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Khoshnaw, Sarkawt Majeed Omar. "Significance of microRNAs and microRNA maturation regulators in breast cancer." Thesis, University of Nottingham, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.594865.
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Background: MicroRNAs (miRNAs) modulate gene expression by targeting mRNAs for cleavage or translational suppression. miRNAs have deregulated expression in human breast cancer (BC), and there is evidence suggesting that they function primarily as tumour suppressors. Dicer and Drosha are two proteins which play key roles in miRNA biogenesis to produce mature miRNAs from their precursor molecules, and are known to be deranged in human Be. The contribution of miRNAs and their maturation regulators in the initiation and progression of human Be can be exploited to achieve novel modifications in the existing diagnostic and therapeutic approaches in Be management. Methods: Protein expression levels of miRNA maturation regu lators, Dicer and Drosha, were assessed immunohistochemically in 2 sets of Be: 1) full-face sections of selected Be cases (n = 24) with normal breast parenchymal tissue (NBPT) and lesions representing different stages of Be progression (Ductal carcinoma in situ, DCIS; invasive breast cancer, IBC; and metastatic breast cancer, MBC) to assess their differential expression, 2) tissue microa rrays (TMAs) comprising a large and well-characterised series of primary IBC (n = 1902) to evaluate their biological, clinicopathological, prognostic and predictive significance. Additionally, miRNA expression profiling was explored in the 4 tissue components (NBPT, DCIS, IBC and MBC) of 7 BC cases using an Agilent microa rray-based miRNA profiling study, to investigate the differential expression of miRNAs in NBPT and different stages of BC progression. Results: Dicer and Drosha protein expression was observed to decrease with BC progression, which implies that loss of these two miRNA maturation regulators might have a role in the process of Be progression. In the IBC series, loss of Dicer expression was associated with features of aggressive behaviour including higher histological grade and loss of ER, PR and BRCA1 protein expression . Moreover, Dicer expression was revealed to be an independent predictor of a shorter disease free interval at 5 years, and predictive of better response to chemotherapy and to endocrine therapy. Loss of Drosha cytoplasmic expression was associated with higher tumour stage and loss of expression of BRCA1 and basal phenotype; and loss of Drosha nuclear expression was associated with larger tumour size, higher tumour grade, and loss of ER, PR, BRCA1, E-cadherin and CK14. Loss of Drosha cytoplasmic expression was associated with shorter Be specific survival, BC recurrence, and distant metastases, and th is correlation was maintained in ER-negative and HER2- negative cases. Loss of Drosha cytoplasmic expression was predictive of better response to systemic therapy in ER-negative patients. Furthermore, we present data revealing that some miRNAs are differentially expressed across the 4 tissue components (NBPT, DCIS, IBC and MBC). The comprehensive survey of miRNA expression profiling, confirmed differential expression of one miRNA previously reported to be deranged in BC, and identified 6 miRNAs downregulated du ring BC progression, 5 downregulated and 1 upregulated, which have not previously been linked to Be. Among the novel downregulated miRNA candidates, 2 (hsa-miR-1181 and hsa-miR-4322) were revealed to gradually reduce in amount across the 4 tissue components. These observatinos support the hypothesis that miRNAs play a role in tumourigenesis and in advancing BC tissues towards acquiring metastatic potential. Conclusions: The intimate involvement of miRNAs and their maturation regulators in malignant transformation, cellular invasion and metastases in BC makes them potentially relevant as future diagnostic, predictive and therapeutic targets. Although the current study is preliminary, we believe the results add to the present knowledge of Significance of miRNAs and their regulators in Be. Therefore, the results would serve as a robust initial set of potential biomarkers for validation in a larger cohort of patients.
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Arseni, Varvara. "MicroRNAs and the canonical microRNA biogenesis pathway in the planarian Schmidtea mediterranea." Thesis, University of Nottingham, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.581999.
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miRNAs are an important class of small non-protein coding RNAs whose specific functions in animals are rapidly being elucidated. miRNAs regulate the expression of many animal genes by post-transcriptional gene silencing and play vital roles in stem cell maintenance, differentiation and apoptosis. In this study, planarians were used as a model system in order to study whether miRNAs have a regulatory role in their stem cell dynamics. Planarians are well known for their remarkable regenerative ability and their amazing capacity for constant re-patterning owing to a population of somatic pluripotent stem cells known as neoblasts. In particular, the aim of the study was to investigate the regulatory role miRNAs may have in planarian stem cell self renewal, proliferation and differentiation. Recently, the differential spatial patterns of expression of miRNAs in whole and regenerating planarians have been characterized by in situ hybridization to nascent miRNA transcripts. These miRNA expression patterns were the first to be determined for a Lophotrocozoan animal. The expression patterns of 42 miRNAs in adult planarians have been characterized, constituting a complete range of tissue specific expression patterns. The majority of planarian miRNAs were expressed either in areas where stem cells (neoblasts) are located and/or in the nervous system. Some miRNAs were definitively expressed in stem cells and dividing cells and moreover miRNAs were found to be expressed in germ stem cells of the sexual strain. Taken the facts that cellular proliferation and differentiation must be controlled during planarian regeneration and tissue homeostasis as well as that miRNAs have been implicated in the control stem cell functions in other organisms, aim of this study was to disrupt the canonical miRNA biosynthesis pathway. In that way, information about the global impact of miRNA regulation in planarian regeneration and tissue homeostasis would be gained.
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Sætrom, Ola. "Predicting MicroRNA targets." Thesis, Norwegian University of Science and Technology, Department of Computer and Information Science, 2005. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-9266.
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MicroRNAs are a large family of short non-encoding RNAs that regulated protein production by binding to mRNAs. A single miRNA can regulate an mRNA by itself, or several miRNAs can cooperate in regulating the mRNAs. This is all dependent on the degree of complementarity between the miRNA and the target mRNA. Here, we present the program TargetBoost that, using a classifier generated by a combination of hardware accelerated genetic programming and boosting, allows for screening several large dataset against several miRNAs, and computes a likelihood of that genes in the dataset is regulated by the set of miRNAs used in the screening. We also present results from comparison of several different scoring functions for measuring cooperative effects. We found that the classifier used in TargetBoost is best for finding target sites that regulate mRNAs by themselves. A demo of TargetBoost can be found on http://www.interagon.com/demo.
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BERTOLAZZI, Giorgio. "MicroRNA Interaction Networks." Doctoral thesis, Università degli Studi di Palermo, 2021. http://hdl.handle.net/10447/498927.
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La tesi di Giorgio Bertolazzi è incentrata sullo sviluppo di nuovi algoritmi per la predizione dei legami miRNA-mRNA. In particolare, un algoritmo di machine-learning viene proposto per l'upgrade del web tool ComiR; la versione originale di ComiR considerava soltanto i siti di legame dei miRNA collocati nella regione 3'UTR dell'RNA messaggero. La nuova versione di ComiR include nella ricerca dei legami la regione codificante dell'RNA messaggero.Bertolazzi’s thesis focuses on developing and applying computational methods to predict microRNA binding sites located on messenger RNA molecules. MicroRNAs (miRNAs) regulate gene expression by binding target messenger RNA molecules (mRNAs). Therefore, the prediction of miRNA binding is important to investigate cellular processes. Moreover, alterations in miRNA activity have been associated with many human diseases, such as cancer. The thesis explores miRNA binding behavior and highlights fundamental information for miRNA target prediction. In particular, a machine learning approach is used to upgrade an existing target prediction algorithm named ComiR; the original version of ComiR considers miRNA binding sites located on mRNA 3’UTR region. The novel algorithm significantly improves the ComiR prediction capacity by including miRNA binding sites located on mRNA coding regions.
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Ammari, Meryem. "Rôle de miR-146a dans la régulation des fonctions monocytaires dans l’arthrite." Thesis, Montpellier 1, 2014. http://www.theses.fr/2014MON1T020.
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Les monocytes sont des leucocytes dérivés de précurseurs de la moelle osseuse pouvant se différencier en macrophages, cellules dendritiques (CD), ou ostéoclastes (OC). Ils jouent un rôle critique dans la persistance de l'inflammation et la destruction des articulations par le biais de mécanismes encore mal connus. Les monocytes sont composés de deux grandes sous-populations chez la souris, discriminées sur la base du marqueur de surface Ly6C. Il a été suggéré que les OC pouvait être préférentiellement différenciés à partir de la sous-population monocytaire Ly6Chigh, dont l'activation excessive et prolongée est une signature clé de nombreuses pathologies inflammatoires. Parmi les acteurs moléculaires responsables de la régulation de l'expression des gènes, les miARNs jouent un rôle majeur dans de nombreux processus physiologiques, dont la réponse inflammatoire, en régulant finement les programmes génétiques au niveau post-transcriptionnel. Leur implication dans l'ostéoclastogénèse est encore mal connue. Par ailleurs, leur expression est perturbée dans de nombreuses pathologies, dont la polyarthrite rhumatoïde qui implique à la fois un dysfonctionnement de la réponse inflammatoire et de l'homéostasie osseuse. Mon projet de thèse vise à mieux comprendre l'implication des sous-populations monocytaires dans la persistance de l'inflammation et de l'activité des OC au travers de l'étude du rôle de miR-146a dans des conditions physiologiques et inflammatoires. J'ai montré que miR-146a est le miARN le plus différemment exprimé entre monocytes classiques Ly6Chigh et non-classiques Ly6Clow, et que son expression est diminuée dans les Ly6Chigh en conditions arthritiques. J'ai également montré que la perte de miR-146a augmente l'ostéoclastogénèse in vitro et l'érosion osseuse in vivo chez les souris arthritiques. Enfin, la surexpression artificielle de miR-146a dans les monocytes Ly6Chigh inhibe la différenciation osteoclastique et la perte osseuse dans l'arthrite expérimentale chez la souris. Mes résultats suggèrent que miR-146a contrôle l'hétérogénéité fonctionnelle des monocytes et qu'une diminution de son expression dans la sous-population Ly6Chigh serait responsable de l'augmentation de l'osteoclastogénèse et de l'érosion osseuse observées en conditions arthritiques. Pour finir, mes résultats montrent également qu'augmenter l'expression de miR-146a dans les monocytes Ly6Chigh présente un intérêt thérapeutique pour contrecarrer la perte osseuse associée à l'arthriteIntroduction : Monocytes represent a prototypic cell type when investigating the interplay between immune and skeletal systems as they can give rise to different cell types including dendritic cells, macrophages and osteoclasts (OC), which play key roles in immunity and bone homeostasis. Circulating monocytes consist of at least two main functional subsets, Ly6Chigh and Ly6Clow monocytes. It has been suggested that OC might develop preferentially from the Ly6Chigh monocyte subset, which excessive and prolonged activation is a hallmark of many inflammatory diseases. Among key molecular rheostats of cell fate, micro(mi)RNAs are a class of regulatory RNAs that control basic biological functions and orchestrate inflammatory responses. Few miRNAs have been involved in osteoclastogenesis. The present study aimed at investigating the role of miRNAs in osteoclastogenesis in the context of monocyte subsets, under steady state and inflammatory conditions. Methods & Results : Using genome-wide miRNA expression study we have identified miRNAs and putative targeted pathways that are differentially expressed between Ly6Chigh and LyC6low FACS sorted mouse monocytes, and common to their human counter parts CD14+CD16- and CD14dimCD16+ monocytes, respectively. Among these, miR-146a showed higher expression in Ly6Clow monocytes when compared to Ly6Chigh monocytes. Under inflammatory arthritis conditions, expression of miR-146a in Ly6Chigh monocytes was down regulated as compared to healthy controls. Using mouse deficient for miR-146a, we showed that knockdown of miR-146a increased OC differentiation in vitro. While no bone phenotype was evidenced in miR-146a deficient mice, nor under steady state or ovariectomized conditions, arthritis-induced bone resorption and bone loss were increased in miR-146a knockout mice. Finally, using a liposomal formulation able to delivermiR-146a mimics to Ly6Chigh monocytes upon intravenous injection, we showed that enforced expression of miR-146a led to decreased number of TRAP positive cells within the synovium of arthritic mice, and efficiently reduced bone erosion in inflammatory arthritis. This effect was associated with decreased RelB expression in miR-146a-overexpressing Ly6Chigh osteoclast progenitors. Conclusion : Overall, our results show that specific over-expression of miR-146a in Ly6Chigh monocytes altered OC differentiation and decreased bone erosion in inflammatory arthritis. These data suggest a novel role for miR-146a in controlling osteoclast fate of Ly6Chigh monocyte progenitors and that reduced miR-146a expression in Ly6Chigh monocytes under arthritic conditions contributes to pathogenic bone loss. Finally, delivery of miR-146a mimics to Ly6Chigh monocytes may offer valuable therapeutic potential to interfere with pathological bone loss
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Dogini, Danyella Barbosa. "Quantificação de diferentes microRNAs no sistema nervoso central = implicações nos mecanismos de desenvolvimento e processos fisiopatologicos." [s.n.], 2010. http://repositorio.unicamp.br/jspui/handle/REPOSIP/309739.
