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1
Weinstein, Earl G. 1974. "MicroRNA cloning and bioinformatic analysis." Thesis, Massachusetts Institute of Technology, 2002. http://hdl.handle.net/1721.1/8390.
Full textAbstract:
Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 2002.Includes bibliographical references.
Part I. Two gene-regulatory noncoding RNAs (ncRNAs), let-7 RNA and lin-4 RNA, were previously discovered in the C. elegans genome. The let-7 gene is conserved across a wide range of genomes, suggesting that these ncRNAs represent a wider class of gene-regulatory RNAs. Both lin-4 and let-7 RNAs are generated from stem-loop precursor RNAs, and share a common biochemical signature, namely 5'-terminal phosphate and 3'-terminal hydroxyl groups. We refer to ncRNAs that share the characteristic size, biochemical signature, and precursor structures of let-7 and lin-4 as microRNAs (miRNAs). The size of this class of genes, and its prevalence in other genomes, are unknown. Therefore, we developed an experimental and bioinformatics strategy to identify novel miRNA genes. We discovered a total of 75 miRNA genes in the C. elegans genome, and orthologues for a majority of these were computationally identified in the C. briggsae, D. melanogaster or H. sapiens genomes. Northern analysis was used to confirm and analyze the expression of these miRNAs. The data set has implications for understanding miRNA gene regulation, miRNA processing, and regulation of miRNA genes. Part II. Directed molecular evolution has previously been applied to generate RNAs with novel structures and functions. This method works because nucleic acids can be selected, randomized, amplified and characterized using polymerase chain reaction (PCR)-based methods. Here we present a novel method for extending directed molecular evolution to the realm of peptide selections by linking a peptide to its encoding mRNA.
(cont.) A proof of principle selection for two different peptides indicates that this tRNA should prove useful in discovering more complex protein molecules using directed molecular evolution.
by Earl G. Weinstein.
Ph.D.
2
Wang, Qi. "Using Imputed Microrna Regulation Based on Weighted Ranked Expression and Putative Microrna Targets and Analysis of Variance to Select Micrornas for Predicting Prostate Cancer Recurrence." Thesis, North Dakota State University, 2014. https://hdl.handle.net/10365/27341.
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Imputed microRNA regulation based on weighted ranked expression and putative microRNA targets (IMRE) is a method to predict microRNA regulation from genome-wide gene expression. A false discovery rate (FDR) for each microRNA is calculated using the expression of the microRNA putative targets to analyze the regulation between different conditions. FDR is calculated to identify the differences of gene expression. The dataset used in this research is the microarray gene expression of 596 patients with prostate cancer. This dataset includes three different phenotypes: PSA (Prostate-Specific Antigen recurrence), Systemic (Systemic Disease Progression) and NED (No Evidence of Disease). We used the IMRE and ANOVA methods to analyze the dataset and identified several microRNA candidates that can be used to predict PSA recurrence and systemic disease progression in prostate cancer patients.
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Goldstein, L. D. "Statistical analysis of microRNA expression and related data." Thesis, University of Cambridge, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.599479.
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The first part of this thesis is concerned with the analysis of miRNA expression data obtained by bead-based flow cytometric profiling. Based on data obtained from 93 human breast cancer samples, we assess the association of individual miRNAs with clinical factors and molecular tumour subtype. We investigate potential mechanisms of miRNA deregulation by analysing matched data on DNA copy number and mRNA expression. We describe an analysis of miRNA and mRNA expression during normal postnatal mouse mammary gland development, a model system for the study of human breast cancer. In the second part of this thesis we are concerned with the analysis of mRNA expression data with a focus on miRNA targets. We develop a statistical method to assess whether predicted miRNA targets show expression levels that are different compared to those of suitably chosen control genes. We find that, across human breast cancers and during mouse mammary gland development, the targets of most miRNAs do not show systematic changes in their expression. In cases where targets are differentially expressed, changes in expression are mostly consistent with miRNA-mediated regulation. We characterize the molecular function of the miRNA miR-124 in the nematode Caenorhabditis elegans. Many targets of miR-124 are coexpressed with and actively repressed by miR-124. Reduced expression levels of miR-124 targets in cells that express the miRNA compared to the rest of the animal are mostly due to direct miRNA-mediated repression in the case of evolutionary conserved targets and due to both direct repression and other regulatory mechanisms in the case of nonconserved targets.
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Alvarez-Saavedra, Ezequiel (Ezequiel Andrès). "Functional analysis of the microRNA genes of C. elegans." Thesis, Massachusetts Institute of Technology, 2008. http://hdl.handle.net/1721.1/42948.
Full textAbstract:
Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2008.This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections.
Includes bibliographical references (p. 231-252).
MicroRNAs (miRNAs) were discovered in C. elegans during studies of the control of developmental timing. MicroRNAs are a large class of short non-coding RNAs found in many viruses, plants and animals that regulate gene expression through sequence-specific base-pairing with target mRNAs. Initial studies since the identification of many miRNAs only six years ago, have revealed their very diverse roles in biology. Yet, few miRNAs have been studied using loss-of-function mutations. We have generated deletion mutations in 87 miRNA genes in C. elegans, and performed an initial characterization of the 95 miRNA mutants available (86% of known C. elegans miRNAs). We found that the majority of miRNAs are not essential for the viability or development of C. elegans, and mutations in most miRNA genes do not result in grossly abnormal phenotypes. Within species, many miRNAs can be grouped into families according to their sequence similarities. We generated a collection of 12 multiply mutant C. elegans strains that each lacks an entire miRNA family. We found that at least four families display synthetic abnormalities, indicating that miRNAs within a family can have redundant functions. While single mutants are superficially wild-type, mutants deleted for all members of the mir-35 or the mir-51 families show embryonic or early larval lethality, mutants deleted for all members of the mir-58 family show an egglaying defect, and mutants deleted for some members of the let-7 family show defects in developmental timing. We developed a microarray technology suitable for detecting microRNAs and used this microarray to determine the profile of microRNAs expressed in the developing mouse brain. We observed a temporal wave of expression of microRNAs, suggesting that microRNAs play important roles in the development of the mammalian brain.
(cont.) We also performed a systematic expression analysis of 334 samples covering diverse human cancers, using a bead-based flow cytometric miRNA expression profiling method we developed. The miRNA profiles reflect the developmental lineage and differentiation state of the tumors, and reveal a general down-regulation of miRNAs in tumors compared to normal tissues.
by Ezequiel Alvarez-Saavedra.
Ph.D.
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Bexon, Kimerley Jane. "Forensic microRNA analysis of body fluids relating to sexual assaults." Thesis, University of Huddersfield, 2017. http://eprints.hud.ac.uk/id/eprint/34347/.
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DNA profiling has become a universal technique for identifying individuals for evidential use in courts of law. In more complex cases such as sexual assaults, identifying the origin of a stain or sample offers valuable information as to the events that occurred. Currently, many ‘in service’ body fluid identification (BFID) techniques are presumptive, require significant sample volumes and generate false positives. As such, the development of a highly specific and reliable BFID technique would be highly beneficial to forensic scientists in provide more informative and reliable evidence. MicroRNAs (miRNA) are short, stable, non-coding RNA’s which modulate gene expression. Expression of some of these miRNA are body fluid specific, making them a potentially robust tool for BFID. The possibility for the integration of a robust, miRNA based BFID technology for forensic casework employing stem-loop reverse transcription and qPCR analysis was the theme of the research presented here. To be incorporated into the workflow of current forensic laboratories, the protocol must be able to be carried out alongside current techniques with limited addition of cost, equipment, analysts and time. A range of custom designed miRNA markers were analysed on vaginal material, menstrual blood, saliva, venous blood, semen, seminal fluid and skin. Screening indicated specificity of hsa mir-124 to vaginal material, hsa-mir-10a, 135a and 888 to semen, hsa-mir-412 and 507 to menstrual blood, hsa-mir-144-3, 144-5, 142 and 451 to blood and although highly expressed in saliva, hsa-mir-205 was also observed in vaginal material. Universal expression was observed in hsa-mir-93, 508, 1260b and SNORD 47 providing a means of normalisation through the designation of these markers as endogenous controls. A combined panel of markers are presented which were capable of identifying all body fluids, excluding skin from single stains. The panel was successful at identifying certain mixtures such as semen within vaginal material but was unable to confirm saliva presence within vaginal material. Screening of hsa-mir 205 within vaginal material uncovered the observation that hsa-mir-205 was impacted by the use of female contraception. Once a full BFID panel was generated the robustness of the markers was further analysed across the menstrual cycle. No significant difference (p > 0.001) was seen in markers highly expressed in vaginal material during screening (hsa-mir-124, 203a, 205). Expression of non specific markers highlighted the importance of the optimisation of input miRNA. Differential extraction of genetic material was found to be detrimental to miRNA sample integrity. As such, total DNA extraction was employed for vaginal swabs obtained from volunteers following unprotected sexual intercourse, markers hsa mir-10a, 135a and 888 were able to successfully detect semen presence for up to 96 hours. The data generated to date has highlighted a number of miRNA markers that provide a platform for BFID. The developed protocol is reliable and robust; requiring minimal optimisation and is capable of integration with current laboratory workflow with minimum implications to time and cost. The markers identified for BFID have been implemented within studies that are representative of real case scenarios, and have demonstrated their ability to be stable, specific and successful in the identification of certain body fluids. Overall, this research showcases a reliable and body fluid specific protocol capable of being performed alongside DNA profiling.
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Lehrbach, Nicolas John. "Genetic analysis of microRNA mechanisms and functions in C. elegans." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609195.
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Moriarty, Charlotte M. Harwood. "Functional Analysis of MicroRNA-10b in Breast Carcinoma: A Dissertation." eScholarship@UMMS, 2009. https://escholarship.umassmed.edu/gsbs_diss/426.
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MicroRNAs (miRNAs) represent a class of small noncoding RNAs that regulate gene expression. Recent studies have shown that miRNAs are mis-expressed in various human cancers and that some miRNAs have the potential to act as tumor suppressors or oncogenes. MiR-10b is one miRNA that has been shown to be deregulated in breast cancer. However, current findings regarding miR-10b’s role in breast cancer are controversial. MiR-10b was originally reported to be downregulated in breast cancer compared to normal breast tissue. Subsequently, miR-10b was argued to be upregulated in metastatic breast cancer cell lines, acting as a potent pro-metastatic agent via regulation of HOXD10. This report was soon challenged by another group who reported that miR-10b expression in a large patient cohort correlated inversely and significantly with tumor size, grade, and vascular invasion, but did not correlate with development of distant metastases or survival. These latter data suggest that miR-10b may impede specific functions associated with breast cancer progression.
