Tesis: "Long non-coding RNA"

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Ozes, Ali Rayet. "Targeting the long non coding RNA HOTAIR in cancer". Thesis, Indiana University, 2016. http://pqdtopen.proquest.com/#viewpdf?dispub=10154781.

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Ovarian cancer (OC) takes the lives of nearly 14,000 US women every year. Although platinum is one of the most effective drugs in treating ovarian cancer, the development of platinum resistance is one of the biggest challenges facing patients. I have shown that the long non-coding RNA HOTAIR contributes to platinum-resistant OC and determined the regulators and targets of HOTAIR during the platinum-induced DNA damage response. My published data supports the role of HOTAIR in contributing to DNA damage induced cellular senescence and secretion of pro-inflammatory cytokines leading to cisplatin resistance. My unpublished work (under review) analyzed the interaction of HOTAIR with the PRC2, its known interacting partner. In this study, I developed a novel strategy blocking HOTAIR-PRC2 interaction and resensitized ovarian tumors to platinum in mouse studies. The results offer a pre-clinical proof of concept for targeting long non-coding RNAs as a therapeutic approach and may represent a strategy to overcome chemotherapy resistance in tumors exhibiting high expression of HOTAIR, a frequent observation in high grade serous OC.

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2

Ard, Ryan Anthony. "Functional long non-coding RNA transcription in Schizosaccharomyces pombe". Thesis, University of Edinburgh, 2016. http://hdl.handle.net/1842/20396.

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Eukaryotic genomes are pervasively transcribed and frequently generate long noncoding RNAs (lncRNAs). However, most lncRNAs remain uncharacterized. In this work, a set of positionally conserved intergenic lncRNAs in the fission yeast Schizosaccharomyces pombe genome are selected for further analysis. Deleting one of these lncRNA genes (ncRNA.1343) exhibited a clear phenotype: increased drug sensitivity. Further analyses revealed that deleting ncRNA.1343 also disrupted a previously unannotated lncRNA, termed nc-tgp1, transcribed in the opposite orientation of the predicted ncRNA.1343 gene and into the promoter of the phosphate-responsive permease gene tgp1+. Detailed analyses revealed that the act of transcribing nc-tgp1 into the tgp1+ promoter increases nucleosome density and prevents transcription factor access. Decreased nc-tgp1 transcription permits tgp1+ expression upon phosphate starvation, while nc-tgp1 loss induces tgp1+ in repressive phosphate-rich conditions. Notably, drug sensitivity results directly from tgp1+ expression in the absence of nc-tgp1 transcription. Similarly, lncRNA transcription upstream of pho1+, another phosphate-regulated gene, increases nucleosome density and prevents transcription factor binding to repress pho1+ in phosphate-replete cells. Importantly, the regulation of tgp1+ and pho1+ by upstream lncRNA transcription occurs in the absence of RNAi and heterochromatin components. Instead, the regulation of tgp1+ and pho1+ by upstream lncRNA transcription resembles examples of transcriptional interference reported in other organisms. Thus, tgp1+ and pho1+ are the first documented examples of genes regulated by transcriptional interference in S. pombe.

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3

Ottway, Charlotte Jane. "Characterisation of Nespas, a non-coding imprinted RNA". Thesis, University of Oxford, 2010. http://ora.ox.ac.uk/objects/uuid:b159c1e9-8d49-460c-a808-d920e8e17779.

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Nespas is the non-coding antisense transcript of the imprinted Gnas cluster; it is expressed from the paternal allele and is located on mouse distal chromosome 2. In this thesis new transcripts of >10 kb and 0.8 kb have been identified. The 0.8 kb transcript is a spliced variant that is retained in the nucleus and its 3’ end lies approximately 30 kb from the start site. Transcription from the Nespas promoter does not proceed beyond this point. A collection of previously known splice variants have also been detected and are exported to the cytoplasm. Nespas is expressed in the embryo during the second half of gestation and peaks at 13.5 dpc. Nespas is imprinted in the placenta at 11.5, 15.5 and 17.5 dpc. The Nespastm4Jop allele, to truncate the Nespas transcript 10.5 kb from the start site, has been transmitted through the germline and a breeding colony established. Preliminary analysis shows Nespas has a regulatory function. A second targeting construct to truncate Nespas 12.5 kb from the start site has been designed and assembled to investigate whether the 3’ end of the Nespas transcript that is transcribed upstream of the Nesp promoter is required for Nespas-mediated silencing of Nesp.

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4

Wijesinghe, Susanne. "Role of long non-coding RNA CCDC26 in gene regulation". Thesis, University of Birmingham, 2018. http://etheses.bham.ac.uk//id/eprint/8441/.

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LncRNAs are increasingly being recognised as functionally important for regulation of biological processes. We have identified an lncRNA which we believe is integral in lineage commitment during haematopoiesis. Here, we report that CCDC26 lncRNA is a regulator of β-globin and c-MYC gene expression. Indeed, CCDC26 silencing in erythroleukemic K562 cells led to several gene expression changes. Upon further investigation of genes linked to erythropoiesis, we observed the upregulation of β-globin expression. Our results suggest that CCDC26 regulates expression of β-globin and other genes by modulating epigenetic changes which could be important in linage-commitment. We have results to suggest the expression of β-globin and c-MYC genes may be synergistic and promotes differentiation of K562. Finally, RNAseq data further supports upregulation of differentiation specific genes further underpinning our hypothesis that this lncRNA may play an important role in lineage commitment.

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5

Cabili, Nataly Moran. "Integrative Characterization of Human Long Non-Coding RNAs". Thesis, Harvard University, 2014. http://dissertations.umi.com/gsas.harvard:11409.

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Since its early discovery as a messenger, RNA has been shown to play a diverse set of regulatory, structural and even catalytic roles. The more recent understanding that the genome is pervasively transcribed stimulated the discovery of a new prevalent class of long non coding RNAs (lncRNAs). While these are lower abundant and relatively less conserved than other class of functional RNAs, lncRNAs are emerging as key players in different cellular processes in development and disease.

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6

Hammel, Alexander John. "Evolutionary conservation of long intergenic non-coding RNA genes in Arabidopsis". Thesis, University of British Columbia, 2013. http://hdl.handle.net/2429/44974.

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7

Keniry, Andrew James. "H19 and miR-675 : a long noncoding RNA conceals a growth suppressing microRNA". Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609990.

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8

Broadbent, Kate Mariel. "The regulatory capacity of long non-coding RNA in Plasmodium falciparum malaria". Thesis, Harvard University, 2014. http://nrs.harvard.edu/urn-3:HUL.InstRepos:13065005.

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The mechanisms underpinning gene regulation in P. falciparum malaria remain largely elusive, though mounting evidence suggests a major role for epigenetic feedback. Interestingly, long non-(protein)-coding RNAs (lncRNAs) have been found to play a dominant role in initiating and guiding the transcriptional, epigenetic, and post-transcriptional status of specific loci across a broad range of organisms. LncRNAs are uniquely poised to act co-transcriptionally on neighboring loci, and/or to remain physically tethered at their site of origin, and through sequence-specific binding activities can impart temporal and spatial specificity to ubiquitously expressed nuclear protein complexes. Proteins, on the other hand, must be translated in the cytoplasm, and hence lose memory of their transcriptional origins. Encouraged by these features of lncRNAs, we set out to investigate the regulatory capacity of P. falciparum lncRNAs on a genome-wide scale. First, we surveyed transcriptional activity across approximately one quarter of the P. falciparum genome using a custom high-density DNA tiling array. We predicted a set of 60 developmentally regulated intergenic lncRNAs, and found that many of these novel loci neighbored genes involved in parasite survival or virulence pathways. Remarkably, upon further analysis of intergenic lncRNA properties, we discovered a family of twenty-two telomere-associated lncRNAs encoded in the telomere-associated repetitive element (TARE) region of P. falciparum chromosome ends. We found that each lncRNA-TARE was encoded adjacent and divergent to a subtelomeric var virulence gene. Moreover, we found that lncRNA-TARE expression was sharply induced between the parasite DNA replication and cell division cycles, that lncRNA-TARE loci contained numerous transcription factor binding sites only otherwise found in subtelomeric var promoter regions, and that the GC content and evolutionary sequence conservation of lncRNA-TAREs was similar to that of P. falciparum ribosomal RNA. Next, we set out to assemble P. falciparum intergenic lncRNA and antisense RNA transcript structures using state-of-the-art deep sequencing and computational tools. Towards this end, we harvested an unprecedented sample set that finely maps temporal changes across 56 hours of P. falciparum blood stage development, and developed and validated strand-specific, non-polyA-selected RNA sequencing methods. This enabled the annotation of over one thousand high-confidence, bona fide lncRNA transcript models, and their comprehensive global analysis. We discovered an enrichment of negatively correlated, tail-to-tail overlapping sense-antisense transcript pairs, suggesting a conserved role for antisense-mediated transcriptional interference in P. falciparum gene regulation. We also discovered a highly correlated spliced antisense counterpart to a gene required for sexual commitment, that the expression of an intriguing subset of antisense transcripts significantly dropped during parasite invasion, and that lncRNA-TARE and 'sterile' var virulence gene transcription was markedly up-regulated during parasite invasion. Lastly, we predicted over one thousand circular RNAs (circRNAs), and validated six circRNA transcript structures. Importantly, this thesis work represents the first focused investigation of lncRNAs in P. falciparum malaria, with the characterization of a compelling family of telomere-associated lncRNAs and numerous antisense RNAs. The data, methods, and results herein offer exceptional technological advancements coupled with compelling insights into the biology of the devastating human pathogen P. falciparum malaria. It is my hope that this work will facilitate future P. falciparum lncRNA functional studies and the strand-specific profiling of additional P. falciparum samples.

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9

Ballantyne, Margaret. "Understanding the role of long non-coding RNA (LncRNA) in vascular pathology". Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8101/.

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Coronary heart disease is a major cause of morbidity and mortality in the Western society. In the case of severe atherosclerosis, percutaneous intervention and coronary bypass grafting remain the preferred form of surgical treatment. However, the patency of both these treatments is limited and several bypass grafts and stents fail due to neointimal formation and in stent restenosis attributable to the proliferation of VSMCs. The resultant luminal renarrowing may manifest clinically with the return of symptoms such as chest pain or shortness of breath and ultimately requires further surgical intervention. Unfortunately, current antiproliferative therapies to inhibit VSMC proliferation have off target effects and can inhibit vessel re-endothelialisation resulting in thrombus formation. As such, novel therapies that specifically target VSMC proliferation but do not affect endothelial growth are urgently required. Long non-coding RNA (lncRNA) are transcripts >200 nucleotides that have been shown to bind DNA, RNA and proteins in order to exert their function. To date a few lncRNAs have been identified that control key aspects of VSMC phenotype, including contraction, proliferation, migration and apoptosis, however, very little is known as to the role of lncRNA in the proliferative and inflammatory phenotype associated with this phenotypic switching. It was therefore hypothesised that lncRNA may be dysregulated in the setting of inflammatory and proliferative vascular pathology and may provide novel therapies to counteract VSMC proliferation and hence vascular disease. The project sought to identify lncRNA expression in quiescent, non-proliferating human saphenous vein (HSV) SMCs, and HSVSMCs that had been treated with the IL1α cytokine and PDGF growth factor. This cytokine and growth factor pair have been implicated in the synergistic activation of the NF-κB transcription factor and in the control of vascular diseases including in stent restenosis, neo intimal formation and atherosclerosis. Using RNA-sequencing, >300 lncRNAs were identified whose expression was altered in HSVSMCs following stimulation with IL1α and PDGF. These lncRNA exhibited distinct expression patterns in a tissue cohort and all showed enrichment in vascular SMCs from either an arterial or venous lineages. Experiments focused on a novel lncRNA (Ensembl: RP11-94A24.1) which showed specific expression in HSVSMCs following treatment but no expression in endothelial cells. This lncRNA was termed smooth muscle induced lncRNA enhances replication (SMILR). Following stimulation, SMILR expression was increased in both the nucleus and cytoplasm, and was detected in conditioned media from dual stimulated HSVSMCs. Furthermore, knockdown of SMILR markedly reduced cell proliferation. Mechanistically, it was noted that expression of genes proximal to SMILR were also altered by IL1α/PDGF treatment possibly indicating that these two genes are under the same promoter control, and HAS2 expression was reduced by SMILR knockdown. Additionally the proliferation of HSVSMCs was increased in a dose dependent manner following administration of a lentivirus containing the full SMILR transcript, confirming the knockdown data. In human samples, increased expression of SMILR was detected in plaque compared to adjacent non-plaque sections by qRT-PCR and following on from the detection of SMILR in conditioned media, SMILR was also detected in plasma samples from patients with inflammatory CVD. Interestingly, the levels of SMILR correlated with plasma C-reactive protein, a current biomarker capable of detecting atherosclerosis progression in patients. These results identify SMILR as a driver of VSMC proliferation and suggest that modulation of SMILR may be a novel therapeutic strategy to reduce vascular pathologies. Additionally the detection of SMILR in plasma highlights the possibility that this lncRNA may have the potential as a biomarker of vascular disease. However, further large cohort studies are required to identify this potential clinical role.

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10

Pettini, Tom. "The role of novel long non-coding RNAs in Hox gene regulation". Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/the-role-of-novel-long-noncoding-rnas-in-hox-gene-regulation(c8e44900-3ac0-40be-8ec6-b50179381d17).html.

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Whole genome transcriptome analysis has revealed that a large proportion of the genome in higher metazoa is transcribed, yet only a small proportion of this transcription is protein-coding. One possible function of non-coding transcription is that it enables complex and diverse body plans to evolve through variation in deployment of a relatively common set of protein-coding genes. Functional studies suggest that long non-coding RNAs (lncRNAs) regulate gene expression via diverse mechanisms, operating in both cis and trans to activate or repress target genes. An emerging theme common to lncRNA function is interaction with proteins that modify chromatin and mediate epigenetic regulation. The Hox gene complexes are particularly rich in lncRNAs and require precise and fine-tuned expression to deploy Hox transcription factors throughout development. Here we identify and functionally characterize two novel lncRNAs within the D. melanogaster Hox complex, in the interval between Scr and Antp. We use nascent transcript fluorescent in-situ hybridization (ntFISH) to characterize the embryonic expression patterns of each lncRNA with respect to flanking Hox genes, and to analyze co-transcription within individual nuclei. We find that the transcription of one lncRNA, ncX, is an initial response to early transcription factors and may activate Scr expression, while transcription of the other lncRNA, ncPRE is consistent with activation and/or maintenance of Scr expression. ntFISH performed in D.virilis embryos revealed the presence of a lncRNA ortholog with highly similar expression to ncX, indicating functional conservation of lncRNA transcription across ~60 million years of evolution. We identify the ncPRE lncRNA locus as a binding site for multiple proteins associated with Polycomb/Trithorax response elements (PREs/TREs) and show that DNA encoding the ncPRE lncRNA functions as a bona fide PRE, mediating trans-interactions between chromosomes and silencing of nearby genes. We find that transcription through the ncPRE DNA relieves silencing, suggesting a role for endogenous transcription of the ncPRE lncRNA in relieving Polycomb-silencing and enabling Scr activation. We demonstrate that both lncRNA transcripts are required for proper Scr expression, and over-expression of either lncRNAs from ectopic genomic loci has no effect on Scr expression, but ectopic expression at the endogenous locus is associated with ectopic Scr activation, indicating that the lncRNA-mediated regulation functions locally at the site of transcription on the chromosome. ncX may mediate transvection effects previously observed at the Scr locus, independent of the protein Zeste. Together our results support a model of competing mechanisms in the regulation of Scr expression - a background of Polycomb repression acting from the ncPRE locus, which in the first thoracic segment is counteracted by lncRNA transcription and Trithorax binding to ncPRE, enabling activation and maintenance of Scr expression. This work provides a functional insight into the complex regulatory interactions between lncRNAs and epigenetic mechanisms, essential to establish and maintain the precise expression pattern of Hox genes through development.

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11

Coyne, Victoria. "Characterization of long non-coding RNAs in the Hox complex of Drosophila". Thesis, University of Manchester, 2017. https://www.research.manchester.ac.uk/portal/en/theses/characterization-of-long-noncoding-rnas-in-the-hox-complex-of-drosophila(733e3dec-3f7b-4d6e-a1bc-674a8786246d).html.