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Orientador: Iscia Lopes-CendesTese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas
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Resumo: MicroRNAs são moléculas recém-descobertas de RNA não-codificadores que possuem de 21 a 24 nucleotídeos e que regulam a expressão após a transcrição dos genes alvo. Essa regulação pode ser realizada através da inibição da tradução ou da degradação do RNA mensageiro. Os miRNAs estão envolvidos em vários processo biológicos como, diferenciação celular e desenvolvimento embrionário, além de apresentarem expressão tecido e tempo-específica. Eles podem regular a expressão de pelo menos 1/3 de todos os genes humanos e estão envolvidos com a regulação do metabolismo e da apoptose. Os miRNAs são a chave como reguladores pós-transcricionais da neurogênese; estudos mostram que eles possuem a expressão associada com a transição entre proliferação e diferenciação e também tem expressão constitutiva em neurônios maduros, evidenciando o envolvimento dessas moléculas com o desenvolvimento do sistema nervoso central (SNC). Outros miRNAs estão sendo estudados e verifica-se que eles agem como reguladores de genes envolvidos em doenças como Alzheimer, Parkinson e, provavelmente, também devam possuir um papel na regulação das epilepsias. No primeiro trabalho, apresentado no segundo capítulo, investigamos o papel dos miRNAs no desenvolvimento do SNC através da quantificação de 104 miRNAs em cérebros em desenvolvimento de camundongos. No segundo trabalho, apresentado no terceiro capítulo, para analisarmos o papel dos miRNAs na epilepsia de lobo temporal, verificamos se havia presença de miRNAs com expressão diferenciada entre tecidos removidos de pacientes que se submeteram a cirurgia de hipocampectomia e tecidos normais provenientes de autópsias. Para ambos os experimentos, foram extraídos os RNAs dos tecidos e quantificados por PCR em tempo real com o kit MicroRNA Assay baseado em iniciadores com estrutura em stem loop. Nos camundongos, análises de bioinformática encontraram quatro cluster de acordo com a expressão dos miRNAs. Um cluster (C1) com 12 miRNAs (miR-9; miR-17- 5p; miR-124a; miR-125a; miR-125b;miR-130a; miR-140; miR-181a; miR-199a; miR-205; miR-214; miR-301) apresentou expressão com diferença significativa durante o desenvolvimento. Nos tecidos dos pacientes, após a análise de bioinformática, encontramos três miRNAs com expressão diferenciada entre pacientes e controle (miR-29b, miR-30d e let-7). Em ambos os experimentos analisamos os possíveis genes alvo desse miRNAs. Nos camundongos, nossos resultados sugerem a presença de um padrão específico de expressão no cluster C1, indicando que esses miRNAs possam ter um papel na regulação de genes envolvidos na neurogênese. Nos tecidos humanos, os genes alvo encontrados estão envolvidos, principalmente, em proliferação celular, neurogênese e apoptose, indicando uma provável atuação dos miRNAs na regulação de genes que estão envolvidos na epilepsia de lobo temporal
Abstract: MicroRNAs are a new class of small RNA molecules (21-24 nucleotide-long) that negatively regulate gene expression either by translational repression or target mRNA degradation. It is believed that about 30% of all human genes are targeted by these molecules. MiRNAs are involved in many important biological processes including cell differentiation, embryonic development and central nervous system formation, besides they showed specific temporal-space expression. They can regulate 1/3 of human genes and are involved in metabolism and apoptosis. miRNAs are the key as neurogenesis postranscriptional regulation; studies previous indicates miRNA expression associate with proliferation and differentiation in development of central nervous system (CNS) and housekeeping expression in mature neurons. They are involved in several diseases as Alzkeimer's and Parkinson and may have a role in epilepsy regulation. In second chapter, we analyze the miRNA expression in mouse brain during four stages of CNS development; in third chapter, we analyze hippocampal tissue of four patients who underwent selective resection of the mesial temporal structures for the treatment of clinically refractory seizures. In addition we used control samples from autopsy (n=4) for comparison. In both experiments, total RNA was isolated from tissues and used in real-time PCR reactions with TaqMan¿ microRNA assays (Applied Biosystems) to quantify 104 (mouse brain) or 157 (human tissue) different miRNAs. In mouse brain analysis, we were able to identified four different clusters (C1, C2, C3 and C4) of miRNAs expression. Significant differences in expression during development were observed only in miRNAs included in C1. Our results suggest the presence of a specific expression pattern in C1, indicating that these miRNAs could have an important role in gene regulation during neurogenesis. We found a significant decrease (p<0,05) in expression of 12 miRNAs (miR-9; miR-17-5p; miR-124a; miR-125a; miR-125b;miR-130a; miR-140; miR-181a; miR-199a; miR-205; miR-214; miR- 301) belonging to cluster C1 in latter stages of development. Computational target identification showed that 10 of the 12 miRNAs present in C1 could be involved in neurogenesis. In human tissues, bioinformatics analyzes identified three miRNAs species which were differently expressed in patients as compared to controls: let7a was over expressed in patients (4 fold increased), miR-29b and miR-30d were down-regulated in patients (2.5 fold and 0.5 fold decreased, respectively). Possible target genes for let-7a are NME6 and NCAM1 (which would be down-regulated in patients); for miR-29b is MCL-1 and for miR30d are CTNND2, LGI1 and SON (which would be up-regulated in patients). We have identified three different miRNA species differently expressed in hippocampal sclerosis. Gene functions related to the possible miRNA targets are involved mainly with cell proliferation, neurogenesis, cell adhesion and apoptosis. Our results indicate new molecular targets which should be explored in additional studies addressing miRNA regulation in hippocampal sclerosis
Doutorado
Neurociencias
Doutor em Fisiopatologia Medica
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Wang, Qi. "Using Imputed Microrna Regulation Based on Weighted Ranked Expression and Putative Microrna Targets and Analysis of Variance to Select Micrornas for Predicting Prostate Cancer Recurrence." Thesis, North Dakota State University, 2014. https://hdl.handle.net/10365/27341.
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Imputed microRNA regulation based on weighted ranked expression and putative microRNA targets (IMRE) is a method to predict microRNA regulation from genome-wide gene expression. A false discovery rate (FDR) for each microRNA is calculated using the expression of the microRNA putative targets to analyze the regulation between different conditions. FDR is calculated to identify the differences of gene expression. The dataset used in this research is the microarray gene expression of 596 patients with prostate cancer. This dataset includes three different phenotypes: PSA (Prostate-Specific Antigen recurrence), Systemic (Systemic Disease Progression) and NED (No Evidence of Disease). We used the IMRE and ANOVA methods to analyze the dataset and identified several microRNA candidates that can be used to predict PSA recurrence and systemic disease progression in prostate cancer patients.
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Kwan, Chun-kit Peter, and 關駿傑. "The expression of microRNA-34c and microRNA processing enzymes in preimplantation embryos." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B44659283.
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D'Ario, G. "IDENTIFICATION OF INTRONIC MICRORNAS ALTERED IN BREAST CANCER THROUGH MICROARRAY HOST GENE EXPRESSION ANALYSIS." Doctoral thesis, Università degli Studi di Milano, 2011. http://hdl.handle.net/2434/157939.
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MicroRNA (miRNAs) are endogenous non-coding RNAs of ∼ 22 nucleotides
in length that function as post-transcriptional regulators of gene and pro-
tein expression through degradation or translation inhibition of the target
messenger RNAs. MiRNAs show altered expression profiles in several hu-
man pathologies, including cancer. They can act as tumour suppressors or
as oncogenes, depending on the characteristics of their target genes.
More than half of the mammalian miRNAs, including several of the miR-
NAs implicated in breast cancer, are localized within the introns of protein-
coding genes, often organized in clusters, and usually transcribed together
with their host gene. It is therefore possible, at least in principle, to iden-
tify novel intronic cancer-regulated miRNAs by examining the expression
profile of their host genes by means of microarrays. For this purpose, we
analyzed the regulation of 253 miRNA host genes in five large breast cancer
microarray data sets comprising more than 950 samples, examining their
association with different clinical and pathological parameters such as tu-
mour grade, estrogen and progesterone receptor status, p53 status, survival,
and occurrence of relapse or metastasis.
We found that MCM7 and SMC4 were the most frequently and significantly
overexpressed genes in high grade tumours. These genes contain two well
known cancer-associated miRNA clusters: miR25-93-106b and miR-15b/16-
2 respectively. In addition we identified six other miRNA host genes that
were significantly downregulated in high grade tumours in all the data sets.
Much less evidence is available in the literature about the involvement in
cancer of the miRNAs contained in these genes (i.e., miR-218-1, miR-342,
miR-483, miR-548f-2, miR-1245 and miR-1266 ).
We measured the expression of the selected miRNAs by Real Time PCR
on an independent cohort of 36 formalin-fixed paraffin-embedded (FFPE)
samples, and we observed reduced expressions level of such miRNAs in high
grade tumours. In particular, we found miR-342-3p, miR-342-5p, miR-483-
3p, and miR-483-5p to be the most significantly downregulated miRNAs.
These miRNAs were also found to correlate with bad prognosis in grade 2
tumours. Finally we provided initial evidence that increased expression of
miR-342-5p, but not miR-342-3p, induces apoptosis in the highly metastatic
MDA-MB-231 breast cancer cell line.
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Smith, Daniel. "Electrochemical detection of microRNA." Thesis, Cardiff University, 2017. http://orca.cf.ac.uk/107718/.
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Members of the recently discovered family of short non-coding RNAs, termed microRNAs (miRNAs), regulate the expression of most genes encoded by the human genome by repressing translation of messenger RNAs to proteins. MiRNAs are stably expressed throughout the body and can be detected robustly and reproducibly by RT-qPCR in body fluids such as blood and urine. Alterations in circulating miRNA profiles have been associated with cancers of the brain, breast and liver, and miRNAs hold great promise as biomarkers of numerous other diseases. However, current methods for miRNA biomarker detection rely on laborious, expensive and expert techniques, and involve invasive biopsy acquisition. The research contained within this thesis focusses on the development of a non-invasive, inexpensive and rapid electrochemical analytical test to quantify miRNA in human urine samples. Therefore we describe how glassy carbon and disposable screen printed carbon electrodes (SPCEs), were modified through electropolymerisation of a naphthalene sulfonic acid derivative. DNA complementary to a target miRNA was attached and the sensor analysed via electrochemical methods using a ferri/ferrocyanide electrolyte. After hybridising with a miRNA target, this analysis was repeated and compared to the original DNA-only analysis to give a corresponding change. This was performed using buffered solutions and shown to be sensitive to 20 fM and selective against sequences with a single mismatch; urine analysis was also performed. The method was then adapted for use with screen printed electrodes, using a new chlorination solvent system, to a lowest detected concentration of 10 fM. The ink materials used for the production of the SPCEs were optimised and a new design developed to allow for multiple analyses on one sensor. A small number of diabetic kidney nephropathy (DKN) patient and healthy control urine samples were then analysed for biomarkers we have recently identified, comparing their relative expression levels.
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Migault, Mélodie. "La séquestration de microARN dans le mélanome métastatique : du mécanisme moléculaire au candidat thérapeutique." Thesis, Rennes 1, 2017. http://www.theses.fr/2017REN1B014/document.
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Les microARN (miARN) sont de petits ARN non-codants dont la principale fonction est de réprimer l’expression génique en s’hybridant par complémentarité de séquence à leurs cibles ARN. L’activité des miARN est également régulée par leurs cibles qui entrent en compétition pour leur liaison. Certains de ces ARN compétiteurs endogènes (ARNce) résistent à la répression induite par le miARN et vont alors les séquestrer. Ils sont appelés éponges à miARN. La dérégulation des réseaux d’ARNce et des éponges à miARN est impliquée dans des processus pathologiques tels que le cancer. Au cours de ma thèse, nous nous sommes intéressés à la séquestration des miARN dans le mélanome cutané. Le mélanome provient de la transformation maligne du mélanocyte, une cellule spécialisée dans la production de pigment. S’il n’est pas pris en charge à temps, des métastases apparaissent et se disséminent rapidement dans l’organisme (ganglions, foie, poumons, cerveau, etc.). Des solutions thérapeutiques existent mais une faible proportion de patients y répondent de manière efficace nécessitant de nouvelles stratégies de traitement. Nous avons mis en évidence que l’ARN messager (ARNm) de TYRP1, gène spécifiquement exprimé dans le mélanocyte et donc le mélanome, porte le rôle d’éponge à miARN dans le mélanome métastatique. Ce rôle est indépendant de la fonction protéique de TYRP1. Nous avons déterminé que l’ARNm de TYRP1 séquestre le suppresseur de tumeurs miR-16 via des sites de liaison (MRE-16) non-canoniques. Les MRE-16 non-canoniques permettent à l’ARNm de TYRP1 de ne pas être dégradé par le miR-16 et le rendent donc plus stable dans la cellule de mélanome. La majorité du pool de miR-16 est ainsi séquestrée et ne peut donc plus réprimer ses cibles intervenant dans la prolifération cellulaire et la croissance tumorale in vivo. Afin de remettre en activité le miR-16 au sein de la cellule de mélanome, nous avons utilisé la technologie du « target site blocker » (TSB), un oligonucléotide antisens modifié ayant une forte stabilité et affinité pour sa cible. Le TSB, spécifique du MRE-16 de l’ARNm de TYRP1, entre en compétition pour la liaison à l’ARNm de TYRP1 avec le miR-16 pour permettre sa libération et son action sur ses cibles effectrices. Nous avons montré in vitro et in vivo via un modèle murin de xénogreffe de tumeur dérivée de patient que la stratégie du TSB est efficace contre le mélanome métastatique. Ces travaux ont permis l’identification d’un nouveau mécanisme oncogénique basé sur la séquestration de miARN et proposent une nouvelle stratégie de thérapie ciblée contre le mélanome métastatiqueMicroRNAs (miRNAs) are small non-coding RNAs. They fine tune gene-expression through specific complementary interaction with their RNA targets. The miRNA repressive function towards a given RNA is highly regulated and in part dependent on the abundance of its other targets competing for miRNA’s binding. Some of these competing endogenous RNAs (ceRNAs) can resist to miRNA-mediated RNA decay thereby sequestering miRNAs. They are named miRNA sponges. Deregulation of ceRNAs and miRNA sponges networks are implicated in many pathologic processes including cancer. My PhD work focused on miRNA sequestration in cutaneous melanoma. Melanomas arise from the malignant transformation of melanocytes; the skin-cell specialized in pigment production. Most melanoma undergoes metastatic evolution, with metastatic cells spreading rapidly in the entire organism (lymph node, liver, lungs, brain, etc.). Early and complete resection of primary in situ melanoma is thus determinant for patient outcome. Since 2010, potent therapeutic options have been developed. Unfortunately, patients ultimately develop resistance while some are non-responders. There is thus an urgent need to develop new therapeutic strategies to treat metastatic melanoma. We have identified that the Tyrosinase Related Protein 1 (TYRP1) mRNA function as a miRNA sponge. TYRP1 is specifically expressed in the melanocytic lineage. TYRP1 mRNA governs melanoma growth endorsing thereby a non-coding function. We demonstrated that TYRP1 mRNA sequesters the tumor suppressor miR-16 via non-canonical miRNA binding sites (MREs-16). Non-canonical miR-16 binding lacks mRNA decay function favoring TYRP1 mRNA stability and miRNA sequestration. Sequestered miR-16 can no more repress its canonical targets involved in cell proliferation and tumor growth. To reset miR-16’s activity and block melanoma growth, we used “Target Site Blocker” (TSB). TSBs are modified antisense oligonucleotides with enhanced stability and affinity to its target. We designed a TSB, named TSB-T3, overlapping specially TYRP1 non-canonical MRE-16. We first showed that TSB-T3 binds to TYRP1 mRNA and competes with miR-16. Freed miR-16 binds to its canonical targets inducing their decay. TSB-T3 blocks melanoma cell growth in vitro and in vivo, using patient-derived tumor xenograft. We thus showed for the first time that TSB’s strategy redirecting a tumor suppressor miRNA is a potent tool to monitor metastatic melanoma growth. Together my PhD work brings out a new oncogenic mechanism based on miRNA sequestration and proposes an original strategy of targeted therapy against metastatic melanoma
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Aggarwal, Neha. "Characterization of a microRNA Harboring Intron for pre-mRNA Splicing and microRNA Processing." Cleveland State University / OhioLINK, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=csu1275407397.