In this thesis, I present my analysis of miR-10b function in breast carcinoma cells, which revealed that it suppresses their migration and invasion. To define a mechanism that accounts for this suppressive function, I identified T-lymphoma invasion and metastasis 1 (TIAM1), a guanine nucleotide exchange factor for Rac1, as a miR-10b target and demonstrated that miR-10b inhibits TIAM1-dependent Rac1 activation, migration, and invasion. In addition, I identified the VEGF receptor fms-related tyrosine kinase 1 (FLT-1) as a second target of miR-10b and discovered a novel function for FLT-1 in promoting breast carcinoma cell migration and invasion. My results show, for the first time, that Rac activation can be regulated by a specific miRNA and provide a novel mechanism for the regulation of TIAM1 and FLT-1 in breast cancer. These data support the conclusion from clinical data that miR-10b expression correlates inversely with breast cancer progression, and suggest that miR-10b functions to impede breast carcinoma progression by regulating key target genes involved in cell motility.
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Woodcock, M. Ryan. "Network Analysis and Comparative Phylogenomics of MicroRNAs and their Respective Messenger RNA Targets Using Twelve Drosophila species." VCU Scholars Compass, 2010. http://scholarscompass.vcu.edu/etd/155.
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MicroRNAs represent a special class of small (~21–25 nucleotides) non-coding RNA molecules which exert powerful post-transcriptional control over gene expression in eukaryotes. Indeed microRNAs likely represent the most abundant class of regulators in animal gene regulatory networks. This study describes the recovery and network analyses of a suite of homologous microRNA targets recovered through two different prediction methods for whole gene regions across twelve Drosophila species. Phylogenetic criteria under an accepted tree topology were used as a reference frame to 1) make inference into microRNA-target predictions, 2) study mathematical properties of microRNA-gene regulatory networks, 3) and conduct novel phylogenetic analyses using character data derived from weighted edges of the microRNA-target networks. This study investigates the evidences of natural selection and phylogenetic signatures inherent within the microRNA regulatory networks and quantifies time and mutation necessary to rewire a microRNA regulatory network. Selective factors that appear to operate upon seed aptamers include cooperativity (redundancy) of interactions and transcript length. Topological analyses of microRNA regulatory networks recovered significant enrichment for a motif possessing a redundant link in all twelve species sampled. This would suggest that optimization of the whole interactome topology itself has been historically subject to natural selection where resilience to attack have offered selective advantage. It seems that only a modest number of microRNA–mRNA interactions exhibit conservation over Drosophila cladogenesis. The decrease in conserved microRNA-target interactions with increasing phylogenetic distance exhibited a cure typical of a saturation phenomena. Scale free properties of a network intersection of microRNA target predictions methods were found to transect taxonomic hierarchy.
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Bracht, John Russell. "Analysis of lin-4 microRNA biogenesis and function in C. elegans." Diss., [La Jolla] : University of California, San Diego, 2009. http://wwwlib.umi.com/cr/ucsd/fullcit?p3378519.
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Thesis (Ph. D.)--University of California, San Diego, 2009.Title from first page of PDF file (viewed Oct. 21, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 139-150).
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Lopes, Ivani de Oliveira Negrão. "Analysis of microRNA precursors in multiple species by data mining techniques." Universidade de São Paulo, 2014. http://www.teses.usp.br/teses/disponiveis/55/55134/tde-19092014-155038/.
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RNA Sequencing has recently emerged as a breakthrough technology for microRNA (miRNA) discovery. This technology has allowed the discovery of thousands of miRNAs in a large number of species. However, despite the benefits of this technology, it also carries its own limitations, including the need for sequencing read libraries and of the genome. Differently, ab initio computational methods need only the genome as input to search for genonic locus likely to give rise to novel miRNAs. In the core of most of these methods, there are predictive models induced by using data mining techniques able to distinguish between real (positive) and pseudo (negative) miRNA precursors (pre-miRNA). Nevertheless, the applicability of current literature ab initio methods have been compromised by high false detection rates and/or by other computational difficulties. In this work, we investigated how the main aspects involved in the induction of predictive models for pre-miRNA affect the predictive performance. Particularly, we evaluate the discriminant power of feature sets proposed in the literature, whose computational costs and composition vary widely. The computational experiments were carried out using sequence data from 45 species, which covered species from eight phyla. The predictive performance of the classification models induced using large training set sizes (≥ 1; 608) composed of instances extracted from real and pseudo human pre-miRNA sequences did not differ significantly among the feature sets that lead to the maximal accuracies. Moreover, the differences in the predictive performances obtained by these models, due to the learning algorithms, were neglectable. Inspired by these results, we obtained a feature set which can be computed 34 times faster than the less costly among those feature sets, producing the maximal accuracies, albeit the proposed feature set has achieved accuracy within 0.1% of the maximal accuracies. When classification models using the elements previously discussed were induced using small training sets (120) from 45 species, we showed that the feature sets that produced the highest accuracies in the classification of human sequences were also more likely to produce higher accuracies for other species. Nevertheless, we showed that the learning complexity of pre-miRNAs vary strongly among species, even among those from the same phylum. These results showed that the existence of specie specific features indicated in previous studies may be correlated with the learning complexity. As a consequence, the predictive accuracies of models induced with different species and same features and instances spaces vary largely. In our results, we show that the use of training examples from species phylogenetically more complex may increase the predictive performances for less complex species. Finally, by using ensembles of computationally less costly feature sets, we showed alternative ways to increase the predictive performance for many species while keeping the computational costs of the analysis lower than those using the feature sets from the literature. Since in miRNA discovery the number of putative miRNA loci is in the order of millions, the analysis of putative miRNAs using a computationally expensive feature set and or inaccurate models would be wasteful or even unfeasible for large genomes. In this work, we explore most of the learning aspects implemented in current ab initio pre-miRNA prediction tools, which may lead to the development of new efficient ab initio pre-miRNA discovery toolsO sequenciamento de pequenos RNAs surgiu recentemente como uma tecnologia inovadora na descoberta de microRNAs (miRNA). Essa tecnologia tem facilitado a descoberta de milhares de miRNAs em um grande número de espécies. No entanto, apesar dos benefícios dessa tecnologia, ela apresenta desafios, como a necessidade de construir uma biblioteca de pequenos RNAs, além do genoma. Diferentemente, métodos computacionais ab initio buscam diretamente no genoma regiões prováveis de conter miRNAs. A maioria desses métodos usam modelos preditivos capazes de distinguir entre os verdadeiros (positivos) e pseudo precursores de miRNA - pre-miRNA - (negativos), os quais são induzidos utilizando técnicas de mineração de dados. No entanto, a aplicabilidade de métodos ab initio da literatura atual é limitada pelas altas taxas de falsos positivos e/ou por outras dificuldades computacionais, como o elevado tempo necessário para calcular um conjunto de atributos. Neste trabalho, investigamos como os principais aspectos envolvidos na indução de modelos preditivos de pre-miRNA afetam o desempenho preditivo. Particularmente, avaliamos a capacidade discriminatória de conjuntos de atributos propostos na literatura, cujos custos computacionais e a composição variam amplamente. Os experimentos computacionais foram realizados utilizando dados de sequências positivas e negativas de 45 espécies, cobrindo espécies de oito filos. Os resultados mostraram que o desempenho preditivo de classificadores induzidos utilizando conjuntos de treinamento com 1608 ou mais vetores de atributos calculados de sequências humanas não diferiram significativamente, entre os conjuntos de atributos que produziram as maiores acurácias. Além disso, as diferenças entre os desempenhos preditivos de classificadores induzidos por diferentes algoritmos de aprendizado, utilizando um mesmo conjunto de atributos, foram pequenas ou não significantes. Esses resultados inspiraram a obtenção de um conjunto de atributos menor e que pode ser calculado até 34 vezes mais rapidamente do que o conjunto de atributos menos custoso produzindo máxima acurácia, embora a acurácia produzida pelo conjunto proposto não difere em mais de 0.1% das acurácias máximas. Quando esses experimentos foram executados utilizando vetores de atributos calculados de sequências de outras 44 espécies, os resultados mostraram que os conjuntos de atributos que produziram modelos com as maiores acurácias utilizando vetores calculados de sequências humanas também produziram as maiores acurácias quando pequenos conjuntos de treinamento (120) calculados de exemplos de outras espécies foram utilizadas. No entanto, a análise destes modelos mostrou que a complexidade de aprendizado varia amplamente entre as espécies, mesmo entre aquelas pertencentes a um mesmo filo. Esses resultados mostram que a existência de características espécificas em pre-miRNAs de certas espécies sugerida em estudos anteriores pode estar correlacionada com a complexidade de aprendizado. Consequentemente, a acurácia de modelos induzidos utilizando um mesmo conjunto de atributos e um mesmo algoritmo de aprendizado varia amplamente entre as espécies. i Os resultados também mostraram que o uso de exemplos de espécies filogeneticamente mais complexas pode aumentar o desempenho preditivo de espécies menos complexas. Por último, experimentos computacionais utilizando técnicas de ensemble mostraram estratégias alternativas para o desenvolvimento de novos modelos para predição de pre-miRNA com maior probabilidade de obter maior desempenho preditivo do que estratégias atuais, embora o custo computacional dos atributos seja inferior. Uma vez que a descoberta de miRNAs envolve a análise de milhares de regiões genômicas, a aplicação prática de modelos preditivos de baixa acurácia e/ou que dependem de atributos computacionalmente custosos pode ser inviável em análises de grandes genomas. Neste trabalho, apresentamos e discutimos os resultados de experimentos computacionais investigando o potencial de diversas estratégias utilizadas na indução de modelos preditivos para predição ab initio de pre-miRNAs, que podem levar ao desenvolvimento de ferramentas ab initio de maior aplicabilidade prática
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Du, Chen. "Global analysis of microrna species in the gall midge Mayetiola destructor." Thesis, Kansas State University, 2015. http://hdl.handle.net/2097/20375.
