose entre hifas e da esporogênese. A maior parte dos micovírus é encontrada como múltiplos fragmentos de dsRNA. Em geral, os micovírus são crípticos (latentes) em relação aos efeitos provocados no fenótipo do fungo hospedeiro, mas podem influenciar a biologia de seu hospedeiro, provocando alterações morfológicas, hiper ou hipovirulência. Por estarem associados ao fenômeno de hipovirulência, os micovírus apresentam uso potencial no biocontrole de fungos fitopatogênicos. O objetivo geral deste trabalho foi caracterizar micovírus presentes em isolados de P. griseola, uma vez que recentemente estes foram detectados, pela primeira vez, nesta espécie de fungo. Para atingir este objetivo, foi realizada a caracterização de dsRNAs presentes em diferentes isolados, a análise da transmissão vertical e a obtenção de linhagens isogênicas por meio da cura de vírus. dsRNAs foram detectados em 31 dos 49 isolados de P. griseola analisados. Neste trabalho, a maioria dos isolados apresentou múltiplos fragmentos de dsRNA, que variaram de zero a 10, com tamanhos estimados entre 0,8 e 4,8 kb. O fragmento de dsRNA de 4,8 kb do isolado 29-3 foi eficientemente transmitido para os esporos assexuais. Entretanto, nem todos os fragmentos de dsRNA, entre um e seis, presentes no isolado Ig848 foram transmitidos para colônias monospóricas. Cicloheximida foi utilizada em concentração de 20 μg/mL a fim de obter a cura de micovírus. Para o isolado 29-3, este tratamento foi ineficaz, pois as três colônias repicadas em cicloheximida durante quatro gerações apresentaram o mesmo perfil de ácidos nucléicos totais presente no isolado selvagem. No caso do isolado Ig848, este mesmo tratamento eliminou os fragmentos de 2,2; 2,0; 1,8; 1,2 e 1,0 kb das colônias Ch2 e Ch4, após sete repicagens sucessivas em meio contendo cicloheximida. Diversos fungos fitopatogênicos são acometidos por infecções virais, sendo que essa variação no perfil de ácidos nucléicos presente nos isolados de P. griseola também é observada em outros patógenos de plantas. A presença de múltiplos fragmentos, em um único isolado, pode ser devido à infecção por vírus com genoma segmentado, RNA satélite, RNA defectivo ou infecções mistas. A eficiência de transmissão por meio dos conídios é variável, dependendo da espécie de fungo considerada, mas geralmente é próxima a 100%, conforme ocorreu para o isolado 29-3 de P. griseola. Tanto a cura de alguns fragmentos de dsRNA quanto a perda espontânea durante a conidiogênese, observada para o isolado Ig848, indicam a infecção por replicons independentes. Estas linhagens isogênicas, com e sem alguns fragmentos de dsRNA, terão o efeito da infecção viral avaliados em aspectos como a taxa de esporulação, o crescimento e a patogenicidade. A caracterização destes vírus, presentes em P. griseola, permitirá estudos posteriores sobre o uso destes no controle biológico da mancha-angular, o que poderá reduzir as perdas econômicas causadas por essa doença na lavoura.
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Darissa, Omar [Verfasser]. "Molecular characterization of a novel segmented dsRNA mycovirus and its association with hypovirulence of Fusarium graminearum / Omar Darissa". Hamburg : Staats- und Universitätsbibliothek Hamburg, 2011. http://d-nb.info/1010759787/34.
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Carvalho, Eudislaine Fonseca de. "Resposta antiviral em células LL5 de Lutzomyia longipalpiscomparativo entre infecção por vírus da Estomatite Vesicular (VSV) e dsRNA". reponame:Repositório Institucional da FIOCRUZ, 2013. https://www.arca.fiocruz.br/handle/icict/13940.
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Previous issue date: 2013Fundação Oswaldo Cruz. Instituto Oswaldo Cruz. Rio de Janeiro, RJ, Brasil
As doenças transmitidas por insetos vetores são de grande importância para saúde pública. No Brasil, as principais doenças compreendem a malária, doença de Chagas, leishmaniose, dengue e febre amarela. O inseto vetor flebotomíneo é o principal transmissor da leishmaniose. Porém, também são vetores de outros agentes patogênicos e hospedeiros de diversos outros microrganismos, tais como bactérias, fungos e arbovírus. Os arbovírus são biologicamente transmitidos entre hospedeiros vertebrados por insetos hematófagos. Sua distribuição ocorre de forma global, porém a maioria é encontrada em áreas tropicais, onde as condições climáticas permitem a transmissão durante todo o ano. Os arbovírus do gênero Vesiculovírus, Orbivírus e Phlebovírus são os mais comuns encontrados em flebotomíneos. Além destes gêneros isolados do próprio inseto, os vírus Mayaro e do Oeste do Nilo também são capazes de infectar uma linhagem celular (LL5) da espécie Lutzomyia longipalpis. A resposta imune é essencial para os artrópodes sobreviverem aos agentes patogênicos, as principais vias envolvidas na resposta imune de artrópodes incluem a via Imd, Toll e Jak/Stat. Os artrópodes possuem diversos mecanismos contra infecção viral, entre eles a apoptose, a via de RNA de interferência (RNAi) e a autofagia. O mecanismo utilizado pela maioria dos vetores é o silenciamento da expressão gênica dos vírus através da via do RNAi Esta via é amplamente conservada em diferentes espécies e se baseia na complementariedade do RNA dupla fita (dsRNA) para degradação de mRNA, sendo assim uma resposta sequência específica. A via Jak/Stat também tem sido associada à resposta antiviral. O gene responsivo a vírus, Vago, atua como um interferon like e é responsável pela ativação de Jak/Stat. A única descrição que existe sobre a resposta antiviral em flebotomíneos é uma resposta inespecífica, que é ativada por qualquer dsRNA. Como não se tem conhecimento preciso dos mecanismos de defesa antiviral neste inseto, nosso trabalho avalia o papel de diferentes componentes do sistema imune no mecanismo de defesa antiviral em células LL5. Realizamos dois modelos eficientes de infecção com vírus VSV e mimetização da infecção, através de transfecção de poly I:C (dsRNA), em células LL5 e em células Aag2 de A. aegypti para o estudo da resposta antiviral. Após a infecção ou transfecção de dsRNA avaliamos o perfil de expressão de alguns genes da resposta antiviral: Dicer 2, Vago, Stat, Defensina e Atg18. LL5 apresentou uma resposta a infecção com VSV diferente das células Aag2, sendo que nestas células a dsRNA é capaz de ativar uma resposta antiviral contra VSV, diferentemente das células LL5. Sugerimos também que a resposta antiviral de LL5 contra infecção com VSV ocorra através do mecanismo de autofagia, pois, outros genes clássicos da via de RNAi e Jak/Stat (Dicer, Vago e Stat) não foram modulados positivamente neste modelo
