To see the other types of publications on this topic, follow the link: Micro RNAs.
Dissertations / Theses on the topic 'Micro RNAs'
Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles
Consult the top 50 dissertations / theses for your research on the topic 'Micro RNAs.'
Next to every source in the list of references, there is an 'Add to bibliography' button. Press on it, and we will generate automatically the bibliographic reference to the chosen work in the citation style you need: APA, MLA, Harvard, Chicago, Vancouver, etc.
You can also download the full text of the academic publication as pdf and read online its abstract whenever available in the metadata.
Browse dissertations / theses on a wide variety of disciplines and organise your bibliography correctly.
1
Amaral, Murilo Sena. "Identificação de RNAs longos não-codificadores de proteínas regulados por micro-RNAs." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/46/46131/tde-22102014-102412/.
Full textAbstract:
Estudos recentes têm revelado que a maior parte dos transcritos gerados em células humanas é composta por RNAs não-codificadores de proteínas (ncRNAs). Uma parte desses ncRNAs compreende a classe de RNAs curtos, que possuem menos que 200 nucleotídeos. Os micro-RNAs (miRNAs) fazem parte dessa classe e têm sido alvo de grande interesse, pois são preditos como possíveis reguladores de mais de 60% dos RNAs mensageiros (mRNAs) humanos. Outra classe dos ncRNAs é composta por ncRNAs longos (lncRNAs, com mais de 200 nucleotídeos), que são transcritos a partir de regiões intergênicas e intrônicas do genoma humano e possuem várias funções, muitas delas relacionadas ao controle da expressão de mRNAs. Recentemente, os lncRNAs têm sido caracterizados quanto à sua estrutura e função. No entanto, muito pouco se sabe sobre os mecanismos pelos quais os lncRNAs são regulados. Este trabalho teve como objetivo avaliar se lncRNAs são regulados por miRNAs em células humanas. Para tanto, identificamos lncRNAs ligados ao complexo de silenciamento induzido por RNA (RISC) em células da linhagem HeLa, utilizando um método aqui desenvolvido de geração de bibliotecas de cDNA direcionadas para sequenciamento em larga escala na plataforma 454/Roche. Em paralelo, sequenciamos os miRNAs ligados ao RISC nestas mesmas células. Os resultados obtidos mostram que centenas de lncRNAs de diversas classes se ligam ao RISC em células HeLa, juntamente com milhares de mRNAs e várias centenas de miRNAs. Entre os miRNAs, encontramos 37 que são preditos como alvejando os lncRNAs detectados. Estes miRNAs constituem possíveis reguladores dos lncRNAs e, portanto, nosso trabalho estabelece um mapa experimental de interações diretas entre lncRNAs e miRNAs. Dentre os lncRNAs identificados ligados ao RISC neste trabalho, destaca-se o TUG1, lincRNA sabidamente envolvido na regulação de genes relacionados à apoptose e ao ciclo celular. Mostramos por ensaio de super-expressão de miRNAs e qPCR que TUG1 é regulado pelo miRNA-148b, um dos miRNAs por nós detectados que possui um sítio alvo altamente conservado em mamíferos localizado na extremidade 3\' de TUG1. Em conjunto, este trabalho contribui para o entendimento da regulação dos níveis de expressão de lncRNAs em células humanas e abre perspectivas para a modulação de miRNAs como estratégia de regulação dos níveis e das funções de lncRNAs.Recent studies have revealed that the largest fraction of the transcripts generated in human cells is composed of non-protein coding RNAs (ncRNAs). A portion of these RNAs encompasses the class of short RNAs, which are less than 200 nucleotides in length. Micro-RNAs (miRNAs) are part of this class and are of great interest, as they are predicted to target over 60% of the human messenger RNAs (mRNAs). Another class of ncRNAs is composed of long ncRNAs (lncRNAs, longer than 200 nucleotides), which are transcribed from intergenic and intronic regions of the human genome and have several functions, many of them related to the control of the mRNA expression. Recently, the structure and function of lncRNAs have been characterized. However, little is known about the mechanisms involved in lncRNA regulation. This work aimed to evaluate whether lncRNAs are regulated by miRNAs in human cells. For this purpose, we identified lncRNAs bound to the RNA-induced silencing complex (RISC) in HeLa cells using a method developed here for the generation of strand-specific cDNA libraries for large scale RNA-sequencing in the 454/Roche plataform. In parallel, we sequenced the miRNAs bound to RISC in these cells. Our results show that hundreds of lncRNAs from diverse classes are bound to RISC in HeLa cells, along with thousands of mRNAs and several hundred miRNAs. Among the miRNAs we identified 37 that are predicted to target the detected lncRNAs. These miRNAs are possible regulators of the lncRNAs, and therefore our work establishes an experimental map of direct interactions between lncRNAs and miRNAs. The lncRNA TUG1, a lincRNA involved in the regulation of genes related to apoptosis and cell cycle, was identified among the lncRNAs bound to RISC. We showed by miRNA over-expression and qPCR that TUG-1 is regulated by the miRNA-148b, which is one of the miRNAs detected in our sequencings and has a binding site highly conserved in mammals located at the TUG1 3` end. Taken together, our results contribute to the understanding of the regulation of the lncRNA expression levels in human cells and open perspectives for the modulation of miRNAs as a strategy to regulate the levels and functions of lncRNAs.
2
Majem, Cavaller Blanca. "Micro-RNAs in ovarian cancer as tools for diagnosis and therapy." Doctoral thesis, Universitat Autònoma de Barcelona, 2017. http://hdl.handle.net/10803/458601.
Full textAbstract:
El càncer d'ovari (CO) és la cinquena causa del càncer en dones i la principal causa de mort entre les malalties ginecològiques. Els símptomes inespecífics i les eines diagnòstiques actualment insuficients no detecten la malaltia en estadis inicials, quan la supervivència és del 90%. Així, al voltant del 70% dels pacients es diagnostiquen en estadis avançats, quan la supervivència és <25%. Mentre que el 80% de les pacients són inicialment quimiosensibles, el 85% d'elles desenvolupen resistència i moren per recurrència. Identificar nous biomarcadors de diagnòstic podria millorar la detecció precoç del CO. A més, desenvolupar noves estratègies terapèutiques eficients és primordial. Els micro-ARNs (miARNs) són ARN no codificants de 22nt que regulen múltiples processos silenciant ARNm diana, i s'han trobat alterats en càncer, essent possibles elements terapèutics. A més, són estables en circulació i, podrien ser eines diagnòstiques no invasives, mitjançant la saliva com a font de biomarcadors. Primer objectiu: identificar nous biomarcadors de diagnòstic per al carcinoma serós d'alt grau (HGSC). Així, 32 salives de pacients en estadis inicials i avançats de HGSC i controls van ser sotmeses a seqüenciació d'ARN. Aproximadament, es va obtenir 100 ng d'ARN per mL de saliva, amb qualitat suficient per a la seqüenciació d'ARN. Les anàlisis bioinformàtiques van mostrar al voltant del 36% de l'alineació amb el genoma humà, donant lloc a la detecció de més de 500 miARNs coneguts. L'anàlisi d'expressió diferencial va mostrar que 49 i 45 miARNs es trobàven alterats significativament en salives de les pacients amb HGSC d’estadi inicial i avançat en comparació amb els controls, respectivament. Curiosament, la família miR-34 apareix comunment alterada, amb tres membres de la família sobreexpressats en les salives de pacients amb HGSC, tant en estadis inicials com avançats, suggerint que podrien ser biomarcadors per millorar la detecció precoç de les pacients amb HGSC. Segon objectiu: identificar noves teràpies basades en miARNs pel CO avançat, ja que les teràpies dirigides s'han convertit en el Sant Grial pel tractament del càncer. Aquí, el miR-654 es va trobar significativament disminuit en teixits de CO comparat amb ovaris benignes. La sobreexpressió d'aquest miARN en línies cel·lulars de CO va disminuir significativament la proliferació cel·lular i va augmentar la mort cel·lular per apoptosis in vitro, acompanyada d'una activació de l’apoptosis a nivell molecular. Cal destacar que l'expressió ectòpica del miR-654 reproduïa in vivo les conseqüències fenotípiques descrites. A més, mitjançant un model preclínic amb cèl·lules derivades d’ascitis de pacients amb HGSC, s’ha demostrat que la sobreexpressió del miR-654 disminuïa la capacitat de formació d’esferes i la seva viabilitat. Mitjançant una anàlisi bioinformàtica dels possibles mRNAs diana del miR-654 es van predir diversos gens, entre els quals HAX1, RAB1B, PBX3, CDCP1 i PLAGL2 van disminuir a nivell de mRNA i proteïna amb la sobrexpressió del miR-654. L’assaig luciferasa va confirmar que el mirR-654 s’uneix directament al 3’UTR dels 5 ARNs diana esmentats. El silenciament d’aquests gens va mostrar que la depleció de CDCP1 i PLAGL2 fenocopia els efectes de la sobreexpressió del miR-654. Curiosament, els nivells de proteina d’ambdós gens estaven reduïts amb la sobreexpressió del miR-654 en el model preclínic utilitzant cèl·lules d’ascitis de pacients, suggerint que l'efecte terapèutic del miR-654 podria ser, en part, conseqüència de la inhibició de CDCP1 I PLAGL2. Finalment, l'anàlisi de microarrays va demostrar que la depleció de CDCP1 i PLAGL2 alterava les vies MYC, Wnt/β-cat, AKT i MAPK. En conjunt, s’ha suggerit que l'expressió ectòpica de miR-654 podria alterar vies importants en OC i que l'ús d'aquest miARN com a eina terapèutica podria millorar les teràpies actuals, potencialment en combinació amb la quimioteràpia estàndard.Ovarian cancer (OC) is the fifth cause of cancer in women and the leading cause of death among gynecological malignancies in developed countries. The unspecific symptoms and currently insufficient diagnostic tools fail to detect the disease at an early stage, when the 5-years survival is 90%. Thus, around 70% of the patients are diagnosed at late stage, when the 5-years survival is <25%. Also, while 80% of the patients are initially chemosensitive, 85% of these develop resistance and die of recurrence. Therefore, identifying new diagnostic biomarkers would potentially improve the early detection of OC. Also, developing novel and efficient therapeutic strategies is paramount. Micro-RNAs (miRNAs) are small non-coding RNAs of 22nt that regulate multiple cellular processes by silencing of the specific target mRNAs, and have been found deregulated in cancer, being potential therapeutic elements. In addition, they are stable in circulation therefore being potential non-invasive diagnostic tools by using saliva as source of biomarkers. The first objective was to identify new miRNA diagnostic biomarkers for high-grade serous carcinoma (HGSC). Thus, 32 salivas were subjected for RNA-sequencing, in particular from early- and late-stage HGSC and control patients. Around 100 ng of RNA was found per 1 mL of saliva from control and HGSC patients, which was of sufficient quality for RNA sequencing. First bioinformatic analyses showed around 36% of alignment with the human genome, thereby resulting in a more than 500 known miRNAs and 65 De Novo miRNAs detected on average in the patients’ cohort. Differential expression analysis showed that 49 and 45 miRNAs were significantly deregulated in salivas from early- and late-stage HGSC patients compared to controls, respectively. Interestingly, miR-34 family appeared commonly altered, with three members of the family overexpressed in saliva from HGSC patients, either from early and late-stages, suggesting that they could be potential biomarkers to improve the early detection of HGSC patients, the most fatal subtype of OC. The second objective was to identify new miRNA-based therapies for late-stage OC, since targeted therapies has became the Holy Grail for cancer therapy. In this study, miR-654 was found significantly under-expressed in OC tissues compared to benign ovaries. Overexpression of this miRNA in clinically relevant OC cell lines resulted in a significant decrease in cell proliferation and marked increased apoptotic cell death in vitro, accompanied by an activation of the apoptotic pathway seen at cellular and molecular level. Importantly, ectopic expression of miR-654 reproduced the described phenotypic consequences in vivo. In addition, a pre-clinical model using 4 patient-derived ascitic cells showed that overexpression of miR-654 decreased the sphere forming capacity and reduced spheroid viability. In silico bioinformatics analysis of putative miR-654 targets predicted several genes, among which HAX1, RAB1B, PBX3, CDCP1 and PLAGL2 decreased at mRNA and protein level. A 3’UTR luciferase reporter assay confirmed that miR-654 is a direct of the 5 targets abovementioned. Additionally, silencing of the direct target genes showed that CDCP1 and PLAGL2 depletion phenocopied the effects of miR-654 overexpression, thereby resulting in a reduced proliferation and in an increased apoptosis. Interestingly, both genes were diminished at protein level upon miR-654-5p in the pre-clinical model using patient-derived ascitic cells, suggesting that the therapeutic effect of the miR-654 could be, in part, due to the inhibition of CDCP1 and PLAGL2. Finally, microarray analysis showed that the depletion of CDCP1 and PLALG2 altered MYC, Wnt/β-cat, AKT and MAPK pathways, which has been confirmed by the overexpression of miR-654. Altogether suggested that ectopic expression of miR-654 impaired canonical pathways in OC and that the use of this miRNA as a therapeutic tool might improve the current therapies, potentially in combination with the standard chemotherapy.
3
Zanca, Almir Samuel. "Identificação de micro RNAs em cana-de-açucar." [s.n.], 2009. http://repositorio.unicamp.br/jspui/handle/REPOSIP/314782.
