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1
Schultes, Stephan. "Nanoparticles for RNA Interference." Diss., lmu, 2009. http://nbn-resolving.de/urn:nbn:de:bvb:19-113293.
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Shah, Samit Friedman Simon H. "Light activated RNA interference." Diss., UMK access, 2007.
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Thesis (Ph. D.)--School of Pharmacy and Dept. of Chemistry. University of Missouri--Kansas City, 2007."A dissertation in pharmaceutical science and chemistry." Advisor: Simon H. Friedman. Typescript. Vita. Description based on contents viewed July 16, 2008; title from "catalog record" of the print edition. Includes bibliographical references (leaves 206-220). Online version of the print edition.
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Marr, Edward John. "RNA interference (RNAi) for selective gene silencing in Astigmatid mites." Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/25722.
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Psoroptic mange, caused by the Astigmatid mite Psoroptes ovis, is an ectoparasitic disease of significant economic importance to agriculture on a global scale and poses a serious welfare concern. With the current chemotherapeutic controls considered unsustainable, there is pressing need for novel control strategies. RNA interference has been proposed as a potential high throughput approach for the identification of novel therapeutic targets with high specificity, speed and at a relatively low cost compared to the existing methods. The presence of the components of the RNA interference (RNAi) pathway in P. ovis was first confirmed through in silico analyses of the P. ovis transcriptome and, following development of a non-invasive immersion method of double stranded RNA (dsRNA) delivery, gene silencing by RNAi was demonstrated in P. ovis. Statistically-significant reduction of transcript level was measured for the three genes targeted: P. ovis mite group 2 allergen (Pso o 2), P. ovis mu class glutathione S-transferase (PoGST-mu1) and P. ovis beta tubulin (Poβtub). This is the first demonstration of gene silencing by RNAi in P. ovis and provides a key mechanism for mining transcriptomic and genomic datasets in the future for novel targets of intervention against P. ovis. The first assessment of gene silencing was also performed in two related Astigmatid mites of high medical importance; the European house dust mite Dermatophagoides pteronyssinus and the scabies mite Sarcoptes scabiei. A statistically-significant reduction in expression of a D. pteronyssinus mu class glutathione S-transferase (DpGST-mu1) transcript was observed. No significant reduction in expression of a S. scabiei mu class glutathione S-transferase (SsGST-mu1) transcript was observed. Additionally, microRNAs (miRNAs) from the related miRNA pathway were identified in a P. ovis small RNA sample and were sequenced and annotated.
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Shoji, Masanobu. "RNA interference during spermatogenesis in mice." Kyoto University, 2006. http://hdl.handle.net/2433/143821.
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Pereira, Tiago Campos. "Estudo de possiveis aplicações médicas da interferencia por RNA." [s.n.], 2005. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316861.
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Orientadores: Iscia Teresinha Lopes-Cendes, Ivan de Godoy MaiaTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-04T19:04:59Z (GMT). No. of bitstreams: 1 Pereira_TiagoCampos_D.pdf: 3895694 bytes, checksum: d999bfc92e9a2e2c757db34bbfc7d7fa (MD5) Previous issue date: 2005
Doutorado
Genetica Animal e Evolução
Doutor em Genetica e Biologia Molecular
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Aitken, Amelia. "Blocking the RNA Interference Pathway Improves Oncolytic Virus Therapy." Thesis, Université d'Ottawa / University of Ottawa, 2017. http://hdl.handle.net/10393/36821.
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Oncolytic viruses are novel candidates for cancer therapy and their efficacy relies on their capacity to overcome the host’s anti-viral barriers. In mammalian cells, the anti-viral response involves a protein-signaling cascade known as the interferon pathway, which alerts the immune system and limits the propagation of infection. Given that most cancer cells have defects in this pathway, they are susceptible to viral infection and responsive to oncolytic virotherapy. For reasons that remain unknown, many cancers are still refractory to oncolytic viruses, which suggests the existence of additional antiviral mechanisms. In this study, we investigate the potential involvement of an alternative antiviral pathway in cancer cells. Given that insects and plants rely on the RNA silencing pathway for their anti-viral protection, we investigated the presence of a similar mechanism in cancer cells. We found viral genome-derived small RNAs in various cancer cell lines upon infection, which is indicative of an RNA-mediated antiviral response. Also, various viruses encode suppressors of the RNA interference pathway. To determine if an oncolytic virus could benefit from such a factor, we engineered an oncolytic virus variant to encode the Nodamura virus B2 protein, a known inhibitor of RNA silencing-mediated immune responses. Using this virus, we observed enhanced cytotoxicity in 33 out of the 38 human cancer cell lines tested. Furthermore, our results show inhibition of viral genome cleavage and altered microRNA processing by our B2-expressing oncolytic virus. Taken together, our data suggests the blockade of RNA silencing antiviral pathways and/or antiviral microRNA processing improves the efficacy of our B2-encoding virus in a cell-line specific manner. Overall, our results establish the improved potential of our novel virus therapy and demonstrate for the first time the involvement of RNA pathways in the antiviral defense of cancer cells.
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Garbutt, Jennifer S. "RNA interference in insects : persistence and uptake of double-stranded RNA and activation of RNAi genes." Thesis, University of Bath, 2011. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.548101.
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RNA interference (RNAi) is a eukaryotic phenomenon where short double-stranded RNA molecules (dsRNAs) repress homologous sequences. In insects RNAi has been widely observed and has proved extremely useful as a reverse genetics tool to elucidate the function of newly identified genes, as well as showing potential as a novel insecticide. Unfortunately, however, not all insect species are equally susceptible to RNAi. This thesis explores whether persistence of dsRNA in insect hemolymph, uptake of dsRNA into insect tissue, or activation of RNAi genes could be limiting factors in RNAi experiments. Trials were conducted with the tobacco hornworm, Manduca sexta, a species in which experimental difficulty has been experienced with RNAi protocols and the German cockroach, Blattella germanica, which is known to be highly susceptible to experimental RNAi. In M. sexta larvae dsRNA disappeared rapidly from the hemolymph in vivo. By comparison, exogenous dsRNA persisted longer in the hemolymph of B. germanica adults. These findings lead me to propose that the rate of persistence of dsRNA in insect hemolymph may be a key factor in determining the susceptibility of insect species to RNAi. Despite such rapid breakdown of dsRNA in M. sexta larvae uptake of exogenous dsRNA into hemocytes, fat body and midgut could be detected by quantitative RT-PCR in vivo and was experimentally investigated in hemocytes in vivo and in vitro using fluorescently labelled dsRNA. Furthermore, quantitative-RT-PCR revealed that the expression of two M. sexta RNAi genes dicer-2 and argonaute-2 (partial sequences of which were isolated during this study) was specifically upregulated in response to injection with dsRNA.
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Lee, Hung-chiu. "Synthetic RNA interference against influenza A virus." Click to view the E-thesis via HKUTO, 2005. http://sunzi.lib.hku.hk/hkuto/record/B35537814.
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Mankin, Danielle N. "MC3R and MC4R Knockdown via RNA Interference." Digital Archive @ GSU, 2012. http://digitalarchive.gsu.edu/biology_theses/37.
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Melanocortins (MCs) play an important role in feeding, metabolism, and energy expenditure. While melanocortin receptor (MCR) mRNA has been found in the mesolimbic dopamine (DA) pathway, the ability of melanocortins to regulate feeding and other behaviors through actions on the mesolimbic DA system have not been examined. Short-hairpin RNAs (shRNAs) were created targeting MC3R and MC4R and were tested via in vitro studies for their ability to knockdown their target receptor. A total of three shRNAs were created targeting each receptor, and each shRNA caused successful knockdown. These shRNAs are tools that can be used for future in vivo studies to examine the various behavioral effects of melanocortins on the mesolimbic DA pathway.
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Lee, Hung-chiu, and 李洪釗. "Synthetic RNA interference against influenza A virus." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B35537814.
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Neofytou, Giannis. "Mathematical models of RNA interference in plants." Thesis, University of Sussex, 2017. http://sro.sussex.ac.uk/id/eprint/67207/.
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RNA interference (RNAi), or Post-Transcriptional Gene Silencing (PTGS), is a biological process which uses small RNAs to regulate gene expression on a cellular level, typically by causing the destruction of specfic mRNA molecules. This biological pathway is found in both plants and animals, and can be used as an effective strategy in defending cells against parasitic nucleotide sequences, viruses and transposons. In the case of plants, it also constitutes a major component of the adaptive immune system. RNAi is characterised by the ability to induce sequence-specific degradation of target messenger RNA (mRNAs) and methylation of target gene sequences. The small interfering RNA produced within the initiated cell is not only used locally but can also be transported into neighbouring cells, thus acting as a mobile warning signal. In the first part of the thesis I develop and analyse a new mathematical model of the plant immune response to a viral infection, with particular emphasis on the role of RNA interference. The model explicitly includes two different time delays, one to represent the maturation period of undifferentiated cells, and another to account for the time required for the RNAi propagating signal to reach other parts of the plant, resulting in either recovery or warning of susceptible cells. Analytical and numerical bifurcation theory is used to identify parameter regions associated with recovery and resistant plant phenotypes, as well as possible chronic infections. The analysis shows that the maturation time plays an important role in determining the dynamics, and that long-term host recovery does not depend on the speed of the warning signal but rather on the strength of local recovery. At best, the warning signal can amplify and hasten recovery, but by itself it is not competent at eradicating the infection. In the second part of the thesis I derive and analyse a new mathematical model of plant viral co-infection with particular account for RNA-mediated cross-protection in a single plant host. The model exhibits four non-trivial steady states, i.e. a disease-free steady state, two one-virus endemic equilibria, and a co-infected steady state. I obtained the basic reproduction number for each of the two viral strains and performed extensive numerical bifurcation analysis to investigate the stability of all steady states and identified parameter regions where the system exhibits synergistic or antagonistic interactions between viral strains, as well as different types of host recovery. The results indicate that the propagating component of RNA interference plays a significant role in determining whether both viruses can persist simultaneously, and as such, it controls whether the plant is able to support some constant level of both infections. If the two viruses are sufficiently immunologically related, the least harmful of the two viruses becomes dominant, and the plant experiences cross-protection. In the third part of the thesis I investigate the properties of intracellular dynamics of RNA interference and its capacity as a gene regulator by extending a well known model of RNA interference with time delays. For each of the two amplification pathways of the model, I consider the cumulative effects of delay in dsRNA-primed synthesis associated with the non-instantaneous nature of chemical signals and component transportation delay. An extensive bifurcation analysis is performed to demonstrate the significance of different parameters, and to investigate how time delays can affect the bi-stable regime in the model.
