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Maniotis, Despina. "Investigation of hammerhead ribozyme function and potential in the central nervous system." Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365768.
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Baughan, Travis Lorson Christian. "Gene therapy in spinal muscular atrophy RNA-based strategies to modulate the pre-mRNA splicing of survival motor neuron /." Diss., Columbia, Mo. : University of Missouri-Columbia, 2008. http://hdl.handle.net/10355/6686.
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The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 10, 2010). Vita. Thesis advisor: Lorson, Christian L. "December 2008" Includes bibliographical references
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Ramachandran, Shyam. "Regulation Of gene expression in cystic fibrosis: implications for biology and therapeutics." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/2613.
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Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that when mutated causes the disease cystic fibrosis (CF). Many obstacles hinder the understanding of CF disease pathogenesis, impeding advancements in understanding how mutations cause disease, and slowing the progress towards new treatments. To this end, we have profiled the transcriptome (mRNA and microRNA) of human and newborn pig CF and non-CF airway epithelia. We show that the use of cross-species transcriptomics allows the identification of genes differentially expressed owing to the loss of CFTR, and not due to confounding environmental or secondary disease progression influences. The identification of reduced OAS1 expression in CF samples is a case in point. We also demonstrate the utility of transcriptome profiling and longitudinal studies in pigs, providing greater understanding of the molecular mechanisms underlying CF disease progression.
MicroRNAs (miRNAs) comprise a large family of ~21-nt long non-coding RNAs that function as key post-transcriptional regulators of gene expression. Very little is known of how CFTR is regulated in the cell, both transcriptionally and post-transcriptionally. We discovered three miRNAs: miR-509-3p, miR-494 and miR-138 with possible CFTR regulatory functions. miR-509-3p or -494 directly target the CFTR mRNA, and decrease CFTR levels when over expressed; while inhibiting them had the opposite effect. Upon stimulating human airway epithelial cells with TNFα or IL-1β, we observed an increase in expression of both miRNAs mediated in part by the NF-κB transcription factor complex, with a concurrent decrease in CFTR expression. Gene ontology classification of predicted targets of miR-509-3p and/or miR-494 expressed in the airway epithelium revealed enrichment for genes in ion transport pathways. To our knowledge, this is the first suggestion of a possible role for miRNAs regulating a broad range of important epithelial electrolyte and fluid transport proteins.
The study of miR-138 mediated regulation of CFTR expression has led to novel discoveries in the field of CFTR transcriptional control. We discovered SIN3A to be a novel transcriptional repressor of CFTR, interacting with CTCF on the CFTR promoter at the -20.9 kb DHS. By validating SIN3A as a conserved target of miR-138, we also discovered miR-138 to be a novel transcriptional regulator/activator of CFTR.
The most common CFTR mutation, ΔF508, causes protein misfolding, degradation, and CF. Manipulating the miR-138/SIN3A regulatory network improved the biosynthesis of CFTR-ΔF508, restoring Cl- transport to human CF airway epithelia. To our knowledge, this is the first example of an individual miRNA having such broad regulatory functions. This discovery also provided novel targets for restoring CFTR function in cells affected by the most common CF mutation. To this end, we are utilizing the molecular signatures of miR-138 over-expression and SIN3A knockdown to identify candidate genes for RNA interference screens, and to identify candidate small molecule drugs that might mimic the effects of these two interventions. The goal of this approach is to develop a new therapeutic agent that restores anion transport to airway epithelia and other cell types and tissues affected by CF.
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PEREIRA, LARISSA M. "Clonagem, expressao, purificacao e caracterizacao estrutural da proteina ribossomal L10 humana recombinante." reponame:Repositório Institucional do IPEN, 2009. http://repositorio.ipen.br:8080/xmlui/handle/123456789/9478.
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Dissertacao (Mestrado)
IPEN/D
Instituto de Pesquisas Energeticas e Nucleares - IPEN-CNEN/SP
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Issa, Mohamed Mahmoud. "Linear and Branched Chitosan Oligomers as Delivery Systems for pDNA and siRNA In Vitro and In Vivo." Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-7376.
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Hamilton, Andrew John. "Inhibiting gene expression with anti-sense RNA." Thesis, University of Nottingham, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.316938.
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Sesto, Nina. "RNA mediated gene expression regulation in Listeria." Paris 7, 2012. http://www.theses.fr/2012PA077233.
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Listeria monocytogenes est une bactérie pathogène responsable de la listériose, une maladie d'origine alimentaire. Alors que de nombreuses protéines de L. Monocytogenes ont été identifiées comme facteurs de virulence, le rôle des petits ARN régulateurs chez Listeria demeure méconnu. Mon projet de thèse avait pour objectif de découvrir de nouveaux concepts sur la régulation de l'expression des gènes impliquant des ARNs régulateurs jouant un rôle dans la virulence de Listeria. Mon travail a été basé sur deux approches distinctes. La première a concerné l'analyse comparative des transcriptomes de L. Monocytogenes et de L. Innocua, une espèce proche non-pathogène. La seconde a concerné la caractérisation de petits ARNs régulateurs et l'identification de leur rôle dans le processus infectieux. L'analyse comparative des deux transcriptomes a révélé une conservation de la majorité des transcrits, à l'exception d'une divergence significative entre les deux espèces pour un sous-ensemble d'ARN régulateurs non-codants. De manière surprenante, nous avons identifié une classe de longs transcrits antisens (lasARNs) qui chevauchent un gène tout en servant de S'UTR au gène adjacent divergent. La transcription du lasARN est à l'origine d'une régulation mutuellement exclusive de l'expression des gènes adjacents avec des fonctions opposées. Cette étude a été la première à comparer deux transcriptomes bactériens avec une résolution d'une seule base. Elle a conduit à la découverte chez L. Monocytogenes de 33 nouveaux petits ARNs et 53 asARNs et à l'identification d'une structure lasRNA /opéron que nous avons appelé « excludon» qui pourrait représenter une nouvelle forme de régulation chez les bactéries. J'ai également effectué une analyse sur plusieurs petits ARNs en combinant des approches de transcriptomes, des modèles d'infection chez la souris et en utilisant des algorithmes de prédiction de cibles des ARNs. J'ai ainsi identifié et caractérisé cinq petits ARNs qui sont importants pour la virulence de Listeria. Au cours de cette analyse, j'ai caractérisé en détail RliB, un ARN à double fonction pouvant agir comme un élément CRISPR et comme un ARN régulateur impliqué dans la régulation de l'homéostasie du fer chez Listeria. Nous avons ainsi réalisé la première étude détaillée d'un élément CRISPR impliqué dans la régulation d'un processus cellulaire fondamental autres que l'immunité contre les bactériophagesListeria monocytogenes is a bacterial pathogen responsible for listeriosis, a food-borne disease. In contrast to the significant advances in identifying proteins involved in virulence, relatively little is known about the role of sRNAs in L. Monocytogenes pathogenesis. The aim of my thesis project was to discover new concepts in RNA-mediated gene expression regulation with a role in Listeria virulence. My work was based on two distinct approaches. The first one involved global transcriptomic studies of L. Monocytogenes and its non-pathogenic relative, Listeria innocua while the second one concerned the characterization of individual sRNAs and the identification of their role in the infectious process. First, our comparative transcriptome analysis revealed conservation across most transcripts, but significant divergence between the species in a subset of non-coding sRNAs. This study was the first to compare two bacterial transcriptomes at a single-base resolution. Pt led to the discovery of 33 new sRNAs and 53 new asRNAs in L. Monocytogenes. Remarkably, we identified a class of long antisense transcripts (lasRNAs) that overlap one gene while also serving as the 5'UTR of the adjacent divergent gene. IasRNA transcription leads to the mutually exclusive regulation of the adjacent genes with opposite functions. This IasRNA/operon structure that we named "excludon" might represent a novel form of regulation in bacteria. Second, we conducted an analysis on several sRNAs by combining transcriptomic approaches, target prediction algorithm and mouse model of infection. I thus identified and characterized five sRNAs that are important for Listeria virulence. Furthermore, I focused on the detailed characterization of RIiB, a dual-function regulatory RNA that acts as a CRISPR element and a regulatory RNA involved in Listeria iron homeostasis regulation, providing the first detailed study of CRISPR element regulating fundamental cellular processes other than acting as a bacteriophage defense system
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Nwokeoha, Sandra. "Lithotripter shock wave induced RNA-based gene therapy." Thesis, University of Oxford, 2017. https://ora.ox.ac.uk/objects/uuid:89b67c7d-4b1c-4254-8e10-86a8e6c6c79d.