Full textAbstract:
Orientadores: Michel Georges Albert Vincentz, Fabio Tebaldi Silveira NogueiraDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
Made available in DSpace on 2018-08-13T11:23:48Z (GMT). No. of bitstreams: 1 Zanca_AlmirSamuel_M.pdf: 11034884 bytes, checksum: 0545b58df6802e07009aef761dda3003 (MD5) Previous issue date: 2009
Resumo: RNAs não-codificantes de 20-27 nucleotídeos (nt) regulam transcricionalmente ou pós-transcricionalmente a expressão de genes endógenos, modelando o transcriptoma e a produção de proteínas. Dentre estes, microRNAs (miRNAs) desempenliam papel chave no desenvolvimento vegetal, observação comprovada pela avaliação fenotípica e molecular de plantas transgênicas e de mutantes defectivos na produção de tais RNAs. MiRNAs são produzidos a partir de precursores longos (pri-miRNAs), os quais são posteriormente processados por enzimas específicas, gerando o miRNA maduro (20-22 nt). O miRNA maduro, por sua vez, guia a clivagem do mRNA de genes-alvo e bloqueia a tradução de proteínas, afetando diversos aspectos do desenvolvimento. O sequênciamento de populações de RNAs regulatórios possibilitou a identificação de miRNAs conservados e específicos em diferentes espécies vegetais, embora estudos em plantas de importância econômica sejam ainda incipientes. Atualmente, existem diversos bancos públicos de sequências ESTs disponíveis. Esses bancos possuem um grande número de sequências não-codíficantes, dentre as quais podem estar presentes pri-miRNAs, os quais são também são moléculas poliadeniladas similares a mRNAs codifícantes. O banco público de ESTs de cana-de-açúcar TIGR Gene Index foi usado como base para uma busca de miRNAs. O processo criado possibilitou a identificação de 20 precursores de miRNAs, agrupados em 15 famílias distintas. No presente trabalho desenvolveu-se também ferramenta para predição de potenciais alvos para os miRNAs encontrados. As famílias de miRNAs de cana-de-áçucar e a ferramenta de predição de genes-alvo estão integrados em banco de dados que estará disponível brevemente. Análise de expressão gênica demonstrou que precursors de miRNAs de cana-de-açúcar acumulam em níveis variáveis em distintos tecidos/órgãos. Além disso, tanto o acúmulo do miRNA maduro quanto a degradação do mRNA-alvo foram avaliados para alguns casos estudados. A caracterização de um miRNA específico de monocotiledôneas (miR528) e a confirmação de seu alvo, um gene comum em angiospermas, predito pela primeira vez neste trabalho, gera um interessante questionamento sobre a regulação desse gene via miRNA apenas em monocotiledôneas
Abstract: No-coding RNAs of 20-27 nucleotides (nt) transcriptional or posttranscriptionally regulate endogenous gene expression, affecting the cellular output of transcripts and proteins. Among these RNAs, microRNAs (miRNAs) play an important role in plant development as confirmed by phenotypic and molecular evaluation of transgenic plants and knockout mutants defective in miRNA biogenesis and function. miRNAs are produced from long precursors (pri-miRNAs), which are processed by specific enzymes into the mature miRNA (20-22 nt). The mature miRNA guides the cleavage of target genes as well as impairs protein translation, affecting several development processes. Deep sequencing of small RNAs identified conserved and species-specific miRNAs. Nevertheless, studies on crops are still in their infancy. Public ESTs databases are an important source of no-coding sequences, in which we can find miRNAs precursors, which are polyadenilated RNAs as messenger RNAs. In this work, the public sugarcane EST database TIGR Gene Index was used to search conserved miRNAs. The pipeline developed in this work made possible the identification of 20 miRNAs precursors, grouped into 15 families. It was also developed a search tool for potential miRNAs targets. Sugarcane miRNAs precursors displayed tissue/organ differential expression profiles. Moreover, a new identified miRNA target was confirmed experimentally. This new target is regulated by a monocot specific miRNA, miR528. Interestingly, this miRNA target is conserved in eudicots and monocots, even though its regulation by miRNA is not. This finding raises the question of why this gene has evolved in having a miRNA-mediated posttranscriptional regulation only in monocots
Mestrado
Bioquimica
Mestre em Biologia Funcional e Molecular
4
Mead, Edward. "Discovery, Characterization, and Functional Analysis of micro RNAs in Culicidae." Diss., Virginia Tech, 2009. http://hdl.handle.net/10919/77433.
Full textAbstract:
MicroRNAs (miRNAs) are non-coding RNAs that often play a fundamental role in gene regulation. Currently, hundreds to over a thousand miRNAs are predicted to be present in many eukaryote species, with many to be discovered; the functions of most are unknown. While much attention has gone towards model organisms, a much greater depth of understanding remains to be gained for the miRNAs of many organisms directly important to humans. There are few verified miRNAs for any mosquito species, despite the role of mosquitoes in many of humanity’s worst diseases. Anopheles gambiae and Aedes aegypti, carriers of malaria and dengue, respectively, are responsible for over a million deaths a year. To date, there are sixty-six microRNAs in An. gambiae in miRBase, a central repository for miRNA sequences. Many of these are based on homology to primarily Drosophila miRNAs. While sequence conservation suggests an important function for these miRNAs, expression has not been experimentally verified for most mosquito miRNAs.
Using small RNA cloning and northern blots, I discovered and analyzed 27 different microRNAs in aged female An. stephensi mosquitoes, the age group responsible for transmission of malarial parasites. Three of these miRNAs are only found in mosquitoes (miR-1889, -1890, and –1891). Cloning and northern analysis revealed an abundance of a miRNA that is linked to longevity in flies, miR-14, across different life stages of mosquitoes. It was also shown that miR-989 was expressed almost exclusively in the adult ovary and its expression fluctuated in response to bloodfeeding, suggesting a possible role in reproduction, an area of great importance to controlling mosquito populations.
Building upon the above cloning experiment, a later high-throughput sequencing effort uncovered 98 miRNA precursors from Ae. aegypti. There are a total of 13 novel miRNAs that have not been found in other organisms by bioinformatic predictions or experiments. These “mosquito-specific” miRNAs may play a role in processes such as blood-feeding or vector-host interactions. A detailed examination of the expression of eight of these miRNAs was conducted in An. gambiae, An. stephensi, Ae. aegypti, and T. amboinensis to determine their expression profile, conservation, and provide hints to their function. My work revealed conserved and sometime stage-specific expression profiles of some of the mosquito-specific miRNAs. I also provided evidence for three lineage-specific miRNAs that may shed light on the divergence of different mosquito lineages.
Extending the finding that miR-989 may be involved in mosquito reproduction, we conducted a detailed analysis of its evolution, expression, possible targets and regulation. miR-989 is conserved in holometabolous insects. miR-989 expression in female An. stephensi and Ae. aegypti dramatically rises following pupal emergence until strong signal is observed, until a blood meal is taken. Expression remains quite strong then begins a steep decline in expression at 32-40 hours post blood meal (PBM), and even by 96 hours PBM, remains weak. Bioinformatic predictions of miR-989 targets coupled with a PCR-based approach uncovered three potential target leads, though preliminary results were artifacts. Although the miR-989 post-emergence expression profile correlates with the expression of Juvenile Hormone, a key reproductive hormone in mosquitoes, no observable induction occurred when abdominal ligation samples were administered methoprene, a JH analog. However, methoprene impacted a number of other miRNAs, with up to a 3.87 fold induction (miR-1891), and a 3.15 fold suppression (miR-9a) of signal. Subsequent northern analysis provided visual confirmation of observable fold changes for miR-1891 and miR-9a, but not for miRNAs that showed changes below two fold. This analysis provides a foundation to study Juvenile Hormone regulation of miRNAs in mosquitoes. In summary, we have expanded the understanding of microRNAs in mosquitoes. An improved understanding of mosquito physiology can assist in efforts to control mosquito-borne infectious diseases.Ph. D.
5
Franklin, Oskar. "Stromal components and micro-RNAs as biomarkers in pancreatic cancer." Doctoral thesis, Umeå universitet, Kirurgi, 2016. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-128000.
Full textAbstract:
Background Pancreatic ductal adenocarcinoma (PDAC) patients have the poorest 5-year survival rates of all cancer forms. It is difficult to diagnose at early disease stages, tumour relapse after surgery is common, and current chemotherapies are ineffective. Carbohydrate antigen 19-9 (Ca 19-9), the only clinically implemented PDAC biomarker, is insufficient for diagnostic and screening purposes. PDAC tumours are characterised by a voluminous stroma that is rich in extracellular matrix (ECM) molecules such as collagens, hyaluronan (HA) and matricellular proteins. These stromal components have been suggested to promote PDAC cell migration, proliferation, evasion of apoptosis and chemotherapy resistance. Those events are mediated via interactions with adhesion receptors, such as integrins and CD44 receptors expressed on cancer cell surfaces. Micro-RNAs (miRNA) post-transcriptionally regulate gene expression in health and disease. At the time of PDAC diagnosis, miRNA levels are altered both in plasma and tumour tissue. Before PDAC diagnosis, tissue miRNA levels are altered in precursor lesions, raising the possibility that plasma miRNAs might aid in early detection. In this thesis, it is hypothesised that stromal components and miRNAs can serve as tissue or blood based biomarkers in PDAC. The aims are: (1) to characterise the expression of stromal components and their receptors in normal and cancerous tissue; (2) to find potential stroma-associated tissue and blood-based biomarkers for diagnosis and prognosis estimates; (3) to determine the cellular effects of type IV collagen (Col IV) in PDAC; (4) to determine if plasma miRNAs that are altered in manifest PDAC can be used to diagnose PDAC earlier. Methods The expression patterns of Col IV, Col IV-binding integrin subunits (α1, α2, β1), Endostatin, Osteopontin (OPN) and Tenascin C (TNC) were analysed in frozen PDAC and normal pancreatic tissue. A tissue microarray (TMA) was constructed using formalin-fixed, paraffin-embedded primary tumours and lymph node metastases. The TMA was used to study the expression levels and associations with survival of the standard CD44 receptor (CD44s), its variant isoform 6 (CD44v6), HA, OPN and Col IV. Circulating levels of HA, Col IV, Endostatin, OPN and TNC were measured in PDAC patients and healthy individuals, and compared with conventional tumour markers (Ca 19-9, CEA, Ca 125 and TPS). The functional roles of Col IV were studied in PDAC cell lines by: (1) growth on different matrices (2) blocking Col IV binding integrin subunits, (3) blocking the Col IV domains 7s, CB3 and NC1, and (4) by down regulation of PDAC cell synthesis of Col IV using siRNA transfection. Plasma miRNAs alterations were screened for in samples from patients with manifest disease, using real-time quantitative PCR (RT-qPCR). To find early miRNA alterations, levels of those miRNAs that were altered at diagnosis were measured in prediagnostic plasma samples. Results High tissue expression of both the standard CD44 receptor (CD44s) and its variant isoform CD44v6 as well as low expression of stromal OPN were associated with poor survival. In addition, high CD44s and low OPN predicted poor survival independent of established prognostic factors. Circulating Col IV, Endostatin, OPN, TNC and HA were increased in preoperative samples from PDAC patients. Preoperatively, higher levels of serum-HA and plasma-Endostatin were associated with shorter survival. Postoperatively, higher levels of Col IV, Endostatin and OPN were associated with shorter survival. On the contrary, only one of the conventional tumour markers was associated with survival (Ca 125). Col IV stimulated PDAC cell proliferation and migration and inhibited apoptosis in vitro, dependent on the collagenous domain (CB3) of Col IV and the Col IV binding integrin subunit β1. Reduced endogenous Col IV synthesis inhibited these effects, suggesting that PDAC cells synthesise Col IV to stimulate tumour-promoting events via a newly discovered autocrine loop. 15 miRNAs were altered in early stage PDAC patients and the combination of these markers outperformed Ca 19-9 in discriminating patients from healthy individuals. However, none of the miRNAs were altered in prediagnostic samples, suggesting that plasma miRNA alterations appear late in the disease course. Conclusions Up regulated stromal components in PDAC tumours are detectable in blood samples and are potential diagnostic and prognostic biomarkers in PDAC. High circulating levels of Col IV, Endostatin, OPN and HA predict poor survival, as well as high expression of CD44s and CD44v6 and low expression of OPN in tumour tissue. PDAC cells synthesise Col IV, which forms BM-like structures close to cancer cells and promote tumour progression in vitro via an autocrine loop. Several plasma-miRNAs are altered in PDAC, but are not useful for early discovery.
6
Sharma, Kanika. "Identification of micro-RNAs and their messenger RNA targets in Prostate cancer and Biological fluids." VCU Scholars Compass, 2014. http://scholarscompass.vcu.edu/etd/3551.
Full textAbstract:
Prostate cancer is the second most common cancer in the United States that affects men today. To better treat this disease accurate biomarkers and successful therapeutic treatments are needed. A novel approach to understand the mechanisms behind prostate cancer tumor formation lies in identifying dysregulated micro-RNAs (miRNAs), which are a class of small (18-24 nucleotides) non-coding RNAs that regulate gene expression posttranscriptionally by either inhibiting protein synthesis or signaling messenger-RNA for degradation. Multiple miRNAs were discovered in our highly tumorigenic and metastatic prostate cancer progression model M12 cell line compared to its weakly tumorigenic P69 parental cell line. Various analyses such as human panel analyses, single-miR analyses and patient tumor biopsy samples were analyzed to determine dysregulated miRNAs that contributed to the progression and metastasis of prostate cancer. Together with performing experiments to identify miRNAs, a de novo next generation sequencing approach was applied to identify miRNAs naturally present in biological fluids of normal and healthy subjects. Since, these miRNAs are highly dysregulated in many diseases, including cancer, they can act as potential biomarkers or therapeutic targets to improve treatments for prostate cancer. Essential miRNAs studied for this research were miR-17-3p that is known to target the ErbB2 mRNA; miR-299-5p that directly targets osteopontin (OPN) mRNA, and miR-147b that directly targets many mRNAs, such as COL4A2, ALDH5A1, NDUFA4, SDHD, and IER5. A wide range of miRNAs were identified in six biological fluids: venous blood, menstrual blood, vaginal fluid, semen, saliva, and feces. There were some miRNAs that were common to all 6 body fluids, some unique to each body fluid, and some miRNAs that literature suggested could potentially be biomarkers or normalizers for body fluid characterization.
7
Júnior, Nelson Gaspar Dip. "Análise de expressão de micro RNA em carcinoma urotelial de bexiga." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/5/5153/tde-10102012-100047/.