Insect-borne diseases have a great importance in public health. In Brazil, the main diseases transmitted by insects include malaria, Chagas disease, leishmaniasis, dengue and yellow fever. Sandflies are the main vectors of leishmania parasites, but may also harbors and even transmit other pathogens such as bacteria, fungi and arbovirus. The arboviruses are biologically transmitted between vertebrate hosts by hematophagous insects. Its distribution occurs globally, but mostly in tropical areas, where climatic conditions may allow transmission throughout the year. The arbovirus of the genus Vesiculovirus, Orbivirus and Phlebovirus are commonly found in sandflies. In addition, the Mayaro virus and West Nile virus are also able of infecting Lutzomyia longipalpis cell line (LL5). The immune response is essential for arthropods to survive the pathogens infection and the major pathways involved in this immune response are Imd, Toll and Jak/Stat pathways. The arthropods have diverse mechanisms against viral infection, including apoptosis, the RNA interference pathway (RNAi) and autophagy. The mechanism used by most of the vectors is the silencing of gene expression through the RNAi pathway. This pathway is conserved among different species and is based on degradation of mRNA complementary to double-strand RNA (dsRNA), thus being a sequence-specific response. The Jak/Stat pathway has also been associated with antiviral response The virus responsive gene, Vago, acts as an interferon-like and is responsible for activation of Jak/Stat. The only information regarding the antiviral response in sandflies is a non-specific response, which is activated by any dsRNA. Since there is no precise knowledge of the antiviral defense mechanism in this insect, our study evaluated the role of different components of the immune system in antiviral defense mechanism in LL5 cells. We used two efficient models of infection with VSV virus and mimicking the infection by transfection of poly I:C (dsRNA) in LL5 cells and Aag2 cells of Aedes aegypti to study the antiviral response. After infection or transfection of dsRNA we evaluated the expression profile of some genes related to antiviral response: Dicer 2, Vago, Stat and Atg18. LL5 and Aag2 showed different responses to VSV infection, and in Aag2 cells the dsRNA is able to activate and antiviral response against VSV, differently in LL5 cells. We also suggest that the antiviral response of LL5 against VSV infection occurs through the mechanism of autophagy, because other classical genes of the RNAi and Jak/Stat pathways were not positively modulated in this model
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Belhouchet, Mourad. "Analysis of an anti-silencing mechanism involved in immune evasion by vector-borne dsRNA animal viruses of family Reoviridae". Thesis, University of Oxford, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.711672.
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Thornley, Thomas B. "IFN-α/β Induction by dsRNA and Toll-Like Receptors Shortens Allograft Survival Induced by Costimulation Blockade: A Dissertation". eScholarship@UMMS, 2006. https://escholarship.umassmed.edu/gsbs_diss/254.
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Costimulation blockade protocols are promising alternatives to the use of chronic immunosuppression for promoting long-term allograft survival. However, the efficacy of costimulation blockade-based protocols is decreased by environmental insults such as viral infections. For example, lymphocytic choriomeningitis virus (LCMV) infection at the time of costimulation blockade treatment abrogates skin allograft survival in mice. In this dissertation, we test the hypothesis that viruses shorten allograft survival by activating the innate immune system through pattern-recognition receptors (PRRs), such as toll-like receptors (TLRs).
To investigate the role of innate immunity in shortening allograft survival, costimulation blockade-treated mice were co-injected with TLR2 (Pam3Cys), TLR3 (polyinosinic:polycytidylic acid, poly(I:C)), TLR4 (lipopolysaccharide, LPS), or TLR9 (CpG DNA) agonists, followed by transplantation with skin allografts 7 days later. Costimulation blockade prolonged skin allograft survival that was shortened in mice coinjected with TLR agonists. To investigate the underlying mechanisms of this observation, we used synchimeric mice, which circulate trace populations of anti-H2b transgenic alloreactive CD8+ T cells. In synchimeric mice treated with costimulation blockade, co-administration of all four TLR agonists prevented deletion of alloreactive CD8+ T cells. These alloreactive CD8+ T cells 1) expressed the proliferation marker Ki-67, 2) upregulated CD44, and 3) failed to undergo apoptosis. We also demonstrate that costimulation blockade-treated CD8α-deficient mice exhibit prolonged allograft survival when co-injected with LPS. These data suggest that TLR agonists shorten allograft survival by impairing the apoptosis of alloreactive CD8+T cells.
We further delineate the mechanism by which TLR agonists shorten allograft survival by demonstrating that LPS and poly(I:C) fail to shorten allograft survival in IFNRI- deficient mice. Interestingly, the ability of poly(I:C) to more potently induce IFN-α/β than LPS correlates with its superior abilities to shorten islet allograft survival and induce allo-specific CTL activity as measured by an in vivo cytotoxicity assay. The ability to shorten allograft survival and induce IFN-α/β is a TLR-dependent process for LPS, but is a TLR-independent process for poly(I:C). Strikingly, the injection of IFN-β impairs alloreactive CD8+T cell deletion and shortens allograft survival, similar to LPS and poly(I:C). These data suggest that LPS and poly(I:C) shorten allograft survival by inducing IFN-α/β through two different mechanisms.
Finally, we present data showing that viruses (LCMV, Pichinde virus, murine cytomegalovirus and vaccinia virus) impair alloreactive CD8+T cell deletion and shorten allograft survival, in a manner comparable to LPS and poly(I:C). Similar to LPS, LCMV and MCMV exhibit an impaired ability to shorten allograft survival in MyD88-deficient mice. These data suggest that the MyD88 pathway is required for certain viruses and TLR-agonists to shorten allograft survival.