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Gene therapy is the process of introducing genes to augment or minimise the expression of absent or defective genes, respectively. Non-viral gene delivery systems for the treatment of genetic diseases, have evolved into highly appreciable nucleic acid-based therapies due to considerably less risk of host immunogenicity and induction of inflammatory responses. However, they are challenged by limited delivery. Thus, more efficient strategies are continually being sought. Lithotripter shock waves (LSW) are powerful acoustic waves that are an attractive choice of delivery system, as they offer a non-invasive, targeted and safe approach. Furthermore, the delivery of messenger ribonucleic acid (mRNA) possesses several advantages over commonly delivered plasmid deoxyribonucleic acids (pDNA), because it does not require opening of the nuclear envelope, thereby reducing the level of cell injury necessary for transfection. This work presents the first investigation on the efficacy of LSW mediated mRNA delivery, based on optimised SW parameters that balance the desired enhanced permeability of cell membranes against undesired cytotoxicity, and maintain the structural and biological stability of the RNA. A transfectability measure that defines the ability of SWs to permeabilise a cell whilst keeping it alive was established for dissimilar cell types, as a function of the acoustic pressure and number of SWs. Statistically significant RNA uptake was recorded in a tissue mimicking system, and using RNA analogues at various concentrations, the SW induced bio-distribution was characterised. In addition to LSW induced gene augmentation using mRNA, it was shown that LSWs could be used to effect gene inhibition through the delivery of siRNA. Kinetic experiments were carried out to measure mRNA uptake during shock wave exposure and indicated that rate of delivery was highest at the start of the SW dose and decreased during treatment. The results also suggested that the enhancement of cell permeability was significantly transient, and that mRNA was highly susceptible to degradation in its naked state. Furthermore, mRNA-based gene expression was shown to be predictive but quantal. The in vitro tissue model was improved from a gel-based system, to one that incorporated multi-cellular spheroids which capture aspects of 3-D tumours. Static overpressure was applied during SW exposure in order to suppress cavitation effects and isolate effects that could be attributed to shear due to cell-to-cell coupling. The results showed that mild overpressure improved RNA uptake the most, but that at higher overpressure, the level of increase in RNA uptake relative to controls, was dependent on the type of RNA nucleotide being delivered. This suggested that a complex interaction between LSW cavitation and direct stress dominates delivery. A final report was on the significant improvement of gene delivery when mRNA was encapsulated within a lipid nanoparticle vector, and SW exposure was assisted by cavitation agents. Also, by exposure to another acoustic stimulus - focused ultrasound (US), direct comparisons were made between SWs and US on the efficiency of delivery and tissue penetration. In conclusion, this thesis has shown that by choosing parameters appropriately, shock waves can be a promising strategy for the delivery of genes to cells.
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Rück, Andreas. "Myocardial gene therapy and gene expression in angina pectoris /." Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-648-4/.
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Preuten, Tobias. "Organellar gene expression." Doctoral thesis, Humboldt-Universität zu Berlin, Mathematisch-Naturwissenschaftliche Fakultät I, 2010. http://dx.doi.org/10.18452/16142.
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Zusätzlich zu der eubakteriellen RNA-Polymerase (RNAP) der Plastiden sind im Zellkern von Arabidopsis thaliana drei weitere, phagentypische RNAP kodiert, die jeweils aus nur einer Einheit aufgebaut sind. Die Enzyme RpoTp und RpoTm werden in die Plastiden, bzw. die Mitochondrien transportiert, während RpoTmp in beiden Organellen zu finden ist. Um die Lichtabhängigkeit der RpoT-Gene zu untersuchen, wurde die lichtinduzierte Akkumulation ihrer Transkripte in 7-Tage alten Keimlingen, sowie 3- bzw. 9-Wochen alten Rosettenblättern mittels quantitativer real-time PCR ermittelt. Die entwicklungsabhängige Regulation der RpoT-Transkript-Akkumulation wurde außerdem während der Blattentwicklung analysiert. Zusätzlich wurde der Einfluss des circadianen Rhythmus untersucht. Es stellte sich heraus, dass die Transkriptakkumulation aller drei RpoT-Gene stark lichtinduziert war und nur marginalen circadianen Schwankungen unterlag. In weiteren Versuchen mit verschiedenen Lichtrezeptor-Mutanten und unterschiedlichen Lichtqualitäten wurde der Einfluss multipler Rezeptoren auf den Prozess der Lichtinduktion gezeigt. In den Zellen höherer Pflanzen finden sich drei Genome. Die Biogenese von Chloroplasten und Mitochondrien, sowie lebenswichtige Prozesse, wie Atmung und Photosynthese setzen oftmals die Aktivität von Genen auf mindestens zwei dieser Genome voraus. Eine intrazelluläre Kommunikation zwischen den verschiedenen Genomen ist daher unumgänglich für einen funktionierenden Stoffwechsel der Pflanze. In dieser Arbeit wurde herausgestellt, dass die Zahl mitochondrialer Genkopien in photosynthetisch inaktiven Arabidopsis-Keimlingen drastisch erhöht ist. Bei der Untersuchung des DNA-Gehaltes in Proben, die Altersstufen von 2-Tage alten Keimblättern bis hin zu 37-Tage alten, seneszenten Rosettenblättern umfassten, fand sich ein deutlicher Anstieg der Kopienzahlen in älteren Rosettenblättern. Außerdem unterschieden sich die Kopienzahlen der untersuchten Gene zum Teil erheblich voneinander.In addition to eubacterial-like multi-subunit RNA polymerases (RNAP) localized in plastids and the nucleus, Arabidopsis thaliana contains three phage-like single-unit, nuclear-encoded, organellar RNAPs. The enzymes RpoTp and RpoTm are imported into plastids and mitochondria, respectively, whereas RpoTmp shows dual targeting properties into both organelles. To investigate if expression of the RpoT genes is light-dependent, light-induced transcript accumulation of RpoTm, RpoTp and RpoTmp was analyzed using quantitative real-time-PCR in 7-day-old seedlings as well as in 3- and 9-week-old rosette leaves. To address the question whether RpoT transcript accumulation is regulated differentially during plant development transcript abundance was measured during leaf development. Additionally, effects of the plants circadian rhythm on RpoT transcript accumulation were analyzed. Transcripts of all three RpoT genes were found to be strongly light-induced even in senescent leaves and only marginally influenced by the circadian clock. Further analyses employing different photoreceptor mutants and light qualities revealed the involvement of multiple receptors in the light-induction process. The biogenesis of mitochondria and chloroplasts as well as processes like respiration and photosynthesis require the activity of genes residing in at least two distinct genomes. There have to be ways of intracellular communication between different genomes to control gene activities in response to developmental and metabolic needs of the plant. In this study, it was shown that gene copy numbers drastically increased in photosynthetically inactive Arabidopsis seedlings. Mitochondrial DNA contents in cotyledons and leaves ranging in age from 2-day-old cotyledons to 37-day-old senescent rosette leaves were examined. A common increase in senescing rosette leaves and drastic differences between individual genes were found, revealing the importance of an integrative chondriome in higher plant cells.
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Busby, Michele Anne. "Measuring Gene Expression With Next Generation Sequencing Technology." Thesis, Boston College, 2012. http://hdl.handle.net/2345/3145.