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Master of ScienceEntomology
Ming-Shun Chen
Robert "Jeff" J. Whitworth
MicroRNA (miRNA) plays a role in nearly all the biological pathways and therefore may provide opportunities to develop new means to combat the Hessian fly, Mayetiola destructor, a destructive pest of wheat. This study presents a comprehensive analysis of miRNA species via deep-sequencing samples from Hessian fly second instar larvae, pupae and adults. A total of 921 unique miRNA species were identified from approximately 30 million sequence reads. Among the 921 miRNA species, only 22 were conserved among Hessian fly and other insect species, and 242 miRNA species were unique to Hessian fly, the remaining 657 share certain sequence similarities with pre-miRNA genes identified from various insect species. The abundance of the 921 miRNA species based on sequence reads varies greatly among the three analyzed stages, with 20 exclusively expressed in adults, two exclusively expressed in pupae and two exclusively expressed in second instar larvae. For miRNA species expressed in all stages, 722 were with reads lower than 10. The abundance of the remaining 199 miRNA species varied from zero to more than eight-fold differences among different stages. Putative miRNA-encoding genes were analyzed for each miRNA species. A single putative gene was identified for 594 miRNA species. Two putative genes were identified for 138 miRNA species. Three or more putative genes were identified for 86 miRNA species. The three largest families had 14, 23 and 34 putative coding genes, respectively. No gene was identified for the remaining 103 miRNA species. In addition, 1516 putative target genes were identified for 490 miRNA species based on known criteria for miRNA targets. The putative target genes are involved in a wide range of processes from nutrient metabolism to encoding effector proteins. Analysis of the expression patterns of miRNA and pre-miRNA for the miRNA family PC-5p-67443, which contains 91 genes, revealed that miRNA and pre-miRNA were expressed differently in different developmental stages, suggesting that different isogenes are regulated by different mechanisms, or pre-miRNAs had other functions in addition to as an intermediate for miRNA biogenesis. The large set of miRNA species identified here provides a foundation for future research on miRNA functions in Hessian fly and for comparative studies in other species. The differential expression patterns between a pre-miRNA and its encoded mature miRNA in a multigene family is an initial step toward understanding the functional significance of isogenes in dramatically expanded miRNA families.
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Samols, Mark Atienza. "Identification and Functional Analysis of Micro-RNAs Encoded by Kaposi’s Sarcoma-Associated Herpesvirus." Case Western Reserve University School of Graduate Studies / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=case1181143062.
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Keung, Man Hong. "The role of MicroRNA in 20(R)-ginsenoside-Rg3-induced anti-angiogenesis." HKBU Institutional Repository, 2010. http://repository.hkbu.edu.hk/etd_ra/1174.
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Avramopoulos, Petros [Verfasser], Stefan [Akademischer Betreuer] Engelhardt, Stefan [Gutachter] Engelhardt, and Arne [Gutachter] Skerra. "Optical analysis of microRNA activity and therapeutic manipulation of microRNA-29 in vivo / Petros Avramopoulos ; Gutachter: Stefan Engelhardt, Arne Skerra ; Betreuer: Stefan Engelhardt." München : Universitätsbibliothek der TU München, 2019. http://d-nb.info/1220320455/34.
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Khan, Nasrin. "Analysis of MicroRNAs in Biological Samples." Thesis, Université d'Ottawa / University of Ottawa, 2015. http://hdl.handle.net/10393/32317.
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MicroRNAs (miRNAs) are a class of small, single-stranded, non-protein coding RNA
molecules that regulate cellular messenger RNA (mRNA) and protein levels by binding to specific mRNAs. Aberrant miRNA expression has been shown to be implicated in several diseases, including cancer. Extracellular miRNAs have been found to circulate in the bloodstream and some of their levels have been associated with different diseases. Furthermore, they hold promise as tissue- and blood-based biomarkers for cancer classification and prognostication. Blood-based biomarkers are attractive for cancer screening due to their minimal invasiveness, relatively low cost and ease of reproducibility. New miRNA analysis techniques will add toward the understanding of their biological functions. In this thesis, I investigate the utility of capillary electrophoresis (CE) and mass spectrometry (MS) for analysis of miRNAs through proof-of-concept experiments. In the fi rst part of this work, we developed a Protein-Facilitated Affinity Capillary Electrophoresis (ProFACE) assay for rapid quantification of miRNA levels in blood serum (see List of publications (6)). We also implemented a capillary electrophoresis with laser induced fluorescence detection (CE-LIF) method with online sample pre-concentration for detection
of endogenous microRNAs in human serum and cancer cells.
3' modification of miRNA is a physiological and common post-transcriptional event
that shows selectivity for specific miRNAs and is observed across species. Recent studies have shown that post-transcriptional addition of nucleotides to the 3' end of miRNAs is a mechanism for miRNA activity regulation. For example, such modifications in plants and C. elegans influence miRNA stability. In humans, effects on miRNA stability and on mRNA target repression have both been observed. Thus, there is a need for miRNA detection techniques which are direct and multiplexed, require minimal sample preparation and provide qualitative information regarding these modifications. We developed a multiplexed miRNA detection technique based on capillary electrophoresis coupled on line with electrospray ionization mass spectrometry (CE-ESI-MS). This method allowed a label-free, direct detection of multiple miRNAs extracted from cancer serum as well as their post-transcriptional modifications with a high mass accuracy.
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Budd, William. "Combinatorial analysis of tumorigenic microRNAs driving prostate cancer." VCU Scholars Compass, 2012. http://scholarscompass.vcu.edu/etd/2848.
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Prostate cancer is the leading non-cutaneous malignancy affecting men in the United States. One in every six men will be affected by prostate cancer. Due to the high incidence of prostate cancer, there is a need to develop biomarkers capable of identifying tumors from benign prostatic lesions. miRNAs are small molecules that regulate protein translation and impact cellular integrity when dysregulated. It is widely thought that miRNAs have the potential to serve as biomarkers. This study utilizes a unique combinatorial analysis of miRNA dysregulation to identify key miRNAs involved in prostate tumor initiation, progression and metastasis. Numerous dysregulated miRNAs potentially influence cancer development. A unique bioinformatically driven, network based approach was used to rank potential miRNAs that drive tumor progression. This study showed that miRNAs preferentially regulate highly connected proteins and transcription factors that affect numerous downstream targets. Thus dysregulation of a single highly connected miRNA could severely impact homeostatic maintenance of the tissue. In combination with miRNA profiling of a cancer cell progression model, the utilization of laser captured microdissection was used to separate cancer specific microRNA portraits from background differences arising from stroma cells, lymphocytes, and remaining normal epithelial cells. Integration of miRNA profiles with information gathered using networks biology and targeted proteomics resulted in the identification of a key miRNA that affects prostate cancer development and may be useful as a novel biomarker for identification/ staging of prostate cancer. Human miR-125b was identified as a potential miRNA suppressor of tumor formation. Previous work has identified miR-125-b as the post-transcriptional regulator of the ErbB2/ ErbB3 growth factor receptor family. Loss of miR-125b drives up expression of ErbB2/ ErbB3 activating downstream PI3K/AKT and RAS oncogene pathways. The level of miR-125b decreases 3-5-fold between benign and tumor epithelium. Further, miR-125b decreases during the development of prostatic intraepithelial neoplasia, which is regarded as an early indicator of prostate cancer. Thus miR-125b may be an ideal marker of early changes indicative of cancer. Restoration of miR-125b into highly tumorigenic, metastatic cells reduces mobility and invasion of underlying tissues. Taken together these data show miR-125b is a tumor suppressor in the healthy prostate.
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Ahmed, Rina [Verfasser]. "Bioinformatic Analysis of microRNA Genes in Free-Living and Parasitic Nematodes / Rina Ahmed." Berlin : Freie Universität Berlin, 2015. http://d-nb.info/1068504803/34.
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Bao, Yu. "Identification and Analysis of Critical Sites in RNA/Protein Sequences and Biological Networks." Kyoto University, 2018. http://hdl.handle.net/2433/235113.
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Muiños, Gimeno Margarita. "Analysis of genetic variation in microrna-mediated regulation and the susceptibility to anxiety disorders." Doctoral thesis, Universitat Pompeu Fabra, 2009. http://hdl.handle.net/10803/7192.
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We have investigated genetic variation in microRNA-mediated regulation as a susceptibility factor for anxiety disorders following two different approaches. We first studied two isoforms of the candidate gene NTRK3 by re-sequencing its different 3'UTRs in patients with Panic (PD) and Obsessive Compulsive disorders (OCD) as well as controls. Two rare variants that altered microRNA-mediated regulation were identified in PD. Conversely, association of a common SNP with OCD hoarding subtype was found. Moreover, we have also studied a possible involvement of microRNAs in anxiety disorders. Consequently, we have analysed the genomic organisation and genetic variation of miRNA-containing regions to construct a panel of SNPs for association analysis. Case-control studies revealed several associations. However, it is worth remarking the associations of miR-22 and miR-488 with PD; two microRNAs for which functional assays and transcriptome analysis after microRNA overexpression showed significant repression of a subset of genes involved in physiological pathways linked to PD development.Hem investigat la variació genètica a la regulació mediada per microRNAs com a factors de susceptibilitat pels trastorns d'ansietat seguint dues aproximacions diferents. Primer vam estudiar dues isoformes del gen candidat NTRK3 mitjançant la reseqüenciació dels seus diferents 3'UTRs a pacients de pànic (TP), a pacients amb trastorn obsessiu compulsiu (TOC) i a controls. Dues variants rares que alteren la regulació mediada per microRNAs foren identificades per TP. D'altra banda, es trobà associació d'un SNP comú amb el subtipus acumulador de TOC. A més, també hem estudiat la possible implicació dels microRNAs als trastorns d'ansietat. Conseqüentment, hem analitzat l'organització genòmica i la variació genètica a regions que contenen microRNAs per construir un panell d'SNPs per fer anàlisis d'associació. Els estudis cas-control van revelar algunes associacions. Tanmateix, val la pena destacar les associacions del miR-22 i el miR-488 amb TP; dos microRNAs pels quals assajos funcionals i anàlisis de transcriptoma després de la seva sobreexpressió han mostrat una repressió significativa d'un grup de gens implicats en vies fisiològiques lligades al desenvolupament del TP.
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Nelson, Brandon John. "MicroRNA analysis of human embryonic stem cell derived cardiomyocytes and neonatal rat ventricular cardiomyocytes." Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2007. http://wwwlib.umi.com/cr/ucsd/fullcit?p1447322.
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Thesis (M.S.)--University of California, San Diego, 2007.Title from first page of PDF file (viewed January 15, 2008). Available via ProQuest Digital Dissertations. Includes bibliographical references (p. 45-48).
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Das, Gupta Mithun [Verfasser], and Jürgen [Gutachter] Löffler. "Analysis of microRNA-profiles in human dendritic cells / Mithun Das Gupta. Gutachter: Jürgen Löffler." Würzburg : Universität Würzburg, 2015. http://d-nb.info/1102829323/34.