In this dissertation, we present data supporting an important role for TLRs and IFN- α/β in shortening allograft induced by costimulation blockade. Our findings suggest that targeting these pathways during the peri-transplant period may enhance the efficacy of costimulation blockade protocols in the clinic.
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Dickerman, Benjamin K. "THE PHYSIOLOGICAL FUNCTION OF THE dsRNA-BINDING PROTEIN PACT/RAX, PROTEIN ACTIVATOR OF PKR AND ITS ROLE IN MOUSE DEVELOPMENT". Case Western Reserve University School of Graduate Studies / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=case1335901974.
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Nathania, Lilian. "Biochemical Analysis of Thermotoga maritima Ribonuclease III and its Ribosomal RNA Substrates". Diss., Temple University Libraries, 2011. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/140013.
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ChemistryPh.D.
The site-specific cleavage of double-stranded (ds) RNA is a conserved early step in bacterial ribosomal RNA (rRNA) maturation that is carried out by ribonuclease III. Studies on the RNase III mechanism of dsRNA cleavage have focused mainly on the enzymes from mesophiles such as Escherichia coli. In contrast, little is known of the RNA processing pathways and the functions of associated ribonucleases in the hyperthermophiles. Therefore, structural and biochemical studies of proteins from hyperthermophilic bacteria are providing essential insight on the sources of biomolecular thermostability, and how enzymes function at high temperatures. The biochemical behavior of RNase III of the hyperthermophilic bacterium Thermotoga maritima is analyzed using purified recombinant enzyme and the cognate pre-ribosomal RNAs as substrates. The T. maritima genome encodes a ~5,000 nucleotide (nt) transcript, expressed from the single ribosomal RNA (rRNA) operon. RNase III processing sites are expected to form through base-pairing of complementary sequences that flank the 16S and 23S rRNAs. The Thermotoga pre-16S and pre-23S processing stems are synthesized in the form of small hairpins, and are efficiently and site-specifically cleaved by Tm-RNase III at sites consistent with an in vivo role of the enzyme in producing the immediate precursors to the mature rRNAs. T. maritima (Tm)-RNase III activity is dependent upon divalent metal ion, with Mg^2+ as the preferred species, at concentrations >= 1 mM. Mn^2+, Co^2+ and Ni^2+ also support activity, but with reduced efficiency. The enzyme activity is also supported by salt (Na^+, K^+, or NH4^+) in the 50-80 mM range, with an optimal pH of ~8. Catalytic activity exhibits a broad temperature maximum of ~40-70 deg C, with significant activity retained at 95 deg C. Comparison of the Charged-versus-Polar (C-vP) bias of the protein side chains indicates that Tm-RNase III thermostability is due to large C-vP bias. Analysis of pre-23S substrate variants reveals a dependence of reactivity on the base-pair (bp) sequence in the proximal box (pb), a site of protein contact that functions as a positive determinant of recognition of E. coli (Ec)-RNase III substrates. The pb sequence dependence of reactivity is similar to that observed with the Ec-RNase III pb. Moreover, Tm-RNase III cleaves an Ec-RNase III substrate with identical specificity, and is inhibited by pb antideterminants that also inhibit Ec-Rnase III. These studies reveal the conservation acrosss a broad phylogenetic distance of substrate reactivity epitopes, both the positive and negative determinants, among bacterial RNase III substrates.
Temple University--Theses
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Elbahesh, Husni M. "Study of Innate Immune Response Components in West Nile Virus Infected Cells". Digital Archive @ GSU, 2011. http://digitalarchive.gsu.edu/biology_diss/94.
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Two cellular innate responses, the dsRNA protein kinase (PKR) pathway and the 2'-5' oligoadenylate synthetase (OAS)/RNase L pathway, are activated by dsRNAs produced by viruses and reduce translation of host and viral mRNAs. PKR activation results in eIF2a phosphorylation. As a consequence of eIF2a phosphorylation, stress granules (SGs) are formed by the aggregation of stalled SG proteins with pre-initiation complexes and mRNA. West Nile virus (WNV) infections do not induce eIF2a phosphorylation despite upregulation of PKR mRNA and protein suggesting an active suppression of PKR activation. Assessment of the mechanism of suppression of PKR activation in WNV-infected cells indicated that WNV infections do not induce PKR phosphorylation so that active suppression is not required.
In contrast to infections with "natural" strains of WNV, infections with the chimeric W956 infectious clone (IC) virus efficiently induce SGs in infected cells. After two serial passages, the IC virus generated a mutant (IC-P) that does not induce SGs efficiently but does induce the formation of NS3 granules that persist throughout the infection. This mutant was characterized.
2'-5' oligoadenylate synthetases (OAS) are activated by viral dsRNA to produce 2-5A oligos that activate RNase L to digest viral and cellular RNAs. Resistance to flavivirus-induced disease in mice is conferred by the full-length 2'-5' oligoadenylate synthetase 1b (Oas1b) protein. Oas1b is an inactive synthetase that is able to suppress the in vitro synthetase activity of the active synthetase Oas1a. The ability of Oas1b to inhibit Oas1a synthetase activity in vivo and to form a heteromeric complex with Oas1a was investigated. Oas1b suppressed 2-5A production in vivo. Oas1a and Oas1b overexpressed in mammalian cells co-immunoprecipitated indicating the formation of heteromeric complexes by these proteins.
Unlike mice, humans encode a single OAS1 gene that generates alternatively spliced transcripts encoding different isoforms. Synthetase activity has previously been reported for only three of the isoforms. The in vitro synthetase activity of additional OAS1 isoforms was analyzed. All tested isoforms synthesized higher order 2-5A oligos. However, p44A only produced 2-5A dimers which inhibit RNase L.
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Bento, Flavia de Moura Manoel. "Silenciamento gênico por interferência de RNA (RNAi) em traça-do-tomateiro, Tuta absoluta (Meyrick), utilizando bactérias expressando dupla fita de RNA (dsRNA)". Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/11/11137/tde-06042018-144427/.