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Huang, Ching-Cheng. "Development of RNA interference in parasitic nematodes." Thesis, Imperial College London, 2010. http://hdl.handle.net/10044/1/5745.
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Exploitation of RNA interference (RNAi) has revolutionised work on Caenorhabditis elegans, and considerable success has been recently reported with plant-parasitic nematodes. It has proven difficult to transfer this technology to animal parasitic species, and previous attempts in this laboratory by feeding Nippostrongylus brasiliensis larvae with Escherichia coli expressing double-stranded RNA (dsRNA) gave no consistent reductions in levels of target transcripts. The aim of this study was to develop methods for RNAi in N. brasiliensis, a rodent strongylid nematode which is closely related to gastrointestinal nematodes of humans and livestock, in order to explore the biological functions of parasite secreted proteins. In order to promote uptake of exogenous macromolecules such as dsRNA and small interfering RNA (siRNA) by infective larvae, the process of activation, whereby larvae are induced to resume feeding and development, was studied in vitro. Activation could be induced solely by exposure of larvae to elevated temperature (37oC), whereas host serum or glutathione had no effect. Neither a membrane permeant analogue of cyclic GMP nor muscarinic receptor agonists promoted activation, suggesting that a cholinergic neural pathway is not involved in the process. Activation at 37oC could be blocked by inhibitors of phosphatidylinositol 3-kinase, Akt protein kinase & cytochrome P450. These data indicate that the early signalling events for larval activation in N. brasiliensis differ substantially from the hookworm Ancylostoma caninum, most probably acting through thermosensory rather than chemosensory neurons, but that they may converge downstream of a step dependent on phosphatidylinositol 3-kinase. Stimulation of protein secretion paralleled resumption of feeding, suggesting that these processes were tightly linked and regulated by similar pathways during activation. Temperature-activated larvae were exposed to dsRNA and siRNA via electroporation or soaking in the presence of a variety of transfection reagents, but RNAi was unsuccessful. Serotonin was demonstrated to increase the rate of uptake of macromolecules, yet larvae exposed to exogenous dsRNA in the presence of serotonin still failed to show RNAi-mediated gene silencing. In addition, RNAi was also observed to be irreproducible in adult N. brasiliensis using the same methods of delivery. The use of mRNA encoding firefly luciferase identified uptake into larvae or adult worms as a major impediment in the process. Venom Allergen Homologue/ASP-Like (VAL) molecules have been identified as major components of secreted proteins from many species of parasitic nematodes. In this study, eight N. brasiliensis VALs were identified and sequenced, and all were shown to be present in products secreted by adult parasites. NbVAL-7 and NbVAL-8 were also detected in secreted products of infective larvae. Structural similarities to other members of the Pathogenesis- Related protein superfamily are discussed, as are possible functions for these proteins in N. brasiliensis.
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Yoon, June-Sun. "THE MECHANISM OF RNA INTERFERENCE IN ARTHROPODS." UKnowledge, 2018. https://uknowledge.uky.edu/entomology_etds/45.
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RNA interference (RNAi) is a useful reverse genetics tool for investigation of gene function as well as for practical applications in many fields including medicine and agriculture. Due to the variability in RNAi efficiency, RNAi-based methods are currently being developed for controlling only coleopteran insects which are known to be amenable to RNAi. The first chapter of my thesis includes findings from research to investigate what are the factors that make coleopteran insects relatively more efficient in RNAi. I used Colorado potato beetle (CPB), Leptinotarsa decemlineata and its cell line (Lepd-SL1) as study models to identify genes that play key roles in RNAi pathway. Five genes including Argonaute-1 (microRNA Argonaute) and Aubergine (PiwiRNA Argonaute) were identified as those required for siRNA (short interfering RNA) RNAi pathway. I also found that RNAi is completely blocked in StaufenC knockdown cells. StaufenC belongs to dsRNA binding protein family and binds to dsRNA as shown by gel mobility shift and the pull-down assays. Interestingly, I also found that StaufenC is downregulated in RNAi resistant cells and StaufenC homologous sequences are present in only coleopteran insects where RNAi works efficiently. These data suggest that StaufenC is a major contributor to efficient RNAi in coleopteran insects and is a potential target for RNAi resistance. The second part of my research is to understand the mechanisms of RNAi in those insects refractory to RNAi. The barriers for successful RNAi include the presence of double-stranded ribonucleases (dsRNase) in the lumen and hemolymph that could potentially digest double-stranded RNA (dsRNA) and the variability in the transport of dsRNA into and within the cells. Recent work in our laboratory showed that the dsRNAs are transported into lepidopteran cells, but they are not processed into siRNAs because they are trapped in acidic bodies. I focused on identification of these acidic bodies in which dsRNAs accumulate in Spodoptera frugiperda Sf9 cells. These studies showed that entrapment of internalized dsRNA in endosomes is one of the major factors contributing to inefficient RNAi. Overall, my research revealed important players involved in successful and unsuccessful RNAi in insects.
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Ramachandran, Pavitra Shyam. "RNA interference therapy for the Spinocerebellar ataxias." Diss., University of Iowa, 2014. https://ir.uiowa.edu/etd/4730.
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The spinocerebellar ataxias are a group of diseases characterized by loss of motor coordination. Spinocerebellar ataxia types 2 and 7 are monogenic, autosomal dominant, late-onset neurodegenerative diseases characterized by ataxia with no effective treatments in the clinic. The most striking feature of these diseases is the degeneration of Purkinje neurons of the cerebellum. Spinocerebellar ataxia type 7 is also characterized by vision loss due to degeneration of the retinal photoreceptors. In this work, we tested the hypothesis that reducing mutant gene expression by RNAi would alleviate disease phenotypes in these two spinocerebellar ataxias.
For spinocerebellar ataxia type 7 (SCA7), we designed and tested RNAi sequences that could reduce the expression of both wildtype and mutant ataxin-7, an approach that would be applicable to all SCA7 patients. We found that AAV1-mediated delivery of a candidate RNAi sequence to the Purkinje neurons of SCA7 mice resulted in long-term sustained reduction of both wildtype and mutant ataxin-7 and resulted in significant improvements in ataxic and neuropathological phenotypes. We also delivered the RNAi sequence (AAV1-mediated) to reduce the expression of both mutant and wildtype ataxin-7 in the SCA7 mouse retina and evaluated retinal function long-term. We observed a preservation of normal retinal function and no adverse toxicity with reduction of wildtype and mutant ataxin-7 alleles. These studies address an important safety concern regarding non-allele specific silencing of ataxin-7 for SCA7 therapy.
To identify therapies for spinocerebellar ataxia type 2 (SCA2), we designed and tested several RNAi sequences to reduce the expression of both wildtype and mutant ataxin-2 in vitro and in vivo. We found that reduction of wildtype ataxin-2 expression in the mouse cerebellum was tolerated 4 months post injection without inducing behavioral deficits or cerebellar pathology. Additionally, we tested other sequences for improved silencing efficacy, and identified a potent RNAi sequence that significantly reduced the expression of both mutant and wildtype ataxin-2 in the cerebellum of a SCA2 mouse model. Ongoing work will establish if long-term reduction of both mutant and wildtype ataxin-2 will provide therapeutic benefit in the SCA2 mouse setting, and the safety of this sequence in normal cerebella.
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Saadat, Angela P. "Identifying Novel Contributors to RNA Interference in Aedes aegypti." Thesis, Virginia Tech, 2015. http://hdl.handle.net/10919/75141.
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Aedes aegypti is an important vector of human pathogens including the viruses yellow fever, dengue and chikungunya. The small interfering RNA (siRNA) pathway is a critical immune response for controlling viral replication in Aedes aegypti. The goal of this research is to identify components of the Aedes aegypti genome that influence this pathway.
A transgenic mosquito strain that reports the status of the siRNA pathway via enhanced green fluorescent protein (EGFP) intensity was employed to differentiate silencing abilities among individuals. Extreme EGFP expression phenotypes, representing efficient and poor silencing abilities, were enriched over five generations.
Transcriptome sequencing and analyses were performed from pools of individuals from each enriched phenotype, revealing potential RNAi contributors. 1,120 transcripts were significantly different (FDR<0.0001) among the extreme phenotypes.
Four genes were chosen, amplified, sequenced for SNP analysis. These analyses were performed on samples obtained by crossing enriched, extreme phenotype F0 individuals, intercrossing their progeny, then selecting individuals representing the extreme phenotypes from the F2 population. Though further verification is needed, findings from these analyses imply the regions of Aedes aegypti, Liverpool strain (AAEL) gene identifiers AAEL005026, AAEL013438 and AAEL011704 amplified do not contribute to the two extreme, opposite RNAi silencing in the sensor strain used here. SNP analyses of AAEL000817 indicate this gene either influences extreme RNAi phenotypes or is closely linked to a gene(s) that contributes to RNAi in Aedes aegypti.