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Long non-coding RNAs (lncRNAs) are often defined as transcripts >200nts that have no discernable protein-coding ability (Quinn and Chang, 2016). Although relatively little is understood about the molecular mechanisms of lncRNA function, they have established roles in regulation of gene expression during development, cell differentiation and pluripotency (Fatica and Bozzoni, 2014; Luo et al., 2016; Quinn and Chang, 2016; Rinn and Chang, 2012) across vastly diverse organisms ranging from plants to humans (Ulitsky and Bartel, 2013). LncRNAs have also been associated with numerous pathological conditions, such as cancers (Brunner et al., 2012), cardiovascular disease and neurodegeneration (Chen et al., 2013). Investigations into lncRNAs in wide ranging organisms, have revealed that many influence gene activity by forming ribonucleoprotein complexes that affect the conformational state of chromatin (Rinn and Chang, 2012). A genomic region that has revealed several functional lncRNAs in diverse organisms is the Hox complex (Pauli et al., 2011; Pettini, 2012; Rinn et al., 2007). The Hox complex encodes a set of transcription factors (TFs), physically clustered in the genome, which provide morphological identity along the anterior to posterior axis of developing embryos (Mallo and Alonso, 2013), throughout the majority of bilatarian animals (Moreno et al., 2011). Misexpression or mutation of Hox genes causes morphological and pathophysiological defects (Quinonez and Innis, 2014). We investigated clustering of lncRNAs throughout the D. melanogaster genome using available annotations and carried out RNA-seq in D. virilis to expand the repertoire of lncRNAs and identify clusters of lncRNAs. We found the Hox complex to be heavily enriched with lncRNAs in both organisms, and syntenic transcripts from D. melanogaster could be identified in D. pseudoobscura and D. virilis. Several lncRNAs aligned with polycomb response elements (PREs); transcription of PREs has previously been linked to a switch in their activity (Herzog et al., 2014). However, we found that transcribed PREs in D. melanogaster move positions relative to the protein-coding genes in other drosophilids, whilst the transcriptional units remain in the same syntenic region. Conservation of syntenic transcripts without evidence of remaining a PRE suggest that the transcription is not linked to PRE function, agreeing with recent findings that transcription of PREs does not affect their function (Kassis and Muller, 2015). We investigated functions of a novel lncRNA and adjacent PRE in the Hox complex by ectopic expression and utilization of other genetic manipulation tools. Overexpression of either the lncRNA or PRE and partial duplication of the lncRNA caused phenotypes such as missing halteres and/or T3 legs, misshaped T3 legs or malformed abdominal segments. The observations that ectopic expression of this lncRNA and an adjacent regulatory element from the Hox complex causes phenotypes that can be linked to adjacent Hox gene misregulation, Antp and Ubx, suggest that they are likely to have roles in the regulation of at least one of these Hox genes.

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12

Schmidt, Benjamin [Verfasser] y Thalia [Akademischer Betreuer] Erbes. "Untersuchungen zur Expression und potentiellen Biomarkerfunktion von long non-coding RNA beim Mammakarzinom". Freiburg : Universität, 2020. http://d-nb.info/1212361199/34.

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13

Yang, Junjie. "The role of H19, a long non-coding RNA in the immune system". Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCC206/document.

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L'empreinte génomique, une régulation épigénétique unique entraînant une expression génique spécifique aux parents d'origine, est essentielle au développement et à la croissance des mammifères. H19 est un ARN long non codant exprimé en milieu maternel qui est un régulateur central du réseau de gènes à empreinte contrôlant le développement. H19 est exprimé pendant le développement embryonnaire dans de nombreux tissus, y compris toutes les cellules hématopoïétiques. Le rôle de H19 au cours du développement embryonnaire n'a été documenté que pour le placenta où il contrôle la croissance. Le rôle de H19 dans la lymphopoïèse n'a pas été étudié. Notre laboratoire a précédemment trouvé H19 comme principal transcrit exprimé sélectivement par les proB du foie foetal (FF), et exprimé de façon différentielle par des immigrants thymiques précoces et tardives. Cependant, le rôle du gèneH19 dans celui du développement des cellules B, ou même dans le système immunitaire, reste méconnu. Ici, nous avons réalisé une caractérisation complète des perturbations du développement et de la fonction du système immunitaire des souris pour lesquelles un grand segment du locus H19 a été supprimé. Dans cette étude, nous avons constaté que le mutant H19 avait un impact spécifique sur le développement des cellules du FF induisant une augmentation sélective du nombre des cellules proB BP1+ présentant des perturbations importantes du réarrangement du locus IgH.On observe également chez les animaux H19-/- adultes une expansion anormale du compartiment B mature. Bien que H19 ne soit plus exprimé après la naissance, les lymphocytes B des souris adultes mutantes présentent un phénotype altéré. On observe en effet des perturbations importantes du profil d'expression du marqueur B220. Les souris H19-/-présentent un défaut d'expansion des lymphocytes B du centre germinatif, ainsi qu'une chute de la production des IgM spécifiques dans le sérum après immunisation. Indiquant une réponse défectueuse des cellules B. De manière cohérente, nous avons trouvé une réactivité réduite au BCR des cellules B naïves H19-/-, associée les expériences de reconstitution compétitive ont mis en évidence un altération cellule-intrinsèque de la réponse humorale chez les animaux mutants. Un défaut d'induction de l'expression des molécules du CMHII, CD40,et CD86, qui pourrait être à l'origine des perturbations de la réponse humorale observée chez les souris H19-/-. Les analyse de transcriptome réalisées sur les lymphocytes B du centre germinal des animaux mutants ont mis en évidence une expression différentielle des gènes impliqués dans la régulation de l'intensité du signal émanant du récepteur B à l'antigène. Au total ce travail nous a permis de démontrer l'activité régulatrice exercée par l'ARN non codant H19 sur le développement et la fonction du système immunitaire
Genomic imprinting, a unique epigenetic regulation resulting in a parent-of-origin specificgene expression, is essential for normal mammalian development and growth. H19 is amaternally expressed long non-coding RNA that is a central regulator of the imprinting gene network controlling development and growth. H19 is expressed throughout embryonic development in multiple tissues including all hematopoietic cells. The role of H19 during embryonic development has only been documented for the placenta where it controls growthand the role of H19 in lymphopoiesis has not been investigated. The laboratory has previouslyfound H19 as the major differentially expressed transcript in two microarrays comparing fetalliver (FL) and bone marrow (BM) derived pro-B cells, as well as between early and latethymic settling progenitors. However, a role for imprinting gene H19 in B cell development,or even in immune system remains elusive. Here we sought to analyze mice where a large segment of the H19 locus has been deleted. In our work, we found that loss of H19 have specific impact on the FL B cell development byproducing increased numbers of BP1+ proB cell. Although BP1+ proB cells from H19-/- FLshowed impaired Ig heavy chain V-D-J rearrangement, that increase resulted in a net enlarged B cell compartment in the adult periphery of H19 mutant. In adult mice, although H19 is notexpressed in B lymphocytes after birth, B cells from H19-/- mice exhibited altered B cellsurface phenotype, represented by an upregulated B220 expression on all B cell subsets. After immunization with different T cell dependent antigens, H19-/- exhibits reduced GC B cells, and impaired specific IgM titer in the serum, indicating a defected B cell response in H19-/-mice. Competitive reconstitution analysis showed a B cell autonomous impairment in the Bcell response. Consistently we found a reduced BCR responsiveness of H19-/- naïve B cells that together with less efficient upregulation of MHCII and CD40 expression after immunization might be responsible for the impaired immune response in H19-/- mice. Genome-wide transcription analysis revealed differential expression of genes involved inregulating the intensity of B cell receptor signaling. This work brings new insights on the regulation role of long non-coding RNA H19 in the early B cell development and immune system

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14

Furió, Tarí Pedro. "Development of bioinformatic tools for massive sequencing analysis". Doctoral thesis, Universitat Politècnica de València, 2020. http://hdl.handle.net/10251/152485.

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[EN] Transcriptomics is one of the most important and relevant areas of bioinformatics. It allows detecting the genes that are expressed at a particular moment in time to explore the relation between genotype and phenotype. Transcriptomic analysis has been historically performed using microarrays until 2008 when high-throughput RNA sequencing (RNA-Seq) was launched on the market, replacing the old technique. However, despite the clear advantages over microarrays, it was necessary to understand factors such as the quality of the data, reproducibility and replicability of the analyses and potential biases. The first section of the thesis covers these studies. First, an R package called NOISeq was developed and published in the public repository "Bioconductor", which includes a set of tools to better understand the quality of RNA-Seq data, minimise the impact of noise in any posterior analyses and implements two new methodologies (NOISeq and NOISeqBio) to overcome the difficulties of comparing two different groups of samples (differential expression). Second, I show our contribution to the Sequencing Quality Control (SEQC) project, a continuation of the Microarray Quality Control (MAQC) project led by the US Food and Drug Administration (FDA, United States) that aims to assess the reproducibility and replicability of any RNA-Seq analysis. One of the most effective approaches to understand the different factors that influence the regulation of gene expression, such as the synergic effect of transcription factors, methylation events and chromatin accessibility, is the integration of transcriptomic with other omics data. To this aim, a file that contains the chromosomal position where the events take place is required. For this reason, in the second chapter, we present a new and easy to customise tool (RGmatch) to associate chromosomal positions to the exons, transcripts or genes that could regulate the events. Another aspect of great interest is the study of non-coding genes, especially long non-coding RNAs (lncRNAs). Not long ago, these regions were thought not to play a relevant role and were only considered as transcriptional noise. However, they represent a high percentage of the human genes and it was recently shown that they actually play an important role in gene regulation. Due to these motivations, in the last chapter we focus, first, in trying to find a methodology to find out the generic functions of every lncRNA using publicly available data and, second, we develop a new tool (spongeScan) to predict the lncRNAs that could be involved in the sequestration of micro-RNAs (miRNAs) and therefore altering their regulation task.
[ES] La transcriptómica es una de las áreas más importantes y destacadas en bioinformática, ya que permite ver qué genes están expresados en un momento dado para poder explorar la relación existente entre genotipo y fenotipo. El análisis transcriptómico se ha realizado históricamente mediante el uso de microarrays hasta que, en el año 2008, la secuenciación masiva de ARN (RNA-Seq) fue lanzada al mercado y comenzó a desplazar poco a poco su uso. Sin embargo, a pesar de las ventajas evidentes frente a los microarrays, resultaba necesario entender factores como la calidad de los datos, reproducibilidad y replicabilidad de los análisis así como los potenciales sesgos. La primera parte de la tesis aborda precisamente estos estudios. En primer lugar, se desarrolla un paquete de R llamado NOISeq, publicado en el repositorio público "Bioconductor", el cual incluye un conjunto de herramientas para entender la calidad de datos de RNA-Seq, herramientas de procesado para minimizar el impacto del ruido en posteriores análisis y dos nuevas metodologías (NOISeq y NOISeqBio) para abordar la problemática de la comparación entre dos grupos (expresión diferencial). Por otro lado, presento nuestra contribución al proyecto Sequencing Quality Control (SEQC), una continuación del proyecto Microarray Quality Control (MAQC) liderado por la US Food and Drug Administration (FDA) que pretende evaluar precisamente la reproducibilidad y replicabilidad de los análisis realizados sobre datos de RNA-Seq. Una de las estrategias más efectivas para entender los diferentes factores que influyen en la regulación de la expresión génica, como puede ser el efecto sinérgico de los factores de transcripción, eventos de metilación y accesibilidad de la cromatina, es la integración de la transcriptómica con otros datos ómicos. Para ello se necesita generar un fichero que indique las posiciones cromosómicas donde se producen estos eventos. Por este motivo, en el segundo capítulo de la tesis presentamos una nueva herramienta (RGmatch) altamente customizable que permite asociar estas posiciones cromosómicas a los posibles genes, transcritos o exones a los que podría estar regulando cada uno de estos eventos. Otro de los aspectos de gran interés en este campo es el estudio de los genes no codificantes, especialmente los ARN largos no codificantes (lncRNAs). Hasta no hace mucho, se pensaba que estos genes no jugaban ningún papel fundamental y se consideraban como simple ruido transcripcional. Sin embargo, suponen un alto porcentaje de los genes del ser humano y se ha demostrado que juegan un papel crucial en la regulación de otros genes. Por este motivo, en el último capítulo nos centramos, en un primer lugar, en intentar obtener una metodología que permita averiguar las funciones generales de cada lncRNA haciendo uso de datos ya publicados y, en segundo lugar, generamos una nueva herramienta (spongeScan) que permite predecir qué lncRNAs podrían estar secuestrando determinados micro-RNAs (miRNAs), alterando así la regulación llevada a cabo por estos últimos.
[CA] La transcriptòmica és una de les àrees més importants i destacades en bioinformàtica, ja que permet veure quins gens s'expressen en un moment donat per a poder explorar la relació existent entre genotip i fenotip. L'anàlisi transcriptòmic s'ha fet històricament per mitjà de l'ús de microarrays fins l'any 2008 quan la tècnica de seqüenciació massiva d'ARN (RNA-Seq) es va fer pública i va començar a desplaçar a poc a poc el seu ús. No obstant això, a pesar dels avantatges evidents enfront dels microarrays, resultava necessari entendre factors com la qualitat de les dades, reproducibilitat i replicabilitat dels anàlisis, així com els possibles caires introduïts. La primera part de la tesi aborda precisament estos estudis. En primer lloc, es va programar un paquet de R anomenat NOISeq publicat al repositori públic "Bioconductor", el qual inclou un conjunt d'eines per a entendre la qualitat de les dades de RNA-Seq, eines de processat per a minimitzar l'impact del soroll en anàlisis posteriors i dos noves metodologies (NOISeq i NOISeqBio) per a abordar la problemàtica de la comparació entre dos grups (expressió diferencial). D'altra banda, presente la nostra contribució al projecte Sequencing Quality Control (SEQC), una continuació del projecte Microarray Quality Control (MAQC) liderat per la US Food and Drug Administration (FDA) que pretén avaluar precisament la reproducibilitat i replicabilitat dels anàlisis realitzats sobre dades de RNA-Seq. Una de les estratègies més efectives per a entendre els diferents factors que influïxen a la regulació de l'expressió gènica, com pot ser l'efecte sinèrgic dels factors de transcripció, esdeveniments de metilació i accessibilitat de la cromatina, és la integració de la transcriptómica amb altres dades ómiques. Per això es necessita generar un fitxer que indique les posicions cromosòmiques on es produïxen aquests esdeveniments. Per aquest motiu, en el segon capítol de la tesi presentem una nova eina (RGmatch) altament customizable que permet associar aquestes posicions cromosòmiques als possibles gens, transcrits o exons als que podria estar regulant cada un d'aquests esdeveniments regulatoris. Altre dels aspectes de gran interés en aquest camp és l'estudi dels genes no codificants, especialment dels ARN llargs no codificants (lncRNAs). Fins no fa molt, encara es pensava que aquests gens no jugaven cap paper fonamental i es consideraven com a simple soroll transcripcional. No obstant això, suposen un alt percentatge dels gens de l'ésser humà i s'ha demostrat que juguen un paper crucial en la regulació d'altres gens. Per aquest motiu, en l'últim capítol ens centrem, en un primer lloc, en intentar obtenir una metodologia que permeta esbrinar les funcions generals de cada lncRNA fent ús de dades ja publicades i, en segon lloc, presentem una nova eina (spongeScan) que permet predeir quins lncRNAs podríen estar segrestant determinats micro-RNAs (miRNAs), alterant així la regulació duta a terme per aquests últims.
Furió Tarí, P. (2020). Development of bioinformatic tools for massive sequencing analysis [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/152485
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15

MOROTTI, ANNAMARIA. "INSIGHTS INTO THE NON-CODING GENOME OF PARATHYROID TUMORS". Doctoral thesis, Università degli Studi di Milano, 2019. http://hdl.handle.net/2434/610124.

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Recently, long non-coding RNAs (lncRNAs) have been implicated in the regulation of several physiological processes such as cell growth, differentiation and proliferation. Although lncRNAs functions in human diseases have not been completely disclosed, some lncRNAs have already been identified as prognostic and diagnostic biomarkers in different tumors. LncRNAs have also a crucial role in normal development of endocrine organs and their role in endocrine cancer pathogenesis is emerging. Parathyroid tumors are rare and heterogeneous diseases characterized by genetic and epigenetic alterations resulting in aberrant expression of both protein coding and non-coding genes. Tumors of the parathyroid glands show a great variability in clinical features such as parathormone (PTH) secretion, in the pattern of cell proliferation and in the genetic background. Mutations in the oncosuppressor CDC73 are key events in most carcinomas whereas alterations in the tumor suppressor Multiple Endocrine Neoplasia 1 (MEN1, located at 11q13.1) occur in up to a third of sporadic adenomas. Although lncRNAs play a regulatory role in endocrine cancer pathogenesis, a lncRNAs profiling in human parathyroid tumors is missing. Therefore, we investigated known lncRNAs expression in a series of normal (PaN) and pathological (adenomatous, PAd, and carcinomatous, PCa) parathyroid glands and correlated their expression with cytogenetic aberration, CDC73 status and MEN1 level. Then, we modulated epigenetic complexes and MEN1 expression to get preliminary insights into HAR1B ncRNA functions in parathyroid pathobiology. We found that a nine-lncRNAs signature distinguishes normal parathyroid glands from parathyroid tumors and also more aggressive tumors from indolent ones. Specifically, KCNQ1OT1 and SNHG6 are enriched in PaNs, HAR1B, HOXA3as, MEG3 and NEAT1 expression characterize PAds, whereas BC200 and HOXA6as are significantly upregulated in PCas compared to PaNs. When this signature was analyzed in a second set of samples, that included also atypical PAds (aPAds), unsupervised analysis showed 3 main samples clusters in which PaNs, all grouped in cluster 2, are distinctly separated from PCas and aPAds, both in cluster 1 and 3. Notably, samples with lncRNAs overexpression, grouped in cluster 3, are characterized by a lower age of tumor onset. Of note, validation analysis highlights a different lncRNAs expression pattern in PCas according to CDC73 gene mutation status. Specifically, CDC73-mutated carcinomas are grouped in cluster 3 and overexpress the majority of the lncRNAs. Moreover, CDC73-mutated PCas display an increment in PTH, calcium serum and ionized calcium levels. Conversely, lncRNAs expression in adenomas is highly heterogeneous. Therefore, we further characterized this tumor type by performing array Comparative Genomic Hybridization (aCGH). This analysis revealed that chromosome 11 loss of heterozygosity (Chr11-LOH) is the main chromosomal aberration among PAds (10 out of 24 PAds; 42%). Interestingly, Chr11-LOH is associated to a significative HAR1B upregulation. Based on these observations, we analyzed possible regulators of HAR1B expression and we found that HAR1B promoter is a target of EZH2, the catalytic subunit of the Polycomb Repressive Complex 2. Selective inhibition of EZH2 with Tazemetostat in HEK293 cells restored HAR1B expression levels. In line with this, PAds with Chr-11 LOH show a downregulation of H3K27me3, the direct target of EZH2 activity, compared to PAds without Chr11-LOH. Moreover, HAR1B levels increase after silencing of MEN1 in primary parathyroid adenoma cultures. Overall, these data shed light on lncRNAs deregulation in human parathyroid tumors and suggest an important role of HAR1B in parathyroid pathobiology.