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O uso da técnica de RNAi vem sendo avaliada em diversos insetos-pragas pois, é uma estratégia inovadora que pode ser integrada no manejo de importantes pragas agrícolas. Os insetos da Ordem Lepidoptera são reconhecidos por apresentarem recalcitrância à técnica de silenciamento utilizando dsRNA. Assim, ajustes devem ser feitos aos métodos de entrega de dsRNA para que haja estabilidade da molécula até atingir o mRNA alvo de silenciamento no inseto. O silenciamento gênico por RNAi possui potencial de uso para o controle da \"traça-do-tomateiro\" Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae), uma das principais pragas do tomateiro no mundo. O objetivo do trabalho foi selecionar e avaliar o silenciamento de genes de T. absoluta, utilizando o método de entrega de dsRNA via bactéria E. coli HT115 (DE3), disponibilizada em dieta artificial. Também, objetivando a aplicabilidade da utilização de bactérias que se desenvolvam no mesmo hábitat de insetos-pragas, estudou-se a colonização das bactérias endofíticas Pantoea agglomerans linhagem 33.1, Burkholderia sp. linhagem SCMS54 e Burkholderia ambifaria linhagem RZ2MS16 em plantas de tomateiro e lagartas de T. absoluta, para posterior transformação e tentativa de utilização como estratégia de entrega de dsRNA para silenciamento de genes alvos de T. absoluta. Foram avaliados, por meio da metodologia de dieta artificial, oito genes de T. absoluta: juvenile hormone inducible protein - JHP; juvenile hormone epoxide hydrolase protein - JHEH; ecdysteroid 25-hydroxylase - PHM; chitin synthase A - CHI; glutathione S-transferase epsilon 2 - GST; carboxylesterase - COE; alkaline phosphatase - AP e; arginine kinase - AK. Por meio de avaliação dos parâmetros biológicos (mortalidade larval; duração da fase larval e peso de pupas) e expressão gênica em cinco períodos de alimentação, comprovou-se a eficiência da metodologia na avaliação do silenciamento gênico por RNAi, sendo possível realizar screening de grande quantidade de genes e avaliar os efeitos do silenciamento gênico no desenvolvimento de T. absoluta. Os genes AK, CHI e JHP apresentaram resultados positivos quanto ao silenciamento gênico e mortalidade larval, sendo promissores para uso de silenciamento por RNAi como estratégia de controle de T. absoluta. Pantoea agglomerans apresentou os melhores resultados de colonização de plantas de tomateiro \"Micro-Tom\" e lagartas de T. absoluta, além de estarem presentes em tecidos preferencialmente utilizados na alimentação das lagartas. Porém, lagartas não apresentaram diferenças na mortalidade larval ao se alimentarem de plantas de tomateiro \"Micro-Tom\" inoculadas com bactérias de P. agglomerans transformadas.The use of RNAi technique has been evaluated in several insect pests because it is an innovative strategy that can be integrated in the management of important agricultural pests. Insects of the Order Lepidoptera are recognized to present recalcitrance to gene silencing using dsRNA. Thus, adjustments should be done to dsRNA delivery methods to have molecule stability until it reaches the mRNA target for silencing in the insect. Gene silencing by RNAi has potential use to control the tomato leafminer Tuta absoluta (Meyrick, 1917) (Lepidoptera: Gelechiidae), one of the main insect pests of tomato crop worldwide. The objective of this work was to select and evaluate the silencing of T. absoluta genes using dsRNA delivery method via E. coli HT115 (DE3) bacterium, offered in artificial diet. Also, aiming the applicability of the use of bacteria growing in the same habitat of insect pests, we evaluate the colonization of the endophytic bacteria Pantoea agglomerans strain 33.1, Burkholderia sp. strain SCMS54 and Burkholderia ambifaria strain RZ2MS16 in tomato plants and T. absoluta larvae for further transformation and potential use as dsRNA delivery strategy for silencing target genes of T. absoluta. We evaluated eight genes of T. absoluta: juvenile hormone inducible protein - JHP; juvenile hormone epoxide hydrolase protein - JHEH; ecdysteroid 25-hydroxylase - PHM; chitin synthase A - CHI; glutathione S-transferase epsilon 2 - GST; carboxylesterase - COE; alkaline phosphatase - AP and; arginine kinase - AK. Evaluating biological parameters (larval mortality, larval stage duration and pupal weight) and gene expression in five feeding periods, we proved the efficiency of the methodology in the evaluation of gene silencing by RNAi, and evaluated the effects of gene silencing on the development of T. absoluta. The genes AK, CHI and JHP presented positive results regarding gene silencing and larval mortality, being promising to use RNAi silencing as a strategy to control T. absoluta. Pantoea agglomerans showed good results colonizing \"Micro-Tom\" tomato plants and T. absoluta larvae, besides being present in tissues preferentially used by larvae for feeding. However, larvae did not show differences in larval mortality when feeding on tomato plants inoculated with transformed P. agglomerans.
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Garcia, Rayssa Almeida. "Validação da estabilidade de estruturas de dsRNA para uso no silenciamento gênico de insetos-praga : avaliação na planta e no inseto alvo". reponame:Repositório Institucional da UnB, 2015. http://dx.doi.org/10.26512/2015.02.D.18462.
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Dissertação (mestrado)—Universidade de Brasília, Programa de Pós-Graduação em Biologia Molecular, 2015.Texto parcialmente liberado pelo autor. Conteúdo restrito: capítulos I, II e III.