Full textAbstract:
Introdução: O câncer de bexiga é a segunda neoplasia maligna mais frequente do trato urinário, com 386.000 casos estimados e 150.000 mortes para 2011 no mundo. Noventa e cinco por cento são carcinomas uroteliais (CUB) papilíferos não músculo-invasivos de baixo grau, que apresentam altas taxas de recidiva, mas raramente progridem. Tumores invasivos de alto grau representam 10-20% dos diagnósticos, são altamente agressivos levando à mortalidade elevada. O conhecimento das vias moleculares envolvidas na carcinogênese dessa neoplasia é importante para a identificação de novos marcadores para diagnóstico, acompanhamento, prognóstico e desenvolvimento de novas terapias alvo. Micro RNA (miRNA) são pequenas sequências não codificantes de RNA que regulam a expressão dos genes inibindo a tradução da proteína ou promovendo a degradação do RNA mensageiro, estando atualmente envolvidos em vários processos celulares fisiológicos e patológicos, incluindo o câncer. Objetivos: Caracterizar o perfil de expressão de miRNA no CUB, relacionando-o com os parâmetros prognósticos clássicos para a doença: grau histológico e estadiamento. Além disso, relacionar esse padrão de comportamento dos miRNA com a recidiva tumoral e sobrevida câncer-específica em pacientes tratados cirurgicamente para CUB. Material e Métodos: Catorze miRNA (miR-100, miR-10a, miR-21, miR-205, miR-let7c, miR- 125b, miR-143, miR-145, miR-221, miR-223, miR-15a, miR-16-1, miR-199a e miR- 452) foram isolados de espécimes cirúrgicos de 60 pacientes divididos em 2 grupos: 30 pacientes com CUB não invasivo (pTa) de baixo grau submetidos à RTU de bexiga, 30 com CUB invasivo (pT2-3) de alto grau submetidos à cistectomia radical. O grupo controle é representado por cinco pacientes portadores de bexiga normal sem CUB que realizaram tratamento cirúrgico aberto para tratamento da hiperplasia prostática benigna (HPB). O processamento dos miRNA envolveu três fases: (1) extração do miRNA com kit específico, (2) geração do DNA complementar e (3) amplificação do miRNA por PCR quantitativo em tempo real (qRT-PCR). A expressão de cada miRNA foi obtida através do cálculo 2- CT e os RNU-43 e RNU-48 foram utilizados como controles endógenos. Testes estatísticos foram aplicados para estudar as variáveis envolvidas e curvas de Kaplan-Meyer foram usadas para avaliar a sobrevida livre de recidiva (SLR) e sobrevida câncer-específica (SCE). Resultados: Dos 14 miRNA estudados a maioria apresentou subexpressão nos dois grupos de tumor analisados, com exceção do miR-10a para o grupo pTa de baixo grau e do miR-100, 21 e 205 para os tumores pT2/pT3 de alto grau, onde demonstraram-se superexpressos. Essas diferenças de expressão de miRNA entre os dois grupos foram estatisticamente. Quando estudamos a relação entre expressão de miRNA e a evolução dos pacientes através de curvas de sobrevida, observamos que maiores níveis de expressão do miR-21 relacionou-se com menor SLR para tumores pTa. Ainda, maiores concentrações de miR-10a e miR-145 se associaram com menor SLR e maiores níveis de miR-10a com menor SCE para tumores pT2-3. Conclusões: Demonstramos um predomínio de subexpressão de miRNA em xv carcinomas de bexiga. Os miR-100, miR-10a, miR-21 e miR-205 demonstraram diferenças no perfil de expressão para grau e estadiamento dentro dos dois grupos de tumor, sendo capazes de diferenciá-los. Maiores níveis de miR-21 se relacionaram com menor SLR para tumores pTa de baixo grau, enquanto maiores concentrações de miR-10a estiveram associadas com menor SLR e SCE para tumores pT2/pT3 de alto grauIntroduction: Bladder cancer (BC) is the second most common malignancy of the urinary tract, with 386,000 cases estimated and 150,000 deaths in 2011. Urothelial carcinomas (UC) represent 95% of BC cases, and knowledge of the molecular pathways associated with BC carcinogenesis is crucial to identify new diagnostic and prognostic biomarkers, and development of new target molecular therapies. MicroRNAs (miRNAs) are short non-coding RNA molecules that play important roles in the regulation of gene expression by acting directly on mRNAs, leading to either mRNA degradation or inhibition of translation, involved in many physiological and pathological processes, including cancer. Objectives: To characterize miRNAs expression profiles in UC, associating with classic prognostic factors: grade and stage. Moreover, correlate miRNA expression with tumor recurrence and survival. Material and Methods: Fourteen miRNAs (miR-100, miR-10a, miR-21, miR-205, miR-let7c, miR-125b, miR-143, miR-145, miR-221, miR-223, miR-15a, miR-16-1, miR- 199a e miR-452) were isolated from surgical specimens from 60 patients classified in two groups: 30 patients with low-grade non-invasive pTa UC that underwent TURB, 30 with high-grade invasive pT2/pT3 UC underwent radical cystectomy. The control group consists in five normal bladder tissue taken from patients that underwent retropubic prostatectomy to treat benign prostatic hyperplasia (BPH). miRNA processing involved three phases: (1) miRNA extraction by specific kits, (2) cDNA generation (3) miRNA amplification through qRT-PCR. Expression profiles were obtained by relative quantification determined by 2-ct method. Endogenous control were RNU-43 and RNU-48. Statistic tests were used to study the prognostic variables and Kaplan-Meyer curves were constructed to analyze disease-free (DFS) and disease-specific (DSS) survivals. Results: All miRNAs were underexpressed in both groups, except miR-10a in pTa and miR-100, 21 and 205 in pT2/pT3 tumors, that where over-expressed. miR-100, miR-21, miR-10a and miR-205 differentialy expressed in both groups and this differences were statistically significant. The Kaplan-Meyer survival curves showed that higher levels of miR-21 were related to shorter DFS for pTa group. Also, higher levels of miR-10a and miR-145 were associated with shorter DFS and higher levels of miR-10a were also related to shorter DSS in pT2/pT3 group. Conclusions: The majority of miRNA were shown to be underexpressed in bladder UC. miR-100, miR-10a, miR-21 and miR-205 were differentially expressed considering tumor grade and stage. The miRNA profile was able to distinguish pTa low grade and pT2-3 high grade tumors. Higher levels of miR- 21 were related to shorter DFS in pTa, while higher levels of miR-10a were associated with shorter DFS and DSS in pT2-3, high grade UC
8
Hayman, Melissa Anne. "Genomic influences on platelet function." Thesis, Queen Mary, University of London, 2018. http://qmro.qmul.ac.uk/xmlui/handle/123456789/36221.
Full textAbstract:
The study of platelet messenger and micro-RNAs is of increasing interest owing to the fact that platelets contain the machinery to splice and translate mRNA into proteins in response to inhibitory or activating signals. However, the relatively small size (roughly 4000-5000 transcripts) and short half-life of the platelet transcriptome makes this a technically challenging aspect of platelet biology to investigate. The aims of these thesis investigations were therefore to optimise protocols for the isolation of platelets for downstream RNA analyses and function testing, to investigate the functional capabilities of platelet subpopulations rich in RNA, and to understand the functional and transcriptomic impact of gene mutations predicted to influence platelet function. I found that the optimal method for isolating platelets from whole blood is to use simple single step centrifugation to obtain platelet rich plasma. This method is as effective as more involved methods at reducing white blood cell contamination whilst causing minimal platelet activation. Using this method in combination with flow cytometric cell sorting techniques I was able to isolate the newly formed reticulated platelet sub-population and to confirm the link between reticulation status and increased RNA content. Furthermore, using a range of platelet function assays I demonstrated that reticulated platelets are more reactive than non-reticulated platelets. By obtaining blood samples from a patient with a PLA2G4A mutation I was able to show that loss of cPLA2α enzymatic activity alters both platelet function and the expression of certain mRNA transcripts. My investigations using samples from a range of patients with bleeding tendencies show the benefit of combining deep platelet phenotyping with next generation sequencing to understand the causation of bleeding disorders. Together these investigations highlight the utility of genomic DNA and platelet specific mRNA studies in providing novel insights in to pathways regulating platelet reactivity.
9
Dinh, Tru-Khang T. "Circulating Micro-RNAs as Biomarkers for Thoracic Radiation Therapy in Lung Cancer." Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:27007756.
Full textAbstract:
Risk of normal tissues toxicity limits the amount of thoracic radiation therapy that can be routinely prescribed for the treatment of non-small cell lung cancer (NSCLC). An early biomarker of response to thoracic radiation may provide a way to predict eventual toxicities during the multi-week treatment regimen. This enables dose adjustment before the symptomatic onset of late effects, such as radiation pneumonitis and esophagitis. Micro-RNAs (miRNAs) are small, non-coding RNAs that regulate gene expression by decreasing the translation of messenger RNAs. miRNAs constitute a major fraction of small RNAs reproducibly found in circulation, in part due to their protective encapsulation within exosomes. They are therefore attractive candidates as serological biomarkers. In this study, we performed miRNA profiling of the blood of 5 NSCLC patients at 5 dose-points during thoracic RT and found 10 miRNAs that correlated well with total radiation dose as well as other common dosimetric parameters. We then assessed these 10 miRNAs in samples from a separate cohort of 21 NSCLC patients receiving RT and identified miR-29a-3p and miR-150-5p as potential, reproducible biomarkers that decreased in circulation with increasing radiation dose. We also conducted in-vitro experiments to measure the expression levels of these miRNAs intracellularly and within exosomes in three NSCLC cell lines and two lung bronchoepithelial and fibroblast lines. The exosomal expression of miR-29a-3p and miR-150-5p decreased with radiation. However, this was concomitant with an increase in intracellular levels, suggesting that exosomal export of these miRNAs may be downregulated in NSCLC and stromal cells as a response to radiation. One may therefore hypothesize that outlier trends in levels of circulating miR-29a-3p and miR-150-5p may predict unexpected responses to radiation therapy, such as toxicity.
10
Wang, Mantian [Verfasser], Erin [Akademischer Betreuer] Schuman, Erin [Gutachter] Schuman, and Michaela [Gutachter] Müller-McNicoll. "Regulation of circular RNAs and micro RNAs in hippocampal neurons / Mantian Wang ; Gutachter: Erin Schuman, Michaela Müller-McNicoll ; Betreuer: Erin Schuman." Frankfurt am Main : Universitätsbibliothek Johann Christian Senckenberg, 2021. http://d-nb.info/123468084X/34.
Full text
11
Samols, Mark Atienza. "Identification and Functional Analysis of Micro-RNAs Encoded by Kaposi’s Sarcoma-Associated Herpesvirus." Case Western Reserve University School of Graduate Studies / OhioLINK, 2007. http://rave.ohiolink.edu/etdc/view?acc_num=case1181143062.
Full text
12
Ewig, Benedikt [Verfasser]. "Tumormarker beim klarzelligen Nierenzellkarzinom: Welches Potential besitzen micro-RNAs? / Benedikt Constantin Jakob Ewig." Bonn : Universitäts- und Landesbibliothek Bonn, 2021. http://d-nb.info/1234550458/34.
Full text
13
Fadhil, Rushdi S. "Salivary micro RNAs as Potential Biomarkers for Oral and Head and Neck Cancers." Thesis, Griffith University, 2020. http://hdl.handle.net/10072/396154.
Full textAbstract:
Head and neck cancer (HNC) could include several squamous cell carcinoma (SCC) cancers that are considered a serious health problem worldwide today. The incidence rate of HNC has seen a sustained increase in Australia. For example, in Queensland, there is a 10.6 % increase of oral squamous cell carcinoma (OSCC) in male annually. Head and neck squamous cell carcinomas (HNSCC) patients may be asymptomatic, therefore, poor prognosis and metastasis are usually common in the patients. Subsequently, late diagnosis has a low rate of survival which most likely results in extreme severity of tumours at the time of diagnosis. The survival rate of patients with HNC is very poor, currently assessed at not more than five years with a high probability of local recurrence (40%). Therefore, a delay in diagnosis is still valid while early diagnostic methods are still needed. Traditional diagnostic methods such as radiology and endoscopic biopsy are major techniques performed for this type of cancer.
However, these clinical methods are stressful, relatively invasive in nature and extremely expensive. Thus, there is an urgent need for more specific, sensitive and straight forward early diagnostic procedures for investigating HNC. Furthermore, there is a necessity to discover novel biomarkers for early HNC diagnosis. MicroRNAs which are small noncoding molecules can be tested in different bodily fluids for e.g. blood, urine, saliva and serum. These have been verified as significant regulators of gene expression in cancer biology and may play a significant role in the early diagnosis of HNC.
Several studies have revealed the importance of miRNAs in the diagnosis of different types of cancer. However, few studies have investigated the expression of salivary profile miRNAs in HNC patients before treatment, and the possible utility of this easily accessible biological fluid as a diagnostic biomarker for HNC. Salivary biomarkers for HNC are still relatively limited compared with other types of cancers such as breast, lung and colon. Regarding existing knowledge, the literature review is inadequate for Australian HNC researchers. Furthermore, conflicting results have been reported in miRNAs expression levels in different types of cancer, including HNC and these might be as a result of contrasting experiment design, validation methods or sample variations. To address this literature gap, this study was designed to profile the expression of different salivary miRNA in HNSSC patients in Australian as compared to healthy individuals; as well as comparing grades and stages of cancer, cancer sites and habit status.
Significant variation in salivary miRNAs expression has been hypothesised between HNC patients and healthy individuals which is invaluable in the diagnosis of HNC. To test the hypothesis, several studies have been carried out. The aims of these studies are: i) To analyse the profile miRNAs expressions and their significance in HNC Australian patients and healthy adults using oral rinse samples; and ii) to test the possible utility of saliva sample as an easily accessible biological fluid and stress free for diagnostic biomarker isolation for HNC.
Chapter one explores the scientific problem and aims of this study. Chapter two provides a comprehensive literature review of the background in this field of work and the methodology used to conduct our research.
Chapter three detected the potential identification of the significant variation of salivary miRNAs in OSCC. Five different miRNAs (miR-21, miR-10b, miR-125a, miR-31 and miR-200a) were selected from the literature review to optimise the conditions of RNA extraction and thermocycles of real time PCR. Besides, these miRNAs have been highlighted in conflicting results both oncogenic and suppressor genic in different types of cancer. However, very little is known about the role and expression of these miRNAs in saliva samples of oral cancer. Thus, the study of this chapter documented the role of these miRNAs that is specifically associated with HNSCC.
In the above study, the result revealed that the overall expression of salivary miR-125a was significantly lower in OSCC patients compared with healthy individuals, while salivary miR-21 in OSCC patients was much higher than healthy individuals. Interestingly, upregulated salivary miR-21 was associated with tumour’s stage of OSCC (p≤0.05). A Receiver Operator Curve (ROC) was also constructed to estimate the discriminatory power of miR- 21 and miR- 125a for their potential to distinguish between healthy adults and the OSCC groups. These two miRNAs were shown to have good discriminatory ability with AUC values of 0.95 and 1, respectively.
In chapter four, next generation sequencing was used to investigate the variation of miRNAs profile expression levels between OSCC patients and healthy individuals, as a more efficient and precise technique. This study aimed to detect potentially significant miRNAs in OSCC. The salivary miRNA expression profiling identified 2565 miRNAs differentially co-expressed. However, 1927 miRNAs expressions are nonsignificantly differentiated between OSCC and the healthy individuals’ group. There are 638 miRNAs significantly co-expressed in the supernatant saliva of OSCC patients compared with healthy individuals group where p-value ≤0.05; 114 miRNAs with a pvalue ≤ 0.001; while 27 miRNAs were significantly different with a p-value ≤ 0.0001.