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Thesis advisor: Gabor MarthWhile a PhD student in Dr. Gabor Marth's laboratory, I have had primary responsibility for two projects focused on using RNA-Seq to measure differential gene expression. In the first project we used RNA-Seq to identify differentially expressed genes in four yeast species and I analyzed the findings in terms of the evolution of gene expression. In this experiment, gene expression was measured using two biological replicates of each species of yeast. While we had several interesting biological findings, during the analysis we dealt with several statistical issues that were caused by the experiment's low number of replicates. The cost of sequencing has decreased rapidly since this experiment's design and many of these statistical issues can now practically be avoided by sequencing a greater number of samples. However, there is little guidance in the literature as to how to intelligently design an RNA-Seq experiment in terms of the number of replicates that are required and how deeply each replicate must be sequenced. My second project, therefore, was to develop Scotty, a web-based program that allows users to perform power analysis for RNA-Seq experiments. The yeast project resulted in a highly accessed first author publication in BMC Genomics in 2011. I have structured my dissertation as follows: The first chapter, entitled General Issues in RNA-Seq, is intended to synthesize the themes and issues of RNA-Seq statistical analysis that were common to both papers. In this section, I have discussed the main findings from the two papers as they relate to analyzing RNA-Seq data. Like the Scotty application, this section is designed to be "used" by wet-lab biologists who have a limited background in statistics. While some background in statistics would be required to fully understand the following chapters, the essence of this background can be gained by reading this first chapter. The second and third chapters contain the two papers that resulted from the two RNA-Seq projects. Each chapter contains both the original manuscript and original supplementary methods and data section. Finally, I include brief summaries of my contributions to the two papers on which I was a middle author. The first was a functional analysis of the genomic regions affected by mobile element insertions as a part of Chip Stewart's paper with the 1000 Genome Consortium. This paper was published in Plos Genetics. The second was a cluster analysis of microarray gene expression in Toxoplasma gondii, which was included as part of Alexander Lorestani et al.'s paper, Targeted proteomic dissection of Toxoplasma cytoskeleton sub-compartments using MORN1. This paper is currently under review. The yeast project was a collaborative effort between Jesse Gray, Michael Springer, and Allen Costa at Harvard Medical School, Jeffery Chuang here at Boston College, and members of the Marth lab. Jesse Gray conceived of the project. While I wrote the draft for the manuscript, many people, particularly Gabor Marth, provided substantial guidance on the actual text. I conceived of and implemented Scotty and wrote its manuscript with only editorial assistance from my co-authors. I produced all figures for the two manuscripts. Chip Stewart provided extensive guidance and mentorship to me on all aspects of my statistical analyses for both projects
Thesis (PhD) — Boston College, 2012
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Biology
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Tigue, P. J. "Hormonal regulation of mammary gene expression." Thesis, University of Nottingham, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.381461.
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Gonçalves, Ângela. "RNA sequencing for the study of gene expression regulation." Thesis, University of Cambridge, 2012. https://www.repository.cam.ac.uk/handle/1810/265548.
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The process by which information encoded m an organism's DNA is used in the synthesis of functional cell products is known as gene expression. In recent years, sequencing of RNA (RNA-seq) has emerged as the preferred technology for the simultaneous measurement of transcript sequences and their abundance. The analysis of RNA-seq data presents novel challenges and many methods have been developed for the purpose of mapping reads to genomic features and expression quantification. In the first part of my thesis I developed an R based pipeline for pre-processing, expression estimation and data quality assessment of RNA-seq datasets, which formed the basis for my subsequent work on the evolution of gene expression regulation in mammals. Since changes in gene expression levels are thought to underlie many of the phenotypic differences between species, identifying and characterising the regulatory mechanisms responsible for these changes is an important goal of molecular biology. For this, I studied the regulatory divergence of liver gene expression and of isoform usage between mouse strains. I demonstrate that gene expression diverges extensively between the strains and propose that the regulatory mechanism underlying divergent expression between two closely related mammalian species is a combination of variants that arise in cis and in trans. Isoform usage diverges to a lesser extent and appears to display a larger contribution of trans acting regulatory elements to its regulation, suggesting that isoform usage may be under different evolutionary constraints. These observations have important implications for understanding mammalian gene expression divergence and for understanding how speciation occurs.
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Brown, Justin Travis. "MRNA degradation in the control of gene expression in yeast." Access restricted to users with UT Austin EID Full text (PDF) from UMI/Dissertation Abstracts International, 2001. http://wwwlib.umi.com/cr/utexas/fullcit?p3024999.
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Taylor, David C. "SELEX targeting mRNAs : the hunt for novel riboregulators /." free to MU campus, to others for purchase, 2001. http://wwwlib.umi.com/cr/mo/fullcit?p3013032.
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Wang, Wen-Hua 1965. "Cytokine gene expression and gene therapy in experimental corneal graft rejection." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=38529.
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It has been proposed that CD4+ T cells and cell-mediated immunity play a central role in corneal allograft rejection. Two subsets of CD4+ T cells, Th1 and Th2 cells, are known to cross-regulate each other through their cytokine pattern and the immune response might be directed predominantly in one or the other direction. As such, it has been hypothesized here that predominant Th1 type immune response could lead to corneal allograft rejection, and that previous inflamed corneal beds (high-risk eyes) might augment the Th1 response and thus accelerate the graft rejection.Reverse transcription of mRNA followed by polymerase chain reaction amplification was used to determine the relative gene expression in ocular tissues (cornea and iris/ciliary body) obtained from syngeneic grafts, low- and high-risk allografts. Compared with the syngeneic grafts, mRNA analysis of the low- and high-risk allografts showed a significantly decreasing expression pattern for the Th3 cytokine TGF-beta2, an early peak followed by a decline in the Th2 cytokines IL-4 and IL-10 expression, and a progressively increasing expression of the Th1 cytokines IL-2 and IFN-gamma and the proinflammatory cytokines IL-1beta and TNF-alpha, which paralleled the course of graft rejection. Prevascularization of the recipient eye (high-risk) significantly accelerated the rejection of corneal allografts and the mRNA levels of the Th1 cytokines IL-2 and IFN-gamma and proinflammatory cytokines IL-1beta and TNF-alpha in high-risk allografts were significantly higher and peaked faster than that in low-risk allografts.
In vivo gene transfer using plasmid DNA encoding cytokines is an attractive alternative to modulate the Th1 inflammatory reaction and immune response. This has led to the hypothesis that transferring the gene encoding Th2 cytokine IL-10 into the recipient could prevent or reduce the subsequent corneal allograft rejection through the suppression of Th1-mediated alloimmune response.
Intramuscular injection with in vivo electroporation of IL-10 plasmid DNA was administered at one week before and at one week after corneal transplantation. Corneal allograft survival was significantly prolonged and the rejection rate was significantly reduced after gene therapy with IL-10 plasmid DNA, compared with that in control groups treated with the empty plasmid vector. In IL-10 treated rats, the mRNA expression for the Th1 cytokines IL-2 and IFN-gamma was depressed, and the IL-10 mRNA expression was significantly increased. However, graft survival was not permanent. (Abstract shortened by UMI.)
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Seow, Yiqi. "Novel Gene Delivery And Expression Protocols For Long Term Gene Therapy." Thesis, University of Oxford, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.526116.
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Chemutai, Patricia. "Gene Expression in Long Term Myoblast /Myocete Cultures: m RNA expression (Acetylcholine Receptor and Galectin-3 gene)." Youngstown State University / OhioLINK, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=ysu1620382476783648.
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Zaghlool, Ammar. "Genome-wide Characterization of RNA Expression and Processing." Doctoral thesis, Uppsala universitet, Institutionen för immunologi, genetik och patologi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-209390.