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Pellegrino, Loredana. "Distinct functional roles of microRNA-23b and microRNA-26a in breast cancer pathogenesis." Thesis, Imperial College London, 2013. http://hdl.handle.net/10044/1/24827.
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Tumour formation and metastasis are distinct processes that arise from cumulative alterations of genomic and epigenetic regulation. Uncontrolled modulation of cell cycle-related genes is crucial to tumour growth and additional genetic modifications provide cancer cells with motile and invasive phenotypes, leading to metastatic dissemination. The cytoskeleton constitutes the structural support to cell motility, invasion and adhesion. Among the best-characterised cytoskeletal modulators are the p21-activated kinases (PAKs). In breast cancer (BC), the HER2 pathway controls the cytoskeletal dynamics and cell motility via PAK activation, through distinct downstream signaling mechanisms. MicroRNAs (miRNAs) are small, non-coding RNAs that modulate gene expression post-transcriptionally. MiRNAs dysregulation can contribute to tumorigenicity, cell motility and metastasis by affecting relevant signaling pathways. We identified PAK2 as target of both miR-23b and miR-26a, implicating a direct role for these miRNAs in cytoskeletal remodeling. Experimentally, expression of miR-23b and miR-26a in BC cells promotes focal adhesions and cell spreading on substrates, but miR-23b alone controls cell-cell junctions and lamellipodia formation. Despite sharing the same target, the two miRNAs show additional distinct functions. MiR-26a overexpression in BC leads to formation of aneuploid cells associated with higher tumorigenicity. On the other hand, miR-23b inhibition enhances BC cell migration, invasion and metastasis in vivo. Clinically, low miR-23b levels correlate with metastatic development in BC patients. Mechanistically, growth factor-mediated signal transductions activate the transcription factor AP-1 and we show that this transcriptionally reduces miR-23b expression thus releasing PAK2 from its translational inhibition. The distinct cellular phenotypes described by the two miRNAs indicate that their global functions depend upon all the genes they regulate. Using RNA-sequencing and luciferase reporter assays, we validated a subset of genes as direct targets of either the two miRNAs. These genes are crucial to distinct molecular pathways and contribute to elucidate the observed phenotypes induced by miR-23b and miR-26a modulation.
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Frackowiak, Agnieszka. "Involvement of microRNA-30 family in metastatic dissemination of breast cancer cells to bone." Thesis, Lyon 1, 2015. http://www.theses.fr/2015LYO10106/document.
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Les métastases osseuses sont des complications fréquentes du cancer du sein, responsables sur le plan clinique d'hypercalcémie, fractures osseuses et douleurs, pour lesquelles, il n'existe que des traitements palliatifs. Les cellules tumorales de carcinomes mammaires qui métastasent au site osseux expriment des gènes qui favorisent le tropisme osseux de ces cellules ainsi que leur ancrage et développement dans la moelle osseuse. Les mécanismes moléculaires sous-jacents à ces processus sont contrôlés par l'expression génique des cellules tumorales qui interagissent avec le microenvironnement et les cellules osseuses. Dans ce contexte, les microARNs en tant que régulateur endogène de l'expression génique, interfèrent avec les différentes étapes de la formation des métastases osseuses, incluant l'échappement des cellules tumorales de la tumeur primaire, la dissémination et l'invasion du site osseux, ainsi que l'apparition de lésions ostéolytiques. Les profils transcriptomiques des microARNs de cellules tumorales mammaires à caractère ostéotropique montrent que l'expression de la famille de microARNs-30s (miRs-30) est inhibée dans ces cellules. En clinique, la faible expression des miRs-30 est associée à un mauvais diagnostique de rechute et au statut hormono-résistant. Dans un modèle animal de métastases osseuses, l'expression forcée des miRs-30 dans des cellules tumorales qui métastasent fortement et spécifiquement à l'os, inhibe la formation des métastases osseuses. Nous montrons que les miRs-30 inhibent l'invasion et stimulent l'ostéoblastogenèse, in vitro et réduisent la charge tumorale et l'ostéoclastogenèse, in vivo. En accord avec ces résultats, l'expression de gènes qui stimulent les métastases osseuses est inhibée par les miRs-30. Parmi ces gènes, l'expression du CTGF (connective tissue growth factor) est augmentée dans les métastases osseuses humaines. Les étapes précoces des métastases osseuses sont étudiées par inoculation de cellules tumorales métastatiques murines dans la glande mammaire de souris. Dans ce modèle, les miRs-30 n'altèrent pas la croissance tumorale et la dissémination métastatique à l'os. Cependant, les miRs-30 inhibent l'invasion et le caractère de cellules souches tumorales de ces cellules métastatiques. Ces résultats suggèrent que les miRs-30, en régulant négativement les métastases osseuses, représentent une thérapie potentielle pour réprimer des gènes cibles qui stimulent les métastases osseusesBone metastasis is a common complication of advanced breast cancers and is clinically responsible of bone fractures, hypercalcemia and pain for which only palliative therapies are proposed. Breast tumor cells that preferentially invade bone express a set of deregulated genes that enhance bone tropism and facilitate bone marrow engraftment which may lead to the formation of overt osteolytic lesions. Molecular pathways underlining these steps are regulated through the tight control of genes expressed by cancer cells interacting with cells from the bone microenvironment. In this context, microRNAs act as regulators of gene expression and control multiple aspects of bone metastasis, including tumor cell escape from the primary site, dissemination, invasion of the bone marrow and secondary outgrowth. MicroRNA transcriptomic profiling of osteotropic breast cancer cell lines identified drastic down-regulation of the miR-30 family (miRs-30). In the clinic, low expression of miRs-30 in breast primary tumors is associated with poor distant metastasis-free survival and hormoneinsensitive status. In a model of human bone metastasis in vivo, the forced expression of miRs-30 in a breast cancer cell line that is highly and specifically metastatic to bone inhibited bone metastasis. We demonstrated that miRs-30 inhibit tumor cell invasiveness and stimulate osteoblastogenesis, in vitro, and reduces tumor burden and osteoclast activity, in vivo. Consistent with that, the expression of several genes that promote bone metastasis were inhibited by miRs- 30. Among these, expression of connective tissue growth factor (CTGF) was up-regulated in human bone metastasis. The early steps of bone metastasis were studied in a mouse model using spontaneously metastatic mouse breast cancer cell lines inoculated in the mammary gland. In this model, miRs-30 did not alter tumor growth or metastatic dissemination to bone. However, miRs-30 inhibited cell invasiveness and cancer stem cell-like phenotype of these metastatic cells. We conclude that miRs-30, by interfering negatively with bone metastasis, represent a potential therapy to repress gene targets that promote bone metastasis
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Robinson, Hollie Christine. "Investigation of the role of microRNA-143 and microRNA-145 in acute vascular injury." Thesis, University of Glasgow, 2014. http://theses.gla.ac.uk/5221/.
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Vascular smooth muscle cell (VSMC) activation leading to proliferation, migration and extracellular matrix (ECM) production is a major cause of neointimal formation after stenting and coronary artery bypass grafting. Drug-eluting stents have reduced clinical incidence of in-stent restenosis by inhibiting this proliferative response but they can also delay vessel re-endothelialisation after injury leading to an increased thrombotic risk. MicroRNAs (miRNAs) are short (~22 nt) non-protein-coding RNAs which act as regulators of gene expression largely through binding to the 3’ untranslated region of target genes and causing degradation or repression of expression. MiR-143 and miR-145 are a miRNA family that are enriched in VSMCs and have been previously shown to influence VSMC phenotype through regulation of their gene targets. Consequently, the aim of this study was to investigate the role that miR-143 and miR-145 play in the neointimal response to stenting. Initial experiments investigated whether modulation of miR-143 or miR-145 expression was capable of significantly altering VSMC phenotype. It was found that modulation of miRNA levels in human saphenous vein (HSV) SMC or endothelial cells (EC) using adenoviruses was not ideal due to transduction and toxicity issues. Use of antimiR miRNA inhibitors and premiR miRNA mimics revealed that modulation of miR-143 or miR-145 levels alone was not sufficient to alter proliferation or migration of HSV SMCs in vitro. Knockdown of miR-143 expression in cells resulted in de-repression of target genes kruppel-like factor 4 (KLF4) and KLF5 but expression levels of other previously identified target genes were unaltered by miRNA modulation. Pre-clinical stenting studies are largely performed in porcine models due to similarities in vessel structure and neointimal formation, however large animal models are not ideal for early investigative studies. In order to examine the role of miR-143 and miR-145 in stent-induced vascular injury we utilised a mouse model where a bare metal stent is deployed in the thoracic aorta of a donor mouse and interposition grafted into the carotid artery of a recipient. This model resulted in the development of a defined neointima over 28 days which consisted largely of VSMCs and ECM, similar to that of the human in-stent restenosis. Vessel expression of miR-143 and miR-145 has been previously shown to be reduced following vascular injury and furthermore overexpression of these miRNA can reduce neointimal formation. MiR-143 and miR-145 knockout (KO) mice have previously been shown to have abnormal VSMC phenotype in their vessel walls including perturbed stress fiber formation, increased presence synthetic machinery and an increased number of synthetic versus contractile VSMCs. MiR-143 and miR-145 KO mice were found to develop significantly less neointimal formation in response to stenting than WT mice indicating that these miRNA are essential for normal vessel response to injury. The reduced neointimal formation following genetic ablation of miR-143 or miR-145 led to the investigation of whether pharmacological knockdown of these miRNA was able to mimic this effect. An antimiR consisting of DNA and locked nucleic acid bases targeted against mature miR-143 expression was used to knockdown miR-143 expression in mice prior to stenting. AntimiR-143-mediated knockdown of miR-143 expression did not significantly alter the degree of neointimal formation seen 28 days following stent deployment when compared to mice that received a control non-targeted antimiR (antimiR-ctl). The neointima of both antimiR-143 and antimiR-ctl mice were comparable and consisted largely of VSMCs and ECM. Re-endothelialisation had occurred by day 28 post-injury in antimiR-143 and antimiR-ctl treated mice indicating that knockdown of miR-143 did not significantly delay EC repopulation in this model. Expression of miRNA and mRNA after vascular injury can be both spatial and temporal. Target de-repression was not detected in the aorta or heart of miRNA KO or antimiR-143 treated mice. This is likely to reflect the complex nature of miRNA gene regulation in vivo which is governed by many different factors including the relative expression of the miRNA and its target, cell stress, and transcriptional activation or inhibition by growth and transcription factors. In summary, the influence of miR-143 and miR-145 on VSMC biology was investigated in vitro using a range of molecular biology techniques and in vivo in a mouse model of in-stent stenosis. Results have extended current knowledge of the degree of influence these miRNA exert over VSMC phenotype and identified that miR-143 and miR-145 are involved in neointimal formation after stenting.
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Kemp, Jacqueline Renee. "Genome-wide Angiotensin II regulated microRNA expression profiling: A smooth muscle-specific microRNA signature." Cleveland State University / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=csu1367845628.
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Azevedo-Pouly, Ana Clara P. "Biological functions of microRNA-216 and microRNA-217 during the development of pancreatic cancer." The Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1374244806.
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Siciliano, Velia. "A microRNA based genetic clock." Thesis, Open University, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.552785.