This study contributes to knowledge of the association between 27 dysregulated salivary miRNAs in OSCC and the potential to use them for OSCC diagnosis (p = 0.0001). 15 of these miRNAs were up-regulated (miR-7703, miR-3928-5p, miR-889- 5p, miR-3147, miR-4474-3p, miR-3170, miR-6895-5p, miR-1238-5p, miR-4521, miR- 548ac, miR-3158-3p, miR-1343-3p, miR-7152-5p, miR-3148, miR-3124-3p, ) and 12 miRNAs were down-regulated (miR-let-7f-5p, miR-let-7a-5p, miR-1247-5p, miR-574- 3p, miR-194-5p, miR-200c-3p, miR-32-5p, miR-6126, miR-99a-5p, miR-345-5p, miR- 301a-3p, miR-101-3p). Also, this study pointed out for the first time that overexpression of miR-7703 is a great and significant biomarker that could be used for cancer diagnosis. Hence, to confirm these results, it is reasonable to validate these miRNAs as signature diagnostic biomarkers in a large majority of patients.
Chapter five was aimed to validate the previous findings of chapter four in relation to the five dysregulated miRNAs in 150 patients with head and neck cancer (HNC) along with 80 healthy individuals. Thus, five dysregulated miRNAs (miR-7703, miR- let-7a- 5p, miR- 345-5p, miR- 3928 and miR- 1470) were selected for the validation study using a real time PCR technique. In this study, significant expression differences of miR-let-7a-5p and miR-3928 (p≤0.05) in HNC patients compared with healthy individuals were confirmed. These miRNAs were revealed to provide a good discriminative ability with AUC values of 0.85 and 0.74, respectively.
Taken together, the data reveals that the availability of significant miRNA could play a great role in HNC early diagnosis. Those studies contribute to knowledge on the correlation of miRNAs expression with OSCC and HNC and improve the availability of current diagnostic methods of cancer. In addition, the literature findings of miRNA studies supported our results and reported consistent results in the role of miRNA in cancer. However, this significance of the dysregulated expressions of miRNAs requires further investigation and additional research.Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
Full Text
14
Raffort, Juliette. "Étude du rôle des monocytes / macrophages et des micro-ARNs dans les anévrismes de l’aorte abdominale." Thesis, Université Côte d'Azur (ComUE), 2018. http://www.theses.fr/2018AZUR4082/document.
Full textAbstract:
L’anévrisme de l’aorte abdominale (AAA) représente un problème majeur de santé publique et est associé à des taux extrêmement élevés de mortalité en cas de rupture aortique. Il est classiquement associé à l’athérosclérose et aux facteurs de risque cardiovasculaires, à l’exception du diabète qui jouerait un rôle protecteur dans la maladie. Bien que certaines études aient montré une infiltration de macrophages et une modification de l’expression des micro-ARNs dans la paroi anévrismale, leurs rôles respectifs dans le développement de l’AAA ne sont pas encore totalement élucidés. Même si les modèles expérimentaux classiques sont utiles pour adresser cette question, ils ne miment pas parfaitement la physiopathologie humaine. Nous avons développé un nouveau modèle d’AAA associant application topique d’élastase et neutralisation systémique du TGFβ permettant de reproduire les principales caractéristiques de l’AAA humain et induisant une rupture aortique. Les objectifs de ce travail étaient de: -1/ Caractériser le phénotype des monocytes/ macrophages dans ce modèle murin d’AAA. -2/ D’étudier l’expression des micro-ARNs dans ce modèle. -3 / Les mécanismes impliqués dans la relation négative entre diabète et AAA étant à ce jour peu connus, le 3ème objectif était de mettre en place une étude clinique afin d’étudier l’expression des micro-ARNs chez des patients diabétiques ayant un AAA.L’application d’élastase associée à la neutralisation du TGFβ chez des souris C57/Bl6j entrainait une augmentation significative de la densité de macrophages infiltrés dans le tissu aortique anévrismal. L’analyse des tissus aortiques a montré des modifications significatives des marqueurs des macrophages pro-inflammatoires dits « M1 » et des marqueurs des macrophages impliqués dans la réparation tissulaire, dits « M2 ». Afin de mieux comprendre le rôle des macrophages dans ce modèle murin, des injections de clodronate ont été réalisées pour dépléter ces cellules. L’injection de clodronate diminuait de façon significative la dilatation anévrismale et prévenait la rupture. Ceci était associé à une préservation de la matrice extracellulaire et une modification de l’expression de certains marqueurs des macrophages, dont l’arginase-1 (ARG1), molécule impliquée dans la réparation tissulaire. La proportion de macrophages exprimant l’ARG1 augmentait en fonction de la sévérité de l’AAA. Enfin, la neutralisation du TGFβ induisait une diminution significative d’un type particulier de monocytes dits « Sat-Mono», impliqués dans la fibrose. Cette étude a ainsi montré le rôle des macrophages dans le développement et la rupture anévrismale avec une modification de leur phénotype. 752 micro-ARNs ont ensuite été analysés dans le tissu aortique, ce qui a permis d’identifier les micro-ARNs dont l’expression était modifiée dans ce modèle par rapport au groupe contrôle. Enfin, l’expression des micro-ARNs a été recherché chez l’homme en mettant en place une étude clinique. Bien que l’AAA chez l’homme soit classiquement associé à l’athérosclérose et aux facteurs de risque cardiovasculaire, il est paradoxalement négativement associé au diabète. Les mécanismes en cause sont encore peu connus. Nous avons comparé l’expression des micro-ARNs entre des patients diabétiques et non-diabétiques présentant un AAA. Cette étude pilote a permis d’identifier des cibles potentielles impliquées dans l’association négative entre diabète et AAAAbdominal aortic aneurysm (AAA) is a major public health concern and is associated with extremely high rates of mortality in case of aortic rupture. AAA is most often associated with atherosclerosis and cardiovascular risk factors, except diabetes that may rather play a protective role in the disease. Even though several studies have highlighted an infiltration of macrophages and changes of the expression of micro-RNAs in the aneurysmal wall, their role in AAA is still not fully understood. While experimental animal models are very useful to address this question, none of them perfectly mimics human pathophysiology. We recently created a new murine model of AAA based on topic application of elastase on the aorta associated with systemic TGFβ neutralization which reproduces the main human features of AAA and leads to fatal aortic rupture. The aims of this study were: -1/ Characterize the phenotype of monocytes/ macrophages in this murine model of AAA. -2/ Study the expression of micro-RNAs in this model. -3/ As the mechanisms involved in the negative association between diabetes and AAA are still poorly known, the third goal was to mount a clinical study to compare the expression of micro-RNAs between diabetic and non-diabetic patients with AAA. Topic application of elastase associated with systemic TGFβ neutralization in C57/Bl6j male mice led to a significant increase of macrophage infiltration in the aneurysmal tissue. This was associated with changes of the gene expression of the markers of the pro-inflammatory macrophages, called “M1” and of the macrophages involved in wound healing, called “M2”. To investigate the role of macrophages in this model, we used liposomes containing clodronate injections to deplete these cells. This led to significant decrease of the aortic dilatation and prevented rupture. This was associated with a better preservation of the extracellular matrix and significant changes in the gene expression of the markers of macrophages including arginase-1 (ARG1), a molecule involved in would healing. The proportion of macrophages expressing ARG1 increased with the severity of the AAA. At last, TGFβ neutralization led to a significant decrease of a population of macrophages involved in fibrosis, called “Sat-Mono”. This study highlighted the role and the phenotypic changes of macrophages during AAA development. We then analyzed the expression of 752 micro-RNAs in the aneurysmal aortic tissue which allowed identifying the micro-RNAs whose expression varied in the murine model. At last, the expression of micro-RNAs was investigated in patients with AAA. We compared the expression of micro-RNAs between diabetic and non-diabetic patients with AAA. This pilot study led to the identification of micro-RNAs that could potentially represent new targets involved in the negative association between diabetes and AAA
15
Moraes, Leonardo Nazario de [UNESP]. "Perfil global de expressão de micro-RNAs no músculo esquelético de ratos com insuficiência cardíaca." Universidade Estadual Paulista (UNESP), 2013. http://hdl.handle.net/11449/92423.
Full textAbstract:
Made available in DSpace on 2014-06-11T19:26:02Z (GMT). No. of bitstreams: 0
Previous issue date: 2013-05-17Bitstream added on 2014-06-13T20:33:35Z : No. of bitstreams: 1
000747177.pdf: 2540909 bytes, checksum: cf2fffb43c647d7ba5a8015e10178111 (MD5)MicroRNAs (miRNAs) constituem uma classe de pequenos RNAs não codificadores de 17 a 25 nucleotídeos de comprimento envolvidos em uma grande variedade de processos celulares. Os microRNAs regulam a expressão gênica pós-transcricionalmente, primariamente pela associação com a região 3' e/ou 5’ não traduzida (3' UTR, 5’ UTR) de seus RNAs mensageiros alvos geralmente reprimindo e, em alguns casos, estimulando a tradução. Atualmente, embora o número de genes relacionados com o desenvolvimento de doenças musculares aumente a cada ano, as vias moleculares envolvidas permanecem pobremente compreendidas. Porém, estudos recentes demonstram a importância da regulação mediada por microRNAs em diversos processos biológicos e em algumas doenças do músculo esquelético. A Insuficiência cardíaca (IC) é uma síndrome clínica associada à disfunção cardíaca, diminuição da expectativa de vida e intolerância aos exercícios físicos. Essa intolerância aos exercícios físicos, bem como a redução da atividade locomotora, estão associados aos principais sintomas dos pacientes com IC: a fadiga e a fraqueza muscular. Esse fenômeno é decorrente, em parte, da mudança no metabolismo de oxidativo para glicolítico e da presença de atrofia e alterações nos tipos de fibras musculares. Nesse estudo, analisamos a expressão de 303 microRNAs no músculo sóleo da ratos Wistar macho com IC induzida por monocrotalina (60 mg/kg). O músculo sóleo dos animais com IC apresentaram uma alteração na expressão relativa de 19 microRNAs, com diminuição na expressão de 05 microRNAs (< 50%; P<0,05) e aumento na expressão de 14 microRNAs (> 150%; P<0,05) quando comparado com o grupo controle. Nossos dados mostram que os miRNAs miR-29b, miR-27a-5p, miR-434-3p, miR-539, miR-489, miR-214, miR-632 e miR-146b são regulados na miopatia esquelética que ocorre na insuficiência cardíaca, sugerindo que essas moléculas possam atuar...
MicroRNAs (miRNAs) are small (~17-25 nt) non-coding RNAs regulating mRNAs involved in various biological processes, including the pathophysiology of striated muscles. miRNAs suppress gene expression through their complementarity to the sequence of one or more mRNAs, usually at a site in the 3′ untranslated region. The formation of an miRNA–target complex results either in inhibition of protein translation or in degradation of the mRNA transcript. There is no doubt that the formation, maintenance, and physiological and pathophysiological responses of skeletal muscles, with all their complex regulatory circuits, are subject to regulation by non-coding RNAs. Heart failure (HF) is characterized by a skeletal muscle myopathy with increased expression of fast myosin heavy chains (MyHCs) and atrophy. The skeletal muscle-specific molecular regulatory mechanisms controlling MyHC expression during HF have not been described and microRNAs may be related to these alterations. Using RT-qPCR TaqMan Low Density Arrays, we measured the expression of 303 microRNAs in soleus skeletal muscle from Wistar rat with monocrotaline (60 mg/kg)-induced HF. RNA isolated from soleus muscle of rat with HF showed increased expression of 14 microRNAs (> 150%; P<0,05) compared with controls. Similarly, HF-induced atrophy were accompanied by reduced expression of 05 microRNAs (< 50%; P<0,05). Interestingly, we outline the miRNAs miR-29, miR-214, and miR-489 that have been found to be dysregulated in diseases associated with skeletal muscle or are changed during muscle adaptation. These microRNAs are involved in important molecular pathways such as the NF-kB-YY1-miR-29 regulatory circuit, the miR-214 and Ezh2 regulatory loop, and the posttranscriptional suppression of oncogene oncogene Dek by the miRNA-489
16
Smith, Nikki. "The role of cellular micro-RNAs in Epstein-Barr virus induced cellular transformation and oncogenesis." Thesis, University of Birmingham, 2011. http://etheses.bham.ac.uk//id/eprint/1344/.
Full textAbstract:
Micro-RNAs (miRNAs) are a class of non-coding RNA which post-transcriptionally regulate gene expression. Epstein-Barr Virus (EBV) transforms resting B-cells in vitro to establish continuously proliferating lymphoblastoid cell lines (LCLs) and is aetiologically linked to lymphomas. Little is known about the contribution of miRNAs to the transformation of B cells. We initially examined the regulation of the oncogenic miR-155, which is highly expressed in Hodgkin’s lymphoma but was reportedly absent in Burkitt’s lymphoma. We found that miR-155 was up-regulated by EBV-LMP1 expression, and that a reported defect of miR-155 processing in Burkitt’s lymphoma was a misinterpretation of data. Next, to identify cellular miRNAs and genes modulated during EBV-induced transformation, we compared the expression profiles of resting B cells and B cells either infected with EBV or stimulated to proliferate with CD40L and IL4. This revealed that a large proportion of miRNAs and genes differentially regulated by EBV and not by CD40L/IL4 were modulated by EBV interaction with its CD21 receptor complex, but these changes were maintained or amplified in LCLs; and included a set of tumour suppressor genes down-regulated by EBV. In addition, bioinformatics analysis indicated that EBV modulates the expression of multiple miRNAs predicted to target the same cellular genes.
17
Moraes, Leonardo Nazario de. "Perfil global de expressão de micro-RNAs no músculo esquelético de ratos com insuficiência cardíaca /." Botucatu, 2013. http://hdl.handle.net/11449/92423.