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NESREEN, HAMAD ABDELGAWWAD HAMAD. "Structural analysis of the interaction between FUS/TLS protein and non-coding RNA". Kyoto University, 2020. http://hdl.handle.net/2433/259065.

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17

Floriot, Océane. "Virus de l’Hépatite B et transcription cellulaire : impact de la protéine HBx et de ses interactions avec les ARNs non-codants". Thesis, Lyon, 2018. http://www.theses.fr/2018LYSE1319/document.

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Le virus de l'hépatite B (VHB) reste un problème de santé majeur dans le monde malgré la disponibilité du vaccin. Le VHB n’est pas éradiqué par les thérapies actuelles et 240 millions de personnes infectées chroniquement restent à risque de développer une cirrhose du foie et un carcinome hépatocellulaire (CHC).Le VHB est un petit virus hépatotrope doté d'un génome à ADN double brin partiel (ADNrc). Après infection l'ADNrc est converti en ADN épisomal (ADNccc) qui est ensuite organisé en minichromosome viral, qui est le modèle pour la transcription et qui initie la réplication. La protéine de l'hépatite B x (HBx) est recrutée sur l'ADNccc pour initier et maintenir la transcription de l'ADN ccc. HBx cible aussi directement des gènes cellulaires impliqué dans le développement du CHC.Nous avons utilisé une approche ChIP-Seq pour identifier toutes les cibles génomiques de HBx dans les cellules qui répliquent le VHB. Les cibles HBx sont à la fois des gènes codant les protéines et des ARNnc (75 miARN et 34 lncRNA). Nous avons montré que HBx réprimait un sous-ensemble de miARNs qui réguleraient négativement la réplication virale (ex : miR-24) et des miARNs impliqués dans le développement du CHC (ex : miR-21). Parmi les lncARNs ciblés pour HBx, nous avons étudié DLEU2, qui est fortement surexprimé dans l’infection par le VHB et le CHC. Nous avons en outre montré que DLEU2 lie à la fois HBx et l’histone méthyltransférase Ezh2, la sous-unité catalytique du complexe répressif PRC2. L'interaction avec DLEU2 et HBx relie les fonctions Ezh2/PRC2 conduisant à l'activation constitutive d'un sous-ensemble de gènes cibles d'Ezh2 qui sont normalement conservés dans un état réprimé. Nous avons également montré que l’interaction de HBx avec DLEU2 se produisait sur le minichromosome de l’ADNccc où elle stimulait la transcription/réplication du virus. Enfin, nous avons caractérisé par ATAC-Seq les changements d'accessibilité de la chromatine imposés par HBV dans les hépatocytes humains primaires
Hepatitis B virus (HBV) remains a major health problem worldwide despite the availability of the vaccine. No cure is available for the 240 million peoples chronically infected with HBV that are at risk to develop liver cirrhosis and hepatocellular carcinoma (HCC). Viral suppression, achieved by long term treatment with nucleotides analogues (NUCs), impacts on liver fibrosis and prevents liver decompensation but HCC risk is not reduced in the first 5 years of treatment. HBV is a small hepatotropic virus with a partially double strand DNA (rcDNA) genome. After hepatocyte infection the rcDNA is converted into the cccDNA episome that is then organized into a viral minichromosome that is the template for all viral transcripts and initiates replication. The hepatitis B x protein (HBx) is recruited on the cccDNA and is required to launch and maintain cccDNA transcription. HBx has also been shown to directly target cellular genes and this has been related to HCC development.We used a ChIP-Seq approach to determine the full repertoire of HBx genomic targets in HBV replicating cells. HBx targets include both protein coding genes and ncRNA (75 miRNAs and 34 lncRNAs). We showed that HBx represses a subset of miRNAs that would negatively regulate viral replication (i.e. miR-24) and miRNAs involved in HCC development (i.e. miR-21). Among the HBx targeted lncRNAs we focused DLEU2, which is strongly upregulated in HBV infection and HCC. We further showed that DLEU2 binds both HBx the Ezh2 histone methyltransferase, the catalytic subunit of the repressive PRC2 complex. The interaction with DLEU2 and HBx re-wires Ezh2/PRC2 functions leading to the constitutive activation of a subset of Ezh2 target genes that are normally kept in a repressed state. We also showed that HBx interaction with DLEU2 occurs on the cccDNA minichromosome where it boosts HBV transcription/replication. Finally, we characterized by ATAC-Seq HBV imposed changes of chromatin accessibility in primary human hepatocytes

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18

Bussotti, Giovanni 1983. "Detecting and comparing non-coding RNAs". Doctoral thesis, Universitat Pompeu Fabra, 2013. http://hdl.handle.net/10803/128970.

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In recent years there has been a growing interest in the field of non-coding RNA. This surge is a direct consequence of the discovery of a huge number of new non-coding genes, and of the finding that many of these transcripts are involved in key cellular functions. In this context, accurately detecting and comparing RNA sequences becomes extremely important. Aligning nucleotide sequences is one of the main requisite when searching for homologous genes. Accurate alignments reveal evolutionary relationships, conserved regions and more generally, any biologically relevant pattern. Comparing RNA molecules is, however, a challenging task. The nucleotide alphabet is simpler and therefore less informative than that of proteins. Moreover for many non-coding RNAs, evolution is likely to be mostly constrained at the structure level and not on the sequence level. This results in a very poor sequence conservation impeding the comparison of these molecules. These difficulties define a context where new methods are urgently needed in order to exploit experimental results at their full potential. These are the issues I have tried to address in my PhD. I have started by developing a novel algorithm able to reveal the homology relationship of distantly related ncRNA genes, and then I have applied the approach thus defined in combination with other sophisticated data mining tools to discover novel non-coding genes and generate genome-wide ncRNA predictions.
En los últimos años el interés en el campo de los ARN no codificantes ha crecido mucho a causa del enorme aumento de la cantidad de secuencias no codificantes disponibles y a que muchos de estos transcriptos han dado muestra de ser importantes en varias funciones celulares. En este contexto, es fundamental el desarrollo de métodos para la correcta detección y comparativa de secuencias de ARN. Alinear nucleótidos es uno de los enfoques principales para buscar genes homólogos, identificar relaciones evolutivas, regiones conservadas y en general, patrones biológicos importantes. Sin embargo, comparar moléculas de ARN es una tarea difícil. Esto es debido a que el alfabeto de nucleótidos es más simple y por ello menos informativo que el de las proteínas. Además es probable que para muchos ARN la evolución haya mantenido la estructura en mayor grado que la secuencia, y esto hace que las secuencias sean poco conservadas y difícilmente comparables. Por lo tanto, hacen falta nuevos métodos capaces de utilizar otras fuentes de información para generar mejores alineamientos de ARN. En esta tesis doctoral se ha intentado dar respuesta exactamente a estas temáticas. Por un lado desarrollado un nuevo algoritmo para detectar relaciones de homología entre genes de ARN no codificantes evolutivamente lejanos. Por otro lado se ha hecho minería de datos mediante el uso de datos ya disponibles para descubrir nuevos genes y generar perfiles de ARN no codificantes en todo el genoma.

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19

Zhang, Zhouwei. "Investigation of DNA and RNA markers by novel technologies demonstrates DNA content intratumoral heterogeneity and long non-coding RNA aberrations in breast tumors". ScholarWorks @ UVM, 2014. https://scholarworks.uvm.edu/graddis/323.

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BACKGROUND: Breast cancer is the most commonly diagnosed cancer and second leading cancer death cause among females in the U.S.A. About 1 in 8 women in U.S will develop invasive breast cancer over the course of her lifetime. In 2013, 234,580 new invasive breast cancer cases are expected to occur in women within the US and approximately 64,640 non-invasive carcinomas in situ were diagnosed in 2013, most of which were ductal carcinoma in situ (DCIS). Along with technological advances, a wide variety of candidate biomarkers have been proposed for cancer diagnosis and prognosis, including DNA content and non-coding RNA. Current techniques for detecting DNA content abnormalities in formalin-fixed, paraffin-embedded (FFPE) tissue samples by flow cytometric analysis have used cells recovered from ≥50Âµm whole tissue sections. Here, in our first study, a novel core punch sampling method was investigated for assessing DNA content abnormalities and intratumoral heterogeneity in FFPE specimens. Secondly, long non-coding RNAs (lncRNAs) has been examined. LncRNA participates in a broad spectrum of biological activities by diverse mechanisms and its dysregulation is associated with tumorgenesis. Some lncRNAs may function as oncogenes (O) and others as tumor suppressor genes (TSG). To date, lncRNA has been investigated primarily by qRT-PCR and RNA sequencing. This study has examined the relationship of lncRNA expression patterns to breast tumor pathology by chromogenic in situ hybridization (CISH). METHODS: Firstly, FFPE breast carcinoma specimens were selectively targeted using 1.0 mm diameter punch needles. Extracted cores were assayed by flow cytometry using a modified-Headley method. Secondly, the lncRNA expression levels of 6 lncRNAs: HOTAIR, H19, KCNQ1OT1, MEG3, MALAT11 and Zfas1, was examined by RNAscope® CISH using FFPE breast tissue microarrays (TMAs) comprising normal adjacent epithelia (NA), DCIS, and invasive carcinoma (IC) from 46 patients. LncRNA associate polycomb complex protein EZH2 was evaluated by immunohistochemistry (IHC). LncRNA data was also compared to standard breast tumor data including ER, PR, Her2 and Ki67 IHC. SYSTAT version 11 statistical package was used to perform for all the tests. RESULTS: Following optimization experiments of the core punch flow cytometric approach, DNA index and percent S-phase fraction intratumoral heterogeneities were detected in 10/23 (44%) and 11/23 (47%) specimens respectively. The lncRNA CISH study utilized a TMA that contained 36 spots of NA breast tissues, 34 DCIS spots and 43 IC spots. HOTAIR CISH staining was significantly stronger in IC than DCIS (p CONCLUSION: Core-punching is an effective alternative to whole specimen sectioning and shows that macro-level genomic heterogeneity is common even within a single FFPE block. The interrelationship of DNA content heterogeneity to other forms of heterogeneity requires further study. RNAscope CISH supports bright-field microscopy investigations of lncRNA expression in FFPE tissue specimens. HOTAIR, H19 and KCNQ1OT1 may be potential breast cancer biomarkers, both HOTAIR and H19 may be a marker for DCIS at increased risk of progression to invasive cancer. HOTAIR, in particular, may be a predictor for invasive cancer grade.

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20

Karlsson, Joakim. "Differential and co-expression of long non-coding RNAs in abdominal aortic aneurysm". Thesis, Uppsala universitet, Institutionen för biologisk grundutbildning, 2014. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-236141.

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This project concerns an exploration of the presence and interactions of long non-coding RNA transcripts in an experimental atherosclerosis mouse model with relevance for human abdominal aortic aneurysm development. 187 long noncoding RNAs, two of them entirely novel, were found to be differentially expressed between angiotensin II treated (developing abdominal aortic aneurysms) and non-treated apolipoprotein E deficient mice (not developing aneurysms) harvested after the same period of time. These transcripts were also studied with regards to co-expression network connections. Eleven previously annotated and two novel long non-coding RNAs were present in two significantly disease correlated co-expression groups that were further profiled with respect to network properties, Gene Ontology terms and MetaCore© connections.

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21

Moreno, Leon Laura. "Étude d'un long ARN non codant induit par l'hypoxie et associé à l’agressivité des adénocarcinomes bronchopulmonaires". Thesis, Université Côte d'Azur (ComUE), 2017. http://www.theses.fr/2017AZUR4144.

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Les cancers bronchopulmonaires non à petites cellules (CBNPC) sont la première cause de décès par cancer, les adénocarcinomes étant la forme la plus fréquente. Malgré une prise en charge précoce, ils constituent, par leur taux de récidive important et leur mauvais pronostic, un véritable problème de santé publique. Nous nous intéressons à l'hypoxie, un facteur agressivité des ADC, et à la famille des longs ARNs non codants (lncARNs), dérégulés dans de nombreux cancers et en réponse à l'hypoxie, mais peu caractérisés sur le plan structural et fonctionnel. Ces transcrits représentent un espoir pour le développement de nouvelles thérapies. Au cours de ma thèse, j'ai identifié une dizaine de lncARNs régulés par l'hypoxie in vitro et in vivo dans des ADC de stades précoces. j'ai caractérisé NLUCAT1, un transcrit nucléaire induit par l'hypoxie. L'invalidation de ce transcrit par le système CRISPR/Cas9 a révélé une diminution de la prolifération et de l'invasion cellulaires, et une augmentation du stress oxydatif et de la sensibilité au cisplatine, traduisant un potentiel rôle pro-oncogénique de ce transcrit dans les ADC. L'analyse du transcriptome a révélé une répression des réseaux de gènes contrôlés par NRF2, HIF et NFkβ dans les cellules déficientes pour NLUCAT1. Nous avons notamment identifié des gènes de la réponse anti-oxydante régulés par NRF2 dont l'ARN interférence mime en partie les conséquences de l'inactivation de NLUCAT1 sur l'apoptose. Nos résultats démontrent que NLUCAT1 exerce des activités pro-tumorales dans les ADC et suggère qu'il pourrait représenter une cible thérapeutique potentielle dans ce type de cancer
Non Small Cell Lung Cancer (NSCLC) is the leading cause of cancer death worldwide, with poor prognosis and a high rate of recurrence despite early surgical removal. It is therefore essential to identify new prognostic markers and new therapeutic targets. We are interested in gene regulation related to hypoxia, a factor associated with relapse of lung adenocarcinomas (LUAD). The roles of long non coding RNAs (incRNAs) in cancer development and hypoxic response are largely unexplored. A transcriptome profiling of early-stage LUAD samples indicated that a set of incRNAs was correlated to a metagene hypoxic signature. Some of these transcripts were also sensitive to hypoxia in LUAD cell lines. We focused on a new "hypoxaLinc", named NLUCAT1 that is strongly up-regulated by hypoxia in vitro and correlated to hypoxic markers and bad prognosis in LUAD samples. Full molecular charactherization of NLUCAT1 showed that LUCAT1 is mainly regulated by NF-kβ and NRF2 transcription factors. Targered deletion of NLUCAT using CRISPR/CAS9 in A549 LUAD cell line, revelated a decrase in proliferative and invasive properties, an increase in oxidative stress and a higher sensisivity to displatin-induced apoptosis. We identified genes of the NRF2-regulated and anti-oxidant response whose RNA interference partially mimicked the consequences of NLUCAT1 inactivation on ROS-dependent caspase activation. Overall, our data strongly demonstrate that NLUCAT1 exerts pro-tumoral activities in early stages hypoxic LUADs ans suggest it could represent a new potential therapeutic target in lung cancer

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22

Surappa-Narayanappa, Ananth Prakash. "The evolution, modifications and interactions of proteins and RNAs". Thesis, University of Cambridge, 2017. https://www.repository.cam.ac.uk/handle/1810/269851.

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Proteins and RNAs are two of the most versatile macromolecules that carry out almost all functions within living organisms. In this thesis I have explored evolutionary and regulatory aspects of proteins and RNAs by studying their structures, modifications and interactions. In the first chapter of my thesis I investigate domain atrophy, a term I coined to describe large-scale deletions of core structural elements within protein domains. By looking into truncated domain boundaries across several domain families using Pfam, I was able to identify rare cases of domains that showed atrophy. Given that even point mutations can be deleterious, it is surprising that proteins can tolerate such large-scale deletions. Some of the structures of atrophied domains show novel protein-protein interaction interfaces that appear to compensate and stabilise their folds. Protein-protein interactions are largely influenced by the surface and charge complementarity, while RNA-RNA interactions are governed by base-pair complementarity; both interaction types are inherently different and these differences might be observed in their interaction networks. Based on this hypothesis I have explored the protein-protein, RNA-protein and the RNA-RNA interaction networks of yeast in the second chapter. By analysing the three networks I found no major differences in their network properties, which indicates an underlying uniformity in their interactomes despite their individual differences. In the third chapter I focus on RNA-protein interactions by investigating post-translational modifications (PTMs) in RNA-binding proteins (RBPs). By comparing occurrences of PTMs, I observe that RBPs significantly undergo more PTMs than non-RBPs. I also found that within RBPs, PTMs are more frequently targeted at regions that directly interact with RNA compared to regions that do not. Moreover disorderedness and amino acid composition were not observed to significantly influence the differential PTMs observed between RBPs and nonRBPs. The results point to a direct regulatory role of PTMs in RNA-protein interactions of RBPs. In the last chapter, I explore regulatory RNA-RNA interactions. Using differential expression data of mRNAs and lncRNAs from mouse models of hereditary hemochromatosis, I investigated competing regulatory interactions between mRNA, lncRNA and miRNA. A mutual interaction network was created from the predicted miRNA interaction sites on mRNAs and lncRNAs to identify regulatory RNAs in the disease. I also observed interesting relations between the sense-antisense mRNA-lncRNA pairs that indicate mutual regulation of expression levels through a yet unknown mechanism.