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This thesis focuses on the design, construction and stable integration in mammalian cells of a natural microRNA-based genetic oscillator. This will help both in better understanding the rules underlying the periodic expression of genes observed in major biological processes, such as the circadian clock and cell-cycle, as well as, in generating new tools to probe and investigate the function of a gene in a cell, by allowing not only its over-expression or knock-down, but also its cyclic expression. The circuit involves a positive feedback loop, consisting of a transcription factor (TF) activating itself, and a negative feedback loop, using a natural micro RNA controlled by the TF, which induces degradation of the TF itself. The circuit was built in a modular way, and implemented it in two lentiviral vectors able to infect both dividing and non-dividing cells, hence suitable for many different applications. Since obtaining stable oscillations is non-trivial, a modified version of the oscillator was engineered, including an intermediate step between the TF and the microRNA, to increase the delay in the negative feedback loop. The oscillatory behavior was tested via in vivo time-lapse fluorescence microscopy in both versions of the oscillator, since both the TF(s) and the microRNA are expressed together with fluorescent reporters.
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Weinstein, Earl G. 1974. "MicroRNA cloning and bioinformatic analysis." Thesis, Massachusetts Institute of Technology, 2002. http://hdl.handle.net/1721.1/8390.
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Thesis (Ph.D.)--Massachusetts Institute of Technology, Dept. of Biology, 2002.Includes bibliographical references.
Part I. Two gene-regulatory noncoding RNAs (ncRNAs), let-7 RNA and lin-4 RNA, were previously discovered in the C. elegans genome. The let-7 gene is conserved across a wide range of genomes, suggesting that these ncRNAs represent a wider class of gene-regulatory RNAs. Both lin-4 and let-7 RNAs are generated from stem-loop precursor RNAs, and share a common biochemical signature, namely 5'-terminal phosphate and 3'-terminal hydroxyl groups. We refer to ncRNAs that share the characteristic size, biochemical signature, and precursor structures of let-7 and lin-4 as microRNAs (miRNAs). The size of this class of genes, and its prevalence in other genomes, are unknown. Therefore, we developed an experimental and bioinformatics strategy to identify novel miRNA genes. We discovered a total of 75 miRNA genes in the C. elegans genome, and orthologues for a majority of these were computationally identified in the C. briggsae, D. melanogaster or H. sapiens genomes. Northern analysis was used to confirm and analyze the expression of these miRNAs. The data set has implications for understanding miRNA gene regulation, miRNA processing, and regulation of miRNA genes. Part II. Directed molecular evolution has previously been applied to generate RNAs with novel structures and functions. This method works because nucleic acids can be selected, randomized, amplified and characterized using polymerase chain reaction (PCR)-based methods. Here we present a novel method for extending directed molecular evolution to the realm of peptide selections by linking a peptide to its encoding mRNA.
(cont.) A proof of principle selection for two different peptides indicates that this tRNA should prove useful in discovering more complex protein molecules using directed molecular evolution.
by Earl G. Weinstein.
Ph.D.
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Torres, Adrian Gabriel. "MicroRNA targeting with oligonucleotide analogues." Thesis, University of Cambridge, 2012. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.610040.
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Morton, Sarah Uhler. "MicroRNA regulation of cardiac development." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3311351.
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Chen, Jianfu Wang Da-zhi. "MicroRNA function in muscle development." Chapel Hill, N.C. : University of North Carolina at Chapel Hill, 2008. http://dc.lib.unc.edu/u?/etd,2013.
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Thesis (Ph. D.)--University of North Carolina at Chapel Hill, 2008.Title from electronic title page (viewed Feb. 17, 2009). "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Cell & Developmental Biology of School of Medicine." Discipline: Cell and Developmental Biology; Department/School: Medicine.
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Hunter, Catriona Mhairi. "MicroRNA regulation of macrophage activation." Thesis, University of Edinburgh, 2017. http://hdl.handle.net/1842/31027.
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Macrophages are mononuclear phagocytic cells that have diverse roles within the body. Tissue specific macrophages, e.g. Kupffer cells, microglia and osteoclasts, have roles in tissue homeostasis, while circulating macrophages play an important role in the innate immune system. Macrophages detect the presence of pathogen associated molecular patterns (PAMPs) via a range of receptors known collectively as pathogen recognition receptors (PRRs). Detection of pathogens causes the macrophages to become ‘activated,’ during which the macrophages undergo extreme morphological and translational changes that enable the pathogen to be neutralised and other immune system components to be recruited. Macrophage activation must be carefully regulated and promptly resolved, as chronic inflammation is damaging to the host. MicroRNAs have emerged as one mechanism by which activation is regulated. MicroRNAs are small, non-coding pieces of RNA that function as a post-transcriptional regulatory mechanism. Their action is exerted through binding with a complementary region in the 3’ untranslated region (3’UTR) of the target mRNA. This binding, facilitated by the ribonuclear protein complex RISC, prevents successful translation of the mRNA into its protein product. MicroRNAs have been shown to function across species, throughout development and during the adult life-span. In the immune system, microRNAs are known to be required for correct formation of germinal centres and normal development of B- and T-cells. MicroRNAs have also been shown to be differentially regulated during macrophage activation with different stimuli. In particular, miR-155, miR-146a and miR-21 are associated with macrophage activation by lipopolysaccharide (LPS). The objective of this work was to further understand the role of microRNAs during macrophage activation with LPS. Two approaches were adopted. Firstly, the regulation of individual microRNAs in LPS-activated bone marrow derived macrophages (BMDMs) was characterised by the use of illumina small RNA sequencing. Secondly, the requirement of the global microRNA population during macrophage biology was investigated through the use of DGCR8 and Dicer knockout systems. In keeping with the large number of changes reported in mRNA translation upon activation, expression of >400 microRNAs were found to be differentially regulated by exposure to LPS. Twelve of these microRNAs were chosen for further study (miR- 142-3p, -146a, -15b, -155, -16, -191, -21, -27b, -30b, -322-5p, -378 and -7a). Individual knock-down of these microRNAs in the RAW264.7 macrophage-like cell line mostly demonstrated subtle, rather than dramatic changes to the activation marker genes studied by RT-QPCR analysis. However, knock-down of miR-146a, -15b, - 155 and -191 were able to significantly alter the expression of the activation marker genes (Tnf-a, Cox2, Cxcl2, Il-6 and Saa3). Interestingly, knock-down of miR-142-3p, miR-146a and miR-155 appeared to show cross-regulation of these microRNAs. The cell index (CI) data suggested that miR-191 and miR-21 influence adhesion in activated macrophages. Studies with the DGCR8 and Dicer knockout systems showed that the global microRNA population was required for successful differentiation of macrophages from embryonic stem cells, and for normal expression of differentiation and activation markers in bone marrow derived macrophages. Overall, these results show that dynamic expression of microRNAs is an integral part of the macrophage response to LPS.
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PERBELLINI, RICCARDO. "I MICRORNA NELLE DISTROFIE MIOTONICHE." Doctoral thesis, Università degli Studi di Milano, 2010. http://hdl.handle.net/2434/150059.
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Myotonic dystrophy (DM), the most common form of muscular dystrophy in adults, is a dominantly inherited disorder with a peculiar and rare pattern of multisystemic clinical features affecting skeletal muscle, the heart, the eye, and the endocrine system.
Classical DM (first described by Steinert and called Steinert’s disease or DM1) has been identified as an autosomal dominant disorder associated with the presence of an abnormal expansion of a CTG trinucleotide repeat in the 3’ untranslated region of the dystrophia myotonica-protein kinase (DMPK) gene on chromosome 19q13.3. Recently, the expansion of a CCTG tetranucleotide repeat located in the intron of the zinc finger 9 (ZNF9) gene on chromosome 19q13.3 was identified as the mutation responsible for DM2. Both mutations lead to the production of mRNA transcripts containing expanded tri- or tetranucleotide repeats (CUG/CCUG) that are retained in muscle nuclei as ribonuclear inclusions and interact with RNA-binding proteins. These interaction are supposed to disrupt the regulation of alternative splicing of several transcripts. Clinical and molecular parallels strongly support that DM1 and DM2 physiopathology is in part the pathogenic consequence of an RNA gain of toxic function.
MicroRNAs (miRNAs) are short non-coding RNAs (~22 nucleotides) regulating gene expression post-trascriptionally either via the degradation of target mRNAs or the inhibition of protein translation. MicroRNAs have been shown to be involved in a range of biological processes, including myogenesis and muscle regeneration. miRNAs are expressed in cardiac and skeletal muscle, and dysregulated miRNA expression has been correlated with muscle-related diseases, including cardiac hypertrophy, cardiac arrhythmias and muscular dystrophy. Given the emerging roles of microRNAs, we have performed miRNAs expression profiling in DM1 and DM2 patients on muscle biopsies and primary cell culture line. Using fast real time PCR, we report here the differences in miRNAs expression profiles between DM1 (n=15), DM2 (n=9) and control subjects (n=14) of 24 specific miRNAs. miRNAs expression profiles in muscle biopsies of DM1 showed up-regulation of miR-1 and miR-335 and down-regulation of miR-29b, miR-29c, miR-33, establishing a provisional DM1 miRNA signature. A similar trend in miRNA modulation was observed in DM2 patients. However, none of the differences reached statistical significance. In order to assess whether DM1 signature miRNA deregulations and DM2 were cell autonomous events, primary cultures of skeletal muscle satellite cells obtained from either DM1 patients (n=5), DM2 patients (n=5) or controls (n=5) were examined. Myoblasts were cultured in growth factor rich medium and then switched to differentiation medium for five days. DM1 and DM2 myoblasts did not display overt morphological alterations of differentiation. When DM1 miRNA signature was examined, we found that miR-29b was strongly down-modulated in differentiated DM1 myotubes. Conversely, miR-335 was enhanced in DM1 myoblasts in growth medium whereas, upon switching to differentiation medium, it increased to a similar level both in DM1 and control myoblasts. When DM2 myoblasts and myotubes was examined, we not found significance statistical differences in miRNAs expression compared with control myoblasts and myotubes.
Furthermore, The cellular localization of DM1 signature miRNAs was assayed by in situ hybridization on cryostat muscle sections derived from DM1 (n=5) and control (n=5) biopsies using digoxigenin labelled LNA probes. We found that miR-29b, -29c, -33 and -335 were either barely detectable or did not show any overt abnormal localization in DM1 compared to control biopsies. Conversely, miR-1 was readily detectable and its intracellular distribution was disrupted. Specifically, in control samples, miR-1 displayed a peculiar enrichment in the perinuclear area. In DM1 sections, centrally nucleated myofibers, a hallmark of DM1, also exhibited a centralization of miR-1 localization. Very small fibers with nuclear clumps, a typical histopathological DM1 alteration, displayed intense miR-1 staining. Certain myofibers displayed an extremely intense and polarized miR-1 accumulation. Atrophic fibers in DM1 muscle are predominantly type I fibers (slow fibers). Aberrant miR-1 distribution was present both in type I and type II myofibers, as assessed by the myosin heavy chain slow isoform counterstaining.
We also tested the cellular localization of two more muscle specific microRNAs, miR-133b and -206, albeit no overt deregulation of their expression was found in whole skeletal muscle RNAs. In control biopsies we found that miR-133b displayed a perinuclear distribution similar to that of miR-1; in keeping with previous findings, miR-206 was barely detectable. In DM1 patients, both miR-133b and -206 exhibited centralization in centrally nucleated myofibers and accumulated in association to small myofibers nuclear clumps. Finally, miRNAs have been shown not only to inhibit protein translation, but also to induce mRNA degradation, at least for certain targets. Thus, in order to assess whether miRNA deregulation was functionally relevant, we examined the impact of the identified miRNAs deregulation on the expression of their potential target genes in DM1 patients. Specifically, we focused on miR-29, that displayed the strongest deregulation, and miR-1, that plays a crucial role in muscle differentiation. Search of the potential targets was performed using Pictar and Targetscan prediction algorithms, given their reported specificity. Indeed, to maximize the accuracy, only targets identified by both softwares were considered. A sub-pool of the identified targets were analyzed, selected among these with a potential link to DM1 physio-pathology. Specifically, selected genes were previously demonstrated to be expressed in skeletal muscle and to be involved in events such as muscle development, atrophy, arrhythmia and splicing. Potential targets were assayed by qPCR and shows that both miR-29 and miR-1 targets were significantly up-regulated in DM1 patients. In conclusion, we identified a small subset of miRNA whose expression and/or localization were deregulated in DM. These findings may improve our understanding of the molecular mechanisms linking (CTG)n/(CCTG)n expansion to disease and may serve as potential prognostic/diagnostic markers.
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Frölich, Anne. "Der Einfluss körperlichen Trainings auf die endotheliale Dysfunktion mit Fokus auf die Rolle der microRNA-21 und microRNA-126 im herzinsuffizienten Mausmodell." Doctoral thesis, Universitätsbibliothek Leipzig, 2015. http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-166793.
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Eine angemessene, regelmäßige sportliche Aktivität wirkt protektiv auf die Erhaltung und Wiederherstellung körperlicher und geistiger Gesundheit.
Die Herzinsuffizienz ist nicht zuletzt durch ihre hohe Prävalenz ein internistisches Krankheitsbild von enormer Relevanz im klinischen Alltag. Im Rahmen einer bestehenden Herzinsuffizienz kann es zur pathophysiologischen Ausprägung einer gestörten Endothelfunktion kommen, welche sich in einer verringerten endothelialen Dilatationsfähigkeit ausdrücken kann.