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The production of fully mature protein-coding transcripts is an intricate process that involves numerous regulation steps. The complexity of these steps provides the means for multilayered control of gene expression. Comprehensive understanding of gene expression regulation is essential for interpreting the role of gene expression programs in tissue specificity, development and disease. In this thesis, we aim to provide a better global view of the human transcriptome, focusing on its content, synthesis, processing and regulation using next-generation sequencing as a read-out. In Paper I, we show that sequencing of total RNA provides unique insights into RNA processing. Our results revealed that co-transcriptional splicing is a widespread mechanism in human and chimpanzee brain tissues. We also found a correlation between slowly removed introns and alternative splicing. In Paper II, we explore the benefits of exome capture approaches in combination with RNA-sequencing to detect transcripts expressed at low-levels. Based on our results, we demonstrate that this approach increases the sensitivity for detecting low level transcripts and leads to the identification of novel exons and splice isoforms. In Paper III, we highlight the advantages of performing RNA-sequencing on separate cytoplasmic and nuclear RNA fractions. In comparison with conventional poly(A) RNA, cytoplasmic RNA contained a significantly higher fraction of exonic sequence, providing increased sensitivity for splice junction detection and for improved de novo assembly. Conversely, the nuclear fraction showed an enrichment of unprocessed RNA compared to when sequencing total RNA, making it suitable for analysis of RNA processing dynamics. In Paper IV, we used exome sequencing to sequence the DNA of a patient with unexplained intellectual disability and identified a de novo mutation in BAZ1A, which encodes the chromatin-remodeling factor ACF1. Functional studies indicated that the mutation influences the expression of genes involved in extracellular matrix organization, synaptic function and vitamin D3 metabolism. The differential expression of CYP24A, SYNGAP1 and COL1A2 correlated with the patient’s clinical diagnosis. The findings presented in this thesis contribute towards an improved understanding of the human transcriptome in health and disease, and highlight the advantages of developing novel methods to obtain global and comprehensive views of the transcriptome.
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Varani, Luca. "NMR studies of RNA and RNA-protein complexes involved in regulation of gene expression." Thesis, University of Cambridge, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.624587.
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Childs, Liam. "Bioinformatics approaches to analysing RNA mediated regulation of gene expression." Phd thesis, Universität Potsdam, 2010. http://opus.kobv.de/ubp/volltexte/2010/4128/.
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The genome can be considered the blueprint for an organism. Composed of DNA, it harbours all organism-specific instructions for the synthesis of all structural components and their associated functions. The role of carriers of actual molecular structure and functions was believed to be exclusively assumed by proteins encoded in particular segments of the genome, the genes. In the process of converting the information stored genes into functional proteins, RNA – a third major molecule class – was discovered early on to act a messenger by copying the genomic information and relaying it to the protein-synthesizing machinery. Furthermore, RNA molecules were identified to assist in the assembly of amino acids into native proteins. For a long time, these - rather passive - roles were thought to be the sole purpose of RNA. However, in recent years, new discoveries have led to a radical revision of this view. First, RNA molecules with catalytic functions - thought to be the exclusive domain of proteins - were discovered. Then, scientists realized that much more of the genomic sequence is transcribed into RNA molecules than there are proteins in cells begging the question what the function of all these molecules are. Furthermore, very short and altogether new types of RNA molecules seemingly playing a critical role in orchestrating cellular processes were discovered. Thus, RNA has become a central research topic in molecular biology, even to the extent that some researcher dub cells as “RNA machines”.
This thesis aims to contribute towards our understanding of RNA-related phenomena by applying Bioinformatics means. First, we performed a genome-wide screen to identify sites at which the chemical composition of DNA (the genotype) critically influences phenotypic traits (the phenotype) of the model plant Arabidopsis thaliana. Whole genome hybridisation arrays were used and an informatics strategy developed, to identify polymorphic sites from hybridisation to genomic DNA. Following this approach, not only were genotype-phenotype associations discovered across the entire Arabidopsis genome, but also regions not currently known to encode proteins, thus representing candidate sites for novel RNA functional molecules. By statistically associating them with phenotypic traits, clues as to their particular functions were obtained. Furthermore, these candidate regions were subjected to a novel RNA-function classification prediction method developed as part of this thesis.
While determining the chemical structure (the sequence) of candidate RNA molecules is relatively straightforward, the elucidation of its structure-function relationship is much more challenging. Towards this end, we devised and implemented a novel algorithmic approach to predict the structural and, thereby, functional class of RNA molecules. In this algorithm, the concept of treating RNA molecule structures as graphs was introduced. We demonstrate that this abstraction of the actual structure leads to meaningful results that may greatly assist in the characterization of novel RNA molecules. Furthermore, by using graph-theoretic properties as descriptors of structure, we indentified particular structural features of RNA molecules that may determine their function, thus providing new insights into the structure-function relationships of RNA. The method (termed Grapple) has been made available to the scientific community as a web-based service.
RNA has taken centre stage in molecular biology research and novel discoveries can be expected to further solidify the central role of RNA in the origin and support of life on earth. As illustrated by this thesis, Bioinformatics methods will continue to play an essential role in these discoveries.Das Genom eines Organismus enthält alle Informationen für die Synthese aller strukturellen Komponenten und deren jeweiligen Funktionen. Lange Zeit wurde angenommen, dass Proteine, die auf definierten Abschnitten auf dem Genom – den Genen – kodiert werden, die alleinigen Träger der molekularen - und vor allem katalytischen - Funktionen sind. Im Prozess der Umsetzung der genetischen Information von Genen in die Funktion von Proteinen wurden RNA Moleküle als weitere zentrale Molekülklasse identifiziert. Sie fungieren dabei als Botenmoleküle (mRNA) und unterstützen als Trägermoleküle (in Form von tRNA) die Zusammenfügung der einzelnen Aminosäurebausteine zu nativen Proteine. Diese eher passiven Funktionen wurden lange als die einzigen Funktionen von RNA Molekülen angenommen. Jedoch führten neue Entdeckungen zu einer radikalen Neubewertung der Rolle von RNA. So wurden RNA-Moleküle mit katalytischen Eigenschaften entdeckt, sogenannte Ribozyme. Weiterhin wurde festgestellt, dass über proteinkodierende Abschnitte hinaus, weit mehr genomische Sequenzbereiche abgelesen und in RNA Moleküle transkribiert werden als angenommen. Darüber hinaus wurden sehr kleine und neuartige RNA Moleküle identifiziert, die entscheidend bei der Koordinierung der Genexpression beteiligt sind. Diese Entdeckungen rückten RNA als Molekülklasse in den Mittelpunkt moderner molekularbiologischen Forschung und führten zu einer Neubewertung ihrer funktionellen Rolle. Die vorliegende Promotionsarbeit versucht mit Hilfe bioinformatorischer Methoden einen Beitrag zum Verständnis RNA-bezogener Phänomene zu leisten. Zunächst wurde eine genomweite Suche nach Abschnitten im Genom der Modellpflanze Arabidopsis thaliana vorgenommen, deren veränderte chemische Struktur (dem Genotyp) die Ausprägung ausgewählter Merkmale (dem Phänotyp) entscheidend beeinflusst. Dabei wurden sogenannte Ganz-Genom Hybridisierungschips eingesetzt und eine bioinformatische Strategie entwickelt, Veränderungen der chemischen Struktur (Polymorphismen) anhand der veränderten Bindung von genomischer DNA aus verschiedenen Arabidopsis Kultivaren an definierte Proben auf dem Chip zu detektieren. In dieser Suche wurden nicht nur systematisch Genotyp-Phänotyp Assoziationen entdeckt, sondern dabei auch Bereiche identifiziert, die bisher nicht als proteinkodierende Abschnitte annotiert sind, aber dennoch die Ausprägung eines konkreten Merkmals zu bestimmen scheinen. Diese Bereiche wurden desweiteren auf mögliche neue RNA Moleküle untersucht, die in diesen Abschnitten kodiert sein könnten. Hierbei wurde ein neuer Algorithmus eingesetzt, der ebenfalls als Teil der vorliegenden Arbeit entwickelt wurde. Während es zum Standardrepertoire der Molekularbiologen gehört, die chemische Struktur (die Sequenz) eines RNA Moleküls zu bestimmen, ist die Aufklärung sowohl der Struktur als auch der konkreten Funktion des Moleküls weitaus schwieriger. Zu diesem Zweck wurde in dieser Arbeit ein neuer algorithmischer Ansatz entwickelt, der mittels Computermethoden eine Zuordnung von RNA Molekülen zu bestimmten Funktionsklassen gestattet. Hierbei wurde das Konzept der Beschreibung von RNA-Sekundärstrukturen als Graphen genutzt. Es konnte gezeigt werden, dass diese Abstraktion von der konkreten Struktur zu nützlichen Aussagen zur Funktion führt. Des weiteren konnte demonstriert werden, dass graphen-theoretisch abgeleitete Merkmale von RNA-Molekülen einen neuen Zugang zum Verständnis der Struktur-Funktionsbeziehungen ermöglichen. Die entwickelte Methode (Grapple) wurde als web-basierte Anwendung der wissenschaftlichen Welt zur Verfügung gestellt. RNA hat sich als ein zentraler Forschungsgegenstand der Molekularbiologie etabliert und neue Entdeckungen können erwartet werden, die die zentrale Rolle von RNA bei der Entstehung und Aufrechterhaltung des Lebens auf der Erde weiter untermauern. Bioinformatische Methoden werden dabei weiterhin eine essentielle Rolle spielen.