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Tan, Yi. "Functional analysis of microRNA-181a : identification of target proteins and application in HCC therapy." Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/24698.
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Hepatocellular carcinoma (HCC), or the cancer of the liver, is of great concern due to its poor patient outcome despite the various treatments available. It is imperative, therefore, that a novel, viable treatment method is developed such that patient survival rates may be improved from current statistics of less than 50%. The role of miRNAs in the regulation of gene expression and cellular development makes it an important player in cancer development process, as it is found that the aberrant expression of miRNAs is a typical feature of cancer cells or even pre-disposed cancer cells. MiR-181a has been shown to be an important miRNA involved in HCC. In this study, we investigated the potential effects of miR-181a in HepG2 cells and the mechanisms in which it works in controlling cell fate. As chemotherapy is widely used in liver cancer treatment, we also study the use of miR-181a along with chemotherapy (i.e. Cisplatin). Using iTRAQ-coupled 2D LC-MS/MS analysis, we report here the study of protein profile of HepG2 cells transfected with miR-181a and its inhibitor respectively. Three main types of cellular proteins including metabolic enzymes, protein binding and stress proteins displayed changes. The changes in the level of proteins (14-3-3σ, Hsp-90β and NPM1) involved in important cancer processes like cell growth were further supported by a Western blot analysis. MiR-181a was subsequently found to significantly increase HepG2 cell viability while inhibiting it displayed the opposite effect. Inhibiting miR-181a also sensitized HepG2 cells to cisplatin treatment and retards cell cycle progression by decreasing the proportion of cells in S and G2/M phases. We next investigated the reasons behind these observations at a molecular level. As miRNAs are known to regulate genes by binding to and targeting mRNAs, we first used bioinformatics to screen out potential cellular targets. Two important genes identified, cyclin-dependent kinase inhibitor 1B (CDKN1β) and transcriptional factor E2F7 (E2F7), which are involved in cell cycle and cell proliferation, were chosen to be further experimentally studied. In vitro validation via surface plasmon resonance (SPR) technique showed a positive binding between miR-181a and the seed regions of the 3'UTRs of the two putative mRNA targets, with dissociation constants being 272.5 ± 0.008 nM and 1.186 ± 0.009 uM for CDKN1β and E2F7 respectively. In vivo luciferase assay studies further validated the miR-181a:mRNA bindings, in both cases displaying significant decrease in luciferase activity when HepG2 cells were co-transfected with the 3'UTR-containing reporter plasmids and miR-181a. A positive binding, however, may not necessarily lead to a lowered expression of protein levels. A Western blot study on the expression levels of the two proteins, however, showed a decrease in the levels of CDKN1β and E2F7. Lastly, to gain an insight into the overall effects miR-181a has in HepG2 cells, a microarray analysis was performed. Cellular pathways important in cancer were studied and results show that miR-181a significantly activated the MAPK/JNK pathway by increasing the expression levels or activity of transcription factor activator protein 1 (AP-1). Inhibiting miR-181a, on the other hand, abolished this observation and significantly decreased expression levels or activity of hypoxia-inducible factors (HIF) and also significantly upregulated the expression levels or activity of SMAD2/3/4 proteins, possibly inducing a cancer-suppressing effect. Overall, miR-181a appears to activate mainly cancer-promoting pathways, and may act as an oncogene in HepG2 cells. Inhibiting it, on the other hand, activates mainly the tumour-suppressing pathways, making it a possible option for therapy. A separate microarray analysis on gene expression showed that one way in which miR-181a could have activated the SMAD, NFκB and MAPK pathways is via the significant increase in gene expression of bone morphogenetic protein receptor type II (BMPR2), a cellular receptor that mediates the signal transduction of these pathways. Our findings provide a new platform of identifying miRNA targets, in the process offering molecular evidence on the mechanism of action of miR-181a, including the beneficial effects of inhibiting miR-181a in HCC therapy.
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Selvaraja, Sudarshan. "Microarray Data Analysis Tool (MAT)." University of Akron / OhioLINK, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=akron1227467806.
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Chow, Hiu Tung. "Arabidopsis miR163 and its target are involved in defense against Pseudomonas syringae." HKBU Institutional Repository, 2016. https://repository.hkbu.edu.hk/etd_oa/312.
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Small RNAs are important regulators for a variety of biological processes, including leaf development, flowering-time, embryogenesis and defense responses. Most ancient miRNAs are conserved among different plant species and well characterized, while young MIRNA genes are considered to be non-conserved, highly species-specific and less well-studied. miR163 is a non-conserved miRNA and its locus has evolved recently by inverted duplication events of its target gene. Previously, we have shown that miR163 acts as a negative regulator of defense response. However, it remains unclear how miR163 and its targets are being regulated in response to pathogen attacks. Here, we further elucidated the molecular controls and the involvement of miR163 and its targets in plant defense response. Elevated level of miR163 was observed by Pst treatment in Arabidopsis thaliana, and this upregulation was found to be important in controlling the accumulation of its targets (PXMT1 and FAMT), to which they were also inducible by Pst treatment. Transcript and protein level analyses in transgenic plants overexpressing miR163-resistant form of PXMT1 or FAMT provided evidence for miR163 in fine-tuning its targets, suggesting that the stress-inducible miR163 and its targets act in concert in affecting defense genes expression. Epigenetically, histone deacetylation was found to involve in the repression of miR163 targets before and after Pst infection. Our findings revealed additional mechanistic insights to the controls and the evolutionary significance of young miRNA in mediating plant defense pathways against biotic stresses.
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Mead, Edward. "Discovery, Characterization, and Functional Analysis of micro RNAs in Culicidae." Diss., Virginia Tech, 2009. http://hdl.handle.net/10919/77433.
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MicroRNAs (miRNAs) are non-coding RNAs that often play a fundamental role in gene regulation. Currently, hundreds to over a thousand miRNAs are predicted to be present in many eukaryote species, with many to be discovered; the functions of most are unknown. While much attention has gone towards model organisms, a much greater depth of understanding remains to be gained for the miRNAs of many organisms directly important to humans. There are few verified miRNAs for any mosquito species, despite the role of mosquitoes in many of humanity’s worst diseases. Anopheles gambiae and Aedes aegypti, carriers of malaria and dengue, respectively, are responsible for over a million deaths a year. To date, there are sixty-six microRNAs in An. gambiae in miRBase, a central repository for miRNA sequences. Many of these are based on homology to primarily Drosophila miRNAs. While sequence conservation suggests an important function for these miRNAs, expression has not been experimentally verified for most mosquito miRNAs.
Using small RNA cloning and northern blots, I discovered and analyzed 27 different microRNAs in aged female An. stephensi mosquitoes, the age group responsible for transmission of malarial parasites. Three of these miRNAs are only found in mosquitoes (miR-1889, -1890, and –1891). Cloning and northern analysis revealed an abundance of a miRNA that is linked to longevity in flies, miR-14, across different life stages of mosquitoes. It was also shown that miR-989 was expressed almost exclusively in the adult ovary and its expression fluctuated in response to bloodfeeding, suggesting a possible role in reproduction, an area of great importance to controlling mosquito populations.
Building upon the above cloning experiment, a later high-throughput sequencing effort uncovered 98 miRNA precursors from Ae. aegypti. There are a total of 13 novel miRNAs that have not been found in other organisms by bioinformatic predictions or experiments. These “mosquito-specific” miRNAs may play a role in processes such as blood-feeding or vector-host interactions. A detailed examination of the expression of eight of these miRNAs was conducted in An. gambiae, An. stephensi, Ae. aegypti, and T. amboinensis to determine their expression profile, conservation, and provide hints to their function. My work revealed conserved and sometime stage-specific expression profiles of some of the mosquito-specific miRNAs. I also provided evidence for three lineage-specific miRNAs that may shed light on the divergence of different mosquito lineages.
Extending the finding that miR-989 may be involved in mosquito reproduction, we conducted a detailed analysis of its evolution, expression, possible targets and regulation. miR-989 is conserved in holometabolous insects. miR-989 expression in female An. stephensi and Ae. aegypti dramatically rises following pupal emergence until strong signal is observed, until a blood meal is taken. Expression remains quite strong then begins a steep decline in expression at 32-40 hours post blood meal (PBM), and even by 96 hours PBM, remains weak. Bioinformatic predictions of miR-989 targets coupled with a PCR-based approach uncovered three potential target leads, though preliminary results were artifacts. Although the miR-989 post-emergence expression profile correlates with the expression of Juvenile Hormone, a key reproductive hormone in mosquitoes, no observable induction occurred when abdominal ligation samples were administered methoprene, a JH analog. However, methoprene impacted a number of other miRNAs, with up to a 3.87 fold induction (miR-1891), and a 3.15 fold suppression (miR-9a) of signal. Subsequent northern analysis provided visual confirmation of observable fold changes for miR-1891 and miR-9a, but not for miRNAs that showed changes below two fold. This analysis provides a foundation to study Juvenile Hormone regulation of miRNAs in mosquitoes. In summary, we have expanded the understanding of microRNAs in mosquitoes. An improved understanding of mosquito physiology can assist in efforts to control mosquito-borne infectious diseases.Ph. D.
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Epanchintsev, Alexey. "Analysis of c-MYC-induced chromosomal instability and generation of a conditional microRNA expression system." Diss., lmu, 2008. http://nbn-resolving.de/urn:nbn:de:bvb:19-83910.
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Vila, Casadesús Maria. "Design of bioinformatic tools for integrative analysis of microRNA-mRNA interactome applied to digestive cancers." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/663087.
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En esta tesis se han desarrollado e implementado distintas herramientas bioinformáticas que permiten el estudio de las interacciones miRNA-mRNA en contextos celulares específicos. oncretamente se ha creado un paquete de R (miRComb) que calcula las interacciones miRNA-mRNA partiendo de expresión de miRNAs y mRNAs, y predicciones bloinformáticas de bases de datos preexistentes. Las interacciones miRNA-mRNA finales son aquellas que muestran una correlación negativa y han estado predichas por al meno una base de datos. Como valor añadido, el paquete miRComb realiza un resumen en pdf con los resultados básicos del análisis (número de interacciones, número de mRNAs target por miRNA, análisis funcional, etc.), que permite comparar los datos de distintos estudios.
Hemos aplicado esta metodología en el contexto de cánceres digestivos. En un primer estudio hemos utilizado datos públicos de 5 cánceres digestivos (colon, recto, esófago, stómago e hígado) y hemos determinado las interacciones miRNA-mRNA comunes entre ellos y específicas de cada uno.