Full textAbstract:
Orientador: Robson Francisco CarvalhoBanca: Francisco Javier Enguita
Banca: Patrícia Pintor Reis
Resumo: MicroRNAs (miRNAs) constituem uma classe de pequenos RNAs não codificadores de 17 a 25 nucleotídeos de comprimento envolvidos em uma grande variedade de processos celulares. Os microRNAs regulam a expressão gênica pós-transcricionalmente, primariamente pela associação com a região 3' e/ou 5' não traduzida (3' UTR, 5' UTR) de seus RNAs mensageiros alvos geralmente reprimindo e, em alguns casos, estimulando a tradução. Atualmente, embora o número de genes relacionados com o desenvolvimento de doenças musculares aumente a cada ano, as vias moleculares envolvidas permanecem pobremente compreendidas. Porém, estudos recentes demonstram a importância da regulação mediada por microRNAs em diversos processos biológicos e em algumas doenças do músculo esquelético. A Insuficiência cardíaca (IC) é uma síndrome clínica associada à disfunção cardíaca, diminuição da expectativa de vida e intolerância aos exercícios físicos. Essa intolerância aos exercícios físicos, bem como a redução da atividade locomotora, estão associados aos principais sintomas dos pacientes com IC: a fadiga e a fraqueza muscular. Esse fenômeno é decorrente, em parte, da mudança no metabolismo de oxidativo para glicolítico e da presença de atrofia e alterações nos tipos de fibras musculares. Nesse estudo, analisamos a expressão de 303 microRNAs no músculo sóleo da ratos Wistar macho com IC induzida por monocrotalina (60 mg/kg). O músculo sóleo dos animais com IC apresentaram uma alteração na expressão relativa de 19 microRNAs, com diminuição na expressão de 05 microRNAs (< 50%; P<0,05) e aumento na expressão de 14 microRNAs (> 150%; P<0,05) quando comparado com o grupo controle. Nossos dados mostram que os miRNAs miR-29b, miR-27a-5p, miR-434-3p, miR-539, miR-489, miR-214, miR-632 e miR-146b são regulados na miopatia esquelética que ocorre na insuficiência cardíaca, sugerindo que essas moléculas possam atuar ...
Abstract: MicroRNAs (miRNAs) are small (~17-25 nt) non-coding RNAs regulating mRNAs involved in various biological processes, including the pathophysiology of striated muscles. miRNAs suppress gene expression through their complementarity to the sequence of one or more mRNAs, usually at a site in the 3′ untranslated region. The formation of an miRNA-target complex results either in inhibition of protein translation or in degradation of the mRNA transcript. There is no doubt that the formation, maintenance, and physiological and pathophysiological responses of skeletal muscles, with all their complex regulatory circuits, are subject to regulation by non-coding RNAs. Heart failure (HF) is characterized by a skeletal muscle myopathy with increased expression of fast myosin heavy chains (MyHCs) and atrophy. The skeletal muscle-specific molecular regulatory mechanisms controlling MyHC expression during HF have not been described and microRNAs may be related to these alterations. Using RT-qPCR TaqMan Low Density Arrays, we measured the expression of 303 microRNAs in soleus skeletal muscle from Wistar rat with monocrotaline (60 mg/kg)-induced HF. RNA isolated from soleus muscle of rat with HF showed increased expression of 14 microRNAs (> 150%; P<0,05) compared with controls. Similarly, HF-induced atrophy were accompanied by reduced expression of 05 microRNAs (< 50%; P<0,05). Interestingly, we outline the miRNAs miR-29, miR-214, and miR-489 that have been found to be dysregulated in diseases associated with skeletal muscle or are changed during muscle adaptation. These microRNAs are involved in important molecular pathways such as the NF-kB-YY1-miR-29 regulatory circuit, the miR-214 and Ezh2 regulatory loop, and the posttranscriptional suppression of oncogene oncogene Dek by the miRNA-489
Mestre
18
Crisafulli, L. "REGULATION OF THE HEMATOPOIETIC SELF-RENEWAL AND LINEAGE CHOICE: ROLE OF PBX1 AND MICRO-RNAS." Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/229560.
Full textAbstract:
Hematopoiesis is a highly regulated process (Orkin and Zon, 2008). For a constant supply of short-lived terminally differentiated blood cells, and for rapid response to hematopoietic stresses, HSCs are endowed with the ability to continuously provide more mature progenitors while properly maintaining their pool size, without exhaustion, throughout life. This equilibrium is finely maintained on one hand by precisely balancing self-renewal and differentiation in HSCs, and on the other hand by regulating proliferation and differentiation also of their downstream progenitors, including lineage choice. Thus, understanding mechanisms of regulation of self-renewal versus differentiation of HSCs and downstream progenitors is a central issue in stem cell field, since tiny alteration of these mechanisms may lead to hematopoietic failure and disease.
Pbx1 is a transcription factor that positively regulates post-natal HSC quiescence. Its absence in HSCs causes an excessive proliferation that leads to their exhaustion, indicating a profound self-renewal defect, and premature myeloid differentiation at the expenses of lymphoid differentiation (Ficara et al., 2008). However, the precise molecular mechanisms through which Pbx1 exerts its function in HSCs and its role in progenitor biology are two still unexplored issues. In particular, it is not known whether Pbx1 function is also mediated by micro-RNAs, crucial new players in the regulation of proliferation and differentiation in several tissues, including the hematopoietic system.
Taking advantage of Pbx1 conditional KO mice, in this study, we demonstrate that Pbx1 functions as a brake on cell differentiation not only in HSCs but also in multi-potent and myeloid-restricted progenitors, to maintain progenitor reservoirs and lymphoid potential. In absence of Pbx1, both myeloid and lymphoid progenitors are able to differentiate into mature progeny but with higher efficiency and premature kinetic for the myeloid lineage and a decreased efficiency in the lymphoid (and erythroid) lineage. Pbx1 acts also upstream to lineage restricted progenitors, affecting lineage choice of multipotent progenitors (MPPs) by restraining myeloid differentiation and allowing lymphoid differentiation.
Moreover, we show that in the absence of Pbx1 HSCs display an altered micro-RNA profile, which resembles the normal MPP profile, suggesting a role for miRNAs in maintaining HSC identity.
Pbx1-null HSCs and MPPs show specific lists of DE miRNAs, with substantial overlaps with the normal HSC-to-MPP transition. Combining miRNA data with transcriptional profile data of the same populations (Ficara et al., 2008) allowed searching for miRNA predicted targets whose change in expression inversely correlates with those of mRNAs. This analysis, coupled with extensive bioinformatics studies (promoter analysis, co-targeting and GSEA) allowed selection of very few candidate miRNAs to be further studied for their specific role in maintaining HSC self-renewal, and their possible regulation by Pbx1. In addition to a list of Pbx1-dependent miRNAs in HSCs and MPPs, we also analyzes for the first time miRNAs characteristic of the normal transition from HSCs to the first MPP stage, which represent an important finding for understanding miRNAs physiologically involved at the apex of hematopoietic system. Overall, this analysis set the basis for the discovery of miRNAs involved in the regulation of self-renewal versus differentiation of HSCs.
19
Raffort, Juliette. "Étude du rôle des monocytes / macrophages et des micro-ARNs dans les anévrismes de l’aorte abdominale." Electronic Thesis or Diss., Université Côte d'Azur (ComUE), 2018. http://theses.univ-cotedazur.fr/2018AZUR4082.
Full textAbstract:
L’anévrisme de l’aorte abdominale (AAA) représente un problème majeur de santé publique et est associé à des taux extrêmement élevés de mortalité en cas de rupture aortique. Il est classiquement associé à l’athérosclérose et aux facteurs de risque cardiovasculaires, à l’exception du diabète qui jouerait un rôle protecteur dans la maladie. Bien que certaines études aient montré une infiltration de macrophages et une modification de l’expression des micro-ARNs dans la paroi anévrismale, leurs rôles respectifs dans le développement de l’AAA ne sont pas encore totalement élucidés. Même si les modèles expérimentaux classiques sont utiles pour adresser cette question, ils ne miment pas parfaitement la physiopathologie humaine. Nous avons développé un nouveau modèle d’AAA associant application topique d’élastase et neutralisation systémique du TGFβ permettant de reproduire les principales caractéristiques de l’AAA humain et induisant une rupture aortique. Les objectifs de ce travail étaient de: -1/ Caractériser le phénotype des monocytes/ macrophages dans ce modèle murin d’AAA. -2/ D’étudier l’expression des micro-ARNs dans ce modèle. -3 / Les mécanismes impliqués dans la relation négative entre diabète et AAA étant à ce jour peu connus, le 3ème objectif était de mettre en place une étude clinique afin d’étudier l’expression des micro-ARNs chez des patients diabétiques ayant un AAA.L’application d’élastase associée à la neutralisation du TGFβ chez des souris C57/Bl6j entrainait une augmentation significative de la densité de macrophages infiltrés dans le tissu aortique anévrismal. L’analyse des tissus aortiques a montré des modifications significatives des marqueurs des macrophages pro-inflammatoires dits « M1 » et des marqueurs des macrophages impliqués dans la réparation tissulaire, dits « M2 ». Afin de mieux comprendre le rôle des macrophages dans ce modèle murin, des injections de clodronate ont été réalisées pour dépléter ces cellules. L’injection de clodronate diminuait de façon significative la dilatation anévrismale et prévenait la rupture. Ceci était associé à une préservation de la matrice extracellulaire et une modification de l’expression de certains marqueurs des macrophages, dont l’arginase-1 (ARG1), molécule impliquée dans la réparation tissulaire. La proportion de macrophages exprimant l’ARG1 augmentait en fonction de la sévérité de l’AAA. Enfin, la neutralisation du TGFβ induisait une diminution significative d’un type particulier de monocytes dits « Sat-Mono», impliqués dans la fibrose. Cette étude a ainsi montré le rôle des macrophages dans le développement et la rupture anévrismale avec une modification de leur phénotype. 752 micro-ARNs ont ensuite été analysés dans le tissu aortique, ce qui a permis d’identifier les micro-ARNs dont l’expression était modifiée dans ce modèle par rapport au groupe contrôle. Enfin, l’expression des micro-ARNs a été recherché chez l’homme en mettant en place une étude clinique. Bien que l’AAA chez l’homme soit classiquement associé à l’athérosclérose et aux facteurs de risque cardiovasculaire, il est paradoxalement négativement associé au diabète. Les mécanismes en cause sont encore peu connus. Nous avons comparé l’expression des micro-ARNs entre des patients diabétiques et non-diabétiques présentant un AAA. Cette étude pilote a permis d’identifier des cibles potentielles impliquées dans l’association négative entre diabète et AAAAbdominal aortic aneurysm (AAA) is a major public health concern and is associated with extremely high rates of mortality in case of aortic rupture. AAA is most often associated with atherosclerosis and cardiovascular risk factors, except diabetes that may rather play a protective role in the disease. Even though several studies have highlighted an infiltration of macrophages and changes of the expression of micro-RNAs in the aneurysmal wall, their role in AAA is still not fully understood. While experimental animal models are very useful to address this question, none of them perfectly mimics human pathophysiology. We recently created a new murine model of AAA based on topic application of elastase on the aorta associated with systemic TGFβ neutralization which reproduces the main human features of AAA and leads to fatal aortic rupture. The aims of this study were: -1/ Characterize the phenotype of monocytes/ macrophages in this murine model of AAA. -2/ Study the expression of micro-RNAs in this model. -3/ As the mechanisms involved in the negative association between diabetes and AAA are still poorly known, the third goal was to mount a clinical study to compare the expression of micro-RNAs between diabetic and non-diabetic patients with AAA. Topic application of elastase associated with systemic TGFβ neutralization in C57/Bl6j male mice led to a significant increase of macrophage infiltration in the aneurysmal tissue. This was associated with changes of the gene expression of the markers of the pro-inflammatory macrophages, called “M1” and of the macrophages involved in wound healing, called “M2”. To investigate the role of macrophages in this model, we used liposomes containing clodronate injections to deplete these cells. This led to significant decrease of the aortic dilatation and prevented rupture. This was associated with a better preservation of the extracellular matrix and significant changes in the gene expression of the markers of macrophages including arginase-1 (ARG1), a molecule involved in would healing. The proportion of macrophages expressing ARG1 increased with the severity of the AAA. At last, TGFβ neutralization led to a significant decrease of a population of macrophages involved in fibrosis, called “Sat-Mono”. This study highlighted the role and the phenotypic changes of macrophages during AAA development. We then analyzed the expression of 752 micro-RNAs in the aneurysmal aortic tissue which allowed identifying the micro-RNAs whose expression varied in the murine model. At last, the expression of micro-RNAs was investigated in patients with AAA. We compared the expression of micro-RNAs between diabetic and non-diabetic patients with AAA. This pilot study led to the identification of micro-RNAs that could potentially represent new targets involved in the negative association between diabetes and AAA
20
Moralles, Patricia Regina Manzine. "Biomarcadores sanguíneos para a doença de Alzheimer : avaliação da expressão gênica da ADAM10 e de micro- RNAs." Universidade Federal de São Carlos, 2016. https://repositorio.ufscar.br/handle/ufscar/7759.
Full textAbstract:
Submitted by Luciana Sebin (lusebin@ufscar.br) on 2016-09-28T18:55:34Z
No. of bitstreams: 2
TesePRMM.pdf: 7211801 bytes, checksum: 788705ca3b95b2f530d64b669f066f88 (MD5)
TesePRMM.pdf: 7211801 bytes, checksum: 788705ca3b95b2f530d64b669f066f88 (MD5)Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-10T18:35:31Z (GMT) No. of bitstreams: 2 TesePRMM.pdf: 7211801 bytes, checksum: 788705ca3b95b2f530d64b669f066f88 (MD5) TesePRMM.pdf: 7211801 bytes, checksum: 788705ca3b95b2f530d64b669f066f88 (MD5)
Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-10T18:35:43Z (GMT) No. of bitstreams: 2 TesePRMM.pdf: 7211801 bytes, checksum: 788705ca3b95b2f530d64b669f066f88 (MD5) TesePRMM.pdf: 7211801 bytes, checksum: 788705ca3b95b2f530d64b669f066f88 (MD5)
Made available in DSpace on 2016-10-10T18:35:56Z (GMT). No. of bitstreams: 2 TesePRMM.pdf: 7211801 bytes, checksum: 788705ca3b95b2f530d64b669f066f88 (MD5) TesePRMM.pdf: 7211801 bytes, checksum: 788705ca3b95b2f530d64b669f066f88 (MD5) Previous issue date: 2016-03-11
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
ADAM10 is an-secretase that cleaves APP in the non-amyloidogenic pathway, thereby inhibiting -amyloid peptide (A ) production in Alzheimer´s disease (AD). Studies have shown decreased ADAM10 platelet levels in AD patients as well as the deregulation of microRNAs (miRNAs) related to molecules involved in the pathophysiology of this disease. The objective was to verify and compare ADAM10 gene expression and micro-RNAs (miRNAs) between AD patients and controls without cognitive impairment. It is a comparative study, based on the assumptions of quantitative research. Biological samples were collected, analyzed and stored in a biorepository. The ADAM10 gene expression in whole blood was studied in 47 AD, 32 healthy controls and 21 mild cognitive impairment (MCI) subjects by RT-qPCR techniques and analyzed by relative expression by 2- Ct. For miRNAs analyses, using MegaplexTM and MirWalk 2.0 database, were analyzed by RT-qPCR ~700 miRNAs in total blood and 21 miRNAs of them were validated in a sample of 21 AD subjects and 17 healthy controls. Statistical association tests, regression and diagnostic accuracy were performed. No significant differences in ADAM10 gene expression were observed between AD and control groups. Therefore, the decrease of ADAM10 protein in platelets of AD patients was not caused by a reduction in mRNA encoding for ADAM10. Mir-144-5p, miR-374 and miR-221 were downregulated in AD subjects, with moderate accuracy diagnosis. However, the association of selected miRNAs expression and Mini Mental State Examination (MMSE) was significantly better as a diagnostic tool compared to their expression separately. The validated miRNAs are involved in the regulation of pathways related to neurodegenerative diseases (beta-amyloid cascade, ubiquitination, transcriptional regulator, synaptic transmission, vesicle trafficking). Specifically, miR-144-5p, miR-374 and miR-221 are relevant for AD, as regulators of APP, BACE1 and ADAM10 translation. To the best of our knowledge, this is the first study to demonstrate a downregulation of these specific miRNAs in total blood of Alzheimer’s disease patients, compared to healthy cognitive controls. These findings are in agreement with AD protein outcomes and place the miRNAs evaluated as potential biomarkers that can be used to improve AD diagnosis.