Das Ziel dieser Dissertation bestand in der Untersuchung der Auswirkungen eines körperlichen Trainings auf die Funktionsleistung der Endothelzellen von herzinsuffizienten Mäusen. Hierzu wurde in einem operativen Eingriff durch die Ligatur des Ramus interventricularis anterior bei jungen Mäusen ein herzinsuffizientes Tiermodell geschaffen und der sekundärpräventive Effekt eines anschließenden zehnwöchigen Laufbandtrainings eruiert. Als Funktionsmaß der Endothelzellen diente dabei ihre endothelabhängige Dilatationsfähigkeit, welche im Organbad erhoben wurde. Weiterhin lag der Fokus auf der Untersuchung des Einflusses der im Endothel exprimierten microRNA-21 und microRNA-126 im trainierten und untrainierten herzinsuffizienten Aortenendothel. Ihre Expression wurde in den verschiedenen Versuchsgruppen quantitativ mittels Real-Time PCR erfasst. In einem weiteren Ansatz bestand das Ziel, den Einfluss einer Angiotensin II- beziehungsweise Zytokinstimulation - als Modell eines mit der Herzinsuffizienz vergesellschafteten Inflammationsgeschehens - auf diese beiden endothelexprimierten microRNAs zu erforschen.
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Massiere, Jessica. "La transition épithélio-mésenchymateuse dans les cellules épithéliales gastriques : rôle des microARN régulés par Helicobacter pylori." Thesis, Bordeaux 2, 2011. http://www.theses.fr/2011BOR21835/document.
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Les microARN sont de petits ARN non codant régulant post-transcriptionnellement l’expression de certains gènes. Du fait de leur fort potentiel régulateur, une modification de leur expression peut conduire à l’apparition de pathologies telles que le cancer ou l’inhibition des mécanismes de défense contre des pathogènes. Notre objectif est de caractériser le rôle de certains miARN dans la formation de cancer gastrique dû à Helicobacter pylori. En effet, cette bactérie peut conduire à l’apparition d’adénocarcinome gastrique et de lymphome du MALT. Sa virulence est essentiellement due à la protéine CagA, injectée dans les cellules de la muqueuse gastrique. Par séquençage à haut débit du contenu en miARN d’une lignée épithéliale gastrique humaine, co-cultivée ou non avec H. pylori, nous avons observé que les niveaux de miR-200b/c sont augmentés par l’infection. Ces miARN sont des inhibiteurs puissants de la transition épithélio-mésenchymateurse (TEM), modification morphologique promotrice d’invasion. Ils ciblent les facteurs de transcription ZEB1/2 avec lesquels ils sont impliqués dans une boucle de rétro-action mutuellement répressive. Le niveau basal élevé de miR-200b/c dans ces cellules réprime totalement ZEB1, tandis que l’infection par H. pylori, sous la dépendance de CagA, promeut une TEM en induisant ZEB1. Paradoxalement, les miR-200b/c sont aussi augmentés lors de l’infection transcriptionnellement. Nous avons pu démontrer que l’augmentation des miR-200b/c dans les cellules infectées a pour rôle de modérer l’induction de ZEB1 via l’activation de NF-kB, constituant ainsi un mécanisme de défense des cellules hôte contre la perte de leur identité épithélialeMicroRNA are small noncoding RNA that post-transcriptionally regulate gene expression. Due to their high regulator potential, a change in their expression may lead to the emergence of diseases such as cancer or inhibition of defense mechanisms against pathogens. Our aim is to characterize the role of miRNA in the response of gastric eptithelial cells to Helicobacter pylori (H. pylori). Indeed, H. pylori promote gastric adenocarcinoma and MALT lymphoma. Its virulence is essentially mediated by CagA, injected into cells of the gastric mucosa. Thanks to high throughput sequencing of miRNA content of a gastric epithelial cell line, infected or not with H. pylori: miR-200b and -200c appeared up-regulated upon infection. These miRNA are potent inhibitors of the “epithelial-to-mesenchymal transition” (EMT), a process that drastically alters cell morphology and promotes cell invasion. MiR-200b/c target the transcription factors ZEB1 and ZEB2, with which they are involved in a mutually repressive feedback loop. In basal conditions, the high levels miR-200b/c in gastric epithelial cells totally silence ZEB1 mRNA whereas H. pylori promotes EMT via ZEB1 expression, on the dependence of CagA translocation into host cells. But, paradoxically, miR-200b/c levels were also up-regulated upon infection. The increased miR-200b/c levels in infected cells moderate ZEB1 induction thanks to NF-kB activation and constitute a self-defense mechanism to thwart the loss of their epithelial phenotype upon infection
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Guérit, David. "Rôle des miR-29a et miR-574-3p au cours de la différenciation chondrocytaire de la cellule souche mésenchymateuse." Thesis, Montpellier 1, 2012. http://www.theses.fr/2012MON1T013/document.
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Avec l'augmentation de l'espérance de vie, les pathologies ostéo-articulaires comme l'arthrose ou la polyarthrite rhumatoïde, caractérisées par la dégradation du cartilage articulaire, deviennent de réels problèmes de santé publique. Les traitements actuels sont essentiellement symptomatiques et aboutissent en ultime recours à la pose de prothèses. En absence de réparation spontanée du tissu et de traitement efficace, des approches d'ingénierie tissulaire du cartilage sont envisagées. Les techniques actuelles reposent sur la transplantation de chondrocytes autologues mais dans la majorité des cas, cette approche n'apporte pas de résultats supérieurs aux techniques chirurgicales utilisées actuellement. Grâce à leurs propriétés de différenciation, les cellules souches mésenchymateuses (CSM) représentent une nouvelle source de cellules ayant des potentiels thérapeutiques intéressants. Cependant, la complexité du processus de différenciation des CSMs vers des chondrocytes articulaires matures rend difficile l'obtention de cartilage fonctionnel après implantation. Il est donc important de mieux comprendre le processus de différenciation de ces cellules afin de mieux contrôler leur devenir in vivo. C'est pourquoi, le laboratoire s'intéresse au rôle des micro-ARNs (miARNs) dans la régulation du processus de différenciation des CSMs. L'objectif de mon projet de thèse a consisté à identifier des miARNs modulés dans la différenciation chondrocytaire des CSM humaines primaires et à étudier leur rôle et leur régulation au cours de la chondrogenèse. Nous avons identifié deux miARNs : miR-29a dont l'expression diminue progressivement au cours de la différenciation et miR-574-3p dont l'expression augmente rapidement puis est maintenue jusqu'à la fin de la différenciation. Ces deux miARNs sont régulés par le facteur de transcription SOX9 mais de manière opposée : SOX9 inhibe miR-29a et induit miR-574-3p. Nous montrons que SOX9 interagit avec YY1 pour réguler miR-29a mais pas miR-574-3p, ce qui pourrait expliquer les effets opposés de SOX9 sur l'expression des deux miARNs. Nous montrons également que ces miARNs sont des inhibiteurs de la différenciation chondrocytaire et avons identifié FOXO3A et RXRα comme cibles respectives de miR-29a et miR-574-3p. L'inhibition de FOXO3A ou RXRα avant l'induction de la différenciation, en utilisant des siARNs spécifiques ou en sur-exprimant les miARNs correspondants, bloque la différenciation des CSM. Ces résultats confirment sur des CSMs adultes, que ces protéines jouent un rôle important dans la chondrogenèse et que miR-29a et miR-574-3p participent aux processus de régulation de la différenciation chondrocytaire. En conclusion, nous avons identifié deux nouveaux miARNs contrôlés par SOX9 et régulant négativement la chondrogenèse grâce à la modulation de deux gènes cibles, dont l'expression est nécessaire avant d'induire la différenciation chondrocytaireRoles of miR-29a and miR-574-3p during the chondrogenic differentiation of mesenchymal stem cells. With the constant increase of the lifespan, osteoarticular pathologies such as osteoarthritis or rheumatoid arthritis, characterized by articular cartilage degradation, are important public health problems. In absence of spontaneous regeneration, cartilage engineering approaches are being considered. Current techniques rely on autologous chondrocyte transplantation but in the majority of cases, this approach gives similar results as current surgeries. Due to their capacity of differentiation toward chondrocytes, mesenchymal stem cells (MSC) represent a new source of cells with therapeutic potential. However, production of a functional cartilage in vivo after implantation of expanded MSC is hampered by the difficulty to reproduce the complexity of the differentiation process to get mature chondrocytes from MSC. The objective of my Ph.D thesis aimed to identify micro-RNAs (miRNAs) modulated during chondrogenic differentiation of primary human MSCs and to study their role as well as their regulation in this process. We identified two miRNAs: miR-29a whose expression decreases progressively during the differentiation and miR-574-3p whose expression rapidly increases and stays constant until the end of the differentiation. Both miRNAs are regulated by the transcription factor Sox9 but in an opposite manner: Sox9 inhibits miR-29a and induces miR-574-3p. We show that YY1 directly interact with Sox9 to regulate miR-29a but not miR-574-3p; this interaction likely explaining the opposite effects of Sox9 on miR-29a and miR-574-3p expression. Moreover we showed that miR-29a and miR-574-3p are both inhibitors of chondrogenesis and we identified FOXO3A and RXRα as their respective targets. In conclusion, we identified two new miRNAs which are regulated by Sox9 and inhibitors of chondrogenesis. They act through the modulation of two target genes, whose role during chondrogenic differentiation of adult MSC was previously not characterized
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Kamrath, Malte [Verfasser]. "Dynamischer biomechanischer Stretch führt zur Regulation von microRNA-146a und microRNA-503 in Kardiomyozyten / Malte Kamrath." Kiel : Universitätsbibliothek Kiel, 2019. http://d-nb.info/1187732788/34.
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Szczyrba, Jaroslaw [Verfasser], and Friedrich [Akademischer Betreuer] Grässer. "Das MicroRNA-Profil des Prostata-Karzinoms und Identifizierung von microRNA-Zielgenen / Jaroslaw Szczyrba. Betreuer: Friedrich Grässer." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2012. http://d-nb.info/1052557279/34.
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Shcheholeva, Iryna. "Synthèse orientée vers la diversité pour l'inhibition de microARN oncogènes." Electronic Thesis or Diss., Université Côte d'Azur, 2024. https://intranet-theses.unice.fr/2024COAZ5006.
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Constituant une majeure partie du génome et compte tenu de leur fonction dans la modulation de l'épigénétique, les ARN non codants (ARNnc) jouent un rôle important dans le développement de plusieurs pathologies, mais restent une classe de cibles biologiques sous-exploitée. Les microARN (miR) sont des ARN à simple brin (ARNsb) constitués de 19 à 25 nucléotides et représentent une famille majeure d'ARNnc, principalement connue pour son rôle dans le contrôle de l'expression des gènes. Chaque microARN inhibe la traduction de plusieurs ARN messagers, et la dérégulation des microARN a été directement liée à plusieurs pathologies dont les cancers. Plus précisément, le microARN-21 est surexprimé dans plusieurs cancers comme montré dans une étude dressant le profil de 540 échantillons cliniques provenant de patients atteints de cancer. L'inhibition du miR-21 représente donc une stratégie prometteuse à la fois comme thérapie efficace seule et comme adjuvant aux traitements existants.L'objectif de ce travail de thèse a été de développer des ligands inhibiteurs de ce microARN oncogène, ciblant l'étape de clivage de Dicer de sa biogenèse. Nous nous sommes concentrés sur ce dernier point pour éviter le défi associé à l'ARNsb en tant que cible biologique, telle qu'une structure secondaire non définie. Dans cette thèse, le précurseur de miR-21, un transcrit long et structuré, a été utilisé comme cible, et des ligands ont été conçus pour se lier à ses régions structurées et empêcher sa reconnaissance par Dicer. Une bibliothèque de ligands a été conçue de novo et synthétisée en appliquant des méthodologies catalytiques efficaces, et leur activité a été évaluée in vitro à l'aide d'essais biochimiques basés sur la fluorescence. L'étude a révélé un certain nombre de nouveaux ligands et inhibiteurs du microARN-21 oncogène avec une activité au niveau micromolaire. Le mécanisme de liaison des meilleurs composés a été étudié avec des méthodes biophysiques et in silico pour établir les relations structure-activité et améliorer l'activité observée. Cette thèse dévoile de nouvelles structures prometteuses qui inhibent la maturation de miR-21 in vitro et fournissent un modèle pour le ciblage de cet ARN non codant avec de petites moléculesConstituting a major part of the transcriptional output and given their function in modulation of epigenetics, noncoding RNAs (ncRNAs) carry an important role in disease development, but remain under-exploited biological targets. MicroRNAs (miRs) are 19 - 25 nucleotide long single-stranded RNAs and represent a major ncRNA family that is primarily known for their role in the control of gene expression. Each microRNA indeed inhibits translation of multiple messenger RNAs, and dysregulation of microRNAs is critical to pathogenesis and oncogenesis in particular. Specifically, microRNA-21 has been in the spotlight after its consistent overexpression in cancers as reported in a study that profiled 540 clinical samples from cancer patients. Thus, inhibition of miR-21 function holds the promise for both an efficient therapy alone and as an adjuvant to the existing treatments.The goal of this PhD work was to develop a small-molecule inhibitor of this oncogenic microRNA, tackling the last step of its biogenesis, an enzymatic cleavage by Dicer. We focused on the latter to mitigate a challenge associated with ssRNA as a biological target, such as the undefined secondary structure. In this thesis, the precursor of miR-21, a longer and structured preceding transcript, was used as a target and small-molecule ligands were designed to bind to its structured regions and impede its recognition via Dicer. The library of the drug-like RNA-focused ligands was designed de novo and synthesised using efficient catalytic methodologies and their activity was assessed in vitro using fluorescence-based biochemical assays with human recombinant Dicer. The study revealed several novel small molecule binders and inhibitors of oncogenic microRNA-21 in the low micromolar range. The binding mechanism of the best compounds was studied with biophysical and in silico methods to establish structure-activity relationships and improve the observed activity. This thesis discloses new promising scaffolds that inhibit miR-21 maturation of miR-21 in vitro and provide a blueprint for targeting this noncoding RNA with small molecules
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Boggs, Rene' Michelle. "MicroRNA expression in canine mammary cancer." Diss., Texas A&M University, 2008. http://hdl.handle.net/1969.1/85979.