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O algodão é uma das principais commodities da economia brasileira, alavancando o País ao posto de quinto maior produtor mundial. Entre as diferentes pragas da cotonicultura, o bicudo-do-algodoeiro é o inseto-praga mais destrutivo. Pela biotecnologia, um dos métodos alternativos de controle de insetos-praga é o silenciamento gênico, por meio do RNA interferente. Contudo, em insetos, absorção do RNA fita dupla (dsRNA) produzidos por plantas é dificultada, principalmente em decorrência da clivagem do dsRNA na planta e da ação de nucleases presentes no intestino dos insetos. Assim, os objetivos do presente estudo foram estudar o papel de nucleases intestinais de Anthonomus grandis na degradação do dsRNA e aumentar a estabilidade das moléculas de dsRNA. Primeiramente, a atividade nucleásica ácida foi detectada no homogenato intestinal de A. grandis. Por meio da busca no transcritoma de A. grandis foram encontradas três contigs codificadores de nucleases, denominados AgNuc1, AgNuc2 e AgNuc3, cujas sequências foram caracterizadas e validadas por RNAi. As três sequências apresentaram similaridade acima de 50 % em relação às outras nucleases de insetos. A análise por qPCR demonstrou que AgNuc2 e AgNuc3 foram altamente expressas no intestino de A. grandis. O silenciamento específico de AgNuc2 culminou na redução da degradação do dsRNA; demonstrando ser a principal nuclease associada à degradação de dsRNA no lúmen intestinal de A. grandis. Além disso, visando aumentar a estabilidade do dsRNA, foram desenhados dsRNAs, baseados na arquitetura de viróide, que são resistentes à ação de nucleases. A análise do movimento de dsRNA-viróide marcado com Cy3 no sistema vascular de Arabidopsis thaliana demonstrou uma localização celular específica para a estrutura do dsRNA. O dsRNA baseado na arquitetura da família Pospiviroidae, foi localizado no núcleo das células, enquanto que o dsRNA, baseado na arquitetura da família Avsunviroidae, foi localizado nos cloroplastos das células. Os dsRNAs estabilizados mostraram uma capacidade de silenciamento gênico 8 vezes superior, quando comparado ao dsRNA linear não estruturado Os dados aqui gerados contribuem para o conhecimento do mecanismo de RNAi em insetos e demonstram que as moléculas de dsRNA estabilizados apresentam grande potencial para a aplicabilidade da tecnologia do RNAi visando o controle de insetos-praga.
Cotton is one of the most important Brazilian commodities and Brazil is the fifth largest world producer. Nonetheless, productivity is constantly crippled by a variety of agricultural pests. Among the different cotton insect pests, cotton boll weevil is the most destructive. By using biotechnology strategies, one of the alternative methods for controlling crop pests is gene silencing through RNA interference. However, in insects, the absorption of double stranded RNA produced by plants is hampered due to dsRNA cleavage in plants tissues and due to the presence of insect gut nucleases. In this context, the objects of this study were to investigate the dsRNA degradation by Anthonomus grandis gut nucleases and improve the stability of dsRNA. Nucleasic activity was detected in A. grandis intestinal homogenate. After searching in A. grandis transcriptome, three contigs codifying nucleases were found, called AgNuc1, AgNuc2 and AgNuc3, whose sequences were characterized and validated by RNAi. The three sequences showed similarity above 50% when compared to other insect nucleases. qPCR analysis showed that AgNuc2 and AgNuc3 are highly expressed in A. grandis midgut. AgNuc2 gene silencing resulted on reduction of dsRNA degradation; thereby, we concluded that AgNuc2 is the main nuclease associated with dsRNA degradation in A. grandis gut lumen. Additionally, in order to increase dsRNA stability, dsRNA with viroid architecture were designed, wich are resistant to plant nucleases. Viroid-dsRNA were marked with Cy3 and analysed regarding their movement in A. thaliana vascular system, wich showed that they are adressed to a specif cellular localization. dsRNA based on Pospiviroidae family architecture was located on cell nuclei while dsRNA based on Avsunviroidae family was located in chloroplasts. Moreover, these stabilized dsRNAs showed high gene silencing activity, eight times higher than linear dsRNA. The data here generated contribute to our understandings of RNAi mechanisms in insects and show that stabilized dsRNAs exhibit great pontential in RNAi applicability aiming crop insect pest control.
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Almeida, João Paulo Pereira de. "O transcritoma antisense primário de Halobacterium salinarum NRC-1". Universidade de São Paulo, 2018. http://www.teses.usp.br/teses/disponiveis/17/17131/tde-15012019-101127/.
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Em procariotos, RNAs antisense (asRNAs) constituem a classe de RNAs não codificantes (ncRNAs) mais numerosa detectada por métodos de avaliação de transcritoma em larga escala. Apesar da grande abundância, pouco se sabe sobre mecanismos regulatórios e aspectos da conservação evolutiva dessas moléculas, principalmente em arquéias, onde o mecanismo de degradação de RNAs dupla fita (dsRNAs) é um fenômeno pouco conhecido. No presente estudo, utilizando dados de dRNA-seq, identificamos 1626 inícios de transcrição primários antisense (aTSSs) no genoma de Halobacterium salinarum NRC-1, importante organismo modelo para estudos de regulação gênica no domínio Archaea. Integrando dados de expressão gênica obtidos a partir de 18 bibliotecas de RNA-seq paired-end, anotamos 846 asRNAs a partir dos aTSSs mapeados. Encontramos asRNAs em ~21% dos genes anotados, alguns desses relacionados a importantes características desse organismo como: codificadores de proteínas que constituem vesículas de gás e da proteína bacteriorodopsina, além de vários genes relacionados a maquinaria de tradução e transposases. Além desses, encontramos asRNAs em genes pertencentes a sistemas de toxinas-antitoxinas do tipo II e utilizando dados públicos de dRNA-seq, evidenciamos que esse é um fenômeno que ocorre em bactérias e arquéias. A interação de um ncRNA com seu RNA alvo pode ser dependente de proteínas, em arquéias, a proteína LSm é uma chaperona de RNA homóloga a Hfq de bactérias, implicada no controle pós-transcricional. Utilizamos dados de RIP-seq de RNAs imunoprecipitados com LSm e identificamos 91 asRNAs interagindo com essa proteína, para 81 desses, o mRNA do gene sense também foi encontrado interagindo. Buscando por aTSSs presentes nas mesmas regiões de genes ortólogos, identificamos 160 aTSSs que dão origem a asRNAs em H. salinarum possivelmente conservados em Haloferax volcanii. A expressão dos asRNAs anotados foi avaliada ao longo de uma curva de crescimento e em uma linhagem knockout de um gene que codifica uma RNase R, possível degradadora de dsRNAs em arquéias. Encontramos um total de 144 asRNAs diferencialmente