The 1,120 genes identified can be validated or eliminated as potential targets in the quest to mitigate the impact of Aedes aegypti.Master of Science in Life Sciences
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Berenjian, Saideh. "Construction of Adenovirus Vectors for Studies of Protein Function and RNA Interference." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Univ.-bibl. [distributör], 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6328.
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Brightwell, Sara. "Identifying novel regulators of reprogramming using RNA interference." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/16156.
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Since Yamanaka and Takahashi first described the isolation of induced pluripotent stem cells (iPSCs) in 2006, researchers have invested a vast amount of time and resources into trying to understand the process of reprogramming. However, the exact mechanisms underlying the induction of somatic cells to pluripotency is still incompletely understood. With this in mind, a screening approach was undertaken to identify shRNA that enhance the reprogramming process. A retrovirus based system was used to knock down candidate genes during reprogramming of mouse embryonic fibroblasts (MEF) containing doxycycline-inducible reprogramming factors and a Nanog-GFP reporter, which is activated when cells become iPSCs. The initial round of screening with over 150 shRNA vectors successfully identified several shRNAs that enhance reprogramming. One of these shRNA vectors exhibited both faster reprogramming kinetics as determined by activation of the Nanog-GFP reporter 2 to 3 days earlier and increased reprogramming efficiency giving rise to >5 fold more GFP+ colonies when compared with a control. Cell surface marker analysis with flow cytometry demonstrated that changes in CD44 and ICAM1 expression, which occur preceding Nanog-GFP expression, were also accelerated. Validation of this shRNA determined that the enhanced reprogramming phenotype is the result of an unknown off-target effect. Microarray and RNA-sequencing analysis was carried out to identify the off target gene with a view to investigate the functional importance of this knock down and its role in establishing the pluripotency transcriptional network during reprogramming.
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Warnock, N. D. "Investigations on RNA interference susceptibility in selected nematodes." Thesis, Queen's University Belfast, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.557851.
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Parasitic nematodes continue to be the causative agents of some of the most prevalent diseases of man and cause significant economic losses to the worldwide agri-food industry. With anthelmintic resistance on the rise and environmental concerns over the use of many nematicides the effectiveness of current control options is diminishing. The only viable alternative is the creation of new, environmentally safe drugs/control options. The reverse genetic technique of RNA interference (RNAi) facilitates the examination of gene function by the application of double stranded (ds)RNA complementary to a gene of interest which triggers endogenous cellular mechanisms that destroy the target gene transcript. Of particular interest here is the application of RNAi to nematode species and how this technology could be harnessed to improve our understanding of nematode biology and, ultimately, exploited to inform drug target selection. This study demonstrates that RNAi remains a potentially useful tool for the validation of gene function despite the pressing need to improve experimental rigor and statistical analysis when validating RNAi induced gene knockdown. Although RNAi failed to silence gene expression in the free-living nematode Panagrellus redivivus and was limited in its efficacy in l3 stage worms of the pig parasite Ascaris suum, it did trigger consistent knockdown of selected genes in A. suum adults. A bioinformatic analysis of the RNAi pathway components in the genomes/transcriptomes of selected nematodes indicated the occurrence of all functional groupings within diverse nematodes, suggesting that RNAi mechanisms are in place broadly in the phylum. There were no RNAi effector deficiencies that specifically mapped to RNAi incompetency. As such there remains hope that with further optimisation, and as culture and delivery techniques continue to improve, RNAi could be an important tool for screening gene function and validating drug targets in nematode parasites.
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Harris, Elizabeth Anne. "Analysis of glucocorticoid receptor function using RNA interference." Thesis, University of Bristol, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.435715.
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Georgiadis, A. "Viral vector-mediated RNA interference in the retina." Thesis, University College London (University of London), 2010. http://discovery.ucl.ac.uk/19985/.
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RNA interference (RNAi) is a highly conserved post-transcriptional gene silencing process triggered by double-stranded RNA (dsRNA) in eukaryotic cells. Elucidation of the RNAi regulatory pathway and its components has led to the identification of endogenous dsRNA molecules, termed microRNAs (miRNAs), which are transcribed as a single hairpin molecule prior to their maturation into a cytoplasmic dsRNA. The efficient gene silencing achieved by these short hairpin RNA (shRNA) molecules and the cumulative understanding of the RNAi pathway has prompted the development of hairpin expression vectors capable of mediating stable gene silencing in vitro and in vivo. The aim of this thesis is to evaluate the efficacy of viral vector-mediated RNAi in the retina using recombinant adeno-associated viruses (AAV) and lentiviruses that contain silencing hairpin cassettes to target four genes in murine photoreceptors and the retinal pigment epithelium (RPE). A detailed assessment of the utility and extend of RNAi in the retina using different viral vectors and hairpin designs is presented in this thesis. Lentiviral and AAV vectors were firstly used to silence GFP in vitro and in vivo as a proof of concept for vector mediated RNAi in the retina. Subsequently, we used lentivirally-mediated RNAi to study disease processes in the retina concentrating on tight junction (TJ) modulators ZO-1 and ZONAB and their role in RPE homeostasis, cell-cycle progression and epithelial-mesenchymal transition (EMT). Here we demonstrated how TJ misregulation can lead to RPE loss, proliferation or dedifferentiation; processes involved in pathological conditions such as atrophic age-related macular degeneration (AMD) and proliferative vitroretinopathy (PVR). Whilst lentivirally-mediated RNAi was used to elucidate aspects of retinal function and disease, AAV-mediated RNAi was used to probe the therapeutic potential of shRNAs by silencing Peripherin-2 (Prph2), the second most abundant retinal protein, using a miRNA-based hairpin. AAV2/8 particles were used to target endogenous Prph2 and evasion of silencing was demonstrated using an engineered Prph2 cDNA that could be used in a suppression and replacement approach for the treatment of dominant retinal disorders.
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Almeida, Ricardo. "RNA interference and heterochromatin formation in fission yeast." Thesis, University of Edinburgh, 2008. http://hdl.handle.net/1842/11198.
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There is strong evidence suggesting that RNAi acts co-transcriptionally in order to promote heterochromatin formation. Thus, it is possible that RITS activity is interlinked with transcription-related processes such as cleavage/poly-adenylation, transcription termination and RNA turnover by the exosome complex. In order to investigate this hypothesis, the integrity of RNAi and heterochromatin was assayed in mutants for factors that are involved in all three pathways. Mutations on dhp1 (termination), pfs2 (cleavage and polyadenylation) dis3 and rrp6 (exosome) had negligible effects on RNAi activity and heterochromatin-mediated silencing with only the exosome showing some involvement in the downstream degradation of centromeric transcripts. Conventional RNAi enforces post-transcriptional repression by targeting mRNA molecules for degradation. This is mediated by the endonuclease activity of Argonaute (“slicing”). Although the key residues for this activity are conserved between human Ago2 and S. pombe Ago1, the importance of this “slicing” activity to heterochromatin assembly was not clear. Mutations were made in putative catalytic residues on the endogenous ago1 gene in order to address this question. These mutations severely affect the activity of RNAi in fission yeast and destabilize the heterochromatin structure at centromeres. Consequently, centromere function is affected and chromosome segregation is deficient. Ago1 localization to the centromeres is impaired in these mutants and cannot nucleate heterochromatin nucleation though siRNA production is not fully abolished. Thus, Ago1 slicing activity is crucial for sustainable RNAi and its role in heterochromatin integrity.
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Rupp, Jessica Lynn Shoup. "RNA interference mediated virus resistance in transgenic wheat." Diss., Kansas State University, 2015. http://hdl.handle.net/2097/20387.
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Doctor of PhilosophyPlant Pathology
John P. Fellers
Harold N. Trick
Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) are two viruses affecting wheat in the Great Plains region of the United States. Genetic resistance is severely limited, requiring management methods focusing on the deployment of resistant varieties and various cultural practices. Evaluation of resistance is complicated by the lack of a standard rating scale. The objective of this work was to develop new avenues to mitigate these challenges. A standardized virus symptom rating scale was developed using historical Kansas rating scales, and validated using multiple wheat populations. Two independent RNA interference (RNAi) expression vectors targeting portions of viral coat protein (CP) of WSMV and TriMV were previously transformed into wheat. T₂ plants and beyond were evaluated using PCR, reverse transcription-PCR and bioassays in which plants were challenged with their respective virus. These lines were evaluated for resistance through the T₆ generation. Crosses were made with the susceptible winter wheat cultivars, ‘Overley’ and ‘Karl 92.’ Real-time PCR results show viral titer was up to 20-fold lower in the T₆ transgenic lines, the F₁, and the BC₁F₁ compared to control plants. This provides evidence that this RNAi silencing method is stable in wheat over multiple generations. WSMV and TriMV use host eukaryotic initiation factors (eIF) in order to facilitate replication of their genomes. Previously created RNAi expression vectors were derived from the sequences of the wheat genes eIF(iso)4E-2 and eIF4G. Evaluation of these lines began in the T₁ generation. Resistance has been demonstrated in three lines of eIF(iso)4E-2 and four lines of eIF4G, derived by single seed descent. T₆ progeny co-infected with WSMV and TriMV continue to be resistant. Crosses have been performed with the winter wheat ‘Karl 92’ and three Kansas elite lines, KS030887K-6, KS09H19-2-3, and KS10HW78-1-1. RNAi construct effectiveness was evaluated using real-time PCR. Results show up to 18-fold reduction in viral titer in the transgenic lines, the F₁, and the BC₁F₁ in comparison to control plants. This research provides the first evidence that a single host transgene can provide resistance to multiple viruses and has great potential benefits to both breeders and producers.