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MicroRNAs (miRNAs) play a vital role in differentiation, proliferation and tumorigenesis by binding to messenger RNAs (mRNA) and inhibiting translation. To initiate an investigation into the identification of miRNAs in the domestic dog, an emerging model for human disease, a comparison of the human and canine genetic databases was conducted. The bioinformatics work revealed significant conservation of miRNA genes between the two species. Proof of principle experiments, including serial dilutions and sequencing, were performed to verify that primers made to amplify human mature miRNAs can be used to amplify canine miRNAs, providing that the mature sequences are conserved. TaqMan® Real-time RT-PCR, a sensitive and specific method, was used to isolate the first miRNA mature products from canine tissues. The expression levels of miR-17-3p, miR-17-5p, miR-18, miR-19a, miR-19b, miR-20, and miR-92 were evaluated in five canine tissues (heart, lung, brain, kidney, and liver). Because miRNAs have been found to act as both tumor suppressors and oncogenes in several different cancers, expression patterns of ten miRNAs (miR-15a, miR-16, miR-17-5p, miR-21, miR-29b, miR-125b, miR-145, miR-155, miR-181b, let-7f) known to be associated with human breast cancer were compared between malignant canine mammary tumors (n=6) and normal canine mammary tissue (n=10). Resulting data revealed miR-29b and miR-21 to have a statistically significant (p<0.05) up-regulation in cancerous samples. Overall expression patterns showed nine of the ten miRNAs follow the same pattern of expression in the domestic dog as the human, while the miR-145 expression does not show a difference between the normal and cancerous samples.
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O'Connor, Anna Maria. "MicroRNA regulation of endothelial calcium signalling." Thesis, Queen's University Belfast, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.602716.
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The Ca2+ signalling pathway is fundamental for many endothelial functions including angiogenesis, regulation of blood vessel tone and barrier selectivity and must therefore be regulated appropriately. MicroRNAs are non-coding RNAs which can regulate the expression of multiple genes at the posttranscriptional level. The aim of this thesis was to investigate the possibility that microRNAs can regulate Ca2+ responses by targeting genes of the IP3-Ca2+ signalling pathway. The microRNA expression profile of primary bovine retinal microvascular endothelial cells (RMECs), a widely used in vitro model for studies of endothelial physiology and angiogenesis, was analysed by high throughput sequencing. Expression of genes involved in IP3-Ca2+ signalling was assessed by RT-PCR. MicroRNA-26a was selected for further analysis because it was highly expressed and predicted to target key genes of IP3-Ca2+ signalling expressed in RMECs, namely IP3 receptor type 1 (ITPRl) and phospholipase C beta 1 (PLCBl). Changes in ITPRl mRNA and protein following overexpression or knockdown of microRNA-26a were consistent with it being a target and luciferase assays confirmed a direct interaction. Uridine triphosphate induced a transient increase in intracellular calcium concentration ([Ca2+]i) measured by fura-2 Ca2+ microfluorimetry that was blocked by PLC and IP3 receptor inhibitors. Overexpression of microRNA-26a decreased this elevation in [Ca2+]i and knockdown of microRNA-26a increased the response. This effect is likely to be at least partially due to the regulation of ITPRl. Many other genes that are modulated by microRNA-26a and may mediate the functional responses in RMECs were detected by high throughput mRNA sequencing. Analysis of the functions associated with these genes supports a role for microRNA-26a in the regulation of vascular physiology. The results suggest that microRNA-26a can modulate the Ca2+ responses of RMECs by targeting the IP3-Ca2+ signalling pathway. Further studies are now warranted to determine the functional significance of microRNA-26a in both retinal microvascular endothelial cell physiology and pathophysiology.
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Tudor, Hannah Rachael. "Characterisation of a spliced tomato microRNA." Thesis, University of East Anglia, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.578250.
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MicroRNAs are short single stranded RNA molecules derived from longer precursors with a hairpin like structure. MiRNAs are taken up into a complex, which can then target mRNA for degradation. Mi.RNAs have been shown to play a role in plant development and stress responses. A novel tomato miRNA has been discovered in our group before I started my project. The secondary structure of the miRNA is unusual as it contains a 600nt intron which is spliced out. We discovered that the miRNA is differentially expressed within the leaves exhibiting a wave like pattern from the base of the leaf to the tip. There are similarities of the expression of this miRNA to sink-to-source transition suggesting that this miRNA may have a role in development from young leaves to mature leaves. The expression of this miRNA is highly increased when the tomato plant is grown in light conditions, and the longer un-spliced version of this miRNA is accumulated at a higher level in plants grown in dark conditions. Therefore we concluded that light controls the splicing of this miRNA. Furthermore looking at the wave-like expression of the miRNA within the leaves and the changes in expression with plants grown in light and dark conditions, it seems plausible that the miRNA is involved in the sink-to-source transition of leaves. Following this hypothesis several constructs of the miRNA have been produced which will be transformed into tomato to test this hypothesis. The discovery of this miRNA and the study of it so far have revealed many avenues of possible investigation. This miRNA seems to be integrally involved in plant growth and development. Further study would show how this miRNA functions in the plant, which may shed light on sink-to-source transition.
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Chow, Wang-ngai, and 周弘毅. "Identification of microRNA in penicillium marneffei." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/198803.
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Penicillium marneffei is the most important thermal dimorphic fungus causing respiratory, skin and systemic mycosis in China and Southeast Asia. While miRNAs are increasingly recognized for their roles in post-transcriptional regulation of gene expression in animals and plants, the existence of miRNAs in fungi was less well studied and their potential roles in fungal dimorphism were largely unknown. Based on available genome sequence of P. marneffei, it is hypothesized that miRNA-like small RNAs (milRNAs) may be expressed in the dimorphic fungus and dicer- or argonuate-like proteins may be involved in dimorphism or virulence in P. marneffei.
I attempted to identify milRNAs in P. marneffei in both mycelial and yeast phase using high-throughput sequencing technology. Small RNAs were more abundantly expressed in mycelial than yeast phase. Sequence analysis revealed 24 potential milRNA candidates, including 17 (2502 reads) candidates in mycelial and seven (232 reads) in yeast phase. Two genes, dcl-1 and dcl-2, encoding putative Dicer-like proteins and the gene, qde-2, encoding Argonaute-like protein, were identified in P. marneffei. Phylogenetic analysis showed that dcl-2 and qde-2 of P. marneffei were more closely related to the homologues in the thermal dimorphic pathogenic fungi, Histoplasma capsulatum, Blastomyces dermatitidis, Paracoccidioides brasiliensis and Coccidioides immitis than to Penicillium chrysogenum and Aspergillus spp., suggesting the co-evolution of dcl-2 and qde-2 among other thermal dimorphic fungi. Moreover, dcl-2 and qde-2 demonstrated higher mRNA expression levels in mycelial than yeast phase by 7 folds and 2 folds respectively (P<0.001). Northern blot analysis confirmed the expression of two milRNAs, PM-milR-M1 and PM-milR-M2, only in mycelial phase. Using dcl-1KO, dcl-2KO, dclDKO and qde-2KO deletion mutants, it was shown that the biogenesis of both milRNAs were dependent on dcl-2 but not dcl-1 or qde-2. While deletion of qde-2, but not the two dcl genes, was found to decrease the virulence level of P. marneffei in mice model, the deaths of the qde-2KO conidia challenged mice were delayed for over 10 days. The qde-2KO conidia have lower recovery rate both in human THP1 and murine J774 macrophage cell lines and also reduced resistance to hydrogen peroxide than the wild type.
This study provided the first evidence for differential expression of milRNAs in different growth phases of thermal dimorphic fungi and shed light on the evolution of fungal proteins involved in milRNA biogenesis and possible role of post-transcriptional control in governing thermal dimorphism. This is also the first study to reveal the relationship between argonuate-like QDE-2 protein and virulence in P. marneffei in mice model. This study provides a foundation for the milRNAs study in pathogenic thermal dimorphic fungi.published_or_final_version
Microbiology
Doctoral
Doctor of Philosophy
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Dawkins, Sam. "MicroRNA release in acute myocardial infarction." Thesis, University of Oxford, 2016. https://ora.ox.ac.uk/objects/uuid:a0a82298-45e5-4f66-b368-446cad9726ae.
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Coronary heart disease (CHD) is the single biggest cause of death in the United Kingdom1. Primary percutaneous coronary intervention (primary PCI) has transformed the early treatment of acute myocardial infarction (MI), improving outcome by rapidly re-opening the occluded coronary artery, with a larger mortality benefit and reduced risk compared with thrombolysis. Despite these advances, and even with the optimal treatment, some patients still sustain substantial myocardial damage leading to mortality and morbidity. MicroRNAs (miRs) are short non-coding RNAs with a role in regulating protein synthesis. Some miRs are cardiospecific, can be detected in plasma after a myocardial infarction and show promise as biomarkers and insights into the mechanisms of myocardial injury. In this work, as part of the Oxford Acute MI (OxAMI) Programme, a cohort of patients recruited at the time of ST elevation MI underwent detailed clinical and microRNA analysis at the time of myocardial infarction. This work was validated using separate discovery and validation cohorts. The source of detected miRs was further analysed in an in-vitro endothelial cell culture model and by measuring miRs released into the venous drainage of the heart, the coronary sinus. In the Discovery Cohort, miRs previously shown to be increased in myocardial infarction (e.g. miR-1, -133a, -499) were detectible in plasma after myocardial infarction, and this was confirmed in the validation cohort. Other miRs with a similar relationship were also identified (e.g. miR-30a, -378a, 125b). Microvascular obstruction was found to be associated with increased infarct size and also with release of microRNAs correlating with infarct size suggesting a link between microvascular obstruction and myocardial necrosis. Analysis of paired coronary artery and coronary sinus samples showed that these miRs increased down the myocardial gradient, suggesting myocardial release. The culmination of this work was to use the experimental findings from circulating plasma, cultured endothelial cells and coronary sinus experiments to design a microRNA panel using a blood sample taken six hours after admission to use in a regression model which was more predictive of final infarct size than troponin alone.
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Thomas, Jordan M. "The role of microRNA in cancer." Thesis, Boston University, 2013. https://hdl.handle.net/2144/21261.
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Thesis (M.A.) PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.Micro ribonucleic acids (miRNAs) are pivotal post-transcriptional regulators of gene expression and if research continues to reveal positive results, they will soon be used as therapeutic targets in the clinical setting for the treatment of a variety of cancers. They are evolutionarily conserved small noncoding RNAs that range from 18 to 24 nucleotides in length. There are over 1,400 miRNAs for which abundant evidence has demonstrated fundamental importance in normal cell development and up- or downregulation when they become deregulated. The deregulation of miRNAs, which can function as tumor suppressors or oncogenes, contributes to the development of cancer, among other diseases. Deregulation of tumor suppressor miRNA can occur in many different tissues of the body and lead to a variety of cancers. Tumor suppressor let-7 negatively regulates expression of an oncogenic mRNA named RAS. In lung cancer, a decrease in let-7 produces an increase in expression of RAS, which contributes to cell proliferation and tumorigenesis. In chronic lymphocytic leukemia, Bcl2 protein becomes overexpressed due to the down-regulation of tumor suppressors miRNA-15 and miRNA-16. MicroRNA-34 plays an important role in neuroblastoma in which its expression is decreased due to mutations that decrease a tumor suppressor protein, p53. Upon deregulation of oncogenic miRNAs, tumor suppressor mRNA expression is decreased and leads to multiple types of cancer. Up-regulation of the miRNA-17-92 cluster in lung cancer leads to increased cell proliferation and contributes to angiogenesis in many cancers. In B cell lymphoma, miRNA-155 becomes up-regulated along with an RNA named BIC. This up-regulation accelerates pathogenesis and up-regulation of an oncogenic protein, c-myc. MicroRNA-21 acts as an anti-apoptotic factor by downregulating apoptosis-related genes and contributing to the development of human glioblastoma. This review summarizes the present understanding of how miRNAs function at the molecular level in the body, how their deregulation contributes to tumor formation, maintenance and metastasis and how they can be used for cancer diagnosis, prognosis and therapy. With the mass amounts of knowledge gained from the current research done on miRNAs, a cancer cure may soon be developed based on the targeting of specific miRNAs. The promise of miRNAs in cancer therapeutics will depend on the development of proper delivery strategies of miRNA mimics and inhibitors, in addition to evaluation of safe usage and toxicity of therapeutic dosages.
2031-01-01
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Jurcevic, Sanja. "MicroRNA expression profiling in endometrial adenocarcinoma." Doctoral thesis, Örebro universitet, Institutionen för hälsovetenskap och medicin, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:oru:diva-41640.
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Hilton, Catriona. "MicroRNA-196a in human adipose tissue." Thesis, University of Oxford, 2015. https://ora.ox.ac.uk/objects/uuid:a1a7a7a6-09a2-422f-b900-e133004193da.