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23

Niemczyk, Malwina. "Epigenetic and transcriptional relationship between a novel long non-coding RNA GNG12-AS1 and imprinted DIRAS3". Thesis, University of Cambridge, 2014. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708234.

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Ahmadov, Ulvi [Verfasser]. "The long non-coding RNA HOTAIRM1 promotes tumor aggressiveness and radiotherapy resistance in glioblastoma / Ulvi Ahmadov". Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2020. http://d-nb.info/1222736969/34.

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25

VINCI, CRISTINA. "THE RELEVANCE OF LONG NON-CODING RNA IN THE BIOLOGICAL AND CLINICAL HETEROGENEITY OF MULTIPLE MYELOMA". Doctoral thesis, Università degli Studi di Milano, 2019. http://hdl.handle.net/2434/615064.

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Il mieloma multiplo (MM) è una proliferazione maligna di plasmacellule del midollo osseo secernenti anticorpi, che rappresenta il 10% di tutte le neoplasie ematologiche. Il MM è caratterizzato da un ampio spettro clinico che va da una presunta condizione pre-maligna chiamata gammopatia monoclonale di significato indeterminato a mieloma extra-midollare/ leucemia plasmacellulare (PCL). Nonostante i notevoli miglioramenti nel trattamento e nella cura dei pazienti, il MM è ancora una patologia incurabile. Negli ultimi anni è stato dimostrato che la stragrande maggioranza (> 90%) della sequenza del genoma umano viene attivamente trascritta, ma solo meno del 2% dei trascritti è rappresentato da RNA messaggero e codifica per proteine. Gli RNA non codificanti (ncRNA) sono coinvolti in vari processi biologici e sono comunemente suddivisi in RNA non codificanti corti (<200nt), come i microRNA (miRNA), e lunghi (> 200 nt, lncRNA). La de-regolazione dei ncRNA è stata evidenziata in tutti i tipi di tumore, compreso il MM. In particolar modo il ruolo dei miRNA nella trasformazione maligna è stato ampiamente esplorato e solo di recente sono stati evidenziati diversi ruoli dei lncRNA sia nelle cellule normali che patologiche. Con l'obiettivo di identificare i lncRNA modulati nel MM, abbiamo messo a punto le condizioni per l'utilizzo del GeneChip® Human Gene 2.0 ST microarray, in grado di distinguere più di 10000 lncRNA, e la metodica dell’RNAseq. Sulla base di queste analisi, effettuate sulla nostra casistica di pazienti (50 tumori primari di MM, 15 PCL e 4 donatori sani), abbiamo evidenziato ST3GAL6-AS1 come unico lncRNA che presenta un significativo incremento di espressione nei campioni patologici rispetto ai controlli sani. I dati GEP hanno evidenziato l’incremento di espressione di questo lncRNA e una correlazione significativa con i livelli di espressione di ST3GAL6, gene adiacente a ST3GAL6-AS1. ST3GAL6 codifica per una proteina che agisce nel processo di produzione di proteine e lipidi glicosilati, ed è stato descritto un suo ruolo nel MM come attore nello spostamento delle cellule (fenomeno dell’“homing”) e nel loro attecchimento nel midollo osseo. Nel nostro studio, ST3GAL6-AS1 e ST3GAL6 hanno mostrato una sovra espressione nelle linee cellulari umane di MM (HMCLs), una localizzazione omogenea nelle frazioni nucleari e citoplasmatiche e una lunga emivita. L'analisi molecolare di ST3GAL6-AS1 nelle HMCLs ha evidenziato la presenza di un polimorfismo a singolo nucleotide (SNP) con codice rs13065271 che interessa un nucleotide fondamentale per gli eventi di splicing. Infatti, la presenza di questo SNP produce 2 eventi di splicing non predetti: la ritenzione dell'introne 3 e il salto dell'esone 3. In particolare, le analisi molecolari su HMCLs omozigoti per l’allele minore hanno mostrato una localizzazione nucleare del lncRNA, un'espressione bassa e un'emivita dei trascritti più breve rispetto alle HMCLs che presentano almeno un allele maggiore. Inoltre, l'analisi dello SNP rs13065271, effettuata su pazienti affetti da MM, ha confermato una significativa riduzione dell’espressione di ST3GAL6-AS1 in pazienti omozigoti per l’allele mutato. Il silenziamento di ST3GAL6-AS1 in HMCLs mediante l’utilizzo di GapmeR ha messo in luce una significativa diminuzione del lncRNA in tutte le HMCLs esaminate. Le HMCLs in omozigosi per l’allele minore dello SNP non hanno presentato effetti biologici; al contrario, le HMCLs con almeno un allele maggiore dello SNP hanno evidenziato effetti biologici in termini di diminuzione della crescita e vitalità cellulare, variazioni della percentuale delle cellule nelle fasi del ciclo cellulare, aumento della percentuale delle cellule apoptotiche e perdita della capacità di formare colonie. L'analisi di microarray con GeneChip® Human Gene 2.0 ST sulle HMCLs silenziate ha rivelato un aumento dell’espressione di FUCA1, gene codificante per un enzima lisosomiale coinvolto nella degradazione delle glicoproteine e dei glicolipidi contenenti fucosio. Inoltre, i livelli di espressione di FUCA1 nella nostra casistica di pazienti di MM sono risultati inversamente correlati, in maniera significativa, con i livelli di espressione di ST3GAL6-AS1. Nel complesso, questi dati suggeriscono l'ipotesi che ST3GAL6-AS1 possa agire sulla regolazione negativa di FUCA1 nel MM.
Multiple myeloma (MM) is a malignant proliferation of antibody-secreting bone marrow plasma cells that accounts for 10% of all hematological malignancies. MM is characterized by a wide clinical spectrum ranging from the presumed pre-malignant condition called monoclonal gammopathy of undetermined significance, to extra-medullary myeloma/plasma cell leukemia (PCL). Despite the remarkable improvements in treatment and patient care, MM remains an incurable disease. In the last few years it has been demonstrated that the vast majority (>90 %) of the human genome sequence is actively transcribed, but only less than 2% of transcripts serve as mRNA to encode protein. The non-coding RNAs (ncRNAs) are involved in various biological processes and are commonly divided into short (<200nt), including microRNAs (miRNAs), and long (>200 nt, lncRNAs) transcripts. The dysregulation of ncRNAs has been reported to occur virtually in all types of cancer including MM. The role of miRNA in malignant transformation has been widely explored and recently different lncRNA roles has been the identified in normal and malignant cells. With the aim to identify lncRNAs modulated in MM, we set up the condition to the use of GeneChip® Human Gene 2.0 ST microarray, able to distinguish more than 10000 lncRNAs, and RNAseq methods. Based on these analyses, on our cohort of MM patients (50 MM primary tumors, 15 PCL and 4 normal donors), we evidenced ST3GAL6-AS1 as the unique lncRNA up-modulated in pathological samples compared to normal controls. GEP data evidenced the up-modulation of this lncRNA and a significant correlation with ST3GAL6, neighboring gene of ST3GAL6-AS1, expression levels. ST3GAL6 codes for a protein able to produce proteins and lipids glycosylation, and a role for ST3GAL6 in homing and engraftment of MM has been described. In our study, ST3GAL6-AS1 and ST3GAL6 were overexpressed in human MM cell lines (HMCLs), and showed equally localization in nuclear and cytoplasmic fractions and a long half-life. Molecular analysis of ST3GAL6-AS1 in HMCLs showed the presence of the splice single nuclear polymorphic (SNP) variation rs13065271 with the production of unpredicted splice events (retention of intron 3 and skipping of exon 3). In particular, homozygous minor allele HMCLs showed a nuclear localization of the lncRNA, a low expression and half-life of transcripts shorter than the HMCLs with at least one major allele. Furthermore, the SNP rs13065271 analysis made on MM patients confirmed the significant down-regulation of ST3GAL6-AS1 expression levels in homozygous minor allele patients. Additionally, silencing of ST3GAL6-AS1 in HMCLs by GapmeR approach evidenced a significant down-regulation of the lncRNA in all the HMCLs investigated. SNP homozygous minor allele HMCLs did not evidenced biological effects; by contrast, HMCLs with at least one SNP major allele evidenced biological effects in terms of decrease of cell growth and viability, changes in percentage of cell cycle phases, increase of percentage of apoptotic cells, and loss of capability to form colonies. GeneChip® Human Gene 2.0 ST microarray analysis on silenced HMCLs evidenced the up-regulation of FUCA1, coding for a lysosomal enzyme involved in the degradation of fucose-containing glycoproteins and glycolipids. Additionally, the expression levels of FUCA1 in our cohort of patients resulted significantly inversely correlated with ST3GAL6-AS1 expression levels. Overall, these data suggest the hypothesis that ST3GAL6-AS1 may act on the down-regulation of FUCA1 in MM.

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Uroda, Tina. "Caractérisation structurale et fonctionnelle de l’ARN long non codant MEG3". Thesis, Université Grenoble Alpes (ComUE), 2019. http://www.theses.fr/2019GREAV014.

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Les ARNs long non codants (ARNlnc) jouent un rôle clé dans les processus cellulaires vitaux, notamment le remodelage de la chromatine, la réparation de l'ADN et la traduction. Cependant, la taille et la complexité des ARNlnc présentent des défis sans précédent pour les études moléculaires mécanistiques, de sorte qu'il s'est avéré difficile jusqu'à présent de relier l'information structurelle à la fonction biologique pour les ARNlnc.Le gène 3 humain exprimé maternellement (de l’anglais "maternally expressed gene 3", MEG3), est un ARNlnc abondant, soumis à empreinte parentale et épissé alternativement. Pendant l'embryogenèse, MEG3 contrôle les protéines Polycomb, régulant la différenciation cellulaire, et dans les cellules adultes, MEG3 contrôle p53, régulant la réponse cellulaire aux stress environnementaux. Dans les cellules cancéreuses, MEG3 est régulé négativement, mais la surexpression ectopique de MEG3 réduit la prolifération incontrôlée, ce qui prouve que MEG3 agit comme un suppresseur de tumeur. Les données suggèrent que les fonctions de MEG3 pourraient être régulées par la structure de MEG3. Par exemple, on pense que MEG3 se lie directement aux protéines p53 et Polycomb. De plus, les différents variants d'épissage de MEG3, qui comprennent différents exons et possèdent ainsi des structures potentiellement différentes, présentent des fonctions différentes. Enfin, la mutagenèse par délétion, basée sur une structure de MEG3 prédit in silico, a permis d’identifier un motif MEG3 supposé structuré impliqué dans l'activation de p53. Cependant, au début de mes travaux, la structure expérimentale de MEG3 était inconnue.Pour comprendre la structure et la fonction de MEG3, j'ai utilisé des sondes chimiques in vitro et in vivo pour déterminer la structure secondaire de deux variants humains de MEG3 qui diffèrent par leurs niveaux d'activation de p53. À l'aide d'essais fonctionnels dans les cellules et de mutagenèse, j'ai systématiquement analysé la structure de MEG3 et identifié le noyau activant p53 dans deux domaines (D2 et D3) qui sont conservés structuralement dans les variants humains et conservés dans l’évolution chez les mammifères. Dans D2-D3, les régions structurales les plus importantes sont les hélices H11 et H27, car dans ces régions, j’ai pu supprimer l'activation de p53 grâce à des mutations ponctuelles, un degré de précision jamais atteint pour les autres ARNlnc jusqu’ici. J'ai découvert de manière surprenante que H11 et H27 sont reliés par des boucles connectées l’une à l’autre (de l’anglais "kissing loops") et j'ai confirmé l'importance fonctionnelle de ces interactions de structure tertiaire à longue distance par mutagenèse compensatoire. Allant au-delà de l’état de l’art, j'ai donc essayé de visualiser la structure 3D d’une isoforme de MEG3 longue de 1595 nucléotides, par diffusion de rayons X à petit angle (SAXS), microscopie électronique (EM) et microscopie à force atomique (AFM). Alors que le SAXS et l’EM sont limités par des défis techniques actuellement insurmontables, l’imagerie par AFM m’a permis d’obtenir la première structure 3D à basse résolution de MEG3 et de révéler son échafaudage tertiaire compact et globulaire. Plus remarquable encore, les mêmes mutations qui perturbent la connexion entre les «boucles» H11-H27 et qui inhibent la fonction de MEG3, perturbent aussi la structure 3D de cet ARNlnc, fournissant ainsi le premier lien direct entre la structure 3D et la fonction biologique pour un ARNlnc.Sur la base de mes découvertes, je peux donc proposer un mécanisme de l’activation de p53 basé sur la structure de MEG3, avec des implications importantes pour la compréhension de la cancérogenèse. Plus généralement, mes travaux prouvent que les relations structure-fonction des ARNlnc peuvent être disséquées avec une grande précision et ouvrent la voie à des études analogues visant à obtenir des informations mécanistes pour de nombreux autres ARNlnc d’importance médicale
Long non-coding RNAs (lncRNAs) are key players in vital cellular processes, including chromatin remodelling, DNA repair and translation. However, the size and complexity of lncRNAs present unprecedented challenges for mechanistic molecular studies, so that connecting structural information with biological function for lncRNAs has proven difficult so far.Human maternally expressed gene 3 (MEG3) is an abundant, imprinted, alternatively-spliced lncRNA. During embryogenesis MEG3 controls Polycomb proteins, regulating cell differentiation, and in adult cells MEG3 controls p53, regulating the cellular response to environmental stresses. In cancerous cells, MEG3 is downregulated, but ectopic overexpression of MEG3 reduces uncontrolled proliferation, proving that MEG3 acts as a tumour suppressor. Evidence suggests that MEG3 functions may be regulated by the MEG3 structure. For instance, MEG3 is thought to bind p53 and Polycomb proteins directly. Moreover, different MEG3 splice variants, which comprise different exons and thus possess potentially different structures, display different functions. Finally, deletion mutagenesis based on a MEG3 structure predicted in silico identified a putatively-structured MEG3 motif involved in p53 activation. However, at the beginning of my work, the experimental structure of MEG3 was unknown.To understand the MEG3 structure and function, I used chemical probing in vitro and in vivo to determine the secondary structure maps of two human MEG3 variants that differ in their p53 activation levels. Using functional assays in cells and mutagenesis, I systematically scanned the MEG3 structure and identified the p53-activating core in two domains (D2 and D3) that are structurally conserved across human variants and evolutionarily conserved across mammals. In D2-D3, the most important structural regions are helices H11 and H27, because in these regions I could tune p53 activation even by point mutations, a degree of precision never achieved for any other lncRNA to date. I surprisingly discovered that H11 and H27 are connected by “kissing loops”, and I confirmed the functional importance of these long-range tertiary structure interactions by compensatory mutagenesis. Going beyond state-of-the-art, I thus attempted to visualize the 3D structure of a 1595-nucleotide long MEG3 isoform by small angle X-ray scattering (SAXS), electron microscopy (EM), and atomic force microscopy (AFM). While SAXS and EM are limited by currently-insurmountable technical challenges, single particle imaging by AFM allowed me to obtain the first low resolution 3D structure of MEG3 and reveal its compact, globular tertiary scaffold. Most remarkably, functionally-disrupting mutations that break the H11-H27 “kissing loops” disrupt such MEG3 scaffold, providing the first direct connection between 3D structure and biological function for an lncRNA.Based on my discoveries, I can therefore propose a structure-based mechanism for p53 activation by human MEG3, with important implications in understanding carcinogenesis. More broadly, my work serves as proof-of-concept that lncRNA structure-function relationships can be dissected with high precision and opens the field to analogous studies aimed to gain mechanistic insights into many other medically-relevant lncRNAs

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27

Cimatti, Laura. "Long Non-Coding RNA Antisense to Uchl1 Increases Its Protein Translation and Identifies a New Class of Protein Translation Activators". Doctoral thesis, SISSA, 2013. http://hdl.handle.net/20.500.11767/3922.