ADAM10 é uma -secretase que cliva a APP através do caminho não amiloidogênico, inibindo desta forma a produção do peptídeo -amiloide (A ) na doença de Alzheimer (DA). Estudos apresentam a diminuição plaquetária da proteína ADAM10 em idosos com DA, assim como a desregulação de microRNAs (miRNAs) relacionados com moléculas envolvidas com a fisiopatologia desta doença. O objetivo geral foi verificar e comparar a expressão gênica da ADAM10 e de miRNAs entre idosos com DA e controles sem alterações cognitivas. Trata-se de um estudo de comparação, baseado nos pressupostos da pesquisa quantitativa. Amostras biológicas foram coletadas, analisadas e armazenadas em um biorrepositório. A expressão gênica da ADAM10 em sangue total foi estudada em 47 sujeitos com DA, 32 controles saudáveis e 21 sujeitos com transtorno neurocognitivo leve (TNCL), através de técnicas de RT-qPCR e analisada pela expressão relativa por 2- Ct. Para análises dos miRNAs, utilizando Megaplex e a base de dados MiRWalk 2.0, foram analisados por RTqPCR ~700 miRNAs no sangue total e 21 deles foram validados em uma amostra de 21 sujeitos com DA e 17 controles. Testes estatísticos de associação, regressão e acurácia diagnóstica foram realizados. Não foi observada diferença significante na expressão gênica da ADAM10 entre sujeitos com DA e controles. Assim, a diminuição dos níveis proteicos da ADAM10 plaquetária em pacientes com DA não foi devido a redução do mRNA codificante para ADAM10. Mir-144-5p, miR-374 e miR-221 estavam menos expressos em indivíduos com DA, com moderada acurácia diagnóstica. Entretanto, a associação da expressão dos miRNAs selecionados com o Mini Exame do Estado Mental (MEEM) foi significativamente melhor como uma ferramenta de diagnóstico em comparação com as análises individuais. Os miRNAs validados estão envolvidos na regulação de vias relacionadas a doenças neurodegenerativas (cascata beta-amiloide, ubiquitinação, reguladores de transcrição, transmissão sináptica, tráfego de vesículas). Especificamente, o miR-144-5p, miR-374 e miR- 221 são relevantes para a DA, como reguladores da tradução da APP, BACE1 e da ADAM10. Segundo nosso conhecimento, este é o primeiro estudo a demonstrar a expressão reduzida desses miRNAs no sangue total de pacientes com DA, em comparação com controles cognitivamente saudáveis. Estes resultados estão de acordo com os resultados proteicos da DA e destacam os miRNAs avaliados como potenciais biomarcadores que podem ser utilizados para o aperfeiçoamento do diagnóstico da DA.
21
Dadalto, Juliane Dias. "Perfil global de expressão de micro-RNAS circulantes como marcadores de resposta terapêutica na leucemia mielóide crônica." Botucatu, 2016. http://hdl.handle.net/11449/138187.
Full textAbstract:
Orientador: Célia Regina NogueiraResumo: A Leucemia Mielóide Crônica (LMC) é uma doença malígna, clonal, da célula tronco hematopoética. A descoberta do cromossomo filadélfia e subsequente identificação do gene BCR-ABL, levaram a compreensão da biologia da doença que culminou com o desenvolvimento de drogas alvo-específicas, assim como o de métodos moleculares para o monitoramento da doença. O foco atual das pesquisas em LMC está voltado para o maior entendimento dos mecanismos moleculares e epigenéticos que levam a resistência terapêutica e progressão da doença. Estudos recentes demonstram que a expressão de micro-RNAs específicos modula oncogenes e genes supressores envolvidos no desenvolvimento de neoplasias. Ao encontro a esta tendência, propomos um estudo que procurou identificar o perfil de micro-RNAs dos pacientes bons respondedores aos tratamentos de primeira linha para LMC. Avaliamos o perfil de micro-RNAs, de 41 pacientes com LMC que atingiram resposta citogenética completa (ausência do cromossomo Filadélfia) após o tratamento com inibidor de tirosinaquinase e transplante alogênico de células progenitoras hematopoéticas, por meio do sistema de micro-RNA PCR arrays (TaqMan® Human Micro-RNA Array A e B). Identificamos uma assinatura de micro-RNA distinta entre os grupos tratados, apesar de se encontrarem no mesmo patamar de resposta clínica e citogenética. Palavras-chave: Leucemia Mielóide Crônica, micro-RNA, imatinibe, transplante, qPCR array.
Abstract: Chronic Myeloid Leukemia (CML) is a malignant clonal hematopoietic stem cell disease. The discovery of the Philadelphia chromosome and subsequent identification of the gene BCR-ABL have led to understanding the biology of the disease and the development of target-specific drugs, as well as molecular methods for monitoring the disease. The current focus of research in the CML is facing the greatest understanding of the molecular and epigenetic mechanisms that lead to therapy resistance and disease progression. Recent studies show that the expression of specific micro-RNAs modulates oncogenes and tumor suppressor genes involved in cancer development.We are proposing a new study in the literature that aims to identify the profile of micro-RNAs of patients good responders to first-line treatments for CML.Evaluated the profile of micro-RNAs in 41 CML patients who achieved a complete cytogenetic response (absence of Philadelphia chromosome) after treatment with tyrosine kinase inhibitor and allogeneic bone marrow transplantation, through the micro-RNA- PCR arrays (TaqMan® Human Micro-RNA Array A e B). We identified a distinct micro-RNA signature between the treated groups, despite being on the same level of cytogenetic and clinical response.Key Words: Chronic Myeloid LeuKemia, micro-RNA, imatinib, bone marrow transplantation, qPCR array.
Mestre
22
Tscheuschler, Maike Karoline [Verfasser]. "Expressionsprofil ausgewählter Micro-RNAs bei Hepatitis B- und Hepatitis C-Virus assoziiertem hepatozellulären Karzinom / Maike Karoline Tscheuschler." Köln : Deutsche Zentralbibliothek für Medizin, 2011. http://d-nb.info/101293361X/34.
Full text
23
Tomiyama, Alberto Hiroyuki. "Micro RNA em adenocarcinoma de próstata: caracterização da expressão em tumores de baixo grau, órgão-confinados." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/5/5153/tde-07022012-112055/.
Full textAbstract:
Introdução: Os micro RNA (miRNA) são formados a partir de RNA precursores de fita dupla que contém entre 60 a 110 nucleotídeos e formam estruturas do tipo hairpin. Imediatamente após sua transcrição pela RNA polimerase II a enzima Dicer promove a clivagem do RNA precursor em seqüências menores contendo 19 a 22 nucleotídeos. Após a clivagem, o miRNA integra-se ao complexo silenciador induzido pelo RNA (RISC) que o conduz ao seu RNA mensageiro (mRNA) homólogo recém transcrito. Esta associação promove a degradação do mRNA, ou interfere na tradução da proteína caracterizando um grande mecanismo de controle da expressão dos genes. Este mecanismo está relacionado ao desenvolvimento de órgãos e tecidos, e está envolvido no processo de carcinogênese. Nosso objetivo é identificar um perfil de expressão de miRNA que defina o adenocarcinoma de próstata de prognóstico favorável e desfavorável considerando os níveis de PSA e dados anatomopatológicos. Materiais e métodos: Foram selecionados 53 pacientes com tumores desfavoráveis (mediana do escore de Gleason igual a 8, 79,2% estadiados pT3, mediana de PSA 10,1 ng/mL e mediana do volume tumoral de 23%) e 45 considerados favoráveis (mediana do escore de Gleason igual a 5, 80% estadiados pT2, mediana de PSA de 7,8 ng/mL e mediana do volume tumoral de 11,5%). O controle foi representado por 7 pacientes com hiperplasia prostática benigna (HPB). Todos os pacientes foram submetidos a prostatectomia radical pelo mesmo cirurgião. Os espécimes cirúrgicos foram examinados na sua totalidade pelo mesmo patologista. A análise dos miRNA foi feita a partir de tecido congelado e tecido incluído em parafina usando a técnica da reação em cadeia da polimerase em tempo real quantitativa (qRT-PCR) utilizando primers e sondas Taqman® específicas. O RNU43 foi usado como controle interno. Resultados: Com exceção dos miRNA 199a, 21, 15a, 16 e 25 que se mostraram subexpressos tanto nos casos desfavoráveis como nos favoráveis, houve uma diminuição global na expressão dos miRNA com redução estatisticamente significativa na expressão dos miRNA 143, 145 e 146a, 191, 218 e Let7c em tumores desfavoráveis em relação aos tumores favoráveis. Conclusão: Demonstramos que no processo de transição entre os carcinomas favoráveis e desfavoráveis de próstata existe uma perda global na expressão de miRNA que podem ser importantes controladores de expressão de uma série de genes relacionados a progressão desta neoplasia. Dados experimentais avaliando o papel desses miRNA devem ser conduzidos para que possamos definir seu papel na evolução do câncer de próstataIntroduction: micro RNA (miRNA) are formed from double-stranded RNA precursors that contain between 60-110 nucleotides and form structures such as hairpin. Immediately after their transcription by RNA polymerase II, the enzyme Dicer promotes the cleavage of precursor RNA sequences containing minor 19-22 nucleotides. After cleavage, the miRNA is part of the RNA-induced silencing complex (RISC) that leads to its messenger RNA (mRNA) newly transcribed counterpart. This association promotes the degradation of mRNA, or interferes with the protein translation characterizing a great mechanism for controlling gene expression. This mechanism is related to the development of organs and tissues, and may be involved in the process of carcinogenesis. Our goal is to identify a miRNA expression profile that distinguishes prostate adenocarcinoma of favorable and unfavorable prognosis considering the PSA and pathological findings. Material and Methods: We studied 53 patients with tumors considered unfavorable (Median of Gleason score 8, 79.2% staged pT3, median of PSA 10.1 ng/mL and median of tumor volume of 23%) and 45 considered favorable (Median of Gleason score 5, 80% staged pT2, median of PSA 7.8 ng/mL and median of tumor volume of 11.5%). The control group was represented by seven patients with benign prostatic hyperplasia (BPH). All patients underwent radical prostatectomy by the same surgeon. The surgical specimen was examined entirely by the same pathologist. The analysis of miRNA was made from frozen and paraffin embedded tissue by quantitative real-time polymerase chain reaction (qRT-PCR) using the Taqman® specific primers and probes. The RNU43 was used as a internal control. Results: Except for miRNAs 199a, 21, 15a, 16 e 25 that were underexpressed by both favorable and unfavorable prostate cancer, there was a global decrease of all miRNAs studied, and some differences were statistically significant as miRNAs 143, 145 e 146a, 191, 218 e Let7c that were underexpressed in unfavorable carcinomas compared favorable tumor. Conclusion: We have demonstrated that in the process of transition between favorable and unfavorable prostate cancer there is a global loss of expression of miRNAs. These molecules can be important controllers of a series of genes related to cancer progression. Experimental studies are needed in order to comprehend the role of these genes in prostate carcinogenesis
24
Pantaleão, Lucas Carminatti. "Reprogramação fenotípica por excesso de glicocorticoides: participação de micro-RNAs no desenvolvimento hepático e possíveis repercussões na vida adulta." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/42/42137/tde-01062015-162513/.
Full textAbstract:
Avaliamos o efeito da RCIU induzida por glicocorticoides sobre a regulação da expressão de miRNAs no fígado de ratos albinos. Animais expostos intrauterinamente à dexametasona apresentaram menor peso ao nascer, fígados relativamente menores dos que os observados em animais controle e menores concentrações hepáticas de PCNA. Em longo prazo, os animais DEX desenvolveram distúrbios metabólicos caracterizados por intolerância à glicose e maior potencial gliconeogênico no desmame e na vida adulta. Ao avaliarmos o perfil de miRNAs no fígado, detectamos aumento da expressão de todo o cluster do miR-322 no período perinatal, com menor conteúdo de alvos preditos desses transcritos (AKT3, CCND1 e INSR). A superexposição ao miR-322-5P reduz a taxa de proliferação em linhagens celulares de hepatocarcinoma e a expressão dos alvos observados no fígado dos animais estudados. Propomos um link entre a expressão aberrante do miR-322-5P e a reduzida taxa de proliferação detectada no tecido hepático em desenvolvimento, contribuindo para o estabelecimento do fenótipo em longo prazo.We evaluated the effects of glucocorticoid induced IUGR on the expression of miRNA on Wistar rat livers. Dexamethasone (DEX) treated animals were lighter and had smaller liver weight:body weight ratio when compared to control animals. Furthermore, liver PCNA expression was downregulated, sugesting a reduction on cell proliferation rate. In the long term, DEX animals developed metabolic disturbances such as glucose intolerance e increased gluconeogenesis rate in a fast state. The analysis of miRNA expression profile showed an upregulation of miR-322 cluster on perinatal period, together with a downregulation of three putative targets: Akt3, CCND1 and INSR. Later, we used in vitro studies to prove that overexpression of miR-322-5P arrests cell cycle and impairs proliferation of HEPG2 cells as well as it downregulates the predicted targets. Based on this data, we suggest a link between overexpression of miR-322-5P and impaired proliferation rate on the developing liver, which affects the phenotype in the long term.