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Unoson, Cecilia. "Small RNA-mediated Regulation of Gene Expression in Escherichia coli." Doctoral thesis, Uppsala universitet, Institutionen för cell- och molekylärbiologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-130885.
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Non-coding RNAs are highly abundant regulators of gene expression in all kingdoms of life that often play important roles in vital cellular functions. In bacteria, small regulatory RNAs (sRNAs) usually act post-transcriptionally by regulating mRNAs through base pairing within ribosome binding sites (RBS), thereby inhibiting translation initiation. tisB encodes a toxin, TisB, whose synthesis is controlled by the sRNA IstR-1. Intriguingly, IstR-1 base pairs far upstream of the RBS but nevertheless inhibits translation initiation. The tisB mRNA is unusual in that ribosomes cannot access the RBS directly, but instead need an unstructured upstream region. This is precisely where IstR-1 exerts its inhibitory effect. We propose this region to serve as a ribosome loading site (standby site) which permits ribosomes to overcome the obstacle of inhibitory RBS-containing structures. Sequence-independent ribosome binding to the standby site allows for efficient relocation to the RBS structure when it is transiently open. Thus, standby sites are translation enhancer elements. I also characterized TisB-mediated toxicity. The hydrophobic protein TisB is targeted to the inner membrane and causes damage. This decreases the intracellular ATP concentration and entails decreased replication, transcription and translation rates. It is likely that this toxin is involved in multidrug tolerance under certain conditions. We identified the sRNA MicF as a negative regulator of lrp expression. Lrp is a global transcription factor that controls genes involved in amino acid metabolism and transport of small molecules. Interestingly, Lrp also downregulates MicF. Thus, this study established that the mutual downregulation of MicF/Lrp creates a positive feedback loop which gives a switch-like behavior important for fast adaptations.
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Khan, Dilshad Hussain. "Role of histone deacetylases in gene expression and RNA splicing." Informa UK Limited, 2012. http://hdl.handle.net/1993/22163.
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Histone deacetylases (HDAC) 1 and 2 play crucial role in chromatin remodeling and gene expression regimes, as part of multiprotein corepressor complexes. Protein kinase CK2-driven phosphorylation of HDAC1 and 2 regulates their catalytic activities and is required to form the corepressor complexes. Phosphorylation-mediated differential distributions of HDAC1 and 2 complexes in regulatory and coding regions of transcribed genes catalyze the dynamic protein acetylation of histones and other proteins, thereby influence gene expression.
During mitosis, highly phosphorylated HDAC1 and 2 heterodimers dissociate and displace from mitotic chromosomes. Our goal was to identify the kinase involved in mitotic phosphorylation of HDAC1 and 2. We postulated that CK2-mediated increased phosphorylation of HDAC1 and 2 leads to dissociation of the heterodimers, and, the mitotic chromosomal exclusions of HDAC1 and 2 are largely due to the displacement of HDAC-associated proteins and transcription factors, which recruit HDACs, from chromosomes during mitosis. We further explored the role of un- or monomodified HDAC1 and 2 complexes in immediate-early genes (IEGs), FOSL1 (FOS-like antigen-1) and MCL1 (Myeloid cell leukemia-1), regulation. Dynamic histone acetylation is an important regulator of these genes that are overexpressed in a number of diseases and cancers. We hypothesized that transcription dependent recruitment of HDAC1 and 2 complexes over the gene body regions plays a regulatory role in transcription and splicing regulation of these genes.
We present evidence that CK2-catalyzed increased phosphorylation of HDAC1 and 2 regulates the formation of distinct corepressor complexes containing either HDAC1 or HDAC2 homodimers during mitosis, which might target cellular factors. Furthermore, the exclusion of HDAC-recruiting proteins is the major factor for their displacement from mitotic chromosomes. We further demonstrated that un- or monophosphorylated HDAC1 and 2 are associated with gene body of FOSL1 in a transcription dependent manner. However, HDAC inhibitors prevented FOSL1 activation independently of the nucleosome response pathway, which is required for IEG induction. Interestingly, our mass spectrometry results revealed that HDAC1 and 2 interact with a number of splicing proteins, in particular, with serine/arginine-rich splicing factor 1 (SRSF1). HDAC1 and 2 are co-occupied with SRSF1 over gene body regions of FOSL1 and MCL1, regardless of underlying splicing mechanisms. Using siRNA-mediated knockdown approaches and HDAC inhibitors, we demonstrated that alternative splicing of MCL1 is regulated by RNA-directed localized changes in the histone acetylation levels at the alternative exon. The change in histone acetylation levels correlates with the increased transcription elongation and results in change in MCL1 splicing by exon skipping mechanism.
Taken together, our results contribute to further understanding of how the multi-faceted HDAC1 and 2 complexes can be regulated and function in various processes, including, but not limited to, transcription regulation and alternative splicing. This can be an exciting area of future research for therapeutic interventions.
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Jackson, Laura. "Oxygen-regulated gene expression in Escherichia coli and hypoxia-targeted gene therapy." Thesis, University of Sheffield, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414676.
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Mordstein, Christine. "Coding-sequence determinants of gene expression in human cells." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/28766.
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The human genome is highly heterogeneous in its GC composition. How codon usage affects translation rates has been extensively studied and exploited to increase protein expression. Although effects on virtually all other steps in gene expression have been reported as well, so far no systematic approach has been taken to quantitatively measure the contribution of each to overall protein levels in human cells. Here, I utilise a library of several hundred synonymous variants of the Green fluorescent protein (GFP) to characterise the influence of codon usage on gene expression in human cells. In an initial small-scale screen, I show that protein levels are largely correlated with codon-usage and particularly GC-content. Additionally, I demonstrate that these changes can already be seen on the RNA level, confirming more broadly previously published data from our lab (Kudla et al., 2006). In order to assess the consequences of randomised codon usage on a larger scale, I established and validated a high-throughput approach for the phenotypic profiling of reporter genes. Using a pool of cells stably expressing >200 GFP variants, I measured multiple parameters simultaneously, such as protein levels, translational state, RNA levels, stability and export. Data from these experiments confirm a strong relationship between GC-content, protein levels, as well as RNA export, reproducibly in two cell lines. Low expression of especially GC-poor variants could not be rescued by splicing, but increased nuclear-to-cytoplasmic RNA ratio, suggesting further mechanisms important for efficient gene expression. These effects are even more pronounced when the distribution of GC is spread evenly along the coding sequence. Interestingly, our data also suggests that high GC within the first 200nt is more predictive of efficient gene expression, contrasting studies performed on bacteria, in which strong secondary folding near the ribosomal binding site was shown to be non-permissive for translation (Kudla et al., 2009). By relating experimentally derived parameters to sequence features known to inhibit expression, I demonstrate that cryptic splicing is a major factor leading to decreased levels of particularly GC-poor GFP variants. An attempt to quantitatively assess the relative contribution of several sequence features (e.g. tAI, GC3, CpG) using multiple regression analysis lead to inconclusive results, leaving the requirement for the exploration of alternative approaches in order to dissect the role of individual parameters, as well as to identify novel determinants of gene expression.