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Thanks to continuous technical advances in the sequencing field nowadays we know that most of the mammalian genome is transcribed. This generates a vast repertoire of transcripts that includes protein-coding messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs) and repetitive sequences, such as Short Interspersed Nuclear Elements (SINEs). A large percentage of ncRNAs is nuclear-enriched with unknown function. LncRNAs may be transcribed in antisense direction and may form sense/antisense pairs by pairing with an mRNA from the opposite strand to regulate chromatin conformation, transcription and mRNA stability. We have identified a nuclear-enriched lncRNA antisense to mouse Ubiquitin Carboxyterminal Hydrolase L1 (Uchl1), a gene expressed in dopaminergic cells and involved in brain function and neurodegenerative diseases. Antisense Uchl1 (AS Uchl1) increases Uchl1 protein synthesis at a post-transcriptional level. AS Uchl1 function is under the control of stress signaling pathways, as mTORC1 inhibition by rapamycin causes an increase in Uchl1 protein that is associated to the shuttling of AS Uchl1 lncRNA from the nucleus to the cytoplasm of dopaminergic cells. AS Uchl1 RNA is then required for the association of the overlapping sense protein-coding mRNA to active polysomes for translation. Moreover, AS Uchl1 activity depends on the presence of a 5’ domain overlapping Uchl1 mRNA and an inverted SINEB2 element embedded along its 3’ sequence. These features are shared by other natural antisense transcripts and among them a lncRNA antisense to Ubiquitously eXpressed Transcript (AS Uxt) increases Uxt protein expression in the presence of stable mRNA level similar to AS Uchl1. These data identified a new functional class of lncRNAs and reveal another layer of gene expression control at the post-transcriptional level. Furthermore, through the replacement of AS Uchl1 5’ overlapping region with an antisense sequence to Green Fluorescence Protein (GFP) we were able to redirect this upregulation of protein synthesis. In fact, the presence of a 5’ overlapping sequence and an embedded inverted SINEB2 element confer to this artificial AS lncRNA to GFP (AS GFP) the capability to induce GFP protein with stable mRNA levels both in cells and in vitro translation assay. Further experiments are needed to set up the in vitro translation assay and to understand the translation enhancement of AS lncRNAs.

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28

Nowicki-Osuch, Karol Piotr. "Identification and characterisation of long non-coding RNAs expressed downstream of EGF-induced signalling programme". Thesis, University of Manchester, 2016. https://www.research.manchester.ac.uk/portal/en/theses/identification-and-characterisation-of-long-noncoding-rnas-expressed-downstream-of-egfinduced-signalling-programme(fd52d235-1a50-4347-bdb1-fdba4fdb912d).html.

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It has recently become apparent that cells encode a large number of novel non-protein-coding genes called long non-coding RNAs (lncRNAs). Whilst the biological function of many lncRNAs remains unknown, recent evidence has suggested that lncRNAs may be important regulators of cellular growth, differentiation and may play a significant role in cancer. Epidermal growth factor (EGF) – an activator of the ERK1/2 signalling cascade – is an important spatio-temporal regulator of transcription and, ultimately, of cellular growth and movement. EGF stimulation triggers a wave-like expression of immediate-early genes (IE genes), followed by delayed-early genes (DE genes) and secondary-response genes (SR genes). Over the years, considerable effort has been made to unravel the regulatory loops downstream of EGF signalling. This study investigated whether lncRNAs are sensitive to EGF signalling and whether they play a role in the transcriptional programme associated with EGF signalling. In order to identify lncRNAs regulated by EGF signalling, I sequenced nuclear RNA in the presence or absence of EGF stimulation. RNA-seq data showed that 173 lncRNAs are upregulated by EGF, of which 89 were intergenic lncRNAs (lincRNAs). The time-dependent expression profile of EGF-upregulated lincRNAs followed the well-established expression pattern of IE genes. Finally, investigation of the expression of lincRNAs in primary breast and lung cancer cells showed that EGF-upregulated lincRNAs were differentially expressed in cancer. The EGF-dependent induction profile and cancer enrichment were particularly strong for one of the transcripts – EGF-induced lncRNA 1 (EIN1) – and I selected it for further studies. Firstly, using bioinformatics and biochemical approaches, I confirmed the non-coding status of the EIN1 transcript. Secondly, I confirmed that EIN1 transcription is ERK1/2-dependent and is independent of protein synthesis. Investigation of EIN1 expression in normal tissues showed its high enrichment in the human cardiovascular system. At the cellular level, the EIN1 transcript was predominantly found in the nucleus. Functionally, the depletion of endogenous EIN1 transcripts (using the newly developed CRISPRi approach) led to changes in the EGF-dependent transcription programme. EIN1 downregulation resulted in the addition of normally EGF-independent genes into the EGF-dependent expression programme. Collectively, these results show that EGF (via the ERK1/2 pathway) can regulate transcription of lincRNAs. The EIN1 example suggests that lincRNAs may play a crucial role in the modulation of the EGF-dependent expression programme by limiting of the scope of the programme.

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Sukubo, N. G. "NO CODING RNAs IN MACROPHAGE POLARIZATION: THE RELEVANCE OF THE "JUNK" RNA". Doctoral thesis, Università degli Studi di Milano, 2016. http://hdl.handle.net/2434/365870.

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Abstract Form the central dogma of the biology formulated by Crick in 1958, to the detection of “junk RNA”, a new prospective has been open. Macrophages are essential cell of the innate immunity system important in onset and resolve inflammation. Moreover they are important in the cross-talk with adaptive immunology. It is also clear that macrophages are involved in many chronic diseases such as atherosclerosis, asthma, rheumatoid arthritis. In these pathologies there is an anomalous prolongation/amplification of the macrophage challenge to lead homeostasis. In this landscape favor macrophage activation and re-programming. Mantovani and colleagues have schematically categorized macrophages in “classical” (M1) activated, triggered with microbial stimuli (e.g. LPS) alone or together with the TLR eliciting; and “alternative” (M2) cell types, activated by IL-4/IL-13. The identification of the underlying molecular mechanisms on the base of this process may suggest new approaches to interfere with chronic inflammation and other inflammatory disease, such as cancer. The aim of this presented work was to characterize the epigenetic mechanism involved in macrophage polarization. To better elucidate this purpose the thesis was divided in three macro chapter: • MicroRNA-135b: the pivot of the macrophage polarization balance; • The “junk” RNA controlled by glucocorticoids: miR-135b and its host gene BLACAT1; • MicroRNA-135b in gouty arthritis. Overall, we identified a specific miRNome in human classic and alternative polarized macrophages (69 differential expressed miRNAs among the subsets). In addition, we pointed out the impact of miR-135b a de novo expressed miRNA in M1 macrophages, and for the first time associated with macrophages in an inflammatory diseases such as gouty arthritis. Indeed we demonstrated that miR-135b locus is activated by the inflammatory stimulus, LPS, which discharge the repressor complex polycomb2. Although, we can classified miR-135 as M1-associated or induced, which damps M2 phenotype in favor of the M1 thru the targeting of important the transcription factor, c-MYC, STAT6 and KLF4, sustaining the inflammation. In addition miR-135b expression is inhibited by the anti-inflammatory cytokine IL-10, to highlight its pro-inflammatory role; the same results was assess for miR-155. We have shown in the model of gouty arthritis, miR-135b is induced during the progression of the inflammation by macrophages. However it provides a negative feedback to limit excessive macrophage response to MSU crystal, thru the targeting of the IL-1β pathway. These results confirm the relevance of miR-135b as an important hinge of macrophage polarity. At the light of these observations, the identification of the underlying process that regulates the expression of miR-135b will be essential. miR-135b is located in the intron of the lncRNA, BLACAT1. We glimpsed that the induction of miR-135b and BLACAT1 is not correlated; on the contrary BLACAT1 is induced by anti-inflammatory stimulation, influencing miR-135b. Although we can speculate a novel type of regulation beyond IL-10, used by the macrophages to control the expression of this miRNA. Probably with the action of the MCP-induced protein 1 (MCPIP1), which is induced downstream LPS and IL-1β pathway and has been shown to suppress miR-135b biosynthesis in cancer.

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Xie, Bingning. "Long non-coding RNA-based mechanisms for the inhibition of cell growth and development by 5 - Fluorouracil". Thesis, Rennes 1, 2016. http://www.theses.fr/2016REN1B046/document.

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Les ARNm codent pour les protéines, tandis qu'un grand nombre d'ARNs nommés longues ARNs non codants (ARNlnc) ne sont pas traduites en protéines. Les deux types d’ARNs existent en isoforms qui se distinguent à cause de l’épissage alternatif. Certains des ARNlnc jouent des rôles importants dans la croissance et différentiation cellulaire. Cependant, leurs fonctions dans la cytotoxicité de la chimiothérapie anti-cancéreuse médicamenteuse utilisant le 5-fluorouracile (5-FU) sont encore inconnues. Pendant mes travaux j'ai trouvé que le traitement par le 5-FU cause l’accumulation des ARNlnc. Ce phénomène est parfois, sous forme d’ARN double brin (ARNds) formé par une paire de transcrits chevauchant, corrélé négativement avec le niveau de la protéine codée par l'ARNm. Cette inhibition potentielle de la traduction des régulateurs du cycle cellulaire clés et les gènes essentiels en formant des l'ARNds peut éventuellement empêcher la progression du cycle cellulaire. Nos analyses prometteuses devraient inspirer des études approfondies des ARNlnc dans la cytotoxicité du 5-FU chez la levure et l’homme afin d’'améliorer la chimiothérapie. J'ai trouvé que la surexpression de RRP6, peut conduire à une résistance accrue au traitement par le 5-FU. Je démontre ensuite que l’ARNlnc MUT1312 forme des ARNds avec RRP6 qui sont négativement corrélés avec le niveau de la protéine Rrp6. Par ailleurs, la surexpression de MUT1312 pendant la mitose et associé avec une diminution d’Rrp6. Ainsi, mon étude suggère que MUT1312 soit impliqué dans la régulation de Rrp6 pendant la differentiation cellulaire. Mes recherches de MUT477/SWI4 indiquent la function importante de la méiose induite à long ARN non codantes en tant que forme d'ARN double brin potentiellement réguler la traduction. J'ai trouvé que SUT200 pourrait inhiber la transcription de CDC6 durant la méiose par read-through. Un cas comparable est MUT1465 et CLN2. J’ai fait un criblage in silico pour trouver des facteurs de transcription qui activent des MUTs durant la méiose. J’ai trouvé que la plupart des MUTs sont induites par Ndt80. MUT1465 est parmi eux : il pourrait être induite par Ndt80 ce qui inhiberait l’expression de CLN2 après l’initiation de la méiose. J’ai trouvé que la répression de certains MUTs par le complexe Ume6/Rpd3 en mitose est différemment régulée entre JHY222 et SK1. MUT100 qui ne possède pas l'élément USR1 fixé par Ume6, et qui est donc une cible indirecte, est déréprimé dans JHY22 ume6 mais pas dans SK1 ume6. Pour la régulation de l'étude de isoforme méiose, Nous avons trouvé que le complexe histone déacétylase Rpd3/Sin3/Ume6, empêche également l'induction de l'isoforme longue de BOI1 dans la mitose par liaison directe de liaison Ume6 à sa cible de URS1. Orc1 est importante pour la réplication de l'ADN. J’ai démontré que mORC1 est une cible directe de l'activateur Ndt80 et que son motif de fixation (MSE) est nécessaire pour l'induction de l’isoforme mORC1 et du gene méiotique SMA2 transcrit de façon divergente. J’ai trouvé qu'une souche incapable d’induire mORC1, contient des niveaux anormalement élevés d’Orc1 pendant la gamétogenèse, ce qui corréle mORC1 avec la baisse de la protéine Orc1. En conclusion, mes études au cours du doctorat révèlent des nouvelles cibles et ainsi offrent des nouvelles perspectives de l’amélioration de la chimiothérapie par le 5-FU. Les mécanismes incluent la formation d'un ARN double brin avec son ARNm anti-sens pour potentiellement inhiber la traduction de l'ARNm, et inhibition en aval de l'ARNm par transcription read-through d’une ARNlnc. Mon travail a également révélé un mécanisme de régulation des ARNlnc et les isoforms d’ARN pendent la croissance et la différentiation cellulaire
RNAs are molecules with important functions in diverse cellular processes. mRNAs encode proteins, while a large number of RNAs called long noncoding RNAs (lncRNAs) are not translated into proteins. Both types of RNAs exist in various isoforms due to alternative splicing.Some of lncRNA play important roles in cell growth and differentiation. However, their functions in the cytotoxicity of the drug anticancer chemotherapy using 5-fluorouracil (5-FU) are still unknown. During my research I found that treatment with 5-FU causes accumulation of lncRNA. Acuumulated antisense lncRNA form double stranded RNA with the mRNAs , negatively correlated with the level of the protein encoded by the mRNA. This potential inhibition of translation of key cell cycle regulators and essential genes by forming dsRNA may possibly prevent the progression of the cell cycle. My results suggest that lncRNA are likely to play an important role in the cytotoxicity of 5-FU. Our promising testing should inspire in-depth studies of lncRNA in the cytotoxicity of 5-FU in yeast and humans to improve chemotherapy.Rrp6 is a 3'-5 'exoribonuclease, which plays an important role in the regulation and modification of rRNA, mRNA and lncRNA. I found that overexpression of RRP6, the homologue of the yeast EXOSC10 gene in mammals, can lead to increased resistance to treatment with 5-FU. I found that the lncRNA MUT1312 form dsRNA with RRP6 that are negatively correlated with the level of Rrp6 protein. Furthermore, overexpression of MUT1312 during mitosis and associated with a decrease of Rrp6. Thus, my study suggests that MUT1312 may involved in the regulation of Rrp6 during cell differentiation. I further explored the function of the double-stranded RNA in meiosis. My research about SWI4/MUT477 indicates the important function of meiosis induced long noncoding RNA as a form of double-stranded RNA potentially regulate translation. Another aspect of the function of lncRNA is to regulate the transcription of downstream mRNA. I found SUT200 could inhibit transcription of CDC6 during meiosis by read-through. A similar case is CLN2/MUT1465. I did an in silico screening to find transcription factors that activate MUTs during meiosis. I found that most MUTs are induced by Ndt80. MUT1465 is among them: it could be induced by Ndt80 which inhibit the expression of CLN2 after initiation of meiosis. I found that repression of certain MUTs by the Ume6 / Rpd3 complex in mitosis is regulated differently between JHY222 and SK1. MUT100 which does not have the Ume6 binding site URS1 element, and is therefore an indirect target is derepressed in JHY22 ume6 but not in SK1 ume6. For the study about regulation of meiosis isoform, we have found that the histone deacetylase complex Rpd3 / Sin3 / Ume6 prevents the induction of long isoform BOI1 in mitosis by direct binding Ume6 binding to its target URS1.Orc1 is important for DNA replication. I have demonstrated that mORC1 is a direct target of the Ndt80 activator and its binding motif (MSE) is required for induction of isoform mORC1 and meiotic gene SMA2 divergently transcribed. I found that a strain incapable of inducing mORC1 contains abnormally high levels of Orc1 during gametogenesis, which correlates with mORC1 declining Orc1 protein. Since eukaryotic genes often encode multiple transcripts with 5'-UTR of variable length, the findings are likely relevant to gene expression during development and disease in higher eukaryotes. In conclusion, my studies during PhD reveal new targets and thus offer new prospects for improving chemotherapy with 5-FU. Mechanisms include (1) the formation of a double strand with its antisense mRNAs to potentially inhibit translation of mRNA, and (2) downstream inhibition of mRNA transcription read-through of a lncRNA. My work also revealed a lncRNA regulatory mechanism and RNA isoforms dangling growth and cell differentiation

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31

Gandhi, Minakshi [Verfasser] y Peter [Akademischer Betreuer] Angel. "Role of long non-coding RNA lincNMR in nucleotide metabolism in cancer / Minakshi Gandhi ; Betreuer: Peter Angel". Heidelberg : Universitätsbibliothek Heidelberg, 2019. http://d-nb.info/1195143826/34.

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32

Musahl, Anne-Susann [Verfasser]. "Transcriptional characterization and functional analysis of long non-coding RNA/protein-coding gene pairs encoded in the human genome / Anne-Susann Musahl". Berlin : Freie Universität Berlin, 2015. http://d-nb.info/1075493706/34.

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33

GIAGNORIO, ELEONORA. "Revealing the involvement of MALAT1, NEAT1, HOTTIP lncRNAs in Amyotrophic Lateral Sclerosis (ALS) via an induced pluripotent stem cell (iPSC)-derived muscle cell model". Doctoral thesis, Università degli Studi di Milano-Bicocca, 2022. http://hdl.handle.net/10281/385034.