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Fransecky, Lars. "Auswirkungen des LRRK2-Knockdown durch RNA-Interferenz auf die murine dopaminerge Zelllinie MN9D." Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2009. http://nbn-resolving.de/urn:nbn:de:bsz:14-qucosa-21600.
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Mutationen im Protein LRRK2 wurden im Zusammenhang mit klinischen Symptomen beschrieben, die dem Idiopathischen Parkinsonsyndrom (IPS) nahezu gleichen. So findet sich neben vielen anderen Mutationen die häufigste pathogene Mutation für das IPS im LRRK2-Gen.
Die Aufklärung der molekularbiologischen Mechanismen, die zur Pathologie der spezifischen Neurodegeneration in der Substantia nigra Pars Compacta (SNpc) und somit zur Idiopathischen Parkinsonssyndrom führen, ist mit der Hoffnung auf kausale und kurative Therapieansätze verbunden.
In dieser medizinischen Doktorarbeit soll daher versucht werden, die biologische Funktion des LRRK2 in einem dopaminergen Mauszellmodell näher zu beschreiben. Hierfür soll die genetische Aktivität des LRRK2 in mesenzephalen, sogenannten MN9D-Zellen reduziert werden, indem der Mechanismus der RNA-Interferenz in vitro durch Transfektion von siRNA angestoßen wird. Durch die Reduktion der LRRK2-Aktivität sollen Veränderungen in den MN9D-Zellen induziert und diese objektiviert werden.
Die Darstellung der Beobachtungen konzentriert sich auf die transkriptionelle Expression von Genen des Zellzyklus sowie der neuralen und dopaminergen Differenzierung (Tyrosinhydroxylase, Nestin und β-Tubulin) durch PCR. Die Proliferation der Zellen vor und nach den RNA-Interferenzexperimenten soll global durch MTT- und BrdU-Test gemessen werden.
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Kok, Kin-hang. "Roles of human double-stranded RNA binding proteins TRBP and PACT in RNA interference." Click to view the E-thesis via HKUTO, 2006. http://sunzi.lib.hku.hk/hkuto/record/B38523218.
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Kok, Kin-hang, and 郭健恆. "Roles of human double-stranded RNA binding proteins TRBP and PACT in RNA interference." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2006. http://hub.hku.hk/bib/B38523218.
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Jagannath, Aarti. "Studies on the RNA interference pathway in mammalian cells." Thesis, University of Oxford, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.711608.
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Hall, Emma Andisi. "Screening for genes involved in cilia formation and function." Thesis, University of Edinburgh, 2012. http://hdl.handle.net/1842/9898.
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Cilia are small microtubule based structures found on the surface of almost all mammalian cells, enclosed in a highly specialised extension of the cell membrane. Components of several key developmental signalling pathways, in particular Hedgehog (Hh) signalling, are enriched in cilia and cells with mutations in cilia structure show aberrant signalling, suggesting cilia act as “antennae” to focus these signalling cascades. A spectrum of human diseases, termed ciliopathies, are caused by problems in cilia formation or cilia function, which display wide ranging phenotypes from embryonic lethality to retinal degeneration, polydactyly to cystic kidneys. Despite recent advances in the understanding of the essential roles cilia play in mammalian development, exactly how these complex structures are put together, how they carry out their diverse functions, and how they are regulated is not well understood. In this thesis, I describe a screen for genes involved in cilia formation and function. While optimising ciliogenesis and immunofluorescence protocols for the screen, the phenotypes of two ciliary mutant cell lines were analysed. Wdr35yet/yet and Dync2h1pol/pol mouse lines were identified in an ENU screen for genes involved in early development, and shown to have gross phenotypes similar to other ciliary mutants (Mill et al. 2011). Intraflagellar transport (IFT) is the active transport of proteins up and down the ciliary axoneme. Dync2h1 is a retrograde IFT motor component, whereas Wdr35 is part of the retrograde IFT-A complex. In this thesis, the cellular phenotypes of mouse embryonic fibroblasts derived from these mutants are described, showing that despite the fact both genes are thought to be involved in retrograde IFT, they show distinct ciliary phenotypes, suggesting novel roles for Wdr35 in mouse ciliogenesis. An siRNA screen was carried out in mouse fibroblasts to identify genes involved in (i) cilia formation, assayed by immunofluorescence for ciliary markers, and (ii) cilia function, assayed by activity of a Hh responsive luciferase transgene as an indirect readout of ciliary function. Although scalable, I initially screened a small test set of thirty-six putative cilia candidates, identified by cross species transcriptomic analysis. We identified several possible hits, many of which were in the ciliome database but also importantly, several genes with no known link to ciliogenesis. Repeats, correlation of phenotype to knockdown efficiencies and localisation studies validated two hits, Ccdc63 and Azi1. Ccdc63 is a novel coiled-coil gene with no previous link to ciliogenesis; the phenotype for this gene was analysed in real time using fluorescently tagged ciliary markers. A second hit, Azi1, was followed up in more detail. The reduction in ciliogenesis upon Azi1 knockdown was confirmed with separate siRNAs, and was rescued by overexpressing siRNA insensitive Azi1-GFP, confirming the phenotype is not due to off-target effects of the siRNAs. Azi1 gene trap mutant mice were generated and confirmed to be null mutations. Surprisingly, the mice survive, showing Azi1 is not essential for mammalian ciliogenesis. However, mutant males are infertile, with highly reduced sperm count and sperm abnormalities indicative of an arrest at Stage IX of spermiogenesis, when the flagellum, a highly specialised motile cilium, forms. The small number of sperm that do get to the epididymus are immotile. We suggest Azi1 is essential to in the formation of the sperm flagella and male fertility.
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Tzelos, Thomas. "RNA interference in parasitic nematodes : from genome to control." Thesis, University of Edinburgh, 2015. http://hdl.handle.net/1842/15906.
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Teladorsagia circumcincta is a parasitic nematode which is a major cause of ovine parasitic gastroenteritis in temperate climatic regions. The parasite has developed resistance to the major anthelmintic drug classes and this challenges its future control. Vaccination is a potential alternative control method since sheep are able to develop protective immunity against this parasite. Although potential vaccine candidates have been revealed, the increasing gene datasets suggest that vaccinetarget selection may be aided by screening methods such as RNAi. This is a reverse genetic mechanism that causes highly specific gene silencing which was initially described and applied to defining gene function in Caenorhabditis elegans. Nevertheless, its application was more difficult than anticipated in parasitic nematodes because of the inconsistency of the silencing effect. In the unsuccessful cases, did the dsRNA penetrate the parasite and activate the RNAi pathway? Thus far, there are no internal controls that indicate the activation of the pathway. Are the RNAi pathway genes constantly transcribed or are they ‘switched on’ in response to the dsRNA exposure? The initial aim of the study was to determine potential marker genes in the RNAi pathway that could indicate the activation of the pathway in C. elegans. After the exposure to dsRNA from two target genes, the transcript levels of three candidate marker genes (Ce-dcr-1, Ce-ego-1 and Ce-rsd-3) were examined and showed that exposure to dsRNA has no effect on the transcript levels of these genes making them inappropriate markers for the activation of the RNAi pathway. The two target-genes were Ce-cpr-4 and Ce-sod-4 which had been proven to be consistently susceptible and refractory to RNAi, respectively. Another aim of the project was to develop an RNAi platform in T. circumcincta for use as a screening method for potential vaccine candidates. The targets selected for the in vitro RNAi included: five members of the Activation-associated Secreted Proteins (ASPs); a Macrophage migration Inhibitory Factor-like (Tci-mif-1) and a Surface Associated Antigen gene (Tci-saa-1), all of which have been associated with vaccine-induced protective immunity. The selection of the ASPs was based on a bioinformatic and transcriptomic analysis of the ASPs in T. circumcincta. The results showed successful knock-down only for three out of five ASP targets after 1 hour of soaking in gene-specific double stranded RNA (dsRNA) which illustrates the inconsistency and the target specificity of RNAi in T. circumcincta which has been observed in the past with other parasitic nematodes. Inconsistencies were also observed within the successful ASP targets with the results not being reproducible after several successful experiments. Potential reasons for the inconsistencies were examined with the duration of larval storage being a critical factor. Larvae stored for a short or long period of time were susceptible and refractory to RNAi, respectively. Experiments were also conducted to investigate how the ASPs relate to extracellular microvesicles (EMVs). These vesicles are considered to play an important role in the intercellular communication between parasites and their hosts, and thus represent potentially useful vaccine and/or drug targets. Transmission electron microscopy (TEM) confirmed that EMVs are excreted / secreted by the parasite and the proteomic analysis revealed several types of proteins within the vesicles such as: ASPs, Actins, Metallopeptidases, and RAB proteins. A comparative analysis of EMVs, EMV-free ES (Excretory / Secretory) and total ES products showed that approximately 35% of the proteins found in the vesicles could also be identified in EMV-free ES and in total ES products, whilst the remaining 65% were present only in EMVs.
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Wen-Hsin, S. L. "Lentiviral-based RNA interference of genes in leukaemic cells." Thesis, University College London (University of London), 2006. http://discovery.ucl.ac.uk/1445137/.