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Yankulov, Krassimir Yankov. "Regulation of transcriptional elongation by RNA polymerase II." Thesis, Open University, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.387189.
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Lin, Chunru. "Functional Role of Dead-Box P68 RNA Helicase in Gene Expression." Digital Archive @ GSU, 2006. http://digitalarchive.gsu.edu/biology_diss/10.
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How tumor cells migrate and metastasize from primary sites requires four major steps: invasion, intravasation, extravasation and proliferation from micrometastases to malignant tumor. The initiation of tumor cell invasion requires Epithelial-Mesenchymal Transition (EMT), by which tumor cells lose cell-cell interactions and gain the ability of migration. The gene expression profile during the EMT process has been extensively investigated to study the initiation of EMT. In our studies, we indicated that tyrosine phosphorylation of human p68 RNA helicase positively associated with the malignant status of tumor tissue or cells. Studying of this relationship revealed that p68 RNA helicase played a critical role in EMT progression by repression of E-cadherin as an epithelial marker and upregulation of Vimentin as a mesenchymal marker. Insight into the mechanism of how p68 RNA helicase represses E-cadherin expression indicated that p68 RNA helicase initiated EMT by transcriptional upregulation of Snail. Human p68 RNA helicase has been documented as an RNA-dependent ATPase. The protein is an essential factor in the pre-mRNA splicing procedure. Some examples show that p68 RNA helicase functions as a transcriptional coactivator in ATPase dependent or independent manner. Here we indicated that p68 RNA helicase unwound protein complexes to modulate protein-protein interactions by using protein-dependent ATPase activity. The phosphorylated p68 RNA helicase displaced HDAC1 from the chromatin remodeling MBD3:Mi2/NuRD complex at the Snail promoter. Thus, our data demonstrated an example of protein-dependent ATPase which modulates protein-protein interactions within the chromatin remodeling machine.
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NandyMazumdar, Monali. "RfaH CONTACTS TO DNA, RNA POLYMERASE AND RIBOSOME ACTIVATE GENE EXPRESSION." The Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1482155208767278.
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Bonella, Henry C. "Growth regulation and gene expression in marine Synechococcus spp." Thesis, University of Stirling, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.484210.
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Firek, Simon. "The promotion of ribosomal RNA transcription in Xenopus laevis." Thesis, University of Portsmouth, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.236392.
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Schultes, Stephan Johannes Baptist. "Nanoparticles for RNA interference : novel preclinical formulations for siRNA mediated gene therapy." München Verl. Dr. Hut, 2010. http://edoc.ub.uni-muenchen.de/11329/.
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Bednarska, Aleksandra. "Artificial systems for in vitro gene expression." Thesis, Université Paris-Saclay (ComUE), 2015. http://www.theses.fr/2015SACLN016/document.
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L’ARN polymérase dépendante d’ADN (RNAP) est une enzyme responsable de la polymérisation de ribonucleotides dans une séquence d'ARN complémentaire de l'ADN de matrice. La famille de RNAP a plusieurs membres, comme de protéines sous-unité unique (par exemple du bactériophage T7) ou multiple sous-unité (bactériennes et eucaryotes). Transcription de l'ARN - un événement crucial dans l'expression des gènes - varie en fonction de l'origine de RNAP. Bien que le processus de transcription est relativement bien caractérisée, de nombreux éléments restent mal compris, surtout par rapport à la dynamique de la reconnaissance de promoteur, d'évasion et de l'allongement dans une contexte de cellule où la densité moléculaire, les concentrations et les effets plus proches environs sont importants. L'objectif de cette thèse était la développement d’une méthode qui permettrait suivre la réaction RNAP in vitro en temps réel dans des conditions très contrôlées. Un axe majeur a été mis pour développer un biocapteur basé surface qui permettrait à la caractérisation des principales étapes de la réaction de transcription. Par conséquent, les interactions entre des molécules d'ADN immobilisés sur une surface du capteur et RNAP libre délivré par un système microfluidique de la surface ont été examinées. Changements de l'indice de réfraction, corrélés avec les changements de masse sur la surface ont été suivis en utilisant l'imagerie par résonance de plasmon de surface (SPRi). SPRi est une technique sensible dédiée à l'analyse des interactions entre deux ligands en temps réel. Les bases du mécanisme sont la détection de légères différences dans la réflexion de la lumière polarisée à un angle fixe qui est associé avec une variation de masse à l'interface. Les données obtenues à partir SPRi sont utilisées pour déterminer la cinétique des interactions. Géométrie d’ADN puces permet de suivre plusieurs échantillons simultanément, qui raccourcit considérablement le temps que de manipulation et améliore la qualité et la reproductibilité des résultats obtenus. Autres biocapteurs optofluidique: résonateur de microring et microscopie de fluorescence par réflexion totale interne (TIRF) ont été développés en parallèle. Nous avons biofunctionalisé et caractérisé des surfaces de capteur (de verre couvert de polymère pour un résonateur de microring et la microscopie TIRF et 50 nm couche mince d’or sur des prismes de SPRi) afin d'immobiliser ADN d'une manière contrôlée, par création d’une monocouche auto-assemblée (SAM). Fonctionnalisation de polymères SU-8 concernées deux méthodes: covalent immobilisation de (bio) molécules et la conjugaison non covalente sur la base de couplage hydrophobe. Pour la fonctionnalisation de surface d’or, quatre stratégies différentes d'immobilisation des molécules ont été comparés: formation de la liaison de thiol - or, la formation des liaisons amide, interactions extrAvidin - biotine et le couplage hydrophobe. Les études de la conjugaison de l'ADN à la surface d'or fonctionnalisé ont été effectuées en ce qui concerne la spécificité et la densité d'ADN immobilisées de longueurs différentes: 50, 500 et 1000 pb. Enfin, les surfaces biofunctionalized ont été utilisées pour suivre en temps réel des réactions de transcription de deux RNAP: bactériophage T7 RNAP monomère et l'holoenzyme d'Escherichia coli RNAP. Les analyses cinétiques de la formation d’un complexe nucléoprotéine et la transcription d'ARN ont été fait par report de la densité et la longueur de l'ADN immobilisé, la position de la séquence du promoteur spécifique. Transcription de l'ARN dans l'appareil SPRi a été confirmée par la collection, la détection et l'analyse des produits ARN.L'objectif final comprends une synthèse de l'ARN contrôlée qui serait une étape intermédiaire d'enquêter en temps réel la production de protéines in vitroDNA-dependent RNA polymerase (RNAP) is an enzyme responsible for the polymerization of ribonucleotides into an RNA sequence complementary to the template DNA. RNAP family has several members being single subunit (e.g. T7 bacteriophage) or multi subunit (bacterial and eukaryote) proteins. RNA transcription – a crucial event in gene expression – differs depending on the RNAP origin. Although the transcription process is relatively well characterized, many elements remain poorly understood, especially with respect to the dynamics of promoter recognition, escape and elongation in a cell like context where molecular density, concentrations and nearest neighbour effects are prevalent.The goal of this thesis was to develop a robust method that would allow real time monitoring of RNAP reaction in vitro in thoroughly controlled conditions. A major axis was to develop a surface-based biosensor that would allow the characterization of the main steps of the transcription reaction. Consequently, interactions between DNA molecules immobilized on a sensor surface and free RNAP delivered through a microfluidic flow system to the surface were examined. Changes in refractive index, correlated with changes in mass at a surface were followed using surface plasmon resonance imaging (SPRi). SPRi is a sensitive technique dedicated to analysis of interactions between two ligands in real time. The mechanism bases on the detection of slight differences in the reflectivity of polarized light at a fixed angle that are associated with a mass variation at the interface. Data obtained from SPRi are used to determine the kinetics of the interactions. Microarray geometry of SPRi allows monitoring several samples simultaneously that significantly shortens manipulation time and improves a quality and reproducibility of obtained results. Other label-free optofluidic biosensors: microring resonator and total internal reflection fluorescence (TIRF) microscopy were developed in parallel.We firstly biofunctionalized and characterized sensor surfaces (polymer coated glass for microring resonator and TIRF microscopy and 50-nm thin layer gold coatings on glass prisms for SPRi) in order to immobilize DNA strands in a controlled manner, using a self-assembled monolayer (SAM). Functionalization of photoresist polymer SU-8 concerned two methods: covalent (bio)molecule grafting and non-covalent conjugation based on hydrophobic coupling. Regarding gold surface functionalization, four different strategies of antifouling (bio)molecule immobilization were compared: thiol – gold bond formation, amide bond formation, extrAvidin – biotin interactions and hydrophobic coupling. Studies of DNA conjugation to the functionalized gold surface were performed with respect to specificity and density of immobilized DNA molecules of different lengths: 50, 500 and 1000 bp.Finally, biofunctionalized surfaces were used for real time monitoring of transcription reactions using two RNAPs: monomeric bacteriophage T7 RNAP and the holoenzyme of Escherichia coli RNAP. Kinetic analyses of nucleoprotein complex formation and RNA transcription were performed as a function of immobilized DNA density, the length of the immobilized DNA, the position of the specific promoter sequence with respect to the point of immobilization and the direction of subsequent transcription. RNA transcription in the SPRi apparatus was confirmed by collection, detection and analysis of relevant products.The future development of biosensors dedicated to in vitro gene expression will include the adaptation of the methods presented above to other optofluidic systems and further development of the technique. The final goal comprises a controlled RNA synthesis that would be an intermediate step to investigate real time in vitro protein production
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Rose, Andrew C. "Studies on the expression of the murine CFTR gene : implications for gene therapy." Thesis, University of Oxford, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365354.