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La SLA è una malattia neurodegenerativa caratterizzata da una progressiva degenerazione dei MN, con conseguente atrofia muscolare, paralisi e morte del paziente. Esistono due forme di SLA, la forma sporadica, nel 90% dei pazienti, e la forma familiare, nel restante 10% dei casi. Diversi geni sono associati alla SLA, come C9ORF72 che è il gene più comunemente associato alla forma familiare di SLA, seguito da TARDBP, SOD1 e FUS. Questi geni influiscono su diverse funzioni cellulari, tra cui lo stress ossidativo, il metabolismo dell’RNA, l’organizzazione del citoscheletro e l’autofagia. I geni associati alla SLA sono espressi in modo ubiquitario e quindi diversi tipi cellulari possono subire alterazioni nella struttura e metabolismo e insieme contribuiscono ai pathways degenerativi della SLA. Oltre ai MN, studi recenti dimostrano che il muscolo scheletrico è coinvolto precocemente durante la patogenesi della SLA. Ad oggi non esistono cure e uno degli obiettivi della ricerca è lo sviluppo di terapie, ottenute tramite una conoscenza specifica degli eventi molecolari che portano alla degenerazione dei MN e del tessuto muscolare. La deregolazione dell’RNA ha un contributo chiave nella patogenesi della SLA. Nel campo dell’RNA non coding, i long non coding RNA (lncRNA) emergono come contribuenti alla patofisiologia della SLA. I lncRNA, lunghi dalle 300 alle centinaia di nucleotidi, sono regolatori dell’espressione di geni muscolari e neuronali, ma il loro contributo alla patogenesi della SLA è ancora ignoto. In questo lavoro abbiamo analizzato i livelli di espressione di MALAT1, NEAT1 e HOTTIP lncRNA coinvolti nello sviluppo e omeostasi del muscolo scheletrico, nel modello di cellule pluripotente indotte umane (iPSC) derivate da pazienti SLA e controlli sani, e differenziate verso un destino miogenico tramite un protocollo basato sulle small molecules. Abbiamo analizzato l’espressione di marcatori dello sviluppo del muscolo scheletrico tramite qPCR. Inoltre, abbiamo predetto in silico e poi validato gli mRNA target dei lncRNA. Abbiamo riportato un diverso pattern di espressione dei lncRNA e target mRNA nelle colture cellulari SLA, rispetto ai controlli, in particolare allo stadio di progenitore mesodermico, miociti e miotubi. Tramite un’analisi di clustering gerarchico abbiamo identificato cluster specifici di lncRNA/geni target che caratterizzano le linee SLA, il che suggerisce che un’alterata espressione di queste molecole può contribuire alla patogenesi della malattia. Le nostre scoperte sulla deregolazione di MALAT1, NEAT1 e HOTTIP e dei loro geni target offre nuovi spunti riguardo le basi molecolari della SLA, suggerendo la possibilità che un alterato sviluppo del muscolo scheletrico, dipendente da queste molecole, possa portare a un’alterazione della massa e funzionalità muscolare durante malattia. Ulteriori studi sono necessari per indagare maggiormente l’effetto sinergico di MALAT1, NEAT1 e HOTTIP sull’insorgenza e/o progressione della malattia, con lo scopo di sviluppare strategie terapeutiche contro la SLA o altre malattie del motoneurone che siano paziente specifiche, basate sui lncRNA
Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative and fatal disease characterized by progressive cortical, bulbar and spinal motor neuron (MN) degeneration, leading to progressive muscle weakness, atrophy, paralysis and, ultimately, death. ALS can occur in two different forms: sporadic ALS (sALS) in ∼90% of individuals and familial ALS (fALS). Different genes have been associated with fALS and/or sALS; C9ORF72–SMCR8 complex subunit (C9ORF72) is the gene most commonly linked to inherited ALS, followed by TAR DNA-binding protein 43 (TARDBP), superoxide dismutase 1 (SOD1) and FUS RNA-binding protein (FUS). Such genes affect several cellular functions, including oxidative stress (SOD1), RNA metabolism (C9ORF72, TARBDP and FUS), cytoskeletal organization [e.g. tubulin alpha-4a (TUBA4A) and profilin 1 (PFN1)] and autophagy [e.g. TANK-binding kinase 1 (TBK1) and optineurin (OPTN). ALS-associated mutant genes are ubiquitously expressed, thus alterations in structure, metabolism and physiology occur in different cell types, synergistically contributing to ALS degenerative pathways. It is generally accepted that ALS is primarily caused by MN death. However, growing evidence has shown that muscle is active and plays a crucial role in the disease onset and progression. Currently, there are no effective treatments for ALS. Indeed, one of the major aims in ALS research is the development of successful therapies, by deepening the knowledge of the molecular events leading to the degeneration of both MNs and muscle tissue. It has become increasingly clear that RNA dysregulation is a key contributor to ALS pathogenesis. Among non-coding RNAs, long non-coding RNA (lncRNAs) are emerging as molecular contributors to ALS pathophysiology because of their role in regulating gene expression. LncRNAs, that are 300 to thousands nucleotides long, being more similar to mRNA than microRNAs, are key MN and muscle gene expression regulators. However, the exact contribution to ALS pathogenesis is still unknown. Here, we analysed the expression levels of MALAT1, NEAT1 and HOTTIP lncRNAs, known to be involved in the development and homeostasis of the skeletal muscle, in a human induced pluripotent stem cell (hiPSC) model differentiated towards a myogenic destiny through a small molecule-based protocol, obtained from ALS patients and healthy controls. The expression of key markers of skeletal muscle development was assessed by qPCR. Further, mRNA targets of the lncRNAs were predicted in silico, and validated by qPCR. We reported a differential lncRNA and mRNA target expression pattern in ALS-mutant cultures compared to controls, particularly at the mesodermal progenitor, early myocyte and myotube stages. Specifically, through hierarchical clustering analysis we identified specific clusters of lncRNA/target gene defining ALS cell lines, suggesting that an altered expression of these molecules might contribute to the disease pathogenesis. Our findings on dysregulation of MALAT1, NEAT1, HOTTIP and their target genes in the iPSC-based ALS in vitro model provide new insights into ALS molecular basis, pointing out the possibility that altered muscle differentiation processes, depending on these lncRNAs, could eventually lead to an altered availability of muscle mass and function in the disease. Further studies in genetically defined, or not defined, ALS patients, and in other motor neuron diseases (MNDs), could help to deeply understand the synergistic effect of MALAT1, NEAT1 and HOTTIP in disease onset and/or progression, towards future development of patient-specific lncRNA-based therapeutic strategies for ALS and other MNDs.

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34

Rontani, Pauline. "Caractérisation du long ARN non codant COSMOC dérégulé dans les troubles du spectre autistique : une approche transcriptomique sur cellules souches olfactives humaines". Thesis, Aix-Marseille, 2018. http://www.theses.fr/2018AIXM0770.

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L’autisme est un syndrome neuro-développemental hétérogène à l’étiologie génétique complexe. Afin d’identifier les dérèglements initiaux responsables de ce mal-développement cérébral, des travaux antérieurs au sein de notre équipe se sont basés sur des cellules représentatives des stades précoces de l’ontogenèse : les cellules souches olfactives. Le gène MOCOS, codant pour la sulfurase du cofacteur à molybdène, a été trouvée sous-exprimé chez la majorité des patients autistes comparés à des sujets contrôles de même âge et du même sexe.Nous avons ensuite postulé que la dissection minutieuse des mécanismes moléculaires pouvant rendre compte de cette dérégulation aiderait à trouver des mécanismes sous-jacents contribuant aux troubles du spectre autistique (TSA). Ceci a conduit à l'identification de COSMOC, un long ARN non codant généré à partir d'une transcription divergente dans la région promotrice de MOCOS, dont l'expression est diminuée chez 10 des 11 patients autistes de notre cohorte. A l’aide de diverses techniques de biologie moléculaire, nous avons montré que la déplétion de COSMOC induit : (1) une sous-expression de MOCOS, (2) une déstabilisation de l'organisation de la chromatine, ce qui suggère une fonction de régulateur transcriptionnel, et (3) une altération du métabolisme des lipides et de l’homéostasie redox de la cellule, deux voies dérégulés dans les TSA. Par ailleurs, COSMOC régule de l’expression de la PTBP2 (polypirimidine track biding protein 2), un facteur d’épissage contrôlant l’expression de nombreuses protéines synaptiques. En conclusion, la dérégulation de COSMOC pourrait expliquer certains des dysfonctionnements observés dans les TSA
Autism is a heterogeneous neuro-developmental syndrome with a complex genetic etiology. In order to unveil the initial disturbances responsible for this brain maldevelopment, previous works in our team relied on cells representative of the early stages of ontogenesis: olfactory stem cells. The MOCOS gene, coding for molybdenum cofactor sulfurase, was found under-expressed in most of autistic patients of our cohort when compared with age- and gender-matched control adults without any neuropsychiatric disorders. We postulated that the meticulous dissection of the molecular mechanisms involved this deregulation would help to unveil pathogenic mechanisms underlying autism spectrum disorders (ASD). This led to the identification of COSMOC, a long non-coding RNA, generated from a divergent transcription in the promoter region of MOCOS, whose expression is decreased in 10 out of 11 autistic patients in our cohort. Using various molecular biological techniques (interference RNA, DNA microarray, qPCR...), we showed that COSMOC depletion induces: (1) an under-expression of MOCOS, (2) a destabilization of chromatin organization, suggesting a transcriptional regulatory function, and (3) an alteration of cellular lipid metabolism and redox homeostasis, two deregulated pathways in ASD. In addition, COSMOC regulates the expression of PTBP2 (polypirimidine track biding protein 2), a splicing factor that controls the expression of many synaptic proteins, including PSD95. In conclusion, the deregulation of COSMOC may explain some of the dysfunctions observed in ASDs

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35

Zhang, Shaoyan. "Overexpression of the Turnip Crinkle Virus Replicase Exerts Opposite Effects on the Synthesis of Viral Genomic RNA and a Novel Viral Long Non-Coding RNA". The Ohio State University, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=osu1595258672390499.

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Furlan, Giulia. "Investigating the contribution of the non-coding gene Ftx to X-chromosome inactivation in mammals". Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCC191/document.

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L’inactivation du chromosome X (XCI) est un mécanisme qui permet l’extinction transcriptionelle d’un des deux chromosomes X chez la femelle. XCI est régulé par une région spécifique nommée centre de l’inactivation du chromosome (Xic), contenant plusieurs gènes produisant de longs ARNs non codants (lncRNAs). Parmi ces lncRNAs, le transcrit Xist est l’effecteur principal pour l’XCI. Xist peut s’accumuler en cis sur le chromosome et recruter la machinerie qui permettra l’initiation et la propagation de l’extinction transcriptionnelle à l’échelle du chromosome.Le laboratoire d’accueil a identifié un nouveau gène du Xic qui produit le lncRNA Ftx. Dans cette étude, on a pu montrer que l’inactivation du chromosome X est fortement perturbée dans les cellules Ftx-/- et s’accompagne par une forte baisse du niveau d’expression et d’accumulation de Xist. Dans ce contexte, certaines cellules parviennent à maintenir l’expression de Xist mais le profil de couverture du chromosome X par Xist est anormal, présentant un profil diffus ; ceci est associé à une extinction transcriptionnelle déficiente des gènes liés à l’X. Dans les lignées hétérozygotes Ftx+/-, l’expression et l’accumulation de Xist est aussi affectée mais dans une moindre mesure, si bien qu’il apparaît que le nombre de copies de Ftx soit important pour sa fonction. Par ailleurs, l’inactivation du chromosome X dans les cellules Ftx+/- est biaisée de telle sorte que le chromosome X portant une copie fonctionnelle de Ftx est préférentiellement inactivé, suggérant un rôle en cis de Ftx. Ces résultats montrent que Ftx est un activateur de Xist et qu’il est essentiel pour la mise en place de l’inactivation
X-chromosome inactivation (XCI) is a female-specific, chromosome-wide regulatory process that, in eutherians, ensures dosage compensation for X-linked genes between sexes. XCI is controlled by a cis-acting locus on the X-chromosome, the X-inactivation center (Xic), enriched in genes producing long non-coding RNAs (lncRNAs). The Xic-linked gene Xist is the master player of XCI, and produces a lncRNA that accumulates in cis on the X-chromosome and recruits the machinery responsible for initiation and propagation of silencing.The laboratory has identified an additional Xic-linked non-coding gene, Ftx. In this study, we could find that, in female Ftx-/- lines, XCI is strongly impaired, with a significant decrease in the levels of Xist expression and in the percentage of cells showing normal Xist accumulation patterns. Importantly, a high proportion of the cells that still retain Xist expression show abnormal X-chromosome coating and a decreased ability to silence X-linked genes. These data reveal that Ftx is a positive Xist regulator and it is required for proper XCI establishment. In female Ftx+/- lines, the levels of Xist expression and the percentage of cells showing normal Xist accumulation patterns are also decreased, albeit to a lower extent compared to Ftx-/- lines, suggesting that Ftx works in a copy-dependent manner. In addition, a high proportion of Ftx+/- cells display skewed X-inactivation, with preferential inactivation of the wild-type X chromosome. This suggests that Ftx role on Xist accumulation is mostly restricted in cis. Taken together, these results demonstrate that Ftx is required for XCI establishment, where it functions as a strong Xist activator

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37

Chehade, Marthe. "Thre Long Noncoding RNA PRINS in Endocrine Cancer: Evaluating its Clinical Significance and the Evidence for its Functional Role". Thesis, The University of Sydney, 2022. https://hdl.handle.net/2123/29641.

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Adrenocortical carcinoma (ACC) and breast cancer are endocrine malignancies with poor prognosis in advanced disease. Long non-coding RNA (lncRNA) have varied roles in the regulation of gene expression and can act as oncogenes and tumour suppressors. Psoriasis-susceptibility associated RNA gene induced by stress (PRINS) is a lncRNA that is under-expressed in ACC tissues in a tumour suppressor pattern and predicts disease recurrence. This thesis aims to evaluate the potential of PRINS as a tumour suppressor in various ACC and breast cancer in-vitro gain-of-function (GoF) models, and explore the cellular changes it effects at the proteome level. The potential of PRINS as a clinical biomarker in breast cancer tissues is also evaluated. Methods: PRINS expression in breast cancer tissues was determined and correlated with tumour and clinical factors. Stable PRINS over-expression models were established in ACC and breast cancer cells for GoF studies. The effect of PRINS over-expression on the proteome of ACC cells was determined using mass spectrometry (MS) and Western Blot (WB). Finally, an ACC CRISPRa PRINS over-expression model was explored. Results: PRINS was under-expressed in breast cancers relative to normal breast tissues and its expression was associated with tumour histological grade. PRINS GoF studies in ACC and breast cancer cell models did not demonstrate tumour suppressor effects. Proteome analysis by MS revealed 28 PRINS-regulated proteins in ACC cells, of which the RNA-binding protein CAPRIN1 was validated with WB. Conclusions: PRINS under-expression was demonstrated as a clinically relevant diagnostic biomarker in breast cancer tissues, but no significant phenotypic effect of PRINS over-expression was demonstrated in in-vitro cancer models. While CAPRIN1 was identified as a target of PRINS, MS analysis did not implicate PRINS in growth-associated cellular pathways. Further interrogation of PRINS potential cellular function is warranted.

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38

Moreno, Leon Laura. "Étude d'un long ARN non codant induit par l'hypoxie et associé à l’agressivité des adénocarcinomes bronchopulmonaires". Electronic Thesis or Diss., Université Côte d'Azur (ComUE), 2017. http://www.theses.fr/2017AZUR4144.

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Les cancers bronchopulmonaires non à petites cellules (CBNPC) sont la première cause de décès par cancer, les adénocarcinomes étant la forme la plus fréquente. Malgré une prise en charge précoce, ils constituent, par leur taux de récidive important et leur mauvais pronostic, un véritable problème de santé publique. Nous nous intéressons à l'hypoxie, un facteur agressivité des ADC, et à la famille des longs ARNs non codants (lncARNs), dérégulés dans de nombreux cancers et en réponse à l'hypoxie, mais peu caractérisés sur le plan structural et fonctionnel. Ces transcrits représentent un espoir pour le développement de nouvelles thérapies. Au cours de ma thèse, j'ai identifié une dizaine de lncARNs régulés par l'hypoxie in vitro et in vivo dans des ADC de stades précoces. j'ai caractérisé NLUCAT1, un transcrit nucléaire induit par l'hypoxie. L'invalidation de ce transcrit par le système CRISPR/Cas9 a révélé une diminution de la prolifération et de l'invasion cellulaires, et une augmentation du stress oxydatif et de la sensibilité au cisplatine, traduisant un potentiel rôle pro-oncogénique de ce transcrit dans les ADC. L'analyse du transcriptome a révélé une répression des réseaux de gènes contrôlés par NRF2, HIF et NFkβ dans les cellules déficientes pour NLUCAT1. Nous avons notamment identifié des gènes de la réponse anti-oxydante régulés par NRF2 dont l'ARN interférence mime en partie les conséquences de l'inactivation de NLUCAT1 sur l'apoptose. Nos résultats démontrent que NLUCAT1 exerce des activités pro-tumorales dans les ADC et suggère qu'il pourrait représenter une cible thérapeutique potentielle dans ce type de cancer
Non Small Cell Lung Cancer (NSCLC) is the leading cause of cancer death worldwide, with poor prognosis and a high rate of recurrence despite early surgical removal. It is therefore essential to identify new prognostic markers and new therapeutic targets. We are interested in gene regulation related to hypoxia, a factor associated with relapse of lung adenocarcinomas (LUAD). The roles of long non coding RNAs (incRNAs) in cancer development and hypoxic response are largely unexplored. A transcriptome profiling of early-stage LUAD samples indicated that a set of incRNAs was correlated to a metagene hypoxic signature. Some of these transcripts were also sensitive to hypoxia in LUAD cell lines. We focused on a new "hypoxaLinc", named NLUCAT1 that is strongly up-regulated by hypoxia in vitro and correlated to hypoxic markers and bad prognosis in LUAD samples. Full molecular charactherization of NLUCAT1 showed that LUCAT1 is mainly regulated by NF-kβ and NRF2 transcription factors. Targered deletion of NLUCAT using CRISPR/CAS9 in A549 LUAD cell line, revelated a decrase in proliferative and invasive properties, an increase in oxidative stress and a higher sensisivity to displatin-induced apoptosis. We identified genes of the NRF2-regulated and anti-oxidant response whose RNA interference partially mimicked the consequences of NLUCAT1 inactivation on ROS-dependent caspase activation. Overall, our data strongly demonstrate that NLUCAT1 exerts pro-tumoral activities in early stages hypoxic LUADs ans suggest it could represent a new potential therapeutic target in lung cancer

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39

Dumbović, Gabrijela 1986. "Molecular and mechanistic characterization of a novel long non-coding RNA derived from NBL2 macrosatellite repeats in colorectal cancer". Doctoral thesis, Universitat Pompeu Fabra, 2017. http://hdl.handle.net/10803/523517.