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Childhood leukemia is a common paediatric cancer in the developed world and the biologically diverse subtypes of this disease are characterised by specific chromosomal translocations that alter the normal proliferative and survival signals of haematopoietic cells. Despite of greatly improved cure rate of childhood leukaemias over the past years, younger patients with acute leukaemias involving E2A-HLF and MLL-ENL translocations still confer a poor prognosis that is associated with a very unfavourable outcome. The incidence of relapse after complete remission seems to crucially depend on a small population of leukaemic stem cells that survive from the initial therapy and sustain the disease. So far it has been unsuccessful to induce long-term gene silencing using siRNA technology in the primary haematopoietic cell lines and leukaemic stem cells. Thus, this project aimed to optimise the current 2nd generation of miR30-based shRNA lentiviral vector to achieve this. The silencing cassettes were delivered to the cells of interest by lentivirus and long-term expression was seen. The result revealed that effective E2A-HLF and MLL-ENL gene silencing was achieved while LM02 gene expression was not significantly knocked down by the predicted LM02 shRNA constructs. The most efficient lentiviral vectors against specific genes will then be used to infect leukaemic cells to test the effect on aspects of the leukaemic phenotype. Understanding of these fusion genes and identification of their downstream target genes in initiating and maintaining transformation events in the leukaemic stem cells may aid the development of revolutionary therapeutics that specifically target leukaemic stem cells. In conjunction with standard therapies, this approach could be more effective in treating MLL-ENL and E2A-HLF patients who tend to have an extremely unfavourable prognosis.
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Zhong, Jing. "Genetics Characterization of Antiviral RNA Interference in Caenorhabditis elegans." Thesis, University of California, Riverside, 2014. http://pqdtopen.proquest.com/#viewpdf?dispub=3644055.
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RNA interference (RNAi) acts as an antiviral defense mechanism in fungi, plants, nematodes, insects, and mammals. In antiviral RNAi, virus-specific double-stranded RNA is processed into small interfering RNAs (siRNAs) to guide specific viral RNA degradation by the RNAi machinery. Although antiviral RNAi is non cell-autonomous in plants, it is unknown if antiviral RNAi is also systemic in animals. In this dissertation, I characterized the nematode Caenorhabditis elegans mutants defective in systemic RNAi in their antiviral RNAi response induced by either the replication of a Flock house virus-derived replicon or the infection of Orsay virus. The results from these genetic studies provided evidence for the first time to support an antiviral function of systemic RNAi in animals. Comparison of the population of viral siRNAs by deep sequencing further revealed that C. elegans mutants with strong defects in systemic antiviral RNAi were all partially defective in the biogenesis of the viral secondary siRNAs. A possible role for the viral siRNAs in systemic antiviral RNAi is discussed.
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Maeshima, Ruhina. "MYCN silencing as therapeutics for neuroblastoma using RNA interference." Thesis, University College London (University of London), 2018. http://discovery.ucl.ac.uk/10043849/.
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Neuroblastoma (NB) is the most common solid tumour in childhood and accounts for 15% of childhood cancer deaths. It is known that high-risk NB is highly correlated with MYCN amplification. Overexpressed MYCN induces proliferation and cell growth and suppresses apoptosis and differentiation pathways in NB cells. Since RNA interference (RNAi) was first described, many research groups have investigated the application of RNAi with the use of short interfering RNA (siRNA). Our aim is to induce apoptosis and differentiation using RNAi as a novel therapeutic strategy for MYCN-amplified NB. Our hypothesis is that MYCN silencing by anti-MYCN siRNA induces apoptosis and differentiation at the mRNA and protein level. We are encapsulating siRNA with liposome and integrin-receptor targeting peptide to deliver MYCN siRNA into NB cells and optimising cationic and anionic polyethylene glycol (PEG)ylated receptor-targeting nanocomplexes (RTNs). In this project, we also aimed to optimise the methods to store RTNs for a long time in trehalose, which is known as a cryoprotectant. As a result, MYCN was silenced by the siRNA at both the mRNA and protein levels, and the siRNA-mediated MYCN reduction induced downstream effects, such as a neuronal differentiation marker TrkA upregulation and the morphological changes of the cells. The anti-MYCN siRNA delivered using RTNs successfully silenced MYCN mRNA in vivo as well. We used an NB cell line with non-functional p53 and resistance toward p53-pathway dependent anti-cancer drugs, probably induced by multiple sessions of chemotherapy and radiotherapy. Therefore, the results are promising for a novel therapy for relapse NB with MYCN amplification. In addition, we successfully demonstrated that trehalose maintains the biophysical properties and the function of RTNs, consisting of either DNA or siRNA at -80 °C. This allows us to make a large amount of RTN for many experiments, store it for the long term, and transport it to a place far from the laboratory.
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White, Melanie Denise. "RNA interference as a therapeutic approach in prion disease." Thesis, University College London (University of London), 2008. http://discovery.ucl.ac.uk/1445182/.
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Prion diseases are fatal, transmissible neurodegenerative disorders characterised by accumulation throughout the brain of PrPSc, an abnormally folded isoform of the normal cellular prion protein, PrP. PrPSc is associated with infectivity but is not directly neurotoxic and targeting it is of limited efficacy in prion therapeutics. However, PrP-null mice are resistant to prion infection and neurotoxicity. Transgene-c mediated depletion of neuronal PrP in mice with established prion infection reverses early spongiosis, neuronal loss and cognitive deficits, and prevents clinical disease progression. Thus, reducing PrPC expression in the brain through extrinsic means is likely to be an effective therapy for prion diseases. RNA interference can be exploited to mediate gene silencing and can be stably achieved in non-dividing cells such as neurons by incorporation of the small interfering RNAs into replication-deficient lentiviruses. The work described in this thesis strongly validates the use of lentiviral-mediated RNA interference as a therapeutic approach in prion disease. Reducing PrPc expression with siRNA duplexes enabled clearance of PrPSc and infectivity from prion-infected cells in vitro. Lentiviruses constructed to express the interfering sequences demonstrated effective reduction of PrPc expression in vitro. Stable expression of the interfering RNA molecules in vivo through lentiviral transduction of the hippocampus reduced local pathology and significantly prolonged survival in a mouse model of prion disease. This represents an important and novel advance in the treatment of established prion disease with relevance for all prion strains.
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Miller, Sherry C. "RNA interference in the red flour beetle Tribolium castaneum." Diss., Kansas State University, 2009. http://hdl.handle.net/2097/1338.
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Doctor of PhilosophyDepartment of Biology
Susan J. Brown
RNA interference (RNAi) is a natural gene-silencing phenomenon triggered by dsRNA (dsRNA). While RNAi is an endogenous process that plays essential roles in regulating gene expression it can also be harnessed as a tool for the study of gene function. Introducing dsRNA that is homologous to target mRNA into a cell triggers the RNAi response causing the destruction of the homologous mRNA and a loss of function phenotype. In some organisms, such as the nematode Caenorhabditis elegans, once dsRNA is introduced into the body cavity, the RNAi effect is seen throughout the organism because the dsRNA is taken up by individual cells and is then spread from cell to cell. This process has been termed the systemic RNAi response. For other organisms, such as the fruit fly Drosophila melanogaster, introduction of dsRNA into the body cavity does not result in a systemic RNAi response. This may be due to the cell’s inability to take up dsRNA or spread that dsRNA from cell to cell. For other organisms, including mammals, introduction of dsRNA into the body cavity does not result in a systemic RNAi response because the immune response causes dsRNA destruction before it can be utilized in the RNAi pathway. For organisms that do not exhibit a systemic RNAi response, complex genetic methods are needed to introduce dsRNA into cells to induce the RNAi response. Therefore, one of the challenges in utilizing RNAi as a genetic tool is introducing the dsRNA into individual cells. In recent years, systemic RNAi responses have been documented in both model and non-model organisms, making RNAi an accessible genetic tool. The red flour beetle, Tribolium castaneum is an emerging model organism that has a robust systemic RNAi response. However, the mechanism of systemic RNAi and the specific parameters required to obtain a strong systemic RNAi response in this organism have not been thoroughly investigated. The aim of this work is to provide data that can allow RNAi to be better utilized as a genetic tool in Tribolium and to use this information as a basis for the use of RNAi in other insects in which it can be performed. Specifically we provide data on the essential parameters necessary to achieve an effective systemic response in Tribolium, we describe differences in the systemic RNAi response between Drosophila and Tribolium, we analyze the conservation and function of RNAi machinery genes in Tribolium and we provide information on the genes critical for a systemic RNAi response in Tribolium.
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Kelly, Amanda. "Vector-mediated RNA interference in zebrafish : a feasibility study." Thesis, University of Manchester, 2010. https://www.research.manchester.ac.uk/portal/en/theses/vectormediated-rna-interference-in-zebrafish-a-feasibility-study(6d028efa-412f-429d-8cf6-c1512e627fd1).html.
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Zebrafish are becoming an increasingly popular model organism in which to model diseases with a genetic component. Their use is hindered however, by the lack of an efficient, reliable, stable and cost-effective method to carry out reverse genetics and model diseases which arise from a loss of function of a gene. RNAi is a method of post-transcriptional gene regulation and has been widely manipulated in other systems to knockdown genes at will. This thesis therefore looks at the feasibility of vector-mediated RNAi in zebrafish by attempting to knockdown green fluorescent protein (GFP) and the Parkinson‟s disease-associated gene PTEN Induced Kinase 1 (PINK1).Initial results in HEK 293 cells and in G0 animals were encouraging, however low expression of the self-reporting vector made the identification of transgenic animals difficult. To improve expression levels the vector was modified to contain a Gal4-VP16/UAS amplification cassette. Inclusion of this cassette led to increased expression and knockdown capabilities of the vector in HEK 293 cells and led to the successful identification of transgenic zebrafish. Despite high level expression however, no knockdown of GFP or PINK1 was detected in transgenic zebrafish larvae expressing the RNAi vectors out to 5 dpf. This lack of knockdown was shown to be despite the expression of the main components of the RNAi pathway and the production of customised miRNAs throughout development and across tissues. Interestingly however, in adult transgenic zebrafish 50% knockdown of PINK1 was detected in brains expressing two independent PINK1 miRNAs compared to the control miRNA and wild type zebrafish brains. This knockdown coincided with increased transcript expression of the RNAi components and increased production of customised mature miRNA in the brain compared to embryos.In an attempt to improve vector-mediated RNAi in zebrafish, the effect of over-expression of components of the RNAi machinery, including Argonaute 2, Dicer, Drosha and Exportin 5 was assessed in zebrafish cells. Of these, only over-expression of Argonaute 2 improved knockdown in HEK 293 cells and resulted in moderate knockdown in two independent zebrafish cell lines, PAC.2 and ZFL cells. This improvement in knockdown was shown to be a result of the RNase activity of Argonaute 2 as mutation of this domain abrogated the effect of Argonaute 2 over-expression. Despite the encouraging results in zebrafish cell lines, injection of Argonaute 2 mRNA into transgenic zebrafish failed to produce knockdown, suggesting perhaps, that in zebrafish embryos other factors apart from Argonaute 2 are also limitingGiven the difficulties of vector-mediated RNAi in zebrafish, this technology is at present not a feasible approach to knocking down genes in zebrafish, at least not to an extent as to model complete loss of gene function.