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Rathjen, Joy. "Expression signals for the phosphoglycerate kinase gene of saccharomyces cerevisiae." Thesis, University of Oxford, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.670320.
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Engdahl, Hilde Merete. "Natural and artificial antisense RNA : a study of inhibition of gene expression /." Uppsala : Swedish Univ. of Agricultural Sciences (Sveriges lantbruksuniv.), 2000. http://epsilon.slu.se/avh/2000/91-576-5784-X.pdf.
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Loushin, Newman Carrie Lee. "Characterization of QKI RNA binding function /." Full text (PDF) from UMI/Dissertation Abstracts International, 2000. http://wwwlib.umi.com/cr/utexas/fullcit?p3004323.
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Tang, Terry, and University of Lethbridge Faculty of Arts and Science. "Mathematical modeling of eukaryotic gene expression." Thesis, Lethbridge, Alta. : University of Lethbridge, Dept. of Chemistry and Biochemistry, 2010, 2010. http://hdl.handle.net/10133/2567.
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Using the Gillespie algorithm, the export of the mRNA molecules from their transcription
site to the nuclear pore complex is simulated. The effect of various structures in the nu-
cleus on the efficiency of export is discussed. The results show that having some of the
space filled by chromatin near the mRNA synthesis site shortens the transport time. Next, the complete eukaryotic gene expression including transcription, splicing, mRNA export, translation, and mRNA degradation is modeled using delay stochastic simulation. This allows for the study of stochastic effects during the process and on the protein production rate patterns. Various protein production patterns can be produced by adjusting the poly-A tail length of the mRNA and the promoter efficiency of the gene. After that, the opposing effects of the chromatin density on the seeking time of the transcription factors for the promoter and the exit time of the mRNA product are discussed.xi, 102 leaves ; 28 cm
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Arevalo, Iracema. "Cutaneous leishmaniasis : iNOS gene expression and a novel immunomodulatory therapy." Thesis, McGill University, 2001. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=31183.
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Nitric oxide (NO) has been shown to be lethal for a variety of intracellular pathogens including Leishmania. In murine models, the inducible nitric oxide synthase gene (iNOS) is expressed in activated macrophages and is involved in the synthesis of NO. Because the role of NO and iNOS in human leishmaniasis was less clear, we examined the expression of the iNOS gene in human macrophages infected with Leishmania in vitro and in biopsy tissue from subjects with cutaneous leishmaniasis.Pentavalent antimony (Sb5+) in the form of Pentostam(TM) or Glucantime(TM) is still the treatment of choice despite its toxicity. Aldara(TM) (5% imiquimod) is an immune-response modifying agent that has been approved by the Food and Drug Administration in the USA for treating genital warts caused by papillomaviruses. We conducted an open-label, prospective study of combined Glucantime(TM) + Aldara(TM) therapy in subjects with CL who had previously failed a complete course of Glucantime(TM) treatment at regular doses. (Abstract shortened by UMI.)
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Burnight, Erin Rae. "Targeting therapeutic vector expression and integration for gene therapy applications." Diss., University of Iowa, 2011. https://ir.uiowa.edu/etd/2831.
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Gene therapy is an attractive treatment for many genetic diseases because rather than treat the symptoms of the disease, it has the potential to correct the underlying defect. Cystic fibrosis and hemophilia A are two monogenic disorders that are particularly well-suited to treatment with gene therapy as a relatively small increase in the function is needed to see improvement. Gene therapy has provided some correction in both diseases using a variety of vector systems but sustained expression and long term correction have yet to be demonstrated in the clinic. It is unclear in which cell type(s) correction of the underlying defect in cystic fibrosis will be most effective. Studies indicate that the majority of CFTR expression is in the submucosal glands and ciliated epithelia – a terminally differentiated cell type (Engelhardt, J.F. et al, 2004, Journal of Clinical Investigation). Therapeutic gene transfer would thus be most effective if achieved in a progenitor cell type. Additionally, the native regulation of CFTR has not been definitively elucidated. To this end, one goal of our studies is to develop a lentiviral vector system with heterologous promoters of varying strengths and cell specificity to aid in our selection of optimal reagents for appropriate CFTR expression. We show that use of novel internal promoters from the human PLUNC and WDR65 genes direct persistent expression in the airway. Additionally, disruption of the nasal epithelium with the detergent polidocanol eliminated reporter expression in mouse airway. Two weeks post-treatment, expression returned indicating targeting of a progenitor cell population with our novel vectors.
Integrating vector systems can treat chronic diseases such as cystic fibrosis because expression can persist long term from these vectors if cells with progenitor capacity are targeted (Sinn, P.L. et al, 2005, Journal of Virology). However, the potential for genotoxicity from vector-related dysregulation is a concern. Thus, a second aim of these studies was to develop a lentiviral vector that can target a specific locus in the genome. We developed a FIV vector in which the integrase was modified with a protein-binding domain that when co-delivered with a fusion consisting of the cognate protein and a DNA binding domain would tether the vector to the appropriate locus. Unfortunately, integrase modification rendered the vector catalytically inactive. Lastly, we hoped to develop a non-viral transposon vector system (piggyBac) for gene transfer applications to the liver for treatment of hemophilia A. The recent demonstration that piggyBac transposase is highly active in mammalian cells warrants further development of this vector as an alternative to other non-viral integrating vector systems currently under investigation. We showed persistent reporter and therapeutic transgene expression in the livers of mice treated with the piggyBac vector. Furthermore, we show for the first time in vivo persistence and increased expression from the recently developed hyperactive transposase. The development of integrating vectors targeted to specific tissues or genomic loci is important in for treatment of the monogenic diseases cystic fibrosis and hemophilia A.
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Turro, Ernest. "Statistcal methods for gene expression analysis using microarray and RNA-Seq data." Thesis, Imperial College London, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.534964.