En un segundo estudio, hemos utilizado la misma metodología para analizar datos de IRNA-mRNA en biopsias de pacientes del Hospital Clínic de Barcelona con cáncer de páncreas. En este estudio hemos descrito interacciones miRNA-mRNA en el contexto de cáncer pancreático y hemos podido validar dos de ellas a nivel experimental.
En resumen, podemos concluir que el paquete miRComb es una herramienta útil para el estudio del interactoma de miRNA-mRNA, y que ha servido para establecer hipótesis biológicas que luego se han podido comprobar en el laboratorio.
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Epanchintsev, Alexey. "Analysis of c-MYC-induced chromosomal instability and generation of a conditional microRNA expression system." kostenfrei, 2007. http://edoc.ub.uni-muenchen.de/8391/.
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Klage, Karsten. "Molecular analysis of the responses of Caenorhabditis elegans (Bristol N2), Panagrolaimus rigidus (AF36) and Panagrolaimus sp. (PS 1579) (Nematoda) to water stress." Diss., Virginia Tech, 2008. http://hdl.handle.net/10919/28215.
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This work provides a comparative and genetic analysis of the responses to water stress in desiccation-tolerant and desiccation-sensitive nematodes. Caenorhabditis elegans, a model organism for the study of development, aging, and cell biology was shown to be a desiccation-sensitive organism that survives relative humidities above 40\% for periods of up to seven days. Transcripts from the desiccation-tolerant species Panagrolaimus rigidus AF36 and sp. PS1579, which were expressed uniquely during separate desiccation and osmotic stresses, as well as during recovery from exposure to the dual stresses, were cloned. These sequences were used to search for similarities in the genome sequence data of C. elegans. Putative anhydrobiotic-related transcripts were identified that potentially encode heat shock protein 70, late embryogenic abundant protein, and trehalose-phosphate synthase. Other putative genes that were identified within eight separate libraries encode proteins involved in transcription (histones), protein biosynthesis (ribosomal proteins, elongation factors), protein degradation (ubiquitin, proteases), and transport and cell structure (actin, collagen). Gene ontology analysis of the cloned transcripts revealed that developmental processes are activated during exposure to the stresses as well as during recovery, which may suggest a â rejuvenationâ process as a key to survival in Panagrolaimus nematodes. Genes that were up-regulated during desiccation stress in C. elegans were classified as belonging either to an early response (until 12 hours of stress), or to a late response (after 12 hours of stress). The early response was characterized by the up-regulation of a large number of genes encoding mono-oxygenases, which may suggest onset of oxidation stress during desiccation of C. elegans. The late response was characterized by the appearance of transcripts encoding proteins of the immune system, heat shock proteins (protein denaturation), and superoxide dismutases (oxidation damage). Genes in C. elegans that were down-regulated in response to desiccation stress include those encoding proteases and lysozymes (metabolic shutdown). Genes that encode channel proteins (water homeostasis) were found among the transcripts up-regulated during recovery of C. elegans. The up-regulation of gpdh-1 and hmit-1.1, two transcripts linked to hyperosmotic stress, suggest that osmotic stress is experienced by C. elegans. Comparison of these data with those obtained from exposure of C. elegans to a range of other stresses showing that the nematode C. elegans uses specific transcripts for the desiccation response; transcripts that are not induced in other stresses such as heat, anoxia or starvation. In addition, transcripts regulated during desiccation stress of C. elegans were also regulated during dauer formation, which may indicate common stress tolerant mechanisms. Recent studies in mammalian cells and C. elegans have shown that microRNAs are able to degrade and to sequester mRNA especially during stress in so called stress bodies. In this study, C. elegans microRNA knock-outs showed a significant decrease in desiccation stress survival compared to wild type C. elegans which may suggest the importance of microRNAs for stress survival in C. elegans and other organisms.Ph. D.
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Youssef, Ninwa. "Analysis of conserved microRNA targets in the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster." Thesis, Södertörns högskola, Institutionen för naturvetenskap, miljö och teknik, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:sh:diva-19211.
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MicroRNA (miRNA) is small regulatory non-coding single stranded RNA molecule that can repress protein expression either at transcriptional or translational level. Since their discoveries in nematodes in the early 1990´s extensive research have shown that this mechanism is conserved across species. Because the miRNA is so small, about 22 nucleotides (nt) long and only requires a minimum of 6nt to interact imperfect with its intended target 3´UTR, therefore a single miRNA could potentially have hundreds of potential targets, which have been suggested by computational prediction. The goal of the project is to experimentally verify three predicted Caenorhabditis elegans mir-2 miRNA targets in cell culture, with as candidate targets fos-1, mek-1 and sel-5. In addition C. elegans mir-2 and its mechanism is conserved in Drosophila Melanogaster, miR-2. We want to elucidate if not only mir-2 miRNA is cross species conserved but also it targets. To test this hypothesis we selected the following predicted mir-2 target candidate genes: C. Elegans iff-1 and Drosophila Melanogaster protein ortholog eIF-5A. Validation of miRNA and its functionality was done by transfecting cells with a luciferase-3´UTR reporter only or luciferase-3´UTR and a miRNA-expression plasmid. After the reporter gene was induced, cells were harvested and the luciferase activity measured and the results normalized and compared. Unfortunately our data were inconclusive and future experiments are needed to give a clear picture.
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Akhbari, Pouria. "Analysis of cellular transcriptomic changes induced by Merkel cell polyomavirus miRNA." Thesis, University of Bradford, 2017. http://hdl.handle.net/10454/15902.
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Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with rising global incidence. Merkel cell polyomavirus (MCV) was discovered in 2008 in 80% of MCC samples and since then a causal link between MCV and the majority of MCC cases has been established. microRNAs (miRNA, miR) are a family of small non-coding RNAs which play a key role in post-transcriptional regulation of gene expression and are considered significant players in disease and development in many species. Whilst the focus of MCV research has thus far been on the oncogenic MCV early proteins, large tumour (LT) and small tumour (sT) antigens, there is a knowledge gap regarding MCV miRNA and its functional significance in MCV pathogenesis. Given the emerging importance of viral miRNAs in virus-host interaction and pathogenesis, the aim of this doctoral research project was to investigate alterations in host cell transcripts induced by MCV miRNA and determine any functional significance these might have on virus-host cell interaction. RNA sequencing (RNA-Seq) in the presence and absence of MCV miRNA uncovered a multitude of downregulated cellular transcripts. Gene ontology analysis revealed that MCV miRNA targets transcripts associated with multiple cellular processes, however, regulation of immune response was overrepresented in our datasets. Validation of RNA-Seq data using MCV miRNA mimics and a synthetic, fully replicative MCV genome (MCVSyn) confirmed RNA-Seq data at mRNA and protein expression level for several targets, including the cytokine stimulating gene, SP100, and the neutrophil stimulator chemokine, CXCL8. Moreover, dual luciferase assays revealed that SP100 and MAPK10 (a member of mitogen-activated protein kinases (MAPK) family which is involved in regulation of CXCL8 expression) are directly and specifically targeted and downregulated by MCV miRNA. The MCV miRNA-dependent dysregulation of CXCL8 secretion is associated with impaired neutrophil migration, suggesting that the virus miRNA may be implicated in evasion of the host immune response.
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Sinha, Pritam Bala [Verfasser]. "Functional analysis of microRNA-130b in bovine oocyte maturation and preimplantation embryo development / Pritam Bala Sinha. Landwirtschaftliche Fakultät." Bonn : Universitäts- und Landesbibliothek Bonn, 2011. http://d-nb.info/1016262914/34.
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Maragkakis, Emmanouil Verfasser], Ivo [Akademischer Betreuer] Grosse, Artemis-Geōrgia [Akademischer Betreuer] [Chatzēgeōrgiu, and Wojciech [Akademischer Betreuer] Makalowski. "Bioinformatics approach for microRNA target prediction and functional analysis / Emmanouil Maragkakis. Betreuer: Ivo Grosse ; Artemis Hatzigeorgiou ; Wojciech Makalowski." Halle, Saale : Universitäts- und Landesbibliothek Sachsen-Anhalt, 2011. http://d-nb.info/1025202783/34.
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Monkeviciute, Aiste. "Analysis of microRNA role in the development of left ventricular hypertrophy in the stroke-prone spontaneously hypertensive rat." Thesis, University of Glasgow, 2014. http://theses.gla.ac.uk/4710/.