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Miller, Sherry C. "RNA interference in the red flour beetle Ttribolium castaneum." Manhattan, Kan. : Kansas State University, 2009. http://hdl.handle.net/2097/1338.
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Ono, Ekaterina Alexandrovna Durymanova. "Silenciamento gênico pós-transicional por interferência por RNA (RNAi) com terapia antiviral para a raiva." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-18082015-153913/.
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A raiva é uma zoonose que afeta todos os mamíferos e causa cerca de 55.000 mortes humanas por ano, causada pelo vírus da raiva. O vírus da raiva pertence à Ordem Mononegavirales, Família Rhabdoviridae e o Gênero Lyssavirus. Ultimamente, o Protocolo de Milwaukee é a base do tratamento humano, com indução do paciente ao coma e uso de massiva terapia antiviral. O protocolo, embora tenha sido utilizado duas vezes com sucesso, inclusive em um caso brasileiro, ainda requer aperfeiçoamentos. Neste sentido, a interferência por RNA (RNAi) é uma nova abordagem para terapia de doenças virais. O objetivo deste trabalho foi avaliar a inibição da replicação do vírus da raiva in vitro e in vivo utilizando RNAi. Para este fim, foram utilizados três siRNAs (siRNA 360, siRNA 652, siRNA 649) com a fita antisenso complementar ao mRNA da fosfoproteína (P) e três siRNAs (Le 1, Le 2, Le 3) contra o RNA líder do vírus da raiva. Para o ensaio in vitro foram utilizadas as amostras PV e 4005 (AgV3) do vírus da raiva e as células de BHK-21 (Baby hamster kidney). As monocamadas celulares foram infectadas com as amostras PV ou 4005 e depois de 2 horas de incubação transfectadas com cada um dos siRNAs em combinação com Lipofectamine 2000TM. Depois de 24 e 48 horas as placas teste e controle foram submetidas à imunofluorescência direta (IFD) com conjugado globulina de coelho anti-ribonucleocapsídeo do vírus da raiva/isotiocianato de fluoresceína (Instituto Pasteur de São Paulo). Os resultados revelaram que os siRNAs contra o RNA líder do vírus não foram capazes de inibir a replicação do vírus. A utilização dos siRNAs contra mRNA P resultaram em títulos de 3,625logTCID50/ml, 3,875logTCID50/ml e 4,125logTCID50/ml para os siRNAs 360, 649 e 652, respectivamente, enquanto que, para a placa controle, o título foi 4,0logTCID50/ml nas placas infectadas com PV e período de incubação de 24h. Nas placas infectadas com a amostra 4005 e tratadas com siRNAs, a maior queda de título viral foi na placa tratada com siRNA 360, de 1,0 log, comparando-se com a placa controle de incubação de 24 para a amostra 4005. Nas placas tratadas com siRNA 649 e siRNA 652, também houve a diminuição de título viral, mas em uma escala menor (0,25log e 0,125log, respectivamente) comparando-se com o controle. Nas placas infectadas com PV e incubadas durante 48h, os títulos apresentados foram de 5,625logTCID50/ml, 4,625logTCID50/ml e 4,75logTCID50%/ml para os siRNAs 360, 649 e 652, respectivamente, enquanto que na placa controle o título foi 6,0logTCID50C%/ml. A placa com período de incubação de 48h com a amostra 4005 e tratada com siRNA 360 apresentou a maior queda de título viral entre os três siRNAs, o que resultou em 1,125log de diferença. Nas monocamadas onde foram administrados siRNA 649 e siRNA 652, observou-se também uma pequena queda de título viral igual a 0,875log e 0,295log, respectivamente, comparando-se com a placa não tratada. Para o ensaio in vivo, foram usados camundongos albino suíços de 21 dias com peso entre 11 e 14g, infectados com a cepa PV e AgV3 em 10DL50% via intracerebral. Duas horas depois da infecção, foi inoculada por via intracerebral uma solução do siRNA 360 com Lipofectamine 2000TM. Os animais com paralisia foram eutanasiados e aqueles sobreviventes foram observados até completar 30 dias de observação quando foram, então, eutanasiados. O sistema nervoso central de todos os animas foi recolhido e submetido a IFD. A utilização do siRNA 360 em camundongos resultou em 30% de animais sobreviventes frente amostra 4005, enquanto que a mortalidade nos animais não tratados foi de 90%. Nos animais inoculados com a amostra PV e tratados com este siRNA, a sobrevivência foi de 40%, enquanto que no grupo controle a mortalidade foi de 100%. O resultado do ensaio in vitro demonstra que os siRNAs utilizados são capazes de inibir a replicação do vírus da raiva, com eficiência mais pronunciada para o siRNA 360. In vivo, este siRNA foi capaz de induzir a proteção parcial dos animais inoculados com as duas variantes virais. Estes resultados, ainda que indiquem a necessidade de mais estudos, permitem concluir que a RNAi é uma tecnologia promissora como antiviral contra a raivaRabies is a zoonotic disease that affects all mammals and causes more than 55.000 human deaths every year, caused by rabies virus (RABV) a virus of the Mononegavirales order, Family Rhabdoviridae and the Lyssavirus genus. After the onset of the symptoms, the illness has a fast progression and the patients feel intense physical suffering. Currently, human rabies treatment has been based on the Milwaukee Protocol which consists on the induction of coma and massive antiviral therapy. Despite this protocol has been successful in two cases, including a Brazilian one, more studies on antivirals for human rabies treatment are required. RNA interference is a new antiviral approach, which gives hope to the possibility of rabies antiviral treatment. The aim of this study was to assess the decrease in titres of rabies virus in vitro and in vivo using short-interfering RNAs. To this end, three siRNAs (siRNA 360, siRNA 652, and siRNA 649) were used with antisense strands complementary to rabies virus phosphoprotein (P) mRNA and three other (Le 1, Le 2, Le 3) to the leader RNA. Pasteur virus strain (PV) and strain 4005 (AgV3) of rabies virus and BHK-21 cells were used, and the monolayers were transfected with each of the RNAs with Lipofectamine-2000 TM. After 22 hours, the siRNA-treated and the control plates were tested by direct fluorescent antibody test (DFAT) with anti-rabies virus nucleocapsid antibody conjugate with fluorescein isothiocianate (Pasteur Insitutte, Brazil). The plates transfected with siRNA against phosphoprotein mRNA were also incubated for 48 hours and subjected to IFD assay. Virus titres were calculated by the Spearman-Karber method. The results showed that siRNAs against virus leader RNA were not able to inhibit the replication of the virus. The use of siRNAs against P mRNA resulting titres of 3.625logTCID50/ml 3.875logTCID50/ml and 4.125logTCID50/ml for siRNAs 360, 649 and 652, respectively, while, for the control plate, the titre was 4.0logTCID50/ml in plates with PV and 24h incubation period. In plates with strain 4005 and treated with siRNAs, the highest viral titre decrease was obtained with siRNA 360, with a 1.0 log difference compared to the control plate of strain 4005 incubated for 24h. The plates treated with siRNA 649 and siRNA 652 have was also shown a decrease in viral titres, but on a smaller scale (0.25log and 0.125log, respectively) compared to the control. The plates infected with PV and incubated for 48 hours showed titre of 5.625logTCID50/ml, 4.625logTCID50/ml and 4.75logTCID50%/ml for siRNAs 360, 649 and 652, respectively, while for the control plate the titre was 6.0logTCID50C%/ml. The plate with strain 4005 and then treated with siRNA360 and incubated for a total of 48h had the highest viral titre decrease among the three siRNAs, which resulted in a 1.125log difference compared to the control plate. In monolayers treated with siRNA649 and siRNA652 there was also a discrete drop in viral titres (0.875log and 0.295log, respectively) compared to the control plate. For the in vivo assay, 21-day old Swiss albino mice weighing between 11 and 14g were intracerebrally inoculated with PV or 4005 strains (10DL50%). Two hours after inoculation, a solution of siRNA360 with Lipofectamine 2000 TM was also intracerebrally injected. Mice presenting paralysis and those that survived the 30 days of observation were euthanized. The central nervous system of all animals was collected and submitted to IFD. The use of siRNA360 in mice resulted in survival of 30% of animals in the group inoculated with strain 4005, whereas 90% mortality was observed in the control group. In animals inoculated with the PV strain, and treated with siRNA360, the survival rate was 40% and in the control group the mortality was 100%. The results of the in vitro assay demonstrate that the siRNAs used are effective in inhibiting the replication of rabies virus with a more intense inhibition regarding siRNA 360. In vivo, this siRNA was able to induce partial protection of animals infected with both viral variants. These results also indicate that, despite the need for further studies, RNAi is a promising technology as antiviral against rabies
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Werth, Stephanie [Verfasser], and Achim [Akademischer Betreuer] Aigner. "Neue Gentargeting-Strategien auf Basis der RNA interference (RNAi) / Stephanie Werth. Betreuer: Achim Aigner." Marburg : Philipps-Universität Marburg, 2013. http://d-nb.info/1038786754/34.