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MicroRNAs are small non-coding RNAs that have been shown to play a role in adipose tissue biology. Adipose tissue depots differ in terms of the metabolic risk that they confer. Therefore, I aimed to identify microRNAs which regulate body fat distribution. An initial microarray screen of abdominal subcutaneous and gluteal adipose tissue, with validatory qPCR, identified microRNA-196a as being more highly expressed in gluteal adipose tissue. These expression patterns were retained in primary and immortalised pre-adipocytes from abdominal subcutaneous and gluteal adipose tissue. The precursor of miR-196a contains a single nucleotide polymorphism, rs11614913. Genotyping was performed on 5,823 individuals from the Oxford Biobank and combined with detailed information on body composition by DXA scanning and miR-196a expression determination in fat biopsies. This revealed that the rs11614913 TT genotype was associated with a 32% reduction in miR-196a expression in abdominal subcutaneous adipose tissue (p=0.002), an expansion in waist-to-hip ratio and an increase in mean adipocyte size in abdominal subcutaneous adipose tissue in males. RS11614913 was also found to be associated with bone mineral density, and the relationship between bone mineral density and body fat distribution was explored using the Oxford Biobank. To establish a functional role for miR-196a, the Pt2 abdominal and gluteal cell lines were validated as models for pre-adipocyte proliferation and adipogenesis. MiR-196aKO pre-adipocyte cell lines were generated. MiR-196aKO was found to reduce proliferation in abdominal subcutaneous (p=0.002) and gluteal (p=0.002) pre-adipocytes, with no effect in adipogenesis. In addition, transcriptomic profiling of miR-196aKO pre-adipocytes revealed enrichment for GO term clusters related to extracellular matrix and angiogenesis, suggesting a mechanism by which miR-196a might regulate regional adipose tissue expansion.
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Kaimal, Vivek. "Computational approaches to study microRNA networks." University of Cincinnati / OhioLINK, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1298041682.
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Pomyen, Yotsawat. "Exploring microRNA biology using integrative bioinformatics." Thesis, Imperial College London, 2014. http://hdl.handle.net/10044/1/24774.
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Deregulation of energy metabolism is one of the emerging hallmarks of cancer required for proliferation and metastasis. MicroRNAs are small RNA molecules that have crucial roles in the regulation of biological processes in organisms, including metabolism. Due to recent discovery of miRNAs in humans, roles of miRNAs in metabolism of tumour cells, and effects these have on cancer patients, are still obscure and in need of expansion. Currently, experimental and computational data on the miRNAs are being analysed by a wide range of statistical methods; however, these methods in their original forms posses many limitations. Therefore, new ways of utilising these statistical methods are needed in order to unravel the roles of miRNAs in cancer metabolism. In this thesis, the roles of a specific miRNA, miR-22, and the three metabolic target genes were investigated through the use of classical statistical methods, revealed that miR-22, the metabolic target genes, and the interactions between them, were beneficial to survival outcome of breast cancer patients. Furthermore, novel combinations of the conventional statistical methods were invented in order to investigate the global miRNA regulations on metabolic target genes. These new procedures were demonstrated by using publicly available data sets. In one analysis, it was found that miRNAs could be divided into six clusters according to the metabolic target genes through a novel combination of statistical methods. A new statistical method was also invented to provide a generalised means to test for clustering based on sets of correlations.
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Adai, Alex Tamas. "Uncovering microRNA function through data integration." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3311333.
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GASPARELLO, Jessica. "Novel trands in microRNA based theranostics." Doctoral thesis, Università degli studi di Ferrara, 2018. http://hdl.handle.net/11392/2478767.
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I miRNA sono una classe di RNA non codificanti in grado di regolare l’espressione genica attraverso l’interazione con la regione 3’UTR del trascritto bersaglio, portando all’inibizione della traduzione o alla degradazione del mRNA bersaglio. A causa della loro breve sequenza (19-25 nucleotidi), i miR sono in grado di riconoscere regioni bersaglio in più trascritti, risultando, coinvolti in più di un processo molecolare. Diverse patologie sono state associate alla disregolazione del profilo di espressione di miR, negli anni sono state quindi elaborate diverse strategie terapeutiche allo scopo di ripristinare i livelli fisiologici di miR.
Molecole in grado di mimare la funzione del miR-210 sono state impiegate nella presente tesi allo scopo di ridurre l’espressione del fattore di trascrizione BCL11A, uno dei principali repressori del gene γ-globinico. Lo studio ha permesso di ottenere due risultati chiave: a) miR-210 riconosce una sequenza presente nella regione codificante del trascritto BCL11A, b) l’incremento dei livelli intracellulari di miR-210 sono associati al decremento di trascritto e proteina BCL11A. I dati suggeriscono che l’impiego di molecole in grado di mimare la funzione di miR-210 possono essere impiegate nel trattamento della β-talassemia.
Le molecole in grado di modulare i livelli intracellulari di miR richiedono un veicolo per essere internalizzate dalle cellule. Nella presente tesi è stata valutata una molecola a struttura calixarenica (ML122) e precedentemente impiegata per la trasfezione di DNA. ML122 è stato testato, sia per la veicolazione di molecole elettricamente cariche (antimiRNA e premiRNA), sia per molecole elettricamente neutre (PNA). I dati hanno dimostrato a) un’elevata efficienza di internalizzazione cellulare delle molecole veicolate da ML122, associata a b) evidenti effetti biologici delle molecole veicolate, che una volta internalizzate, sono rilasciate dal complesso formato con ML122 e svolgono il loro ruolo biologico.
Come dimostrato da Chim nel 2008 i miR sono presenti in diversi fluidi biologici, tra cui il plasma, dove risultano particolarmente stabili, rendendoli ottimi marcatori nell’ambito della diagnostica non invasiva. Partendo da questo assunto i cmiRNA sono stati impiegati come marcatori diagnostici in due differenti ambiti.
Nella prima parte i miR sono stati impiegati come marcatori per la diagnosi di carcinoma del colon-retto giungendo alle seguenti conclusioni: a) i miR sono fisiologicamente rilasciati dalle cellule e ogni linea cellulare presenta un proprio profilo di ‘secrezione’ che risulta indipendente dalle concentrazioni intracellulari di miR, b) la comparazione di due diverse metodiche PCR per la quantificazione di miRNA (RTqPCR e ddPCR) ha dimostrato risultati comparabili, nonostante la ddPCR risulti maggiormente performante, specialmente in campioni molto diluiti c) l’analisi di 3 miR selezionati sulla base di analisi riportate in letteratura, in pazienti affetti da CRC e donatori sani ha dimostrato diversi limiti derivanti dall’impiego di un numero limitato di miR suggerendo la necessità di considerare panelli più ampi di miR.
L’analisi di miRNA circolanti è stata inoltre applicata al campo dell’identificazioni di frodi sportive, in particolar modo i miR sono stati proposti come marcatori di autoemotrasfusione. L’analisi di un numero seppur limitato di campioni, con metodica microarray ha permesso di identificare due diverse liste di miR candidati marcatori di autoemotrasfusione. Partendo dall’assunto che durante i 2 processi chiave dell’ABT: prelievo del sangue e reinfusione, generano un’alterazione dei livelli di ossigeno, è stata stilata una prima lista di 8 miR, la cui espressione risulta modulata in risposta alla variazione dei livelli di ossigeno. Traendo vantaggio dai dati di microarray è stata inoltre, proposta una seconda lista di miR, la cui espressione è modulata in seguito alla reinfusione.MiRNAs are a class of small non-coding RNA of about 19-23 nucleotides in length able to act as regulators of gene expression thanks to their ability to bind the 3’UTR which results in inhibition of translation or in mRNA degradation. Due to their short sequence, they can bind more than one transcript, so they may be involved in more than one biological pathway. Since their first identification in 1993, they have been associated to a long list of physiological or pathological conditions. The dysregulation of miRNA profile may be associated to several diseases, so therapies based on the restore of physiological miRNA levels may have huge impact on several pathologies, for this reason molecules able to both increase or decrease miRNA levels have been recently developed. Through miRNAs levels regulation is possible to indirectly regulate their targets levels. This evidence was investigated in this thesis to reduce levels of BCL11A, one of the principal repressor of γ-globin gene. The following key results: a) miR-210 interaction with BCL11A coding region was demonstrated with SPR-based analysis, b) the increase of miR-210 intracellular levels leads to a decrease of BCL11A transcript and protein, encouraged us to consider the employment of miR-210 mimicking molecules as possible therapeutic approach to reduce BCL11A expression in the field of β-thalassemia treatment. Modulation of miRNAs levels into cells can be achieve with different kinds of molecules and most of them generally require an appropriate vehicle. At this propose we investigated with encouraging results a calixarene-based structure compound called ML122, previously used for DNA delivery, to vehicle miRNA-based molecules and PNAs. a) High transfection efficiency, associated with b) evident biological effects obtained when both premiRNA molecules and PNAs are vehicled with ML122, allow us to consider ML122 as a possible alternative to commercial available transfection agents. MiRNAs are present not only into cells but as demonstrated by Chim and colleagues they were found also in several body fluids, including plasma, in which have been demonstrated to be very stable. Several groups employed circulating miRNAs at diagnostic or prognostic propose opening a new important issue in the field of the non-invasive diagnostic techniques. In the present thesis we employed the non-invasive diagnostic technique in two different fields. First, we considered circulating miRNA in colorectal cancer diagnosis and management. In this section, three important massages were obtained a) miRNA are normally released by cells, the release pattern is different in each cell line and often differs from the miRNA pattern into cells, b) a comparison between two different techniques, RTqPCR and ddPCR, demonstrates how the two techniques gave comparable results, even if ddPCR for technical issue is more suitable for miRNA quantification in plasma samples, c) analysis of the three selected miRNAs in CRC patients and healthy controls demonstrates how additional miRNAs (at 6 or 7 miRNAs) are needed to identify patterns associated to CRC patients. The analysis of cmiRNAs was also, not applied to a health problem but to identify an illicit practice in sport such as autologous blood transfusion. The analysis of a limited number of samples using a high-throughput miRNA analysis method, i.e. microarray, allow us to identify two different list of miRNAs possible biomarkers for the detection of ABT: (a) a list of 8 miRNAs which modulation may be related to different oxygen availability immediately after the two key steps of ABT practice i.e. blood withdrawal and blood reinfusion. Moreover, a second list (b) was obtained considering all expressed miRNAs in our plasma samples. Despite the number of analysed samples is limited (6 ABT trained subjects and 3 control pools) preliminary encouraging data were obtained, which of course need to be confirmed increasing the samples size.
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SOKOLOVA, VIKTORIJA. "MICRORNA INVOLVEMENT IN COLON CANCER PROGRESSION." Doctoral thesis, Università degli Studi di Milano, 2012. http://hdl.handle.net/2434/214611.
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Loss of response to TGF-β occurs in many cancers and disruption of its regulatory circuitry appears as a central event in the genesis of colorectal cancer (CRC) malignancy. Lack of an inhibitory response to TGF-β is common to most colon cancer cell lines. Recently, abrogation of the TGF-β growth inhibitory response mediated by different miRNAs has been reported. By searching for miRNAs in regions showing copy number changes and concordant gene expression in 36 sporadic CRCs compared to their normal counterpart, we identified the miR-17-92 cluster localized on the 13q31 locus, gained and highly expressed at early stages of CRC. We selected the TGF-β sensitive FET colon carcinoma and investigated the relationship between enhanced expression of miR-20a and TGF-β mediated growth inhibition. We found that miR-20a affects p21 levels and has a negative and significant effect on the cytostatic response mediated by TGF-β. We confirmed that p21 down-modulation is addressed by direct binding of its 3’-UTR by miR-20a. Moreover, we observed also that miR-20a is able to block the transactivation of the 2.3-kb CDKN1A promoter upon TGF-β stimulation, but not the activation of the Smad3/4-reponsive reporter. We also found that two of the c-MYC repressor genes, E2F5 and KLF11, are directly targeted by miR-20a, thus resulting in abrogation of the TGF-β mediated repression of c-MYC. Our experiments suggest for miR-20a an interference with the TGF-β homeostasis in colon cancer addressing the up-regulation of p21 expression, through mechanisms involving more effectors of the TGF-β cascade.
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Stuchi, Nathália Maciel Maniezzo [UNESP]. "Avaliação da Anexina A1, FPR1, FPR2 e miRNAs em adenocarcinoma gástrico." Universidade Estadual Paulista (UNESP), 2016. http://hdl.handle.net/11449/141940.