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MicroRNAs (miRs) are a group of short non-coding RNAs, on average 22 nucleotides in length, that form an important axis of post-transcriptional regulation of gene expression. They have been identified as major modulators of all biological processes including development, cell differentiation, growth and apoptosis as well as diseases such as cancer, diabetes and cardiovascular disease (CVD). In the developed world CVD remains the leading cause of morbidity and mortality, and a substantial burden on healthcare. Left ventricular hypertrophy (LVH) is defined as an increase in thickness of the myocardium and is an important risk factor in CVD. The stroke-prone spontaneously hypertensive rat (SHRSP) is an animal model of essential hypertension used in research of CVD together with a normotensive reference strain Wistar-Kyoto (WKY). The SHRSP animals exhibit an increase in the size of myocardium prior to the onset of hypertension and have established LVH at 16 weeks of age thus are a good model for investigating the genetics of this condition. The aim of this project was to identify signature expression patterns of novel and previously implicated microRNAs and to investigate their role in the development of LVH in the SHRSP. Furthermore, potential gene targets of candidate selected microRNAs were identified to investigate biological pathways involved in the disease process. MicroRNA microarray profiling was performed by Dr. McBride in the hearts of 5 week old SHRSP and WKY male rats using the LC Sciences (LCS) multispecies chip based on Sanger miRBase 11.0. The data were analysed (Drs. McBride and McClure) using Rank Product (RP) analysis method and evaluated in combination with the statistical analysis provided by LC Sciences (LCS). LCS data indicated 103 microRNAs differentially expressed at 5 weeks of age, 64 at 16 weeks of age, with 9 in common. The RP analysis identified 72 microRNAs differentially expressed between WKY and SHRSP at 5 weeks of age and 51 at 16 weeks of age, and 21 microRNAs were differentially regulated at both time points. Both methods identified a subset of 35 microRNAs in 5 week old hearts and 8 in 16 week old samples. TaqMan® microRNA assays were used to confirm these expression patterns. Based on these data and published literature candidate microRNAs – miR-195, miR-329 and miR-451 were selected for further experimental investigation. Expression of candidate microRNAs (miR-195, miR-329 and miR-451) in neonatal hearts of SHRSP and WKY rats was also investigated. It was found that all three candidate microRNAs were differentially expressed at this time point and there were significantly increased levels in the SHRSP compared to WKY. Cardiac cell line H9c2 AngII model of hypertrophy was used to investigate the effect of AngII on our candidate miRNA expression levels. A 96 hour stimulation of H9c2 cell with AngII resulted in a significant increase in cell size. Levels of miR-195 and miR-329 were not affected by addition of AngII; expression of miR-451 was significantly down-regulated immediately post stimulation, however levels were increased at the final assessment at 96 hours. Adenoviral vectors over-expressing miR-195, miR-329 and miR-451 were designed and generated. These vectors were used to investigate if overexpression of each individual miR could affect cell size in the selected in vitro model of cardiomyocyte hypertrophy. It was found that all candidate microRNAs reduced AngII mediated hypertrophic cell growth at higher doses. Identifying pathways and specific gene targets affected by changes in microRNA levels is of paramount importance. Availability of such data not only provides information about regulation of cardiac homeostasis, but also possible therapeutic approaches for treatment and prevention. Target prediction algorithms (DIANAmT, miRanda, miRDB, miRWalk, PICTAR5, PITA, RNA22, RNAhybrid and Targetscan) were used to identify potential gene targets for candidate microRNAs. To refine these lists to genes relevant to the experimental design Ingenuity Pathway analysis (IPA 9.0) software was used to overlay microRNA microarray data with results of heart mRNA gene expression data (M. McBride, personal communications) from the same cardiac tissue and to relate these to appropriate pathways and cellular functions. A list of 12 genes was generated: similar to CG4768-PA (RGD1309748), KN motif and ankyrin repeat domains 1 (Kank1), sterile alpha motif domain containing 4B (Samd4b), dual specificity phosphatase 10 (Dusp10), follistatin-like 3 (secreted glycoprotein) (Fstl3), jun D proto-oncogene (JunD), forkhead box M1 (Foxm1), SIN3 homolog A transcription regulator (yeast) (Sin3a), cyclin-dependent kinase 1 (Cdk1), kinesin family member 23 (Kif23), bone morphogenetic protein receptor type IA (Bmpr1a) and sestrin 1 (Sesn1). Expression of these candidate targets was assessed in heart tissues from neonates, 5 and 16 week old rats. Six out of ten of these targets were differentially expressed at one or more time points. To further investigate the proposed targeting of these genes by candidate microRNAs, expression levels were measured in each of the predicted targets in H9c2 cell transduced with miR over-expressing viruses. The expression patterns of Cdk1, Kif23, Kank1 and Sin3a were consistent with overexpression of the targeting microRNA, i.e. expression of each gene was down-regulated. In summary, data presented in this thesis elucidate the role of miR-195, miR-329 and miR-451 in the development of LVH in the SHRSP. Understanding the underlying cause for differential expression of these candidate microRNAs, confirming gene targets and identifying relevant pathways will improve the understanding of LVH at the molecular level. It will also help explain the pathophysiology of cardiovascular disease development in this rat model of human hypertension providing a basis for the development of novel therapeutic approaches to treat or prevent LVH.
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Ricci, Pierbruno. "The Renal Cysts and Diabetes syndrome : from transcriptional profiling and functional analysis of a novel mouse model to biomarkers evaluation in human patients." Thesis, Sorbonne université, 2018. http://www.theses.fr/2018SORUS111/document.
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Les mutations hétérozygotes du gène codant pour le facteur de transcription HNF1B sont à l'origine d'un syndrome multisystémique complexe connu sous le nom de « Renal Cysts and Diabetes » (RCAD). Un modèle de souris généré dans notre laboratoire s'est avéré reproduire plusieurs caractéristiques de la maladie humaine. Nous avons réalisé un séquençage ARNm-microARN à différents stades de développement (E14,5 ; E15,5 ; E17,5) de ce modèle. Nous avons montré que les gènes les plus dérégulés étaient impliqués dans les processus métaboliques de transport, de lipides et d’acides organiques et étaient exprimés dans les tubules proximaux et, dans une moindre mesure, dans l’anse de Henlé et les canaux collecteurs. Nous avons sélectionné quatre microARN (miR-802, 194-2, 192 et -30a), régulés à la baisse et potentiellement contrôlés par HNF1B. Des expériences de transactivation de gène rapporteur dans des cellules HEK-293 ont montré que HNF1B était capable de transactiver la transcription de ces microARN via des sites de liaison présents dans les séquences régulatrices de ces gènes. En utilisant des microARN MIMICS nous avons par la suite montré que mir-802, mir-194-2 et mir-192 étaient capables d'inhiber l’expression d’un gène rapporteur contenant la région 3'UTR de HNF1B. L'analyse d'échantillons d'urine de 22 patients RCAD et de 22 contrôles sains a permis d'identifier 146 peptides excrétés de manière différentielle et associés au syndrome. En utilisant ces résultats dans un modèle mathématique, classificateur prédit efficacement le syndrome RCAD avec une sensibilité de 91.7% et une spécificité de 91.1% sur une large population de patientsHeterozygous mutations in the gene encoding the transcription factor HNF1B are the cause of a complex multisystem syndrome known as Renal Cysts And Diabetes (RCAD). A mouse model generated in our laboratory was shown to reproduce several features of the human disease. We performed high-throughput mRNA-microRNA sequencing at different developmental stages (E14.5, E15.5, E17.5). We showed that the most down-regulated genes were involved in transport, lipid and organic acid metabolic processes and expressed in proximal tubules and to a lesser extent in the loop of Henle and collecting ducts. We then selected four microRNAs (mir-802, 194-2, 192 and -30a), which were down-regulated and potentially controlled by HNF1B. Luciferase assays in HEK-293 cells showed that HNF1B was able to specifically transactivate in a dose response mode these microRNAs through binding HNF1B-binding sites in their regulatory promoter/enhancer upstream sequences. We subsequently showed by luciferase assays using miRNA MIMICS that mir-802, mir-194-2 and mir-192 were able to inhibit luciferase vectors containing the 3’UTR of Hnf1b. Analysis of urine samples from 22 RCAD patients and 22 healthy controls led to the identification of 146 peptides differentially excreted and associated with RCAD including a similarity regarding collagen and uromodulin fragments with the RCAD mouse model. Combining the peptides into a mathematical model we used independent cohorts of patients to validate the prediction of the RCAD syndrome. Our classifier efficiently predicted RCAD syndrome with 91.7% sensitivity and 91.1% specificity on a wide population
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Abd-el-Naby, Walaa Slouma Hamouda [Verfasser]. "Expression analysis of regulatory MicroRNA in bovine cumulus oocyte complex and preimplantation embryos / Walaa Slouma Hamouda Abd El Naby." Bonn : Universitäts- und Landesbibliothek Bonn, 2012. http://d-nb.info/1043054782/34.
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Theiss, Julia. "Analysis of Sex Myoblast Migration in mir-44/45 C. elegans Mutants." Scholarship @ Claremont, 2019. https://scholarship.claremont.edu/scripps_theses/1327.
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microRNAs are single-stranded small RNAs that function as post-transcriptional regulators of gene expression. We are studying the mir-44 family, specifically mir-44 and mir-45, which have identical sequence. Loss of mir-44 and mir-45 results in defects that suggest that the mir-44 family acts to negatively regulate the MAPK pathway. The MAPK pathway regulates sex myoblast migration, a process which is required for normal egg laying. We hypothesized that the mir-44 family of microRNAs is necessary for normal sex myoblast migration and subsequent formation of the functional egg laying structure in the hermaphrodite. We created a mutant that had mutations in both mir-44 and mir-45 and a transgene that expresses GFP in the sex myoblast cells. Then we observed the migration and division of the sex myoblasts in wild-type and mutant worms using fluorescence microscopy. In all cases, the mutant worms displayed a greater percent difference from average sex myoblast migration and division. However, a two-tailed two-proportions z-test found no significant difference between wild type and mutant sex myoblast migration (p=0.9148), nor in mutant sex myoblast division along the axial (p=0.4205) and sagittal (p=0.3583) planes of the body. This allows us to conclude that mir-44 and mir-45 are unlikely to be responsible for the migration nor division of the sex myoblasts, and the defects are likely due to interference with a different biological mechanism.
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Lombe, Chipampe Patricia. "Analysis, expression profiling and characterization of hsa-miR-5698 target genes as putative dynamic network biomarkers for prostate cancer: a combined in silico and molecular approach." University of the Western Cape, 2019. http://hdl.handle.net/11394/7026.
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Philosophiae Doctor - PhD2018, the International Agency for Research on Cancer (IARC) estimated that prostate cancer (PCa) was the second leading cause of death in males worldwide. The number of deaths are expected to raise by 50 % in the next decade. This rise is attributed to the shortcomings of the current diagnostic, prognostic, and therapeutic biomarkers used in the management of the disease. Therefore, research into more sensitive, specific and effective biomarkers is a requirement. The use of biomarkers in PCa diagnosis and management takes advantage of the genetic alterations and abnormalities that characterise the disease. In this regard, a microRNA, hsa-miR-5698 was identified in a previous study as a differentiating biomarker between prostate adenocarcinoma and bone metastasis. Six putative translational targets (CDKN1A, CTNND1, FOXC1, LRP8, ELK1 and BIRC2) of this microRNA were discovered using in silico approaches. The aim of this study was to analyse via expression profiling and characterization, the target genes of hsa-miR-5698 in order to determine their ability to act as putative dynamic network biomarkers for PCa. The study was conducted using a combined in silico and molecular approach. The in silico part of the study investigated the putative transcriptional effects of hsa-miR-5698 on the promotors of its translational targets, the correlation between hsa-miR-5698 and mRNA expression profiles as well as the co-expression analysis, pathway analysis and prognostic ability of the target genes. A number of computational software were employed for these purposes, including, R Studio, Trident algorithm, STRING, KEGG, MEME Suite, SurvExpress and ProGgene. The molecular part of the study involved expression profiling of the genes in two PCa cell line LNCaP and PC3 via qPCR.
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Jan, Michael. "Novel Mechanisms Underlying Homocysteine-Suppressed Endothelial Cell Growth." Diss., Temple University Libraries, 2014. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/264103.
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PharmacologyPh.D.