expressos ao longo da curva de crescimento, para 56 desses o gene sense também está diferencialmente expresso, caracterizando possíveis mecanismos de regulação em cis por esses RNAs. Na linhagem knockout, encontramos cinco asRNAs diferencialmente expressos e apenas para um desses o gene sense também está diferencialmente expresso, resultado que não nos permitiu inferir um possível papel de degradação de dsRNAs da RNAse R em H. salinarum NRC-1. Nesse trabalho apresentamos um mapeamento completo do transcritoma antisense primário de H. salinarum NRC-1 com resultados que consistem em um importante passo na direção da compreensão do envolvimento da transcrição antisense na regulação gênica pós-transcricional desse organismo modelo do terceiro domínio da vida.Antisense RNAs (asRNAs) constitute the most numerous class of non-coding RNAs (ncRNAs) detected by transcriptome highthroughput methods in prokaryotes. Despite this abundance, little is known about regulatory mechanisms and evolutionary aspects of these molecules, mainly in archaea, where the mechanism of double-strand RNA (dsRNA) degradation remains poorly understood. In this study, using dRNA-seq data, we identified 1626 antisense transcription start sites (aTSSs) in the genome of Halobacterium salinarum NRC-1, an important model organism for gene expression regulation studies in Archaea. By integrating gene expression data from 18 RNA-seq paired-end libraries, we were able to annotate 846 asRNAs from mapped aTSSs. We found asRNAs in ~21% of annotated genes including genes related to important characteristics of this organism, such as: gas vesicle proteins, bacteriorhodopsin, translation machinery and transposases. We also found asRNAs in type II toxin-antitoxin systems and using public dRNA-seq data, we show evidences that this phenomenon might be conserved in archaea and bacteria. The interaction of a ncRNA with its target may depend on intermediary proteins action. In archaea, the LSm protein is a RNA chaperone homologous to bacterial Hfq, involved in post-transcriptional regulation. We used RIP-seq data from RNAs immunoprecipitated with LSm and identified 91 asRNAs interacting with this protein, for 81 of these the mRNA of the sense gene is also interacting. We searched for aTSSs present in the same region of orthologous genes in the Haloferax volcanii. We found 160 aTSSs that originated asRNAs in H. salinarum NRC-1 that might be conserved in this two archaea. The expression of annotated asRNAs was analyzed over a growth curve and in a knockout strain for RNase R gene. We found 144 asRNA differentially expressed over the growth curve, for 56 of these the sense gene was also differentially expressed, characterizing possible cis regulators asRNAs. In the knockout strain we found five differentially expressed asRNAs and only one asRNA/gene pair, this result does not allow us to infer a dsRNA degradation in vivo activity for this RNase in H. salinarum NRC- 1. This work contributes to the discovery of the antisense transcriptome in H. salinarum NRC- 1 a relevant step to uncover the post-transcriptional gene regulatory network in this archaeon.
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Shi, Zhongjie. "Biochemical properties and substrate reactivities of Aquifex Aeolicus Ribonuclease III". Diss., Temple University Libraries, 2012. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/213666.
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ChemistryPh.D.
Ribonuclease III is a highly-conserved bacterial enzyme that cleaves double-stranded (ds) RNA structures, and participates in diverse RNA maturation and decay pathways. Essential insight on the RNase III mechanism of dsRNA cleavage has been provided by crystallographic studies of the enzyme from the hyperthermophilic bacterium, Aquifex aeolicus. However, those crystals involved complexes containing either cleaved RNA, or a mutant RNase III that is catalytically inactive. In addition, neither the biochemical properties of A. aeolicus (Aa)-RNase III, nor the reactivity epitopes of its cognate substrates are known. The goal of this project is to use Aa-RNase III, for which there is atomic-level structural information, to determine how RNase III recognizes its substrates and selects the target site. I first purified recombinant Aa-RNase III and defined the conditions that support its optimal in vitro catalytic activity. The catalytic activity of purified recombinant Aa-RNase III exhibits a temperature optimum of 70-85°C, a pH optimum of 8.0, and with either Mg2+ or Mn2+ supports efficient catalysis. Cognate substrates for Aa-RNase III were identified and their reactivity epitopes were characterized, including the specific bp sequence elements that determine processing reactivity and selectivity. Small RNA hairpins, based on the double-stranded structures associated with the Aquifex 16S and 23S rRNA precursors, are cleaved in vitro at sites that are consistent with production of the immediate precursors to the mature rRNAs. Third, the role of the dsRBD in scissile bond selection was examined by a mutational analysis of the conserved interactions of RNA binding motif 1 (RBM1) with the substrate proximal box (pb). The individual contributions towards substrate recognition were determined for conserved amino acid side chains in the RBM1. It also was shown that the dsRBD plays key dual roles in both binding energy and selectivity, through RBM1 responsiveness to proximal box bp sequence. The dsRBD is specifically responsive to an antideterminant (AD) bp in pb position 2. The relative structural rigidity of both dsRNA and dsRBD rationalizes the strong effect of an inhibitory bp at pb position 2: disruption of one RBM1 side chain interaction can effectively disrupt the other RBM1 side chain interactions. Finally, a cis-acting model was developed for subunit involvement in substrate recognition by RNase III. Structurally asymmetric mutant heterodimers of Escherichia coli (Ec)-RNase III were constructed, and asymmetric substrates were employed to reveal how RNase III can bind and deliver hairpin substrates to the active site cleft in a pathway that requires specific binding configurations of both enzyme and substrate.
Temple University--Theses
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Saunders, Caroline Ginny. "A study of orchid mycorrhizal fungi : an examination of the influence of dsRNA elements on symbiosis and the application of molecular methods for fungal identification". Thesis, University of Hull, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.389288.
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羽者家, 宝. "植物内在性dsRNAによる全身性の免疫系活性化効果とその応用". Kyoto University, 2019. http://hdl.handle.net/2433/245330.
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Xie, Bingning. "Long non-coding RNA-based mechanisms for the inhibition of cell growth and development by 5 - Fluorouracil". Thesis, Rennes 1, 2016. http://www.theses.fr/2016REN1B046/document.