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Kandan-Kulangara, Febitha. "Poly(ADP-ribose) polymerase-1 (PARP-1) and RNA interference (RNAI) during cell death." Doctoral thesis, Université Laval, 2013. http://hdl.handle.net/20.500.11794/25972.
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L’activation de la poly(ADP-ribose) polymérase-1 (PARP-1) en réponse aux dommages à l’ADN est impliquée dans diverses réponses cellulaires, de la réparation de l’ADN à la mort cellulaire. Dans l'annexe I, nous avons décrit différentes techniques indispensables pour détecter le métabolisme de PARP-1 en réponse aux dommages à l’ADN in vitro et in vivo. Les travaux de cette thèse se concentrent sur le rôle de PARP-1 dans la mort cellulaire. PARP-1 est clivée et inactivée par des caspases pendant l’apoptose ; j’ai donc utilisé une PARP-1 non-clivable pour étudier le rôle de l’activation et de la fragmentation de PARP-1 dans la mort cellulaire induite par les UVB. Nous avons observé que, contrairement aux fibroblastes de peau humaine exprimant la PARP-1, les fibroblastes avec un "knockdown" de PARP-1 sont résistants à l’apoptose induite par les UVB, phénotype pouvant être totalement inversé par ré-expression de PARP-1 sauvage mais pas de PARP-1 non-clivable par les caspases, suggérant un rôle significatif du clivage de PARP-1 en réponse à la mort cellulaire induite par les UVB (chapitre 2). Dans ce contexte, nous avons récemment passé en revue comment les substrats non clivables par des caspases peuvent être utilisés comme outil important pour démystifier le rôle de ce clivage pour la mort comme pour la vie, avec l’exemple spécifique de PARP-1 non-clivable par les caspases (chapitre 3). Curieusement, en utilisant l’ARNi comme outil d’étude du rôle de PARP-1 dans la mort cellulaire, nous avons observé que l’ARNi stable (shRNA) de nombreux gènes, incluant PARP-1, échoue lors de l’apoptose, en raison de l’inactivation catalytique par clivage par une caspase de l’endoribonucléase Dicer-1, indispensable pour la régulation de l’ARNi et des miARN (chapitre 4). Cependant, nous avons découvert que l’ARNi transitoire persiste plusieurs jours même après induction de l’apoptose, soulignant des différences entre les ARNi stable et transitoire dans la dynamique de "knockdown" génétique et dans la dépendance de la fonction de Dicer-1 (chapitre 5). En résumé, mon travail a permis la découverte des avantages et des limites de l’ARNi durant l’apoptose et le rôle de PARP-1 dans la mort cellulaire induite par les UVB.
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Hönemann, Mario. "RNA Interferenz unter Verwendung eines lentiviralen Vektosystems zur Modifikation einer persistierenden Masernvirusinfektion." Doctoral thesis, Universitätsbibliothek Leipzig, 2011. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-77084.
Full textAbstract:
Die Vorliegende Arbeit beschäftige sich mit der Etablierung eines lentiviralen Vektorsystems, mit dem es möglich ist die RNA-Interferenz experimentell zu nutzen. Dafür wurden SEC Sequenzen in den Vektor pGJ3-eGFP kloniert. Nach Optimierung der Transfektions und Transduktionsschritte wurden im Anschluss rekombinante virale Partikel hergestellt. Zur Überprüfung der Effektivität der Induzierten RNA-Interferenz erfolgte die Transduktion einer persistierend mit Masern infizierten Zelllinie (C6SSPE). Ziel der siRNA Sequenzen war dabei die mRNA des N-Proteins, welches eine zentrale Rolle im viralen Replikationszyklus spielt. Die Reduktion der mRNA wurde über quantitative real time RT-PCR nachgewiesen.
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Pretorius, Ashley. "Functional analysis of the mouse RBBP6 gene using Interference RNA." Thesis, University of the Western Cape, 2007. http://etd.uwc.ac.za/index.php?module=etd&action=viewtitle&id=gen8Srv25Nme4_4435_1264363734.
Full textAbstract:
The aim of this thesis was to investigate the cellular role of the mouse RBBP6 gene using the interference RNA (RNAi) gene targeting technology and also to understand the relevance of two promoters for the RBBP6 gene.
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Dudley, Nathaniel Ray Goldstein Robert P. "Understanding the molecular mechanisms of RNA interference in Caenorhabditis elegans." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2006. http://dc.lib.unc.edu/u?/etd,63.
Full textAbstract:
Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2006.Title from electronic title page (viewed Oct. 10, 2007). "... in partial fulfillment of the requirement for the degree of Doctor of Philosophy in the Department of Biology." Discipline: Biology; Department/School: Biology.
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Munir, Alia. "RNA interference as therapy in a model of Cushing's disease." Thesis, University of Sheffield, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.577520.
Full textAbstract:
Hypothesis: That siRNAs could be translated into systemic therapy for Cushing's disease in man. Background and importance: Cushing's disease is an uncommon but devastating condition, with a mean age of onset of 36 years, and a five-fold excess mortality when inadequately treated. Major complications include diabetes, hypertension, osteoporosis, life-threatening infection, depression and psychosis. No established ACTH-Iowering medical therapy exists. 95% of cases are caused by a tiny benign corticotrope adenomas of the pituitary gland, often only millimetres in diameter, and in 40% of cases are invisible on MR!. These adenomas express the prohormone 'Pro-opiomelanocortin' (pOMC), which undergoes post-translational processing to adrenocorticotrophin (ACTH), before secretion into the systemic circulation. ACTH drives the adrenal glands to synthesise and secrete excessive amounts of the hormone cortisol, which causes the clinical condition of Cushing's disease. Invasive neurosurgery aimed at removal of the pituitary tumour, causes long-term remission in only 50% of patients with Cushing's disease and results in hypopituitarism in approximately half. Hypopituitarism is associated with a two-fold excess mortality. Bilateral adrenalectomy is effective treatment but is complicated by life-long hypoadrenalism (two-fold excess mortality) and the development of an expanding, and pituitary mass (Nelson's syndrome) in up to 30% of cases. Use of adrenal steroidogenesis inhibitors is not usually effective in long-term management.
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Weinberg, David E. (David Eric). "Discovery and biochemical characterization of RNA interference in budding yeast." Thesis, Massachusetts Institute of Technology, 2013. http://hdl.handle.net/1721.1/80887.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2013.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Cataloged from student-submitted PDF version of thesis. Vita.
Includes bibliographical references.
RNA interference (RNAi) is a eukaryotic pathway for the post-transcriptional regulation of gene expression. In the simplest form of RNAi, a double-stranded RNA (dsRNA) trigger is converted into small-RNA duplexes by the Dicer enzyme. These duplexes are then loaded into the effector protein Argonaute to guide the cleavage of target transcripts. RNAi and related RNA-silencing pathways are found in plants, animals, fungi, and protists, suggesting origins in an early eukaryotic ancestor and selective pressures to maintain the pathway. A prominent exception to this widespread conservation of RNAi is the budding yeast Saccharomyces cerevisiae, which lacks homologs of Dicer and Argonaute. Indeed, RNAi had been presumed lost in all budding yeasts. Motivated by the presence of Argonaute homologs in some budding-yeast species, we examined whether these species contain a functional RNAi pathway. High-throughput sequencing led to the identification of endogenous small RNAs that are generated by a novel Dicer enzyme. In Saccharomyces castellii, these Argonaute-bound small RNAs serve as guides to repress mRNA targets, which are predominantly repetitive elements. RNAi can be restored to S. cerevisiae by introducing the genes encoding S. castellii Dicer and Argonaute, and the reconstituted pathway silences endogenous transposons. Budding-yeast Dicer has a different domain architecture than canonical Dicer yet generates siRNAs of a similar length. In contrast to canonical Dicer, which successively removes small-RNA duplexes from the dsRNA termini, budding-yeast Dicer molecules bind cooperatively to the interior of dsRNA substrates, with the distance between adjacent active sites determining product length. These distinct mechanisms impart corresponding substrate preferences and product characteristics that are important for Dicer function. Structural studies of budding-yeast Argonaute yielded a crystal structure of the functional Argonaute-guide complex. Eukaryotic Argonaute makes extensive sequence-independent interactions with the guide RNA to maintain the seed region in a helical conformation with the base edges accessible for target binding. An invariant glutamate residue, which is only positioned in the catalytic pocket after guide-RNA binding, constitutes the previously missing component of a ribonuclease H-like active site.
by David E. Weinberg.
Ph.D.
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Spellman, Rachel Claudine Helen. "Investigation of polypyrimidine tract binding protein function by RNA interference." Thesis, University of Cambridge, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.614210.
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Kamath, Ravi Shanker. "Functional analysis of the C. elegans genome using RNA interference." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620522.
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Samarasinghe, Buddhini. "Analysis of RNA interference in the parasitic nematode Haemonchus contortus." Thesis, University of Glasgow, 2010. http://theses.gla.ac.uk/1957/.