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DNA hypomethylation of repeats has been found in almost every tumor type studied. Hypomethylation of repetitive elements was shown to increase the likelihood of homologous recombination of these repeated sequences and thus contribute to genome instability. In our group we have found a macrosatellite repeat called NBL2 hypomethylated in a subset of colorectal cancers (CRCs). NBL2 somatic hypomethylation in CRC associated with genomic damage, especially in tumors having wild type P53. Here, we describe the genomic location of NBL2 by thorough in silico analysis, metaphase FISH and PCR on DNA from human-rodent somatic cell hybrids. We find that NBL2 repeats are dispersed on multiple chromosomes, however tandem arrays are placed close to centromers in the short arms of acrocentric chromosomes 13, 14, 15 and 21. Furthermore, we mechanistically addressed NBL2 transcription in an effort to elucidate the possible roles NBL2 RNA might have. We describe a novel lncRNA transcribed from NBL2 repeats upon DNA hypomethylation and histone acetylation only from chromosomes 13, 14, 15 and 21. We characterize NBL2 lncRNA as several high-molecular weight transcripts (4.2, 10 and 13 kb). By applying single molecule RNA FISH, we demonstrate those transcripts are retained in the nucleus, where they are widely distributed with preferential accumulation in the perinucleolar region. In order to study the molecular pathway in which NBL2 lncRNA might be involved, we analyzed its putative protein interacting partners by RNA affinity purification followed by proteomic analysis. We find that NBL2 RNA binds with high affinity to several proteins, mainly involved in RNA metabolism. Among those proteins is RNA binding protein CELF1 that regulates pre-mRNA splicing and mRNA stability/decay. Based on our results, we propose a model where, in addition to the contribution of somatic DNA hypomethylation of NBL2 repeats to genomic damage, NBL2 lncRNA contributes to instability by sequestering proteins involved in RNA metabolism, such as CELF1, consequently destabilizing their mRNA targets. NBL2 repeats may thus have a role in the generation of genomic instability underlying CRC pathogenesis by increasing cancer risk upon its somatic demethylation in cancer precursor cells, and/or by enhancing instability during tumor progression. The abnormal transcription of these repeats we observed in the form of NBL2 lncRNA may play a role in either or both of these pathways.
La hipometil·lació de l'ADN en elements repetitius s'ha trobat en gairebé tots els tipus de tumor estudiats. Aquesta hipometil·lació augmenta la probabilitat de recombinació homòloga d'aquestes seqüències repetides i per tant contribueix a la inestabilitat del genoma. El nostre grup ha descobert que un element macrosatèl.lit repetit en tàndem, anomenat NBL2 es troba desmetil.lat en un subgrup de càncers colorectals (CCR) i la seva hipometil.lació somàtica en el CCR s'associa amb el dany genòmic, especialment en tumors que no tenen mutat el gen de la P53. A continuació, es descriu la localització genòmica de NBL2 mitjançant una anàlisi in silico detallada, realitzant hibridacions de fluorescència in situ en metafases i monitorant per PCR la seva presència en l'ADN genòmic procedent de cèl.lules híbrides humanes / de rosegadors. Hem trobat que els elements NBL2 estan dispersos en diversos cromosomes, però les concatenacions en tàndem es troben a prop dels centròmers i més concretament en els braços curts dels cromosomes acrocèntrics 13, 14, 15 i 21. D'altra banda, s'ha caracteritzat la transcripció procedent de NBL2 i s'han intentat esbrinar les possibles funcions que l'ARN de NBL2 podria tenir en el context del CCR. Hem determinat l'existència d'un nou ARN llarg no codificant (lncRNA) que es transcriu quan el seu ADN es desmetil.la i presenta les seves histones acetilades només a partir dels cromsomes 13, 14, 15 i 21. La transcripció de l'element NBL2 genera tres lncRNA amb un d'alt pes molecular (4,2, 10 i 13 kb). Aquests transcrits són retinguts al nucli, en el qual s'acumulen preferencialment en la regió perinucleolar. Per tal d'estudiar les vies intracel.lulars on l'ARN de NBL2 podria estar involucrat, i determinar la seva possible funció, es va monitorar la seva interacció amb proteïnes realitzant una purificació per afinitat seguida d'una anàlisi proteòmica. Hem determinat que l'ARN de NBL2 s'uneix amb alta afinitat a diverses proteïnes implicades principalment en el metabolisme de l'ARN. Entre aquestes proteïnes destaca CELF1, una proteïna que regula el processament del pre-ARNm i l'estabilitat de l'ARNm. D'acord amb els resultats, es proposa un model en el qual, no només la hipometil.lació somàtica de l'ADN de NBL2 contribuiria al dany genòmic, sinó que també l'ARN de NBL2 podria jugar un paper pertorbador a través de segrestar proteïnes implicades en el metabolisme de l'ARN, com per exemple CELF1, i en conseqüència, desestabilitzar els seus ARNm diana. Així doncs, els elements repetits NBL2 podrien tenir un paper en la generació de la inestabilitat genòmica subjacent a la patogènesi del càncer de còlon per mitjà de l'increment del risc a causa d' una desmetil.lació somàtica present en les cèl.lules precursores del càncer, i/o afavorint la inestabilitat durant la progressió tumoral. La transcripció aberrant que hem observat a partir d'aquests elements NBL2 repetitius en forma de lncRNA podria contribuir en ambdues vies.

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40

Barcelos, Rafael Mazioli. "Is Rickettsia amblyommii able to regulate long non-coding RNA expression in Amblyomma sculptum tick? An in silico approach". Universidade Federal de Viçosa, 2017. http://www.locus.ufv.br/handle/123456789/12399.

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O carrapato Amblyomma sculptum pertence ao complexo Amblyomma cajennnense, taxon mais importante na transmissão e como hospedeiro da bactéria Rickettsia rickettsii, principal agente etiológico da Febre Maculosa Brasileira e da Febre Maculosa das Montanhas Rochosas nos EUA. Como membro deste complexo de espécies, o carrapato A. sculptum é atualmente muito importante sob o aspecto médico veterinário atuando como vetor deste agente zoonótico no Brasil. Seu papel no ciclo biológico da riquétsia, bem como a relação Rickettsia-carrapato, precisam ser bem compreendidos pois estudos recentes encontraram esta espécie de bactéria no carrapato A. sculptum. Como parte desta relação, as funções de RNAs não codificantes de cadeias longas (lncRNA) são desconhecidas e precisam ser descobertas. Os lncRNAs estão envolvidos em uma infinidade de atividades celulares tais como transcrição, silenciamento gênico e abertura cromossômica, por exemplo. Dessa forma, apresentamos aqui uma abordagem in silico da modulação de lncRNAs pela Rickettsia amblyommii em carrapatos da espécie A. sculptum. Tomando como dados trabalhos publicados anteriormente nós identificamos e avaliamos possíveis lncRNAs para expressão diferencial em intestinos e ovários desta espécie de carrapato. Dois montadores de sequencias foram testados, Trinity e CLC Genomics, para construção de contigs e um filtro a partir de um pipeline caseiro para selecionar apenas sequências com características de lncRNA. Um total de 31.888 e 23.733 contigs foram montados pelo Trinity e CLC Genomics, respectivamente. Nós anotamos mais de 500 sequências possíveis de lncRNA contra os bancos de dados RefSeq RNA, RNA Central e NONCODE. Nossos resultados sugerem que a R. amblyommii está induzindo expressão diferencial de lncRNAs nos tecidos dos intestinos e ovários. Nosso trabalho contribui para o aumento do banco de dados de lncRNA de carrapatos bem como fornecer idéias iniciais de quais sequências de lncRNA estão envolvidas na relação Rickettsia- carrapato
The Amblyomma sculptum tick belongs to Amblyomma cajennnense complex, the most important taxon in transmission and host for Rickettsia rickettsii bacteria, the main etiologic agent of Brazilian Spotted Fever in Brazil and Rocky Mountain Spotted Fever in USA. As a member of this species complex, A. sculptum tick is actually very important under human and veterinary subject acting as vector for this zoonotic agent in Brazil. Its role in biological cycle of rickettsia strains, as well Rickettsia-tick interactions, still need to be better understood since recent studies found these strains infecting A. sculptum ticks. As putative modulators of these pathogen-host interaction, the roles of long non-coding RNAs (lncRNA) of ticks are unknown and need to be discovered and characterized. The lncRNAs are involving in modulation of a plethora of cell activities as transcription, gene silencing and chromosome opening, for example. Thus, herein we present an in silico approach for analyze the modulation of lncRNAs by R. amblyommii in A. sculptum tick. Using previously published data sets of A. sculptum transcriptomes, we identified putative lncRNAs and evaluated for differential expression in midgut and ovaries in this tick specie. Two assemblers were tested, Trinity and CLC Genomics, to construct the contigs and a pipeline to select the sequences with lncRNA characteristics. We obtained 31,888 and 23,733 contigs of putative lncRNAs using Trinity and CLC Genomics, respectively. We further identified more than 500 sequences of putative lncRNA that significantly aligned to sequences of RefSeq RNA, RNA Central and NONCODE databases. The transcriptome analysis further suggests that R. amblyommii is inducing differential expression of putative lncRNAs in midgut and ovary tissues. The work herein contributes for tick lncRNA database increasing and the initials insights of which lncRNA sequences are involving in Rickettsia-tick relationship

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41

CAO, YU. "Role of lncRNA in cancer development and progression". OpenSIUC, 2017. https://opensiuc.lib.siu.edu/dissertations/1429.

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PART1, TITLE: A p53-inducible long non-coding RNA PICART1 mediating cancer cell proliferation and migration. Long non-coding RNAs (lncRNAs) function in the development and progression of cancer, but only a small portion of lncRNAs are characterized thus far. A novel lncRNA transcript with 2.53 kb in length was identified by a transcriptome sequencing analysis, named p53-inducible cancer-associated RNA transcript 1 (PICART1). This PICART1 is upregulated by p53 through a p53-binding site at -1808 to -1783bp. In breast and colorectal cancer cells and tissues, PICART1 expression was decreased. Ectopic expression of the PICART1 suppressed growth, proliferation, migration, and invasion of MCF7, MDA-MB-231 and HCT116 cells whereas silencing of PICART1 stimulated the cell growth and migration. In these cells, the expression of PICART1 lowered down the levels of p-AKT (Thr308 & Ser473) and p-GSK3β (Ser9), and accordingly, β-catenin, cyclin D1 and c-Myc expression were decreased, but p21cip1/Waf1 expression was increased. Together these data suggest that PICART1 is a novel p53-inducible tumor suppressor lncRNA, functioning through the AKT/GSK3β/β-catenin signaling cascade. PART2, TITLE: The novel long non-coding RNA PANCR is a tumor suppressor gene in breast cancer. Long non-coding RNAs (lncRNAs) function as oncogenes or tumor suppressors in development and progression of cancer. Chromosome 16q22.1 region is frequently deleted in breast cancer, which may contribute to breast carcinogenesis by inactivation of tumor suppressor genes. This study characterized a new lncRNA tumor suppressor, named p53 activating non-coding RNA (PANCR), located in this Chromosome 16q22.1 region. This PANCR lncRNA consists of 1.5kb in length. Our data showed that PANCR was downregulated in breast cancer tissues and cell lines. In the breast cancer cell lines, PANCR expression appeared reversely correlated with cell malignancy, and in breast cancer tissues, PANCR was downregulated over 2 times in 31 (62.0%) of 50 cases when compared to adjacent normal breast tissues. In breast cancer cells MCF7 cells, ectopic expression of PANCR suppressed cell proliferation in culture, but in contrast, shRNA–mediated silencing of PANCR promoted cell growth and proliferation.

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42

Oldoni, E. "THE ROLE OF CELLULAR AND EXOSOMAL NEURAL-DERIVED LONG NON CODING RNA (LNCRNA) IN MULTIPLE SCLEROSIS: POTENTIAL BIOMARKERS OF DISEASE SUSCEPTIBILITY AND PROGRESSION". Doctoral thesis, Università degli Studi di Milano, 2018. http://hdl.handle.net/2434/540356.

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Long non-coding RNAs (lncRNAs) are a novel class of transcripts that are pervasively transcribed in the genome. Several lines of evidence correlate dysregulation of different lncRNAs to human diseases including neurological and autoimmune disorders, but their expression has not been exhaustively investigated in MS so far. The main aim of this study was to identify a specific signature of cellular and neural-derived exosomal lncRNA expression. Regarding lncRNA expression levels from Peripheral Blood Mononuclear Cells (PBMC), we studied a discovery cohort of MS patients who were compared against controls. Results were validated in a larger cohort and further replicated in an independent Belgian population. LncRNA PCR arrays from System Bioscience (SBI) containing 90 common lncRNAs were used to screen lncRNA expression levels in PBMC from 5 patients with Relapsing Remitting (RR)-MS, 5 with Primary Progressive (PP)-MS and 5 age-matched controls. Results were validated by real time PCR in a further independent Italian cohort consisting of 30 PBMC samples from MS patients and 30 controls. Best hits were replicated using droplet digital PCR in a Belgian cohort consisting of 24 MS patients and 23 controls. In particular, in the Italian validation cohort ANRIL, TUG1, XIST (p<0.0001) and SOX2OT (p<0.001) were strongly down-regulated in RR-MS versus controls, while GOMAFU, HULC (p<0.0001) and BACE-1AS (p<0.001) showed a robust down-regulation both in RR and Progressive MS in comparison with controls. NRON and TUG1 downregulation in MS patients, compared with controls (p<0.05 and p<0.0001 respectively), was confirmed in the Belgian population. In addition, a protocol for the extraction and characterisation of neural-derived exosomes has been developed in order to investigate exosomal lncRNA expression levels. Using two types of commercial arrays, the human RT2 lncFinder array (QIAGEN) and the human RT2 lncRNA inflammation response and autoimmunity array (QIAGEN), generalised deregulation in exosomal lncRNA was observed. Moreover, the expression pattern of these molecules was different in RR-MS and in PP-MS. Precisely, results from the human RT2 lncFinder array (QIAGEN) analysis led to the identification of 7 most significantly deregulated lncRNAs, precisely AIRN (5.30-fold increase over controls, p=0.04); FAS-AS1 (4.76-fold increase over controls, p=0.02); HOTAIR (4.47-fold increase over controls, p=0.03); NAMA (13.24-fold increase over controls, p=0.01); TRERNA1 (5.84-fold increase over controls, p=0.01) and HOXA-AS2 (0.56-fold increase over controls, p=0.04). Six lncRNA were significantly deregulated in the RR-MS subgroup, precisely AIRN (10.77-fold increase over controls, p=0.04); DLX6-AS1 (46.95-fold increase over controls, p=0.01); FAS-AS1 (11.37-fold increase over controls, p=0.001); HOTAIR (9.31-fold increase over controls; p=0.02); and TRERNA1 (6.61-fold increase over controls, p=0.003). In PP-MS only SOX-2OT showed a significant upregulation (8.95-fold increase over controls, p=0.02). When we used the array containing lncRNA linked with inflammation and autoimmunity, MZF-AS1 (0.47-fold decrease over controls, p=0.03), CEP83-AS1 (0.15-fold decrease over controls, p=0.02), RP11-282O18.3 (0.27-fold decrease over controls, p=0.02), RP11-84C13.1 (0.28-fold decrease over controls, p=0.04), SNHG7 (0.064-fold decrease over controls, p=0.04) and TP73-AS1 (0.48-fold decrease over controls, p=0.04) were significantly downregulated in MS, regardless of the subtype, while RP11-38P22.2 (19.5-fold increase over controls, p=0.04) showed an upregulation. Considering the disease subgroups, RR-MS patients showed a significant downregulation in RP11-363G2.4 (0.07-fold decrease over controls, p=0.008) and in TP73-AS1 (0.76-fold decrease over controls, p=0.02), while RP11-38P22.2 levels were upregulated (22.32-fold increase over controls, p=0.04). We found a general downregulation in lncRNA expression analysed in PP-MS, in particular FGF14-IT1 (0.08-fold decrease over controls, p=0.007) and RP11-282O18.3 (0.14-fold decrease over controls, p=0.04) were significantly altered. Some important forms of dysregulation were observed, considering the expression levels of lncRNAs known to be involved in brain function and in neurological and autoimmune disorders. The rationale of this study might then be used to set up a future study with the purpose of selecting potential biomarkers for disease aggressiveness and possible response to therapy.