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Les ARNm codent pour les protéines, tandis qu'un grand nombre d'ARNs nommés longues ARNs non codants (ARNlnc) ne sont pas traduites en protéines. Les deux types d’ARNs existent en isoforms qui se distinguent à cause de l’épissage alternatif. Certains des ARNlnc jouent des rôles importants dans la croissance et différentiation cellulaire. Cependant, leurs fonctions dans la cytotoxicité de la chimiothérapie anti-cancéreuse médicamenteuse utilisant le 5-fluorouracile (5-FU) sont encore inconnues. Pendant mes travaux j'ai trouvé que le traitement par le 5-FU cause l’accumulation des ARNlnc. Ce phénomène est parfois, sous forme d’ARN double brin (ARNds) formé par une paire de transcrits chevauchant, corrélé négativement avec le niveau de la protéine codée par l'ARNm. Cette inhibition potentielle de la traduction des régulateurs du cycle cellulaire clés et les gènes essentiels en formant des l'ARNds peut éventuellement empêcher la progression du cycle cellulaire. Nos analyses prometteuses devraient inspirer des études approfondies des ARNlnc dans la cytotoxicité du 5-FU chez la levure et l’homme afin d’'améliorer la chimiothérapie. J'ai trouvé que la surexpression de RRP6, peut conduire à une résistance accrue au traitement par le 5-FU. Je démontre ensuite que l’ARNlnc MUT1312 forme des ARNds avec RRP6 qui sont négativement corrélés avec le niveau de la protéine Rrp6. Par ailleurs, la surexpression de MUT1312 pendant la mitose et associé avec une diminution d’Rrp6. Ainsi, mon étude suggère que MUT1312 soit impliqué dans la régulation de Rrp6 pendant la differentiation cellulaire. Mes recherches de MUT477/SWI4 indiquent la function importante de la méiose induite à long ARN non codantes en tant que forme d'ARN double brin potentiellement réguler la traduction. J'ai trouvé que SUT200 pourrait inhiber la transcription de CDC6 durant la méiose par read-through. Un cas comparable est MUT1465 et CLN2. J’ai fait un criblage in silico pour trouver des facteurs de transcription qui activent des MUTs durant la méiose. J’ai trouvé que la plupart des MUTs sont induites par Ndt80. MUT1465 est parmi eux : il pourrait être induite par Ndt80 ce qui inhiberait l’expression de CLN2 après l’initiation de la méiose. J’ai trouvé que la répression de certains MUTs par le complexe Ume6/Rpd3 en mitose est différemment régulée entre JHY222 et SK1. MUT100 qui ne possède pas l'élément USR1 fixé par Ume6, et qui est donc une cible indirecte, est déréprimé dans JHY22 ume6 mais pas dans SK1 ume6. Pour la régulation de l'étude de isoforme méiose, Nous avons trouvé que le complexe histone déacétylase Rpd3/Sin3/Ume6, empêche également l'induction de l'isoforme longue de BOI1 dans la mitose par liaison directe de liaison Ume6 à sa cible de URS1. Orc1 est importante pour la réplication de l'ADN. J’ai démontré que mORC1 est une cible directe de l'activateur Ndt80 et que son motif de fixation (MSE) est nécessaire pour l'induction de l’isoforme mORC1 et du gene méiotique SMA2 transcrit de façon divergente. J’ai trouvé qu'une souche incapable d’induire mORC1, contient des niveaux anormalement élevés d’Orc1 pendant la gamétogenèse, ce qui corréle mORC1 avec la baisse de la protéine Orc1. En conclusion, mes études au cours du doctorat révèlent des nouvelles cibles et ainsi offrent des nouvelles perspectives de l’amélioration de la chimiothérapie par le 5-FU. Les mécanismes incluent la formation d'un ARN double brin avec son ARNm anti-sens pour potentiellement inhiber la traduction de l'ARNm, et inhibition en aval de l'ARNm par transcription read-through d’une ARNlnc. Mon travail a également révélé un mécanisme de régulation des ARNlnc et les isoforms d’ARN pendent la croissance et la différentiation cellulaireRNAs are molecules with important functions in diverse cellular processes. mRNAs encode proteins, while a large number of RNAs called long noncoding RNAs (lncRNAs) are not translated into proteins. Both types of RNAs exist in various isoforms due to alternative splicing.Some of lncRNA play important roles in cell growth and differentiation. However, their functions in the cytotoxicity of the drug anticancer chemotherapy using 5-fluorouracil (5-FU) are still unknown. During my research I found that treatment with 5-FU causes accumulation of lncRNA. Acuumulated antisense lncRNA form double stranded RNA with the mRNAs , negatively correlated with the level of the protein encoded by the mRNA. This potential inhibition of translation of key cell cycle regulators and essential genes by forming dsRNA may possibly prevent the progression of the cell cycle. My results suggest that lncRNA are likely to play an important role in the cytotoxicity of 5-FU. Our promising testing should inspire in-depth studies of lncRNA in the cytotoxicity of 5-FU in yeast and humans to improve chemotherapy.Rrp6 is a 3'-5 'exoribonuclease, which plays an important role in the regulation and modification of rRNA, mRNA and lncRNA. I found that overexpression of RRP6, the homologue of the yeast EXOSC10 gene in mammals, can lead to increased resistance to treatment with 5-FU. I found that the lncRNA MUT1312 form dsRNA with RRP6 that are negatively correlated with the level of Rrp6 protein. Furthermore, overexpression of MUT1312 during mitosis and associated with a decrease of Rrp6. Thus, my study suggests that MUT1312 may involved in the regulation of Rrp6 during cell differentiation. I further explored the function of the double-stranded RNA in meiosis. My research about SWI4/MUT477 indicates the important function of meiosis induced long noncoding RNA as a form of double-stranded RNA potentially regulate translation. Another aspect of the function of lncRNA is to regulate the transcription of downstream mRNA. I found SUT200 could inhibit transcription of CDC6 during meiosis by read-through. A similar case is CLN2/MUT1465. I did an in silico screening to find transcription factors that activate MUTs during meiosis. I found that most MUTs are induced by Ndt80. MUT1465 is among them: it could be induced by Ndt80 which inhibit the expression of CLN2 after initiation of meiosis. I found that repression of certain MUTs by the Ume6 / Rpd3 complex in mitosis is regulated differently between JHY222 and SK1. MUT100 which does not have the Ume6 binding site URS1 element, and is therefore an indirect target is derepressed in JHY22 ume6 but not in SK1 ume6. For the study about regulation of meiosis isoform, we have found that the histone deacetylase complex Rpd3 / Sin3 / Ume6 prevents the induction of long isoform BOI1 in mitosis by direct binding Ume6 binding to its target URS1.Orc1 is important for DNA replication. I have demonstrated that mORC1 is a direct target of the Ndt80 activator and its binding motif (MSE) is required for induction of isoform mORC1 and meiotic gene SMA2 divergently transcribed. I found that a strain incapable of inducing mORC1 contains abnormally high levels of Orc1 during gametogenesis, which correlates with mORC1 declining Orc1 protein. Since eukaryotic genes often encode multiple transcripts with 5'-UTR of variable length, the findings are likely relevant to gene expression during development and disease in higher eukaryotes. In conclusion, my studies during PhD reveal new targets and thus offer new prospects for improving chemotherapy with 5-FU. Mechanisms include (1) the formation of a double strand with its antisense mRNAs to potentially inhibit translation of mRNA, and (2) downstream inhibition of mRNA transcription read-through of a lncRNA. My work also revealed a lncRNA regulatory mechanism and RNA isoforms dangling growth and cell differentiation
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Hahn, Sabine. "Virologische Untersuchungen an Stieleichen (Quercus robur L.) zum verursachenden Pathogen der pfropfübertragbaren chlorotischen Ringflecken". Doctoral thesis, Humboldt-Universität zu Berlin, Landwirtschaftlich-Gärtnerische Fakultät, 2006. http://dx.doi.org/10.18452/15457.