Cardiovascular disease (CVD) is the leading cause of death worldwide, and is projected to remain so for at least the next decade. Ever since its discovery in the urine and blood of children with inborn errors of metabolism, homocysteine (Hcy) at elevated plasma concentrations has been associated with CVD clinically and epidemiologically. Observational studies and meta-analyses have noted that changes in plasma Hcy by 5μM increase the odds ratio of developing coronary artery disease by 1.6-1.8 among other CVD. Clinical trials aimed at reducing plasma Hcy for benefit against development of subsequent cardiovascular events have had unconvincing results, but have moreover failed to address the mechanisms by which Hcy contributes to CVD. Recommendations from national agencies like the American Heart Association and the United States Preventive Services Task Force emphasize primordial prevention as a way to combat CVD. Reducing plasma Hcy as secondary and primary interventions does not fulfill this recommendation. In order to best understand the role of Hcy in CVD, an investigation into its mechanisms of action must be undertaken before measures of primordial prevention can be devised. Numerous experimental studies in the literature identify vascular endothelium as a target for the pathological effects of Hcy. Endothelial injury and impairment are contributory processes to atherosclerosis, and Hcy has been demonstrated to inhibit endothelial cell (EC) growth and proliferation through mechanisms involving cell cycle arrest, oxidative stress, and programmed cell death in vitro. Animal models have also confirmed that high levels of Hcy accelerate atherosclerotic plaque development and lead to impairment of vascular reendothelialization following injury. Hcy has been shown to have the opposite effect in vascular smooth muscle cells (SMC), causing their proliferation and again contributing to atherosclerosis. The cell-type specificity of Hcy remains to be understood, and among the aims of this research was to further characterize the effects of Hcy in EC. The overarching goal was discovery in order to direct future investigations of Hcy-mediated pathology. To begin, the first investigation considered the transcriptional and regulatory milieu in EC following exposure to Hcy. High-throughput screening using microarrays determined the effect of Hcy on 26,890 mRNA and 1,801 miRNA. Two different in vitro models of hyperhomocysteinemia (HHcy) were considered in this analysis. The first used a high dose of 500µ Hcy to mimic plasma concentrations of patients wherein the transsulfuration pathway of Hcy metabolism is impaired as in inborn cystathionine-ß-synthase deficiency. The other set of conditions used 50µ Hcy in the presence of adenosine to approximate impairment of the remethylation pathway of Hcy metabolism wherein s-adenosylhomocysteine accumulates, thus inhibiting s-adenosylmethionine formation and methylation reactions. These distinctions are important because most clinical trials do not distinguish between causes of HHcy, thereby ignoring the specific derangements underlying HHcy. mRNA and miRNA expression changes for both sets of treatment conditions identified CVD as a common network of Hcy-mediated pathology in EC. Moreover, methylation-specific conditions identified cell cycle modulation as a major contributory mechanism for this pathology, which agrees with recent findings in the literature. Analysis of significant mRNA changes and significant miRNA changes independently identified roles for Hcy in CVD and cell cycle regulation, thereby suggesting that miRNA may mediate the effects of Hcy in addition to gene expression changes alone. To investigate the role of Hcy in the cell cycle further, the next set of investigations considered the effect of Hcy under conditions approximating impaired remethylation in early cell cycle events. Previous studies have demonstrated that Hcy inhibits cyclin A transcription in EC via demethylation of its promoter. Conversely, Hcy induces cyclin A expression in SMC, again making the case for a cell type-specific mechanism in EC. Preceding cyclin A transcription and activation, canonical events in the early cell cycle include D-type cyclin activation, retinoblastoma protein (pRB) phosphorylation, and transcription factor E2F1 activation. In a series of in vitro experiments on EC, it was seen that Hcy inhibits expression of cyclin D2 and cyclin D3, but not cyclin D1. Next, pRB phosphorylation was seen to be decreased following treatment with Hcy. This also led to decreased E2F1 expression. However, this series of events could be reversed with E2F1 supplementation, allowing the cell cycle to proceed. As Hcy exerts a number of its effects via regulation of gene transcription, a final series of investigations aimed to predict potential targets of Hcy by examining patterns of transcription factor binding among known targets of Hcy regulation. Gene promoters of Hcy-modulated genes were analyzed in order to determine common transcription factors that potentially control their regulation. The locations of CpG-rich regions in promoters were identified to determine which regions would be most susceptible to regulation by DNA methylation. Next, high-throughput next-generation sequencing (NGS) and bisulfite NGS was performed for DNA from EC treated with Hcy in order to determine methylation changes after Hcy treatment. A number of potential transcription factors and their binding sites were identified as potential mediators of Hcy-mediated gene regulation. Taken together, these investigations represent an exploration of Hcy-mediated pathology in CVD, by focusing upon novel regulatory mechanisms in EC. Objective high-throughput arrays identified roles for Hcy in CVD and cell cycle pathways regulated by miRNA and gene expression, which were confirmed experimentally in vitro. These observations led to an investigation and identification of common transcription factors that potentially regulate Hcy-altered gene expression. This framework may be used to guide future investigations into the complex pathological network mediating the effects of Hcy in CVD. First, identification of a role for miRNA in mediating the effects of Hcy represents a novel regulatory mechanism, heretofore largely unexplored. Next, expanding the role of Hcy in EC cell cycle regulation to identify upstream mediators greatly adds to the published literature. Finally, noting that these changes center upon transcriptional and post-transcriptional regulation gives import to developing methods to characterize promoter and transcription factor regulation. The investigations presented herein and their results provide evidence that the future of Hcy research is vibrant, relevant, and not nearly surfeit.
Temple University--Theses
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Hu, Haiyang [Verfasser]. "Computational and Statistical Analysis of Sequence and Expression Features of MicroRNA and Long Noncoding RNA in Primate Brains / Haiyang Hu." Berlin : Freie Universität Berlin, 2016. http://d-nb.info/1093404175/34.
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Paluschinski, Martha [Verfasser]. "The Functional Analysis and Characterization of the Liver-Specific MicroRNA miR 122 and of its Associated Target Genes / Martha Paluschinski." Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2021. http://d-nb.info/1225555760/34.
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Stokowy, Tomasz, Markus Eszlinger, Michał Świerniak, Krzysztof Fujarewicz, Barbara Jarząb, Ralf Paschke, and Kurt Krohn. "Analysis options for high-throughput sequencing in miRNA expression profiling." Universitätsbibliothek Leipzig, 2014. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-144393.
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Background: Recently high-throughput sequencing (HTS) using next generation sequencing techniques became useful in digital gene expression profiling. Our study introduces analysis options for HTS data based on mapping to miRBase or counting and grouping of identical sequence reads. Those approaches allow a hypothesis free detection of miRNA differential expression. Methods: We compare our results to microarray and qPCR data from one set of RNA samples. We use Illumina platforms for microarray analysis and miRNA sequencing of 20 samples from benign follicular thyroid adenoma and malignant follicular thyroid carcinoma. Furthermore, we use three strategies for HTS data analysis to evaluate miRNA biomarkers for malignant versus benign follicular thyroid tumors. Results: High correlation of qPCR and HTS data was observed for the proposed analysis methods. However, qPCR is
limited in the differential detection of miRNA isoforms. Moreover, we illustrate a much broader dynamic range of HTS compared to microarrays for small RNA studies. Finally, our data confirm hsa-miR-197-3p, hsa-miR-221-3p, hsa-miR-222-3p and both hsa-miR-144-3p and hsa-miR-144-5p as potential follicular thyroid cancer biomarkers.
Conclusions: Compared to microarrays HTS provides a global profile of miRNA expression with higher specificity and in more detail. Summarizing of HTS reads as isoform groups (analysis pipeline B) or according to functional criteria (seed analysis pipeline C), which better correlates to results of qPCR are promising new options for HTS analysis. Finally, data opens future miRNA research perspectives for HTS and indicates that qPCR might be limited in validating HTS data in detail.
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Herráiz, Yebes Alba. "Molecular analysis of ovarian resorption by hydric stress in Blatella germanica." Doctoral thesis, Universitat Pompeu Fabra, 2014. http://hdl.handle.net/10803/299212.
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In the present thesis we studied the effects of hydric stress in the ovary of the cockroach Blattella germanica. At morphological level, we described ovarian resorption induced by water deprivation. At a molecular level, we looked for markers differentially expressed under hydric stress. First, we focused on aquaporins given their role as water channel proteins. We cloned, sequenced and functionally characterised an aquaporin that transports water and urea. Then, we obtained a library of microRNAs (small non-coding RNAs that regulate gene expression post transcriptionally) present in the ovary under stress conditions. We analysed the differential expression of 13 of them and we observed that the majority were overexpressed under hydric stress. We selected miR-34-5p to perform a deeper study, including a study of their potential target mRNAs. Among the possible targets, we studied the function of Windei, a factor that resulted necessary for the trimethylation of lysine 9 in histone H3 and for chorion formation. Finally, we selected CARMER (an arginine methyltransferase) as hydric stress marker candidate. We assessed that it is overexpressed in response to hydric stress and that it is necessary for the dimethylation of arginine 17 in histone H3.En esta tesis estudiamos los efectos del estrés hídrico en el ovario de la cucaracha Blattella germanica. A nivel morfológico describimos el proceso de reabsorción ovárica tras la privación de agua. A nivel molecular buscamos marcadores diferencialmente expresados en respuesta a este estrés. Primero nos centramos en las acuaporinas por su papel como proteínas que forman un canal transportador de agua. Clonamos, secuenciamos y caracterizamos funcionalmente una acuaporina que transporta agua y urea. Después obtuvimos una biblioteca de microRNAs (RNAs cortos no codificantes que regulan la expresión génica post-transcripcionalmente) presentes en el ovario en situación de estrés. Analizamos la expresión diferencial de 13 de ellos y observamos que la mayoría se sobreexpresaban en situación de estrés. Elegimos miR-34-5p para hacer un estudio más profundo, incluyendo un análisis de sus potenciales mRNA diana. De entre ellos, estudiamos la función de Windei y comprobamos que dicho factor es necesario para la trimetilación de la lisina 9 en la histona H3 y para la formación del corion. Por último, seleccionamos CARMER (una arginina methyltransferasa) como candidato a marcador de estrés hídrico. Comprobamos que CARMER se sobreexpresa en respuesta a estrés y que es necesario para la dimetilación de la arginina 17 en la histona H3.
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Rosewick, Nicolas. "Next-generation transcriptome analysis of deltaretrovirus induced leukemia: from microRNAs to macroRNAs." Doctoral thesis, Universite Libre de Bruxelles, 2015. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209102.