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Parekh, Swati [Verfasser], and Wolfgang [Akademischer Betreuer] Enard. "Optimising gene expression profiling using RNA-seq / Swati Parekh ; Betreuer: Wolfgang Enard." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2018. http://d-nb.info/1189067013/34.
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McGrath, Susan. "The development of an RNA assay for Clostridium botulinum toxin gene expression." Thesis, University of Ulster, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.326309.
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Chiu, Ya-Lin. "HIV-1 Gene Expression: Transcriptional Regulation and RNA Interference Studies: a Dissertation." eScholarship@UMMS, 2003. https://escholarship.umassmed.edu/gsbs_diss/118.
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Gene expression of human immunodeficiency virus type-1 (HIV-1), which causes Acquired Immunodeficiency Syndrome (AIDS), is regulated at the transcriptional level, where negative factors can block elongation that is overcome by HIV Tat protein and P-TEFb. P-TEFb, a positive elongation transcription factor with two subunits, CDK9 and Cyclin T1 (CycT1), catalyzes Tat-dependent phosphorylation of Ser-5 in the Pol II C-terminal domain (CTD), allowing production of longer mRNAs. Ser-5 phosphorylation enables the CTD to recruit mammalian mRNA capping enzyme (Mce1) and stimulate its guanylyltransferase activity. This dissertation demonstrates that stable binding of Mce1 and cap methyltransferase to template-engaged Pol II depends on CTD phosphorylation, but not on nascent RNA. Capping and methylation doesn't occur until nascent pre-mRNA become 19-22 nucleotides long. A second and novel pathway for recruiting and activating Mce1 involved direct physical interaction between the CTD, Tat and Mce1. Tat stimulated the guanylyltransferase and triphosphatase activities of Mce1, thereby enhancing the otherwise low efficiency of cotranscriptional capping of HIV mRNA. These findings imply that multiple mechanisms exist for coupling transcription elongation and mRNA processing at a checkpoint critical to HIV gene expression.
To elucidate P-TEFb's function in human (HeLa) cells, RNA interference (RNAi) was used to degrade mRNA for hCycT1 or CDK9. Down-regulation of P-TEFb expression by RNAi can be achieved without causing major toxic or lethal effects and can control Tat transactivation and HIV replication in host cells. High-density oligonucleotide arrays were used to determine the effect of P-TEFb knockdown on global gene expression. Of 44,928 human genes analyzed, 25 were down-regulated and known or likely to be involved in cell proliferation and differentiation. These results provide new insight into P-TEFb function, its potent role in early embryonic development and strong evidence that P-TEFb is a new target for developing AIDS and cancer therapies.
To fulfill the promise of RNAi for treating infectious and human genetic diseases, structural and functional mechanisms underlying RNAi in human cells were studied. The status of the 5' hydroxyl terminus of the antisense strand of short interfering RNA (siRNA) duplexes determined RNAi activity, while a 3' terminus block was tolerated in vivo. A perfect A-form helix in siRNA was not required for RNAi, but was required for antisense-target RNA duplexes. Strikingly, crosslinking siRNA duplexes with psoralen did not completely block RNAi, indicating that complete unwinding of the siRNA helix is not necessary for RNAi in vivo. These results suggest that RNA amplification by RNA-dependent RNA polymerase is not essential for RNAi in human cells.
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Ismail, Said. "Development of novel MoMLV gene transfer systems by exploiting retroviral RNA processing." Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.365806.
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Eriksson, Jonas. "Gene therapy tools: oligonucleotides and peptides." Doctoral thesis, Stockholms universitet, Institutionen för neurokemi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-132271.
Full textAbstract:
Genetic mutations can cause a wide range of diseases, e.g. cancer. Gene therapy has the potential to alleviate or even cure these diseases. One of the many gene therapies developed so far is RNA-cleaving deoxyribozymes, short DNA oligonucleotides that specifically bind to and cleave RNA. Since the development of these synthetic catalytic oligonucleotides, the main way of determining their cleavage kinetics has been through the use of a laborious and error prone gel assay to quantify substrate and product at different time-points. We have developed two new methods for this purpose. The first one includes a fluorescent intercalating dye, PicoGreen, which has an increased fluorescence upon binding double-stranded oligonucleotides; during the course of the reaction the fluorescence intensity will decrease as the RNA is cleaved and dissociates from the deoxyribozyme. A second method was developed based on the common denominator of all nucleases, each cleavage event exposes a single phosphate of the oligonucleotide phosphate backbone; the exposed phosphate can simultaneously be released by a phosphatase and directly quantified by a fluorescent phosphate sensor. This method allows for multiple turnover kinetics of diverse types of nucleases, including deoxyribozymes and protein nucleases. The main challenge of gene therapy is often the delivery into the cell. To bypass cellular defenses researchers have used a vast number of methods; one of these are cell-penetrating peptides which can be either covalently coupled to or non-covalently complexed with a cargo to deliver it into a cell. To further evolve cell-penetrating peptides and understand how they work we developed an assay to be able to quickly screen different conditions in a high-throughput manner. A luciferase up- and downregulation experiment was used together with a reduction of the experimental time by 1 day, upscaling from 24- to 96-well plates and the cost was reduced by 95% compared to commercially available assays. In the last paper we evaluated if cell-penetrating peptides could be used to improve the uptake of an LNA oligonucleotide mimic of GRN163L, a telomerase-inhibiting oligonucleotide. The combination of cell-penetrating peptides and our mimic oligonucleotide lead to an IC50 more than 20 times lower than that of GRN163L.
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Wagner, Laura A. "Silencing mutant Huntingtin by RNA interference for the treatment of Huntington Disease." Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/937.
Full textAbstract:
Huntington Disease (HD) is a dominantly inherited neurological disease attributed to a CAG expansion within the HD gene. The HD mutation gives rise to a polyglutamine expansion in exon 1 of the protein huntingtin (Htt). Since the discovery of the HD mutation in 1993, various HD gene mouse models have been developed to contain either fragments or full-length copies of the mutant HD gene. The existence of these HD mouse models enables focused therapeutic testing to develop potential treatments for HD. RNA interference (RNAi) therapy is a targeted gene silencing approach whereby synthetic RNA constructs are shuttled into the cell by viral vectors and used by the cell’s endogenous RNAi machinery to silence a gene of interest. RNAi therapy holds promise for mutant huntingtin (muHtt) allele-specific silencing as a treatment for HD. The purpose of this thesis was to develop the tools for pre-clinical testing of RNAi-mediated gene silencing of human muHtt in the YAC128 mouse model of HD. First, AAV serotypes were compared for delivery to striatal neurons, the neurons most affected in HD. From this work AAV serotype 1 was selected as the most effective serotype for construct delivery. Second, synthetic RNAi constructs including short-hairpin RNA (shRNA) and microRNA-based constructs (miR-shRNAs) were compared for silencing of human muHtt expression in vivo. Here, miR-shRNAs were found to have increased gene silencing and improved tolerance in avoiding immune activation compared to shRNAs. Alternatively, the shRNAs induced dramatic immune activation and morbidity in some cases. Ultimately these findings will contribute to a pre-clinical trial in YAC128 mice investigating Htt RNAi-mediated gene silencing in the treatment of HD, which is also discussed in this thesis. This future work provides proof-of-principle for muHtt allele-specific silencing as a treatment of HD.
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Powell, Robert. "Analysis of HIV-1 tat-TAR RNA interactions in vivo." Thesis, University of Oxford, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.239314.
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Muralidhar, Mandayam G. "Regulation of the DHFR gene in mouse cells : processing of HnRNA and expression of a minigene /." The Ohio State University, 1987. http://rave.ohiolink.edu/etdc/view?acc_num=osu148732966214487.
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Groot-Wassink, Thomas Georg. "In-vivo detection of gene expression and gene therapy for malignancy using sodium / iodide symporter gene transfection." Thesis, Imperial College London, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.412885.
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Howe, Whitney M. "The mechanism of gene expression regulation by the ykkCD putative riboswitch." Muncie, Ind. : Ball State University, 2009. http://cardinalscholar.bsu.edu/652.
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