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Regelmäßige Bonituren haben gezeigt, dass virusverdächtige Symptome an Stieleichen, die zu etwa 90 % als chlorotische Ringflecken auftreten, im nord- und mitteldeutschen Raum weit verbreitet sind. In der vorliegenden Arbeit sollte der Erreger dieser Symptome isoliert und näher charakterisiert werden. Aus zwei Blattproben mit chlorotischen Ringflecken konnten stäbchenförmige Viruspartikeln mit einer Länge von ca. 450 nm isoliert und auf krautige Indikatoren übertragen werden. In einer RT-PCR mit Hüllprotein bzw. Transportprotein-sequenzspezifischen Primern wurden diese als Tobacco mosaic virus (TMV)- bzw. Tomato mosaic virus (ToMV)- Isolate identifiziert. Eine Infektion der Stieleichen mit weiteren bekannten Viren von Gehölzen, wie dem Cherry leaf roll virus (CLRV) oder dem Erreger der Ebereschenringfleckigkeit konnte mittels ELISA und RT-PCR ausgeschlossen werden. DsRNAs der Größen 1.5 und 1.6 kb sowie 1.8 und 2.0 kb konnten symptomunabhängig aus Rindengewebe, Knospen und Blättern von Stieleichen isoliert werden. Mit Hilfe der RT-DOP-PCR und der cDNA-Klonierung gelang es, Teile des 1.5/1.6 kb dsRNA-Moleküls zu charakterisieren. Die Sequenz von 479 Aminosäuren (1437 Nukleotiden) wies eine Identität von 56 % zur RNA-abhängigen RNA-Polymerase (RdRp) des Beet cryptic virus 3 (BCV 3) auf. Der spezifische Nachweis dieser Sequenz gelang mittels RT-PCR sowohl in dsRNA-Proben, als auch in angereicherten Nukleokapsiden symptomloser und symptomatischer Stieleichen. In Nested-PCR-Analysen konnte das Fragment jedoch nicht nur in Gesamt-RNA von Stieleichen, sondern auch in Gesamt-RNA und DNA verschiedenster gesunder Pflanzen amplifiziert werden. Phylogenetische Vergleiche mit ausgewählten RdRps viralen und pflanzlichen Ursprungs zeigten die engste Verwandtschaft der Stieleichen-dsRNA-Sequenz zu den Partitiviren, zu denen sich neben BCV 3 auch die endogene dsRNA aus Pyrus und aus Chloroplasten von Bryopsis gruppiert. Diese Erkenntnisse lassen in der charakteristischen Doppelbande von 1.5/1.6 kb das Vorliegen einer endogenen dsRNA vermuten. Hiermit ist in dieser Arbeit das Auftreten verschiedener Viren in Eichen nachgewiesen worden, von denen die meisten höchstwahrscheinlich nicht im direkten ursächlichen Zusammenhang mit der chlorotischen Ringfleckigkeit der Eiche stehen.Ratings of oak populations revealed that around 90 % of all oak trees affected by viruslike symptoms showed chlorotic ringspots and that these symptoms are widely spread in oaks in north and central Germany. In this study the putative agent of these symptoms should be isolated and specified. Rod-shaped particles with a length of 450 nm were recovered from two different samples of leaves displaying chlorotic ringspots by mechanical inoculation of herbaceous indicator plants. These particles were identified to be Tobacco mosaic virus (TMV)- and Tomato mosaic virus (ToMV)- isolates by RT-PCR analyses of the coat- and movement protein genes. Infections with other well known viruses of forest trees, like Cherry leaf roll virus (CLRV) and the agent causing ringspots in European mountain ash, were excluded by ELISA and RT-PCR. DsRNA fragments of 1.5 and 1.6 kb as well as 1.8 and 2.0 kb were extracted from leaves, inner bark and bulbs of all symptomatic and asymptomatic samples of common oak. The nucleotide sequence of the 1.5 and 1.6 kb dsRNA fragment was partially characterised by reverse transcription degenerated oligonucleotide primed (DOP)-PCR and cDNA cloning. The obtained nucleotide sequence of 1437 nt encoding a putative protein of 479 amino acids revealed an identity of 56 % with the RNA-dependent RNA polymerase (RdRp) of Beet cryptic virus 3 (BCV 3). PCR amplification of the RdRp coding nucleotide sequence was possible using a number of different dsRNA samples as well as concentrated nucleocapside preparations. The same sequence was also amplified successfully by Nested-PCR not only in total RNA extracted from symptomatic and asymptomatic oak samples but also from total RNA and DNA of diverse plants. Phylogenetic analysis revealed further similarities to RdRp´s of endogenous dsRNA of Pyrus and chloroplasts of Bryopsis, both members of the Partitiviridae as well as BCV 3. These results strongly indicate that the 1.5/1.6 kb dsRNA of oak is endogenous dsRNA. In summary, it has been shown that oaks in Germany are commonly infected by a variety of different viruses most of them possibly unrelated to the wide-spread ringspot symptoms of oaks.