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Plus de 20 million de personnes à travers le monde sont infectées par le virus T-lymphotrope humain de type 1 (HTLV-1), causant des leucémies à cellules T dans ~5 % des individus infectés. Le virus de la leucémie bovine (BLV), structurellement et fonctionnellement proche de HTLV-1, induit des leucémies à cellules B dans des modèles animaux suite à une infection naturelle (bovin) ou expérimentale (mouton). Les mécanismes moléculaires responsables du potentiel oncogène de ces deux virus restent largement incompris. Dans les deux maladies, leucémies T chez l’homme, leucémies B chez l’animal, le site intégration du virus dans les cellules leucémiques est très variable. Il est donc généralement admis que le potentiel oncogène du provirus est principalement lié à l’activité de l’oncoprotéine virale Tax. De manière paradoxale cependant, ni HTLV-1 ni BLV n’expriment de protéines virales au stade tumoral. Dans ce travail, nous avons étudié le transcriptome non codant des leucémies induites par BLV et HTLV-1 par séquençage à haut débit. Dans la première partie, nous démontrons que le provirus BLV n’est en fait pas silencieux dans les cellules tumorales. BLV produit un ensemble de dix microARNs (miRNAs) très abondants et extrêmement conservés dans toutes les tumeurs. Cette observation constitue la première description de miRNAs encodés par un rétrovirus. Les microARNs encodés par BLV sont transcrits par la RNA Polymérase III, stratégie qui permet leur production de façon indépendante de celle des messagers viraux ainsi que leur expression abondante dans le contexte tumoral caractérisé par l’absence d’activité RNA Polymérase II. Nous avons ensuite montré que, comme HTLV-1, BLV produit des transcrits encodés par le brin négatif du provirus. L’analyse par séquençage ARN à haut débit (RNA-Seq) de tumeurs induites par BLV montre l’absence d’expression virale à partir du promoteur viral situé dans le LTR 5’. Cependant, elle révèle la présence de deux transcrits viraux anti-sens non codants (AS1 et AS2) produits par le LTR 3’. Nous avons identifié ces transcrits dans toutes les tumeurs BLV analysées. Enfin, l’analyse RNA-Seq de tumeurs induites par HTLV-1 et BLV a révélé la présence d’interactions transcriptionnelles virus-hôte. Les gènes hôtes affectés sont significativement enrichis en gènes liés au cancer. Ces résultats suggèrent que les transcrits HTLV hbz et BLV AS1 jouent un rôle essentiel dans la tumorigenèse en interagissant avec le génome de l’hôte. Nous avons également détecté ce type de perturbation à des temps précoces dans le modèle expérimental BLV chez le mouton. Ces observations suggèrent que ces interactions virus-hôte constituent des événements précoces qui procurent un avantage sélectif aux clones associés, mais que d’autres altérations -génétiques et/ou épigenetiques- sont nécessaires à l’établissement de la tumeur. En conclusion, nos travaux vont permettre de mieux comprendre le rôle des interactions virus-génome hôte dans l’oncogenèse ainsi que la fonction de transcrits non codants dans le développement des cancers qu’ils soient ou non d’étiologie virale.More than 20 million people are infected by Human T-cell Lymphotropic Virus type 1 (HTLV-1) worldwide and this will cause T-cell leukemia in 5% of them. Yet the molecular mechanisms that underlie the oncogenic potential of this virus remain largely unknown. Bovine Leukemia Virus (BLV) is closely related to HTLV1 and causes a very similar B-cell leukemia in cattle and sheep. As for HTLV1, the oncogenic mechanisms underlying BLV-induced leukemia remain poorly understood. In both diseases, leukemic cells harbor mainly one integrated provirus, yet the integration sites are very variable. As a consequence, it is generally assumed that the oncogenic effect of the provirus is largely mediated by the virally encoded Tax protein. Paradoxically, however, both HTLV1 and BLV proviruses are found to be epigenetically silenced in tumor cells. Thus Tax, as any other virally encoded protein, is not expressed in leukemic cells suggesting that other factors are involved in tumorigenesis. In this study we made three observations that might dramatically change the prevalent dogma of HTLV1 and BLV-induced leukemia. First, we demonstrated that the BLV provirus is not silent at all in tumor cells. A cluster of BLV-encoded microRNAs (miRNAs) is highly expressed, accounting for 40% of the miRNAs present in leukemic cells. This finding is the first description of retroviral-encoded miRNAs. BLV miRNAs are transcribed from five independent RNA Pol III units and are exceedingly conserved across BLV isolates (more than the protein coding genes), strongly supporting an essential yet still unknown function. Next we showed that – as HTLV1 – BLV strongly expresses antisense RNAs. High-throughput sequencing of RNA libraries (RNA-seq) from BLV associated tumors, as expected, showed no expression of viral mRNA from the 5’ LTR. However, it did reveal the presence of two novel non-coding antisense transcripts originating in the 3’ LTR of BLV. Finally, RNA-Seq analysis of HTLV-1 and BLV-induced tumors revealed that the viral 3’ LTR-driven antisense RNAs produced by both viruses interact with host genes localized in the vicinity of proviral integration. Enrichment analysis of affected host genes suggests a significant bias towards cancer-related genes. Host gene perturbations were also found at early stages post-infection in the BLV experimental model in sheep, suggesting that provirus-dependent cancer driver gene perturbations trigger initial amplification of the corresponding clones, requiring additional genetic and/or epigenetic changes to develop full blown leukemia. Overall, our findings reveal an unexpected role for BLV and HTLV antisense transcripts and contribute to the understanding of non-coding RNA-mediated mechanisms in leukemogenesis.
Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished
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Bissels, Ute [Verfasser]. "Combined analysis of microRNA and mRNA signatures in human hematopoietic stem and progenitor cells using a novel microarray quantification system / Ute Bissels." Aachen : Hochschulbibliothek der Rheinisch-Westfälischen Technischen Hochschule Aachen, 2011. http://d-nb.info/1016243669/34.
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Zillmer, Stephanie [Verfasser], Michaela [Akademischer Betreuer] [Gutachter] Nathrath, and Stefan [Gutachter] Burdach. "Integrative analysis of microRNA and mRNA expression profiles in osteosarcoma cell lines / Stephanie Zillmer. Betreuer: Michaela Nathrath. Gutachter: Michaela Nathrath ; Stefan Burdach." München : Universitätsbibliothek der TU München, 2016. http://d-nb.info/1106382161/34.
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Preusse, Martin [Verfasser], Fabian J. [Akademischer Betreuer] [Gutachter] Theis, and Hans-Werner [Gutachter] Mewes. "Analysis of microRNA function using systemic regulatory features and graph models / Martin Preusse ; Gutachter: Fabian J. Theis, Hans-Werner Mewes ; Betreuer: Fabian J. Theis." München : Universitätsbibliothek der TU München, 2016. http://d-nb.info/1144483271/34.
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Koliha, Nina [Verfasser], Falk [Gutachter] Nimmerjahn, and Andreas [Gutachter] Baur. "Analysis of the microRNA profile and origin of exosomes in plasma of melanoma patients and healthy individuals / Nina Koliha ; Gutachter: Falk Nimmerjahn, Andreas Baur." Erlangen : Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU), 2016. http://d-nb.info/1116712822/34.
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Thibord, Florian. "Variation génétique et plasmatique des microARNs : impact sur les paramètres biologiques de l’hémostase OPTIMIR, a novel algorithm for integrating available genome-wide genotype data into miRNA sequence alignment analysis A Genome Wide Association Study on plasma FV levels identified PLXDC2 as a new modifier of the coagulation process." Thesis, Sorbonne université, 2019. http://www.theses.fr/2019SORUS379.
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Les microARNs (miARNs) sont les membres d’une classe de petits ARNs non codants d’environ 22 nucléotides, dont le mécanisme principal est de réguler l’expression des gènes dans le cytoplasme. Leurs importance est telle qu’il est estimé que la majorité des gènes humains sont régulés par ces petits ARNs, et ils sont ainsi potentiellement impliqués dans le développement de nombreuse pathologies. La séquence des miARNs peut-être soumise à des variations post- transcriptionnelles et des variations génétiques générant alors des séquences isoformes appelées isomiRs. Afin de détécter et quantifier précisemment l’expression des miARNs à partir de données de séquençage, cette hétérogénéité intra-miARN, due aux isomiRs, doit être prise en compte, tout comme l’homogénéité inter-miARN due aux miARNs paralogues. Le pipeline optimiR, développé dans le cadre de cette thèse, permet de surmonter ces challenges grâce notamment à l’intégration de l’information génétique des échantillons analysés, ainsi qu’à une stratégie d’alignement originale, qui permettent de détecter les isomiRs tout en distinguant les miARNs paralogues. Les données analysées lors de cette thèse proviennent de la cohorte MARTHA, composée de patients ayant développé une thrombose veineuse (VTE), parfois avec récidive. L’expression normalisée de 162 miARNs obtenue pour 344 patients a ensuite été utilisée afin d’analyser: 1) les déterminants génétiques de l’expression de ces miARNs; 2) l’association des miARNs avec le risque de récidive pour la VTE; 3) les corrélations avec certains paramètres biologiques de l’hémostase. Collectivement, ces analyses m’ont permis d’identifier des microARNs d’intérêt pour la recherche sur la thrombose veineuse et sur l’hémostaseMicroRNAs (miRNA) are small non coding RNAs with an average size of 22 nucleotides, mainly known to regulate gene expression in the cytoplasm. These small RNAs are estimated to regulate the majority of human genes, and are potentially involved in several diseases. MiRNA sequences might contain genetic variants and can undergo post-transcriptional variations, which generate miRNA isoforms called isomiRs. In order to accurately detect and quantify miRNA expression, isomiRs as well as paralogous miRNAs must be accounted for. The optimiR pipeline developed during this project overcome these challenges by integrating genetic information and by implementing an original strategy based on local alignement. Sequencing data were obtained from the MARTHA cohort, which is composed of french unrelated patients who experienced venous thrombosis (VTE). Normalized expression of 162 miRNAs from 334 patients were used to analyze: 1) the genetic determinants of miRNA expression; 2) the association of miRNA expression levels with VTE recurence; 3) the correlations between miRNA expression levels and hemostatic traits. As a whole, these analyses allowed me to identify miRNAs of interest for the study of VTE and hemostasis
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Moss, Tiffanie. "CHARACTERIZATION OF STRUCTURAL VARIANTS AND ASSOCIATED MICRORNAS IN FLAX FIBER AND LINSEED GENOTYPES BY BIOINFORMATIC ANALYSIS AND HIGH-THROUGHPUT SEQUENCING." Case Western Reserve University School of Graduate Studies / OhioLINK, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=case1333648149.
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