25
Lagadinos, Alexander N. "Human Cytomegalovirus Reprograms the Expression of Host Micro-RNAs whose Target Networks are Required for Viral Replication: A Dissertation." eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/683.
Full textAbstract:
The parasitic nature of viruses requires that they adapt to their host environment in order to persist. Herpesviruses are among the largest and most genetically complex human viruses and they have evolved mechanisms that manipulate a variety of cellular pathways and processes required to replicate and persist within their hosts. Human cytomegalovirus (HCMV), a member of the β- herpesvirus sub-family, has the capacity to influence the expression of many host genes in an effort to create an optimal environment for infection. One mechanism utilized by HCMV to alter gene expression is the host RNA interference (RNAi) pathway. This is evidenced by a requirement of host factors to process viral micro-RNAs (miRNAs) and by the dynamic expression of host miRNAs during infection.
The work presented in this dissertation demonstrates that productive HCMV infection reprograms host miRNA expression in order to positively influence infection. I was able to identify a cohort of infection-associated host miRNAs whose change in expression during infection was highly significant. Using the enhancer-promoter sequences of this panel of host miRNAs, I statistically enriched for the presence of functional transcription factor binding sites that regulated the expression of two highly conserved clusters of host miRNAs: miR132/212 and miR143/145. Given that inhibiting their infection-associated change in expression during infection was detrimental to viral replication, it suggests that HCMV mechanistically influences the expression of these miRNA clusters. In order to determine the functional relevance of these miRNAs, I assembled a cohort of potential miRNA target genes using gene expression profiles from primary fibroblasts. By statistically enriching for miRNA recognition elements (MRE) in the respective 3’-UTR sequences, I generated a miRNA target network that includes thousands of host genes. I evaluated the efficacy of our novel miRNA target prediction algorithm by confirming the functionality of enriched MREs present in the 3’-UTR of KRas and by confirming anecdotal miRNA targets from published studies. Gene ontology terms enriched from infection-associated host miRNA target networks suggest that the utility of host miRNAs may extend to multiple host pathways that are required for viral replication. The targeting of multiple miRNAs to shared genes increased the statistical likelihood of target site enrichment. I propose that identifying cooperative miRNA networks is essential to establishing the functional relevance of miRNAs in any context. By combining contextual data on the relative miRNA/mRNA abundance with statistical MRE enrichments, one will be able to more accurately characterize the biological role of miRNAs.
26
Lagadinos, Alexander N. "Human Cytomegalovirus Reprograms the Expression of Host Micro-RNAs whose Target Networks are Required for Viral Replication: A Dissertation." eScholarship@UMMS, 2008. http://escholarship.umassmed.edu/gsbs_diss/683.
Full textAbstract:
The parasitic nature of viruses requires that they adapt to their host environment in order to persist. Herpesviruses are among the largest and most genetically complex human viruses and they have evolved mechanisms that manipulate a variety of cellular pathways and processes required to replicate and persist within their hosts. Human cytomegalovirus (HCMV), a member of the β- herpesvirus sub-family, has the capacity to influence the expression of many host genes in an effort to create an optimal environment for infection. One mechanism utilized by HCMV to alter gene expression is the host RNA interference (RNAi) pathway. This is evidenced by a requirement of host factors to process viral micro-RNAs (miRNAs) and by the dynamic expression of host miRNAs during infection.
The work presented in this dissertation demonstrates that productive HCMV infection reprograms host miRNA expression in order to positively influence infection. I was able to identify a cohort of infection-associated host miRNAs whose change in expression during infection was highly significant. Using the enhancer-promoter sequences of this panel of host miRNAs, I statistically enriched for the presence of functional transcription factor binding sites that regulated the expression of two highly conserved clusters of host miRNAs: miR132/212 and miR143/145. Given that inhibiting their infection-associated change in expression during infection was detrimental to viral replication, it suggests that HCMV mechanistically influences the expression of these miRNA clusters. In order to determine the functional relevance of these miRNAs, I assembled a cohort of potential miRNA target genes using gene expression profiles from primary fibroblasts. By statistically enriching for miRNA recognition elements (MRE) in the respective 3’-UTR sequences, I generated a miRNA target network that includes thousands of host genes. I evaluated the efficacy of our novel miRNA target prediction algorithm by confirming the functionality of enriched MREs present in the 3’-UTR of KRas and by confirming anecdotal miRNA targets from published studies. Gene ontology terms enriched from infection-associated host miRNA target networks suggest that the utility of host miRNAs may extend to multiple host pathways that are required for viral replication. The targeting of multiple miRNAs to shared genes increased the statistical likelihood of target site enrichment. I propose that identifying cooperative miRNA networks is essential to establishing the functional relevance of miRNAs in any context. By combining contextual data on the relative miRNA/mRNA abundance with statistical MRE enrichments, one will be able to more accurately characterize the biological role of miRNAs.
27
Cordeiro, Santanach Anna. "ARNs no codificants petits en Limfoma de Hodgkin: regulació epigenètica de micro RNAs i importància de la via PIWI/piRNA." Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/401705.
Full textAbstract:
El limfoma de Hodgkin (LH) és una neoplàsia de cèl·lules B que es caracteritza per tenir un 1% de cèl·lules tumorals, anomenades cèl·lules de Hodgkin i Reed-Sternberg (HRS), envoltades per un microambient reactiu. Per això, l’estudi de la funció i del paper pronòstic dels ARNs no codificants petits en gangli sencer, i no únicament en les cèl·lules tumorals, pren un gran significat.
Els miRNAs participen en els processos fisiològics però també intervenen en el desenvolupament de malalties, incloent el càncer. Poden actuar d’oncogens o de gens supressors de tumors depenent del seu ARN missatger diana. La identificació dels miRNAs inhibits durant el procés de limfomagènesi per metilació ens permetrà identificar miRNAs amb funció de supressors tumorals en el LH clàssic. Després de tractar amb un agent desmetilant, el 5-Aza-dC, dues línies cel·lulars de LH, aquestes reexpressaven diversos miRNAs, dels quals 10 dels quals eren comuns i tenien una illa CpG com a màxim a una kilobase del lloc d'inici de la transcripció. Mitjançant una PCR específica de metilació (MSP), vam observar que, entre d’altres, el miR-34a i el miR-203, coneguts supressors tumorals en altres neoplàsies, estaven metilats en quatre línies cel·lulars de LH. A continuació, mitjançant MSP in situ vam avaluar l’estat de metilació d’aquests dos microRNAs en mostres de teixit de pacients. Vam observar que el miR-34a i el miR-203 mostraven marcatge ens els nuclis de les cèl·lules HRS quan utilitzàvem el primer que detectava les seqüències metilades i cap senyal utilitzant el primer per a les seqüències desmetilades. Per últim vam avaluar els efectes del 5-Aza-dC in vitro després de tractar les cèl·lules amb dosis creixents de fàrmac, el qual tenia un efecte citotòxic a dosis elevades però el màxim efecte de reexpressió dels miRNAs miR-34a i miR-203 es trobava a dosis baixes i intermèdies.
La via PIWI/piRNA es creia que només estava activa en cèl·lules germinals i embrionàries però actualment hi ha evidències que també pot jugar un paper clau en el procés de carcinogènesi, ja que la seva desregulació provoca inestabilitat genòmica entre d’altres funcions. A més, recentment s’ha identificat el valor pronòstic dels nivells d’expressió de les proteïnes PIWI, principals reguladores del procés de biogènesi dels piRNAs. Creiem que aquesta via pot trobar-se desregulada en el LH clàssic i jugar un paper clau en el procés de limfomagènesi. Es detecten nivells baixos de PIWIL1 en les línies cel·lulars de LHc, però no en les cèl·lules B controls. En les mostres de pacients, detectem PIWIL1 en el citoplasma de les cèl·lules HRS en un terç dels pacients analitzats. PIWIL2 i PIWIL4 es troben infraexpressats en les línies de LH en comparació amb les cèl·lules B. En totes les mostres analitzades de pacients detectem PIWIL2 i PIWIL4. Els 3 piRNAs seleccionats (piR-651, piR-20365 i piR-20582) s’expressen en totes les mostres analitzades i estan significativament sobreexpressats en LHc en comparació als GRs. De forma interessant, els pacients amb nivells baixos de piR-651 presenten una menor taxa de resposta complerta (p=0.002) i una pitjor supervivència global (p=0.02). L’anàlisi de piR-651 en mostres de sèrum mostra que es troba infraexpressat al diagnòstic (p=0.01) però recupera els nivells normals a l’assolir la resposta complerta (p=0.05). Mitjançant hibridació in situ podem veure que el piR-651 s’expressa de forma elevada en el citoplasma de les cèl·lules HRS.Classical Hodgkin lymphoma (cHL) is a B cell neoplasia that comprises 11% of all lymphomas and is characterized by the presence of few tumoral cells, the so called Hodgkin Reed-Sternberg (HRS) cells, surrounded by a reactive microenvironment. Studying the functions and the prognostic role of small non-coding RNAs in the whole lymph node and not only in the tumoral cells is of great importance. Epigenetic mechanisms are crucial for the inactivation of key genes related to the survival of HL cells, and methylation is a frequent epigenetic mechanism of microRNA silencing. We have examined the methylation-induced silencing of tumor suppressor microRNAs in HL cell lines and confirmed our results in patient lymph nodes. In addition, we evaluated the in vitro effectiveness of 5-aza-2-deoxycytidine (5-Aza-dC) in HL cell lines. Ten microRNAs containing CpG islands in their promoter region were re-expressed in both the L-428 and L-1236 cell lines. Interestingly, miR-34a and miR-203, both known tumor suppressor microRNAs, were found to be methylated in cell lines and in patient samples. 5-Aza-dC treatment resulted in a dose-dependent antiproliferative effect at 72 h in all the HL cell lines. In summary, 5-Aza-dC treatment of HL cell lines inhibits proliferation at high doses and produces re-expression of the tumor suppressor microRNAs at low-intermediate doses. PiwiRNAs, small non-coding RNAs processed by Piwi proteins, are involved in maintaining genome stability in germline cells. Recently, piwiRNA expression has been identified in some tumors. We have examined the potential reactivation of the Piwi/piwiRNA pathway in cHL. We found that Piwi proteins and three selected piwiRNAs, including piR-651, were expressed in cHL patients and cell lines, indicating that the Piwi/piwiRNA pathway is active in cHL. Interestingly, low levels of piR-651 were associated with lack of complete response to first-line treatment, as well as shorter disease-free and overall survival in a cohort of 94 cHL patients. At diagnosis, piR-651 was underexpressed in cHL serum samples compared to healthy controls, while after complete remission, piR-651 levels increased to levels similar to healthy controls. This is the first evidence that piwiRNAs are active in tumor and serum samples and impact prognosis in cHL.
28
Fluck, Petra [Verfasser], and Friedrich [Akademischer Betreuer] Grässer. "Untersuchung zur Funktion der EBV-kodierten micro RNAs miR-BHRF1-2 und miR-BHRF1-3 / Petra Fluck. Betreuer: Friedrich Grässer." Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2011. http://d-nb.info/1051434718/34.
Full text
29
Viana, Nayara Izabel. "O papel dos micros RNAs 143 e 145 e seus genes alvo na etiopatogenia da hiperplasia prostática benigna." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/5/5153/tde-09012013-163954/.