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43

David, Antoine. "Rôle du long ARN non-codant CRNDE dans le myélome multiple". Thesis, Université de Paris (2019-....), 2019. http://www.theses.fr/2019UNIP7104.

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En dépit des avancées thérapeutiques récentes, le MM reste à ce jour une maladie incurable, dont les voies moléculaires impliquées dans l’hétérogénéité clinique, la progression, et l’agressivité du MM restent encore relativement peu connues. Au cours de cette dernière décennie, le rôle des lncRNAs a été de plus en plus étudié. En effet, les données génomiques, les études globales d’expression spatio-temporelle et l’existence de plusieurs lncRNAs démontrés comme impliqués dans différentes voies physiologiques suggèrent que les lncRNAs sont des régulateurs transcriptionnels et post-transcriptionnels importants, et la dérégulation de leur expression a maintenant été observée dans de nombreux cancers. Dans ce contexte, l’objectif principal de ma thèse a été de d’identifier les lncRNAs potentiellement impliqués dans le MM, et de caractériser les voies moléculaires via lesquelles ils exercent leur fonction. Pour étudier le rôle des lncRNAs dans le MM, nous avons dans un premier temps utilisé une analyse de données transcriptomiques publiées de plasmocytes tumoraux de patients atteints de MM au diagnostic, afin d’extraire les données concernant spécifiquement les lncRNAs. Grâce à cela, nous avons pu identifier 105 lncRNAs dont l’expression est dérégulée dans le MM, dont CRNDE, un lncRNA précédemment identifié comme oncogène dans plusieurs cancers.CRNDE est surexprimé dans les plasmocytes de patients atteints de MM comparé aux plasmocytes contrôle dans notre propre cohorte de patients de l’hôpital Saint-Louis. De plus, nous avons observé qu’une forte expression de CRNDE est corrélée à un mauvais pronostic. En utilisant une approche de délétion par la technique de CRISPR/Cas9 dans une lignée de MM, nous avons pu démontrer que CRNDE régule la prolifération, l’apoptose, ainsi que la tumorigénicité de ces cellules. Ce modèle cellulaire nous a également servi pour montrer que CRNDE régule l’expression du récepteur à l’IL6, et que la diminution de la prolifération observée dans les clones CRNDEΔ/Δ semble être en grande partie due à une perte de réponse à l’IL6, une cytokine jouant un rôle central dans la physiologie du MM.L’ensemble de ces résultat ont permis de démontrer un rôle important d’un lncRNA dans le MM, CRNDE, ainsi que de déterminer le mécanisme d’action par lequel il exerce son effet
Multiple myeloma (MM) is a malignancy of antibody-secreting plasma cells which remains incurable. MM is characterised by a wide clinical and prognostic spectrum, even within groups bearing the same primary cytogenetic event, for which the secondary molecular mechanisms responsible are still incompletely understood. Long non-coding RNAs (lncRNAs) are now recognised as an important class of regulatory molecules which are increasingly implicated in tumorigenesis and cancer progression. While recent studies have demonstrated prognostically relevant changes in the lncRNA expression profile in MM, the functional significance and molecular pathways downstream of these changes remain poorly characterised. In this study we have undertaken a thorough functional and molecular characterisation of the effect in MM cells of Colorectal Neoplasia Differentially Expressed (CRNDE), a known oncogenic lncRNA which has been previously implicated in diverse solid and haematological malignancies. CRNDE is overexpressed in plasma cells of MM patients, where it is a poor prognostic marker. CRISPR-mediated deletion of the CRNDE locus decreases proliferation and adhesion properties of MM cells in vitro and reduces tumour growth in an in vivo xenograft model. Transcriptomic profiling in CRNDE-deleted cells demonstrated that CRNDE activates expression of a number of genes previously implicated in the aetiology of MM, including the gene encoding the receptor of IL6 (IL6R), a cytokine critical for MM cell proliferation and survival. We further demonstrate that deletion of the CRNDE locus impacts upon IL6 signalling and proliferative responses in MM cells. Altogether this study reveals a novel mechanistic pathway by which the lncRNA CRNDE impacts upon MM growth and disease progression, by regulating the expression of IL6R and thus controlling response to IL6 signalling

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44

Smith, Jenna E. "Investigation of the mRNP and Transcriptome Regulated by Nonsense-Mediated RNA Decay". Case Western Reserve University School of Graduate Studies / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=case1421428941.

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45

Horn, Jessica [Verfasser], Thomas [Gutachter] Rudel y Cynthia [Gutachter] Sharma. "Molecular and functional characterization of the long non-coding RNA SSR42 in \(Staphylococcus\) \(aureus\) / Jessica Horn ; Gutachter: Thomas Rudel, Cynthia Sharma". Würzburg : Universität Würzburg, 2019. http://d-nb.info/1176972960/34.

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46

Trembinski, Dorotée Julia [Verfasser], Stefanie [Akademischer Betreuer] Dimmeler, Stefanie [Gutachter] Dimmeler y Michaela [Gutachter] Müller-McNicoll. "Long non-coding RNA sarrah: Staying young at heart / Dorotée Julia Trembinski ; Gutachter: Stefanie Dimmeler, Michaela Müller-McNicoll ; Betreuer: Stefanie Dimmeler". Frankfurt am Main : Universitätsbibliothek Johann Christian Senckenberg, 2019. http://d-nb.info/1202297943/34.

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47

Reis, André Anversa Oliveira. "Estudo da regulação por microRNAs do RNA longo não codificador de proteínas TUG1 envolvido em processos tumorigênicos". Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-15092016-080815/.

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No final do século passado, avanços ocorridos no campo da Biologia Molecular levantaram questionamentos sobre como organismos complexos, com poucos genes a mais que organismos relativamente mais simples, regulariam seu desenvolvimento e funções celulares tão mais intrincadas. A descoberta dos RNAs não codificadores de proteínas e suas funções lançou nova luz ao entendimento da regulação da expressão gênica em organismos superiores. Apesar do conhecimento adquirido nos últimos anos, pouco ainda é sabido sobre a regulação destes RNAs. MicroRNAs, por outro lado, são uma espécie bem estudada de pequenos RNAs preditos como possíveis reguladores de mais de 60 % dos genes codificadores de proteínas no genoma humano, considerados importantes reguladores da expressão gênica em nível pós-transcricional. O presente projeto estudou a possível regulação do gene não codificador de proteínas TUG1, envolvido em proliferação celular e apoptose, por miRNAs e o papel desta regulação em processos tumorigênicos. Para isto foram utilizadas técnicas que superexpressaram e silenciaram miRNAs e técnicas de PCR quantitativo em tempo real para medir o nível de TUG1 nas amostras tratadas. Verificou-se a possibilidade de regulação do TUG1 por microRNAs em diferentes linhagens celulares sendo que, no entanto, esta regulação não parece ser importante em nível fisiológico
At the end of the last century, advances occurred in the Molecular Biology field raised questions about how complexes organisms, with few more genes than relatively simpler organisms, regulate it so intricate development and cellular functions. The discovery of long non-protein coding RNAs and it functions gave light to the understanding of gene expression regulation in superior organisms. Despite the knowledge acquired in the last years, few is yet known about the regulation of these RNAs. MicroRNAs, other way, are a well-studied tiny RNAs specie. They are predicted as possible regulators of more than 60 % of protein-coding genes in the human genome and considered important regulators of gene expression regulation at post-transcriptional level. This project studied the possible regulation of the non protein-coding gene TUG1, involved in cell proliferation and apoptosis, by microRNAs and the role of this regulation in tumorigenic processes. In order to do this we used techniques that superexpressed and silenced miRNAs and techniques of real time quantitative PCR to measure the TUG1 levels in the treated samples. We find the possibility of regulation of TUG1 by microRNAs in different cell lineages but this regulation does not seems to be important in a physiologic context.

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48

Riquier, Sébastien. "Dans les abysses du transcriptome : découverte de nouveaux biomarqueurs de cellules souches mésenchymateuses par analyse approfondie du RNAseq". Thesis, Montpellier, 2019. http://www.theses.fr/2019MONTT004.

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Le développement du séquençage ARN, ou RNAseq, a permis l'essor de la recherche intensive de biomarqueurs dans de nombreux domaines de la biologie. L’information complète du transcriptome contenue dans les données de sorties, permet à un bioinformaticien assidu de dépasser les connaissances actuelles et d’accéder, grâce à des pipelines informatiques avancés, à d’innombrables signatures d’intérêts inédites. Dans cette thèse nous mettons en avant que ces marqueurs potentiels, essentiellement explorés pour répondre à des problématiques clinique en conditions pathologiques, peuvent être utilisés pour affiner la caractérisation de types de cellules sans marqueurs strictement spécifiques. Nous nous sommes intéressés aux cellules souches mésenchymateuses (MSCs), un type de cellules souches adultes multipotentes, fortement utilisées en clinique mais ne possédant pas de marqueurs positifs strictement spécifiques.Notre étude se concentre sur la recherche des ARN longs non-codants non annotés. Ces ARNs, aussi nommés "lncRNA", constituent une classe émergente de transcrits encore peu explorée à ce jour. De plus, cette catégorie démontre une spécificité conditionnelle et tissulaire élevée. Nous avons élaboré un pipeline d’analyse RNAseq optimisé pour la reconstruction et la quantification de lncRNAs non annotés.En utilisant les données publiques de RNAseq, venant de différentes sources de MSCs et d'autres types de cellules, nous avons identifié de nouveaux lncRNA non annotés exprimés spécifiquement dans les MSCs.Nous avons développé pour ce projet Kmerator.jl, un outil qui permet de décomposer un transcrit en sous séquences spécifiques (k-mers) afin de chercher et quantifier plus rapidement la signature de nos candidats dans un grand nombre de données RNAseq. Kmerator a également été utilisé dans d'autres applications pour tester la qualité des données RNA-seq disponibles en accés public.Après validation de ces nouveaux biomarqueurs de MSCs par qPCR, nous avons eu recours à plusieurs outils informatiques pour prédire leurs fonctions potentielles. Enfin, nous avons analysé des données RNAseq « single-cell » pour aborder l’hétérogénéité d’expression au sein des populations MSCs
The development of RNA sequencing, or RNAseq, have opened the path of intensive biomarkers research in many areas of biology. The complete information of the transcriptome contained in the output data, allows a bioinformatician to surpass the current knowledge and to access, thanks to advanced computer pipelines, to signatures of new interest. In this thesis, we are showing that these potential markers, classically used in clinical and pathological conditions, can be used to characterize cell types without extensive markers profile. We have studied mesenchymal stem cells, a type of adult multipotent stem cells, strongly used in clinics but without strickly specific positive markers. Our study mainly focuses on the search for non-annotated, long non-coding RNAs. These RNAs, also called "lncRNA", constitute an emerging class of transcripts and are still lightly explored.In addition, this category presents a highly tissue-related specificity. We have developed an optimized RNAseq pipeline for the reconstruction and quantification of non-annotated lncRNAs.Using public data from RNAseq, coming from different sources of MSC and other cell types, we have identified new non-annotated lncRNAs clearly and specifically expressed in MSCs. to complete this project, we developed Kmerator.jl, a bioinformatical tool that allows to decompose a transcript in k-mer, and select specific sub-sequences, in order to search and quantify at a faster rate the signature of our candidates in a large number of RNAseq dataset. After validation of these new biomarkers of MSCs by qPCR, we used several computer tools to predict their potential functions. Finally, we analyzed single-cell RNAseq data to address the heterogeneity of expression within MSC populations

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Blank-Giwojna, Alena [Verfasser] y Ingrid [Akademischer Betreuer] Grummt. "Long non-coding RNA KHPS1 drives a regulatory cascade which activates expression of the protooncogene SPHK1 / Alena Blank-Giwojna ; Betreuer: Ingrid Grummt". Heidelberg : Universitätsbibliothek Heidelberg, 2019. http://d-nb.info/1201414059/34.

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Korostowski, Lisa. "Transcript Regulation within the Kcnq1 Domain". Diss., Temple University Libraries, 2012. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/235191.

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Molecular Biology and Genetics
Ph.D.
Epigenetics was a term first coined to understand how cells with the same genetic make up can differentiate into various cell types. Elegant research over the past 30 years has shown that these mechanisms include heritable marks such as DNA methylation and histone modifications along with stable expression of non- coding RNAs. Within the realm of epigenetics is a phenomenon known as genomic imprinting. Imprints are marks that distinguish the maternal from the paternal chromosomes in the form of methylation. Methylation marks can influence transcript expression, resulting in only one allele being expressed. One imprinted domain is the Kcnq1 domain located on chromosome 11p15.5 in humans and chromosome 7 in the mouse. This domain is thought to be under the control of a paternally expressed long noncoding RNA (ncRNA) Kcnq1ot1. The Kcnq1ot1 ncRNA is expressed on the paternal chromosome due to a differentially methylation region located within its promoter. The promoter is methylated on the maternal allele thus inhibiting ncRNA expression, whereas the promoter is unmethylated on the paternal allele. In the placenta, a most of the genes located within a one mega-basepair region are exclusively expressed from the maternal chromosome, whereas the transcripts on the paternal chromosome are silenced by the ncRNA. The placenta seems to follow the classic idea of an imprinted domain. However, in the embryo and more specifically, in the embryonic heart, this is not the case. In the embryonic heart, only a 400kb region is restricted to maternal expression. In addition, one the genes, Kcnq1, starts out expressed exclusively from the maternal allele in early development but switches to biallelic expression during mid-gestation. The purpose of my research is to determine the underlying complexities that are involved in the regulation of transcripts within the Kcnq1 domain. This involves the Kcnq1 gene itself, which has been shown to transition from mono- to biallelic expression during mid-gestation and the Kcnq1ot1 ncRNA per se. I hypothesize that regulation by the Kcnq1ot1 ncRNA is not occurring in a uniform manner in the embryo; rather, the amount of regulation by the ncRNA is dependent on the developmental stage and specific tissue. In addition, this regulation involves complex interactions between enhancers, insulators and other regulatory elements to control the amount of silencing by the Kcnq1ot1 ncRNA. First, through a series of experiments looking at the Kcnq1 promoter, the mechanism of Kcnq1 paternal expression was determined. It was confirmed that Kcnq1 becomes biallelic during mid-gestation in the heart. Bisulfite mutagenesis and methylation sensitive chromatin immunoprecipitation were used to test the hypothesis that the Kcnq1 promoter was methylated in early development and then lost its methylation mark. However, a lack of methylation disproved this mechanism of paternal Kcnq1 activation. Rather, chromosome conformation capture (3C) determined that the Kcnq1 promoter interacts in a tissue-specific manner with regions within the domain that have enhancer activity. The role of the ncRNA within our system was also investigated. Interestingly, when Kcnq1ot1 allelic expression was profiled throughout development in heart, it transitioned to biallelic expression during heart development but remained monoallelic in the liver and brain. Several possibilities could account for this phenomenon, including loss of promoter methylation and/or an alternative transcript start site. Both of these options were explored using bisulfite mutagenesis and 5' RACE. However, the Kcnq1ot1 promoter region retained its methylation mark even after the maternal transcript was turned on, disproving this idea. Rather, a maternal specific transcript was found in the heart to start downstream of the CpG islands. Lastly, to gain a better understand of the Kcnq1ot1 ncRNA, experiments were carried out on a mutant mouse in which a truncated form of the ncRNA was transmitted paternally; this is dubbed the "Kterm" mouse. Unexpectedly, Kcnq1 still followed the same mono- to biallelic transition as seen in the wild-type, whereas the head and body counterparts from the same stage embryos were biallelic for Kcnq1. Also, the immediate upstream genes, Cdk1nc and Slc22a18, lost their mono-allelic expression in neonatal heart, liver and brain when the Kterm mutation was transmitted. This suggested that Kcnq1ot1 did not function as a silencer for Kcnq1 paternal expression in the heart, but rather had an alternative and previously unknown function. From qRT-PCR, 3C and ChIP assays, it was determined that the Kcnq1ot1 ncRNA plays a role in regulating Kcnq1 gene expression in the heart by limiting its interaction to specific cis-acting enhancers. When the ncRNA was absent, the Kcnq1 promoter interacted with non-native sites along the domain, possibly causing the increase in transcript expression. This phenomenon was specific to the heart and was not seen in other tissues. These findings showed that Kcnq1 paternal expression is the result of strong developmental and tissue specific enhancers. Chromatin interactions in cis put a strong enhancer in contact with the Kcnq1 promoter to increase its expression in later development. In addition, a truncation mutation model identified a key role for the Kcnq1ot1 ncRNA in regulating Kcnq1 expression. Instead of regulating the imprinting status of Kcnq1, the ncRNA regulates the amount of Kcnq1 transcript being produced in the heart by regulating chromatin interactions. Finally, these studies identified a maternally expressed Kcnq1ot1 transcript whose role in heart development is still not fully understood. Taken together, these findings support a model where an inhibitory factor(s) silence the paternal Kcnq1 transcript and maternal Kcnq1ot1 transcript and in later development, this factor is released allowing for expression and chromatin interactions to occur.
Temple University--Theses

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Tesis sobre el tema "Long non-coding RNA"

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