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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
Apesar do declínio da incidência, o câncer gástrico ocupa ainda a terceira posição em causa de morte por câncer no mundo, tendo como principal fator de risco a bactéria Helicobacter pylori. Esta bactéria pode levar a uma inflamação persistente através da produção de citocinas pró-inflamatórias e de espécies reativas de oxigênio e nitrogênio, estimulando a proliferação celular bem como outros processos envolvidos na carcinogênese. Ainda envolvidos nestes processos tem sido observada a participação de microRNAs, que exercem papel importante na regulação pós-transcricional, influenciando processos fisiológicos normais da célula bem como aqueles ligados às doenças, como por exemplo o câncer gástrico. Alguns miRNAs podem atuar como oncogenes, genes supressores de tumor e biomarcadores para diversas patologias, podendo alvejar genes relacionados com inflamação e câncer como o gene ANXA1 (Anexina-A1). A Anexina-A1 é uma proteína anti-inflamatória e com ação anti-proliferativa, que se liga à receptores do tipo formil peptídeo como por exemplo FPR1 e FPR2, ambos sabidamente relacionados com a progressão de doenças como o câncer. Desta forma o presente trabalho teve como objetivos avaliar a expressão da proteína Anexina A1 e seus receptores FPR1 e FPR2, bem como avaliar a expressão do RNAm da ANXA1 e de miRNAs que possam modular a expressão desse gene (hsa-mir-27a, hsa-mir-196a e hsa-mir-222) em adenocarcinoma gástrico e correlacionar estes resultados com os aspectos clínico-patológicos. Foram avaliadas 31 amostras de adenocarcinoma gástrico, assim como as regiões metaplásica ou normal adjacentes ao tumor. A quantificação relativa (RQ) do RNAm da ANXA1 e miRNAs foi realizada por PCR quantitativo em tempo real utilizando ensaio TaqMan, e a expressão proteica da AnxA1, FPR1 e FPR2 por imuno-histoquímica e análise densitométrica. Em relação à expressão gênica relativa foram observados o aumento da expressão da ANXA1 nos tumores (RQ=1,374; p<0,001), assim como do miR-196a que apresentou aumento de expressão tanto no tecido metaplásico (RQ= 4,784; p=0,0016) e tumoral (RQ=16,99; p< 0,001) em relação ao tecido normal. O miR-27a não se apresentou diferencialmente expresso nos diferentes tecidos e houve diminuição da expressão do miR-222 (RQ=0,687; p=0,01) no tecido tumoral em relação ao tecido normal. Apenas o miR-196a e a ANXA1 apresentaram correlação significantemente inversa (r= -0,55; p=0,003), e este miRNA foi o único que apresentou associação com o sexo feminino, devido aumento de expressão em mulheres. Quando se comparou a expressão relativa aos parâmetros clínico-patológicos e tumorais não foram encontradas diferenças significativas. Quanto à expressão proteica o FPR2 não apresentou marcação no tecido normal, metaplásico e tumoral. Contudo a AnxA1 e FPR1 apresentaram imunomarcação positiva amplamente distribuída no tecido tumoral e positivamente correlacionados tanto no epitélio (r=0,87; p<0,0001) como estroma (r=0,62; p=0,004). Portanto, nossos resultados sugerem que a ANXA1 é modulada pelo miR-196a em câncer gástrico, e evidenciam que tanto a AnxA1 como o FPR1 também estão envolvidos no processo de carcinogênese gástrica. Tais achados podem abrir possibilidades para futuros estudos sobre novos alvos terapêuticos já que AnxA1 e FPR1 são possíveis alvos farmacológicos, e as terapias baseadas em microRNAs tem sido amplamente pesquisadas.
Gastric cancer still ranks third in cause of cancer death worldwide, despite the decline in its incidence, being Helicobacter pylori the main risk factor for this disease. This bacterium can lead to persistent inflammation via the production of proinflammatory cytokines and reactive oxygen and nitrogen species, stimulating cell proliferation and other processes involved in carcinogenesis. Still involved in this process, it has been observed the participation of miRNAs, which play an important role in post- transcriptional regulation, influencing normal physiological processes of the cell as well as those linked to diseases such as gastric cancer. Some miRNAs can act as oncogenes, tumor suppressor genes and biomarkers for various diseases, can targetting genes linked to inflammation and cancer such as ANXA1 gene (Annexin A1). Annexin A1 is an anti-inflammatory protein with anti-proliferative action that binds to formyl peptide receptors such as, FPR1 and FPR2, both known to be related to the progression of diseases such as cancer. Thus, the present study aimed to evaluate the expression of Annexin A1, FPR1 and FPR2 receptors, and the expression of mRNA ANXA1 and miRNAs (hsa-mir-27a, hsa-mir-196a and hsa-miR 222) in gastric adenocarcinoma, also correlate these results to the clinicopathological features. 31 adenocarcinoma samples were evaluated, as well as normal or metaplastic region adjacent to the tumor. Relative quantification (RQ) of miRNA and mRNA ANXA1 was evaluated by TaqMan assay and protein expression AnxA1, FPR1 and FPR2 by immunohistochemistry and densitometry analysis. Regarding the relative expression the following results were observed, increased expression in tumors of ANXA1 (RQ = 1.374; p < 0.001) and miR- 196a that showed increased expression it he metaplastic tissue (RQ = 4.784; p = 0.0016) and tumor (RQ = 16.99; p < 0.001) compared to normal tissue. The miR- 27a did not appear differentially expressed in different tissues and there was a decrease of miR -222 expression (RQ = 0.687; p = 0.01) in tumor tissue compared to normal tissue. Only the miR-196a and ANXA1 presented a significant inverse correlation (r = -0.55; p = 0.003), and this miRNA was the only one that showed association to female gender, due to increased expression in women when comparing the relative expression to clinicopathological parameters and tumor features. The FPR2 was not expressed in normal, metaplasic and tumor tissue. However, the AnxA1 and FPR1 were widely distributed positive immunostaining in tumor tissue and positively correlated both in the epithelium (r = 0.87 ; p < 0.0001) and stromal (r = 0.62 ; p = 0.004). Thus, our results suggest that ANXA1 is modulated by miR-196a in gastric cancer, and evidencing that both AnxA1 and FPR1 are also involved in gastric carcinogenesis process. These data open the possibility for future studies on new therapeutic targets since AnxA1 and FPR1 are adjustable pharmacological targets, and microRNAs ` therapies have been widely researched.
FAPESP: 2012/15036-8
CNPq: 474776/2013-1
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Bertoni, Natália. "Perfil de expressão de microRNAs circulantes em carcinomas de fígado, pâncreas e vias biliares." Botucatu, 2017. http://hdl.handle.net/11449/148864.
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Orientador: Patricia Pintor dos ReisResumo: Introdução: Os carcinomas hepato-pancreato-biliares (HPB) são carcinomas agressivos, com células progenitoras comuns, sugerindo que mecanismos moleculares de tumorigênese podem ser compartilhados entre estes tumores. Os microRNAs (miRNAs) são importantes reguladores da expressão gênica e têm sido investigados como potenciais biomarcadores diagnósticos, prognósticos e de resposta a tratamento de pacientes com câncer. O objetivo deste estudo foi identificar o perfil de expressão de miRNAs no plasma de pacientes com carcinomas HPB e potenciais processos biológicos envolvidos na tumorigênese destes carcinomas.Pacientes e Métodos: Foram analisadas 12 amostras de plasma, sendo 4 obtidas de pacientes com carcinoma hepatocelular, 4 com adenocarcinoma de pâncreas e 4 com colangiocarcinoma e 10 amostras de plasma de indivíduos saudáveis (grupo de referência). A expressão de miRNAs plasmáticos foi determinada por meio do ensaio TaqMan® Array Human MicroRNA Cards (card A, v3.0). Análises de predição de mRNAs-alvo potencialmente regulados pelos miRNAs identificados e redes de interação miRNAs-mRNAs-alvo foram geradas.Resultados: Dos 42 miRNAs com expressão desregulada no plasma de pacientes com carcinomas HPB comparados ao grupo de indivíduos saudáveis, 28 miRNAs (67%) são compartilhados entre os três subtipos tumorais, sendo: 19 com expressão diminuída e 9 com expressão aumentada. Os mRNAs-alvo preditos dos miRNAs com expressão alterada nos carcinomas HPB estão associados com importa... (Resumo completo, clicar acesso eletrônico abaixo)
Mestre
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Davis, M. P. "Generation of a murine ES cell system deficient in microRNA processing for the identification of microRNA targets." Thesis, University of Cambridge, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598389.
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I have developed a system in mouse embryonic stem (ES) cells to simply and rapidly derive gene lists enriched for miRNA targets. I have disrupted miRNA processing by the targeted insertion of a gene trap cassette into the second allele of Dgcr8 in cell lines that already bear a gene trap within their first allele. This led to a broad reduction of miRNA processing in these cells. As a consequence of the disruption of this locus I was able to identify a number of miRNAs that appear to be processed in DGCR8 independent manner. I proceeded to transfect these cells with Es-cell-expressed miRNA mimics. I used microarrays to identify transcripts that are down regulated as a consequence of the miRNA reintroduction. By comparing transcripts that had been up regulated upon the depletion of Dgcr8 to this set I was able to create miRNA target lists for mmu-miR-25 and mmu-miR-291a-3p. These lists should be enriched for functionally relevant, co-expressed targets, moderated for miRNA mimic over expression and to a large extent devoid of interference from target saturation and combinatorial regulation. The system should also not be susceptible to problems associated with functional redundancy. In total I identified 25 target candidates for miR-291a-3p and 40 candidates for miR-25. Amongst these genes are a number of oncogenes and tumour suppressor genes in addition to genes involved in cell cycle regulation and extra-cellular signal transduction. In conclusion it appears that miRNAs play a fundamental role in the regulation of the ES cell transcriptome and as such are deserving of considerable future research. It is my belief that the method presented in this thesis could contribute significantly to this effort by providing substantial and experimentally derived miRNA candidate target lists upon which to base future hypotheses.
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Hart, Martin [Verfasser], and Friedrich [Akademischer Betreuer] Grässer. "Das komparative MicroRNA-Profil zunehmend maligner Prostatakarzinome und Identifizierung von microRNA-Zielgenen / Martin Hart. Betreuer: Friedrich Grässer." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2014. http://d-nb.info/105372585X/34.
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Kuwabara, Yasuhide. "Increased MicroRNA-1 and MicroRNA-133a Levels in Serum of Patients With Cardiovascular Disease Indicate Myocardial Damage." Kyoto University, 2012. http://hdl.handle.net/2433/157428.
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Shenoy, Shamika. "Exosomal microRNA as a sepsis biomarker : Assessing different volumes of plasma for possible quantification of exosomal microRNA." Thesis, Högskolan i Skövde, Institutionen för biovetenskap, 2019. http://urn.kb.se/resolve?urn=urn:nbn:se:his:diva-17523.
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Sepsis is a medical emergency and it arises from extreme response of the host to an infection. Current diagnosis in sepsis relies on nonspecific clinical signs and culture-based analysis, which is time-consuming. It is critically important for clinicians to follow a protocol to identify sepsis and administer antibiotic therapy without any delay. Sepsis-specific biomarkers are being assessed for early diagnosis and thus improving the outcome of the sepsis patient. Many cellular molecules have been proposed to be sepsis-specific biomarkers. However, these molecules lack specificity and sensitivity. MicroRNA expression in biological fluids, particularly plasma and other tissues is very specific to disease state and are found to be promising diagnostic biomarkers in sepsis. Therefore, it is essential to extract qualitative and sufficient amount of microRNAs from human plasma for the downstream application of two-tailed RT-qPCR method microRNA needed for detection of sepsis patients. The aim of this study was to find optimal volume of plasma required to measure microRNA as sepsis biomarker. The study also included isolating exosomal microRNA from blood samples to check whether blood can be used for extraction. The study was conducted with healthy donor samples and the extraction is performed using Plasma/Serum Exosome Purification (product 58300, Norgen Biotek Corporation, Canada) and RNA Isolation Mini Kit and Total RNA Purification Kit (product 37500, Norgen Biotek Corporation, Canada). The samples were assessed for its quantity and quality by Qubit® and Nanodrop™ technology. Based on the comparison of amount of exosomal microRNA extracted from different plasma volumes, it can be concluded, that increasing volume of plasma may not give higher quantity of microRNA.
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Pers, Yves-Marie. "Effet thérapeutique des cellules souches mésenchymateuses dans l'arthrose : mécanismes et translation clinique." Thesis, Montpellier, 2018. http://www.theses.fr/2018MONTT045.
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Les cellules souches mésenchymateuses (CSM) sont des cellules stromales présentes dans différents types de tissus. En plus de leur capacité à se différencier en plusieurs lignées (chondrocytes, adipocytes et ostéoblastes), les CSM présentent également des propriétés immunosuppressives. Bien que ces mécanismes soient loin d'être entièrement compris, leur capacité immunosuppressive a récemment été démontrée comme étant modulée par des miARN. L'arthrose est la forme la plus courante de maladies articulaires sans traitement curatif et se caractérise principalement par la dégradation du cartilage articulaire, avec des altérations osseuses sous-chondrales et une inflammation synoviale. Les CSM pourraient offrir un potentiel thérapeutique intéressant pour le traitement de l'arthrose.Nos travaux ont montré qu'une injection autologue de CSM d'origine adipeuse (ASC) dans une articulation arthrosique améliore la douleur et les niveaux fonctionnels chez les patients. Nous avons souligné la tolérance immunitaire systémique induite à la suite d'injections intra-articulaires d'ASC. Enfin, nous avons étudié le profil d'expression des miARN des CSM humaines lors de leur stimulation par des cellules mononuclées du sang préalablement activés. Nous avons identifié le miR-29a et le PSAT1 comme de nouveaux candidats pour réguler l'activité immunosuppressive médiée par les CSMMesenchymal Stem Cells (MSCs) are stromal cells present in a number of different tissue types. In addition to their ability to differentiate into multiple lineages (chondrocytes, adipocytes and osteoblasts), MSCs also display immunosuppressive properties. Whilst these mechanisms are far from fully understood, their immunosuppressive capacity has recently been shown to be modulated by miRNAs. OA is the most common form of joint diseases without curative treatment and mainly characterized by the degradation of articular cartilage, with subchondral bone alterations and synovial inflammation. MSC might provide therapeutic potential for treatment of OA.Here, we showed that an autologous injection of adipose-derived MSC (ASC) into an osteoarthritic joint improved pain and function levels in patients. We underscored the systemic immune tolerance induced following intra-articular injections of ASCs. Finally, we investigated the miRNA expression profile of human MSCs upon their stimulation by peripheral blood mononuclear cells. We identified miR-29a and PSAT1 as new candidates to regulate immunosuppressive activity mediated by MSCs