Full textAbstract:
Parasitic nematode infections worldwide cause a significant impact on human health, as well as economic and welfare losses to the animal and agriculture industries. The principal method of control for parasitic nematode infections is currently limited to repeated treatments with anthelmintic drugs, but widespread resistance to all major classes of these drugs is a growing problem. As a result, there is an urgent need for alternative methods of controlling these infections, and the development of molecular vaccines and novel drugs represent two possible approaches. However, both these approaches require a deeper understanding of gene function in order to identify suitable control targets. This project examines RNA interference (RNAi) in the parasitic nematode Haemonchus contortus to determine if this could be developed as a functional tool and advance the discovery of novel control targets for parasitic nematodes. RNAi has proven less effective in parasitic nematodes than in the free-living model nematode Caenorhabditis elegans and it is unclear why this is so. This project examined the reliability of RNAi in H. contortus, and several genes were successfully silenced using RNAi. Further analysis of RNAi susceptible genes revealed that RNAi silencing appears to be related to the site of expression of the target gene; genes expressed in tissues which are accessible to the environment such as intestine, excretory cell and amphids were silenced by RNAi. Upstream promoter regions of RNAi susceptible genes were examined for the presence of motifs which may regulate spatial gene expression, an approach that could be used to predict gene susceptibility to RNAi. RNAi treated larvae were subsequently used to infect sheep in the first in vivo RNAi study, resulting in a significant impact on worm survival in vivo. In addition, several components of the RNAi pathway in H. contortus were characterised in this project, demonstrating the presence of a functional RNAi pathway that is capable of reliably silencing genes. In conclusion, the findings presented in this project suggest that RNAi may be used in the future to evaluate the function of a novel vaccine or drug target for controlling H. contortus infections in sheep.
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Parrish, Susan N. "Characterization of the mechanism of RNA interference in caenorhabditis elegans." Available to US Hopkins community, 2002. http://wwwlib.umi.com/dissertations/dlnow/3068195.
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Schwarz, Dianne S. "Biochemical Mechanism of RNA Interference in Higher Organisms: A Dissertation." eScholarship@UMMS, 2005. https://escholarship.umassmed.edu/gsbs_diss/186.
Full textAbstract:
RNA interference (RNAi) is an evolutionarily conserved, sequence-specific gene silencing pathway found in eukaryotes, in which 21-nucleotide, small interfering RNAs (siRNAs) guide destruction of a corresponding target mRNA. RNAi is a natural mechanism for both genome surveillance and gene regulation. Moreover, siRNAs can be transfected into cultured mammalian cells, causing the sequence-specific ‘knock down’ of an mRNA. My work in the Zamore lab has centered around the Drosophilain vitro system and cultured mammalian cells to study the RNA interference (RNAi) pathway. small interfering RNAs (siRNAs) are incorporated into the RNA-induced silencing complex (RISC), which culminates in the cleavage of a complementary target mRNA. Previous work proved that certain structural features of siRNAs are essential for RNAi in flies, including the requirement for 5´ phosphates and 3´ hydroxyl groups. In cultured mammalian cells, the requirement for a 5´ phosphate also holds true, but we found no evidence to support the necessity for 3´ hydroxyls in either system. In addition, siRNAs can act as single strands entering the pathway downstream of double-stranded siRNAs, both of which are competent in directing the cleavage of its cognate mRNA at a single site.
While these key features are a requirement for functional siRNAs, alone they do not determine the efficiency to which an siRNA can enter the RISC. In fact, both strands of an siRNA can enter RISC to a different degree as determined by the stabilities of the 5´ ends of the siRNA strand, a phenomenon termed ‘functional asymmetry’. This characteristic is also reflected in another class of small RNAs involved in gene silencing known as microRNAs (miRNAs), which are processed from long hairpin RNA structures into mature, single-stranded non-coding RNAs. The asymmetric loading of siRNAs suggests that miRNAs are initially generated from siRNA-like duplexes cleaved from the stem of the hairpins. The strand whose 5´ end is less tightly paired will be processed into the mature miRNA, while the other strand is destroyed. By applying the rules of siRNA asymmetry it is possible to predict which side of the stem will be processed into the mature miRNA, a finding verified experimentally by our lab and others. This discovery also has additional implications in designing highly effective siRNAs and in reducing siRNA off-target effects.
We used these results to design siRNAs that target the single nucleotide polymorphism in superoxide dismutase that causes the familial form of amyotrophic lateral sclerosis (ALS), but leave the wild-type mRNA intact and functional. Our experiments have helped define the ‘rules’ for creating SNP-specific siRNAs. In particular, we found that only siRNAs with a purine:purine mismatch to the allele not intended for destruction show good discrimination. The placement of the mismatch in a tiled set of siRNAs shows that mismatches located in the 5´ region of the siRNA, a region shown to be responsible for siRNA binding, can not discriminate between alleles. In contrast, mismatches in the 3´ region of the siRNA, the region contributing to catalysis, discriminate between wild-type and mutant alleles. This work is an important step in creating allele-specific siRNAs as therapeutics for dominant negative genetic diseases.
But how does RISC cleave its target? By isolating both the 5´ and 3´ cleavage products produced by RISC in the Drosophila in vitro system, we discovered that RISC acts as a Mg2+-dependent endonuclease that cleaves a single phosphodiester bond in the mRNA target, leaving 5´ phosphate and 3´ hydroxyl groups. These findings were a critical step in the demonstration that Argonaute, a protein known to be a component of RISC, is the RNAi endonuclease.
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Di, Bella Sebastiano. "RNA-interference e Farmacogenomica: dall'analisi dell'RNA agli effetti dei farmaci." Doctoral thesis, Università di Catania, 2013. http://hdl.handle.net/10761/1392.
Full textAbstract:
Le interazioni tra i geni insieme con i loro livelli di espressione determinano un fenotipo in un dato organismo. Tuttavia pochi sforzi sono stati fatti per usare i dati fenotipici che capire le singole relazioni genotipo-fenotipo. L'origine genetica di una malattia è spesso scoperta una volta che il suo fenotipo è stato chiaramente definito, in aggiunta i tratti complessi delle malattie comprendono una varietà di fenotipi e meccanismi biologici, tali da rendere difficile determinare i geni da studiare.
Per molto tempo, i fenotipi sono stati visti unicamente come indicatori di cambi nei
genotipi o nelle malattie. L'abilità di interferire in maniera sistematica nei componenti
genetici ha aumentato l'importanza dei fenotipi come tool per comprendere i processi
biologici a livello molecolare.
A livello qualitativo il data set di mutazione umana supera quella di qualsiasi altra specie
poichè i fenotipi umani possono essere facilmente individuati e descritti dalle loro
caratteristiche osservabili e l'esistenza di specifici gruppi di fenotipi di malattie suggerisce
che la fenomica è possibile negli umani.
Molti fenotipi umani possono essere trovati nel database OMIM
(Online Mendelian Inheritance in Man), in cui sono espressi in linguaggio naturale, ma che non
contiene un sistema standardizzato per lo scoring dei fenotipi.
Un particolare problema con l'analisi dei fenotipi è la mancanza di un comune vocabolario
per descriverli, a causa di questa eterogeneità l'analisi dei fenotipi è scoraggiante ed ancora
un processo relativamente inesplorato. Un altro ostacolo deriva dalla scarsa disponibilità
di data set di phenotipi con i rispettivi geni.
Eshmun è un web tool che integra informazioni eterogenee il cui scopo è quello di annotare un set di geni tramite l'uso di network, basandosi sulle rappresentazioni dei fenotipi, microRNA, grug, proteine, effetti collraterali e fattori di trascrizione. Tale ricchezza di informazioni è una valida risorsa per i ricercatori biomedici.
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Martin, Janine Nicole. "Developing RNAi therapy For DYT1 dystonia." Diss., University of Iowa, 2011. https://ir.uiowa.edu/etd/1019.
Full textAbstract:
DYT1 dystonia is an early onset central nervous system-based movement disorder characterized by uncontrolled sustained muscle contractions that can lead to debilitating abnormal postures. Though a genetic mutation in the gene TOR1A is responsible for most DYT1 cases, the low penetrance of the disease implicates additional genetic and environmental modifiers. Current therapeutic options for DYT1 dystonia are limited to symptomatic treatments with variable effectiveness. Currently, the underlying pathogenesis of this disease and the role of torsinA (torA), the protein product of TOR1A, in the development of this disease have yet to be established.
In the first part of this thesis we aimed to further understand the effects of the TOR1A mutation at the molecular, cellular and organismal level in order to identify disease associated biomarkers that can be later used to measure the effectiveness of novel therapies. We found that expression of mutant torsinA (torA(ÄE)) in a cellular and an animal model of DYT1 had no significant effect on global transcription, despite its interaction with nuclear envelope proteins. Recent research has unearthed a role for microRNAs (miRNAs) in neuronal development and maturation. Consequently we explored whether torA(ÄE) expression in murine neural tissue was associated with changes in miRNA expression in young DYT1 knockin (KI) mice. Since the primary sight of dysfunction is still being debated, we profiled miRNA expression of the two strongest candidates, the striatum and cerebellum, both of which have well established roles in the control and coordination of muscle movements. We have identified several microRNAs that were uniquely altered in either the striatum or cerebellum and further research will be conducted to determine their usability as disease biomarkers. Finally, we were unable to identify motor phenotypes in either a DYT1 (KI) mice or a novel DYT1 transgenic model in open field, rotarod or staircase forepaw reaching tests.
In the second part of this thesis we aimed to develop and evaluate the safety and efficacy of viral therapeutic RNAi constructs for in DYT1 murine models. DYT1 is an ideal candidate for this form of therapy due to its dominant inheritance, common mutation and potentially reversible phenotype. Virally delivered short-hairpin RNAs (shRNA) designed to knockdown torA(ÄE) in either an allele-specific or nonallele-specific manner were injected into the striatum of DYT1 transgenic or KI mice respectively. Unexpectedly, we found widespread lethal toxicity and behavioral abnormalities in mice injected with either therapeutic or control shRNAs that weren't observed in mice injected with no shRNAs. Further studies found that regions where toxic shRNAs were expressed corresponded with neuronal loss and glial activation. Finally, we found evidence that the severity of toxicity was influenced in part by the genetic background of the mice.
In summary, the studies completed in this thesis contribute important information to the fields of dystonia pathogenesis and therapeutics, and more broadly pertain to the development of therapeutic gene silencing for neurological disease.