Full textAbstract:
Introdução: A hiperplasia prostática benigna (HPB) é apontada como um dos mais comuns problemas de saúde associado ao envelhecimento dos homens. A incidência da doença começa a aumentar a partir dos 40 anos de idade, e se torna frequente em cerca de 50% dos homens com 50 anos e em quase 90% dos homens após a oitava década. A etiopatogenia da HPB ainda não foi totalmente elucidada, mas sabe-se que alguns fatores aumentam os riscos de aparecimento do problema. Seu melhor entendimento contribuiria substancialmente para o estabelecimento de um consenso terapêutico para a doença. Nesse sentido, os marcadores moleculares têm sido pesquisados na tentativa de auxiliar ou mesmo suplantar a eficiência dos métodos tradicionais. MicroRNAs (miRNAs) são uma classe de pequenos RNA regulatórios (19-25 nucleotídeos), não codificadores que possuem um papel fundamental no controle da expressão gênica. Essas pequenas moléculas estão envolvidas em vários processos celulares fisiológicos e patológicos. Alguns estudos demonstraram que os miRNAs 143 e 145 têm papel fundamental na diferenciação e proliferação celular da musculatura lisa. A ação de miRNAs na HPB ainda foi pouco explorada. Objetivos: Analisar a expressão do miR-143 e de seus genes e proteínas alvo ERK5 e KRAS e do miR-145 e dos seus genes e proteínas alvo MAP3K3 e MAP4K4, em pacientes portadores de HPB e comparar os perfis de expressão destes com parâmetros clínicos dos pacientes. Material e Métodos: Para análise dos miRNAs e dos seus genes alvo, foram estudados 44 pacientes diagnosticados com HPB submetidos à ressecção transuretral da próstata ou prostatectomia aberta. A análise da expressão foi realizada pela técnica de RT-PCR quantitativo. O grupo controle foi composto de tecido prostático normal de dois pacientes jovens doadores de órgãos. A análise proteica foi feita a partir de 38 pacientes, pela técnica de Western Blotting. Resultados: Os miRNA 143 e 145, apresentaram superexpressão em 62,5% e 73,8% dos casos, respectivamente. O gene ERK5 apresentou subexpressão de 59,4%, evidenciando um possível controle negativo por parte do miR-143. O gene MAP4K4 apresentou subexpressão na totalidade das amostras estudadas, demonstrando um possível controle por parte do miR-145. Os genes KRAS e MAP3K3 apresentaram superexpressão de 79,4% e 61,5% das amostras, respectivamente. De acordo com a expressão proteica, encontramos perfis semelhantes aos da expressão gênica. Foi encontrada maior expressão das proteínas KRAS e MAP3K3. Considerando a expressão gênica e proteica comparadas aos parâmetros clínicos, somente a proteína ERK5 apresentou significância (p=0,019), estando mais presente em pacientes com próstatas > 60 gramas. Conclusão: A superexpressão dos miRNAs 143 e 145 encontrada neste estudo pode estar envolvida na etiopatogenia da HPB alterando a homeostase do tecido fibromuscular principalmente, controlando proliferação e diferenciação. A superexpressão gênica e a forte expressão proteica de KRAS também pode estar envolvida na etiopatogenia da HPB, já que esta molécula quando ativada é responsável pelo estímulo de vias que resultam na proliferação, sobrevivência, motilidade celular e tráfego intracelular. A superexpressão do MAP3K3 pode estar sendo responsável pela angiogênese que ocorre em HPB. A subexpressão de MAP4K4, neste caso possivelmente controlada por miR-145, pode estar deixando de regular negativamente mTOR, gerando uma proliferação celular irregular responsável pela HPB. A detecção das proteínas em níveis semelhantes aos que foram encontrados na expressão gênica reforça estas hipóteses.Introduction: Benign prostatic hyperplasia (BPH) is one of the most common male health problems associated with aging. The incidence of disease starts to increase after 40 years, and compromises half of men in the fifths and almost 90% over 80 years. The pathogenesis of BPH has not been fully elucidated, but it is known that some factors increase the risk of occurrence of the problem. A better understanding of BPH pathogenesis would substantially contribute to the establishment of a therapeutic consensus for the disease. Thus, molecular markers have been investigated attempting to help or even overcome the efficiency of traditional methods. MicroRNAs (miRNAs) are a class of small non-coding regulatory RNA (19-25 nucleotides) that plays a key role in gene expression control. These small molecules are involved in various physiological and pathological cellular processes. Some studies have shown that miRNAs 143 and 145 play a fundamental role in cellular differentiation and proliferation of smooth muscles. The action of miRNAs in BPH has been poorly explored. Objectives: Analyze the expression of miR-143 and its target genes and proteins ERK5 and KRAS and miR-145 and its target genes and proteins MAP3K3 and MAP4K4, in patients with BPH and compare their expression profiles with clinical parameters of patients. Material and Methods: For analysis of miRNAs and its target genes, we studied 44 patients diagnosed with BPH undergoing transurethral resection of the prostate or open prostatectomy. The expression analysis was performed by quantitative RT-PCR. Control group consisted of normal prostate tissue from two young patients organ donors. Protein analysis was done from of 38 patients using Western Blotting technique. Results: miR-143 and 145 presented overexpression in 62.5% and 73.8% of cases, respectively. The ERK5 gene demonstrated underexpression in 59.4%, indicating a possible control by the miR-143. MAP4K4 gene showed underexpression in 100% of samples and could be under a potential control by the miR-145. KRAS and MAP3K3 genes revealed overexpression of 79.4% and 61.5% of cases, respectively. According protein expression, to find similar profiles of gene expression. We found an increased expression of the proteins MAP3K3 and KRAS. Considering the gene expression and protein compared to clinical parameters, only the protein ERK5 showed significance (p = 0.019), being more present in patients with prostates > 60 grams. Conclusions: Overexpression of miRNAs 143 and 145 found in this work may be involved in the pathogenesis of BPH mainly by changing the fibromuscular tissue homeostasis, controlling proliferation and differentiation. Overexpression of KRAS may also be involved in the pathogenesis of BPH, since this molecule when activated can trigger cell proliferation, survival, intracellular trafficking and motility. Overexpression of MAP3K3 can be responsible for BPH angiogenesis. The MAP4K4 underexpression, in this case possibly controlled by miR-145 overexpression, could inhibit mTOR pathway, leading to irregular cell proliferation responsible for disease. Detection of proteins at similar levels to those found in gene expression reinforce our hypothesis.
30
Fantinatti, Bruno Evaristo de Almeida [UNESP]. "Elucidação dos efeitos de cromossomos supernumeráveis na espécie de ciclídeo africano Astatotilapia latifasciata, com base na análise de expressão de micro-RNAs." Universidade Estadual Paulista (UNESP), 2015. http://hdl.handle.net/11449/144074.
Full textAbstract:
Made available in DSpace on 2016-09-27T13:40:03Z (GMT). No. of bitstreams: 0
Previous issue date: 2015-07-31. Added 1 bitstream(s) on 2016-09-27T13:45:14Z : No. of bitstreams: 1
000868844_20170731.pdf: 356252 bytes, checksum: 38bf2107cf66a4b3cb426b24a0184a36 (MD5) Bitstreams deleted on 2017-08-07T14:09:10Z: 000868844_20170731.pdf,. Added 1 bitstream(s) on 2017-08-07T14:10:14Z : No. of bitstreams: 1
000868844.pdf: 8016009 bytes, checksum: a5ffc89b1b5a01ff1b019ba237d0b05e (MD5)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
CAPES: 3127-14-1
CNPq: 151697/2001-6
FAPESP: 13/04533-3
31
Fantinatti, Bruno Evaristo de Almeida. "Elucidação dos efeitos de cromossomos supernumeráveis na espécie de ciclídeo africano Astatotilapia latifasciata, com base na análise de expressão de micro-RNAs." Botucatu, 2015. http://hdl.handle.net/11449/144074.
Full text
32
Cavé-Radet, Armand. "Évolution de la tolérance aux Hydrocarbures Aromatiques Polycycliques (HAPs) chez les spartines polyploïdes : analyses physiologiques et régulations transcriptomiques par les micro-ARNs." Thesis, Rennes 1, 2018. http://www.theses.fr/2018REN1B064/document.
Full textAbstract:
Cette étude vise à explorer les mécanismes de tolérance des plantes aux xénobiotiques organiques de la famille des HAPs (phénanthrène), à travers l’analyse de l’impact des évènements de spéciation par hybridation et duplication génomique (allopolyploïdie). Nous avons pour cela mené une approche comparative sur un modèle de spéciation allopolyploïde récente, constitué des espèces parentales hexaploïdes S. alterniflora et S. maritima, et de l’allopolyploïde S. anglica qui résulte de la duplication du génome de leur hybride F1 S. x townsendii. Une approche intégrative basée sur des analyses physiologiques et moléculaires nous a permis de montrer que chez Spartina l’hybridation et le doublement du génome augmentent la tolérance aux xénobiotiques. Le parent paternel S. maritima se montre particulièrement sensible au phénanthrène par rapport au parent maternel S. alterniflora. Différentes analyses transcriptomiques ont permis l’identification de novo de transcrits spécifiquement exprimés en condition de stress, et l’annotation des petits ARNs (miARNs, leurs gènes cibles, et siARNs) agissant en tant que régulateurs de l’expression des gènes et la régulation des éléments transposables. Les analyses d’expression différentielle en réponse au stress ont permis de générer un modèle de régulation (miARN/gènes cibles) en réponse aux HAPs, testé par validation fonctionnelle en système hétérologue chez Arabidopsis. Un travail exploratoire de profilage du microbiome de la rhizosphère des spartines exposées au phénanthrène a été réalisé pour préciser les mécanismes de dégradation des xénobiotiques dans l’environnement en vue d’une application dans les stratégies de remédiation verteWe explored mechanisms involved in tolerance to organic xenobiotics belonging to PAHs (phenanthrene), in the context of allopolyploid speciation (hybrid genome duplication). We developed a comparative approach, using a recent allopolyploidization model including the hexaploid parental species S. alterniflora and S. maritima, and the allopolyploid S. anglica, which resulted from genome doubling of the F1 hybrid S. x townsendii. Integrative approach based on physiological and molecular analyses highlights that hybridization and genome doubling enhance tolerance to xenobiotics in Spartina. The paternal parent S. maritima exhibits higher sensitivity compared to the maternal parent S. alterniflora. Various transcriptomic analyses were performed, to identify de novo stress responsive transcripts, and to annotate small RNAs (miRNAs, their target genes, and siRNAs) involved in gene expression and transposable element regulations. Differential expression analyses in response to stress allowed us to develop a putative miRNA regulatory network (miRNA/target genes) in response to PAH, functionally validated in Arabidopsis as heterologous system. An exploratory profiling of Spartina rhizosphere microbiome exposed to phenanthrene was also performed to characterize environmental degradation abilities, in the perspective of optimizing green remediation strategies
33
Diez, de Revenga Delia Blaya. "Estudio de los microRNAs en la fisiopatología de la hepatitis alcohólica." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/586375.
Full textAbstract:
Las enfermedades hepáticas por alcohol son una de las mayores causas de mortalidad y uno de los principales motivos para el trasplante de hígado en Europa y Norte América. La hepatitis alcohólica (HA) es un episodio agudo que aparece en pacientes con una enfermedad hepática alcohólica. Las formas severas de la HA tienen una mayor mortalidad a corto plazo, además estos pacientes tienen una respuesta insuficiente a los tratamientos actuales. La patofisiología de la HA está caracterizada por: infiltrado de células inflamatorias, daño hepatocelular, esteatosis, fibrosis, reacción ductular y colestasis, aunque mucho aspecto son todavía desconocidos.
Los microRNAs (miRNAs) son moléculas de RNA no codificantes para proteínas, que tienen aproximadamente 22 nucleótidos. Son reguladores post-transcripcionales de la expresión génica uniéndose al RNA mensajero por complementariedad de secuencia. Son capaces de inhibir la traducción a proteína o de provocar la degradación del RNA mensajero. Cada miRNA es capaz de unirse a varios RNA mensajeros diana, pero a su vez cada RNA mensajero puede estar siendo regulado por diversos miRNAs. La expresión global de miRNAs y de genes es contexto dependiente, siendo diferente en cada tipo celular, tejido o condición fisiológica. Esta ampliamente descrito que existe una desregulación de la expresión de los miRNAs en prácticamente todas las enfermedades en las que se han estudiado.
El objetivo de esta tesis doctoral era identificar los miRNAs más relevantes en la fisiopatología de la hepatitis alcohólica y evaluar su función.
Esta tesis doctoral muestra que la HA está caracterizada por un importante cambio en la expresión hepática de los miRNAs. Mediante array de miRNA se analizó la expresión en hígado de pacientes con HA respecto a individuos controles. Se identificaron 117 miRNAs desregulados. Seguidamente se realizó una comparativa del perfil de miRNAs en la HA con los perfiles de otras enfermedades hepáticas crónicas para identificar miRNAs específicamente desregulados en la HA y por tanto potencialmente involucrados en aspectos propios de la fisiopatología de la HA. Hemos identificado 18 miRNAs desregulados específicamente en la HA. A continuación, gracias a un análisis integrativo de la expresión de miRNA y la expresión génica identificamos que los genes potencialmente regulados por los miRNAs están involucrados en el metabolismo y el transporte de los ácidos biliares.
De entre los miRNAs específicamente desregulados en la HA hemos identificado al miR-182 por su alta expresión en los pacientes con HA y posteriormente mostramos la correlación de su expresión con parámetros clínicos, con índices de severidad de la enfermedad hepática y con una mayor mortalidad. Posteriormente evaluamos la expresión del miR-182 en varios modelos animales que representan características clave de la HA como fibrosis, endotoxemia, inflamación, toxicidad por alcohol o reacción ductular. Nuestros resultados mostraron que el miR-182 está principalmente sobreexpresado en un modelo animal con reacción ductular y colestasis, además de estar principalmente expresado por las células de la reacción ductular. Consecutivamente, se realizó un estudio in vivo de pérdida de función del miR-182 en un modelo de reacción ductular y colestasis. Nuestros resultados revelaron una bajada del contenido hepático de ácidos biliares, una bajada de las transaminasas hepáticas y una menor expresión de genes de mediadores inflamatorios. Por otro lado, se estudiaron in vitro los efectos de una sobreexpresión del miR1-82 en diversos tipos celulares. Este estudio mostró que la sobreexpresión del miR-182 tiene mayor impacto sobre colangiocitos provocando una mayor expresión de mediadores inflamatorios. En cambio los efectos sobre hepatocitos y macrófagos fueron menores. Conjuntamente estos resultados sugieren que el miR-182 está involucrado con el daño colangiolar y la inflamación hepática.
Seguidamente, se estudió el papel del miR-155, un miRNA ampliamente descrito por su relación con el sistema inmune y las células inflamatorias. La expresión hepática del miR-155 se encuentra sobreexpresada en el hígado de pacientes con HA, así como en el hígado de pacientes con hepatitis autoinmune (HAI). Sorprendentemente encontramos una reducción de la expresión del miR-155 en las células inflamatorias circulantes de los pacientes con HA y con HAI. A continuación, evaluamos el efecto de la deficiencia del miR-155 en dos modelos animales de daño hepático bien diferenciados. En el modelo de intoxicación por paracetamol no observamos cambios significativos en los animales deficientes en el miR-155. En cambio, en un daño por concanavalina los animales deficientes en miR-155 mostraron un mayor nivel de transaminasas hepáticas y mayor expresión de citoquinas proinflamatorias. Además observamos una reducción del reclutamiento de células CD4+CXCR3+, que podrían representar células T reguladoras infiltradas. Consecutivamente realizamos un trasplante de medula ósea capaz de expresar el miR-155 en animales deficientes; estos animales mostraron una reducción de la expresión de los mediadores inflamatorios, así como menor muerte hepatocelular. Todos estos resultados muestran que la restauración de la expresión del miR-155 en las células inflamatorias podría ayudar a aliviar el daño hepático.
En conclusión, esta tesis doctoral demuestra que existe una desregulación hepática de los miRNAs y que esta desregulación está relacionada con la fisiopatología de la enfermedad, revelando así que los miRNAs son potenciales diana terapéuticas en el tratamiento de las enfermedades hepáticas en general y la HA en particular.Alcoholic hepatitis (AH) develops in patients with underlying ALD and heavy alcohol intake and has a high short-term mortality. The pathogenesis of AH is still poorly understood; it is characterized by inflammatory cell infiltration, steatosis, fibrosis, ductular reaction expansion and cholestasis. MicroRNAs (miRNAs) are small non-coding RNA known to participate in the regulation of important pathophysiological pathways in liver diseases. The main aim of this doctoral thesis was to identify the most relevant miRNAs in the pathophysiology of the AH and evaluate its function. Our results reveal that in the liver of AH patients there are an important dysregulation in the miRNA expression, as compared to normal liver. In addition, 18 miRNAs were found specifically expressed in AH compared to other chronic liver diseases. Furthermore, functional analysis of the potential target genes of these miRNAs showed its involvement in the metabolism and transport of biliary acids. We selected miR-182 since it was the most highly expressed miRNA in AH. The miR-182 showed a positive correlation with severity scores and with short-term mortality. miR-182 is mainly expressed in an animal model of ductular reaction and colestasis; and is mainly expressed in ductular reaction cells. In vivo study of loss-of-function of miR-182 shows a decrease in liver damage, bile acid contents and a reduction in the expression of inflammatory mediators. In addition in vitro studies overexpressing the miR-182 showed a high impact in human cholangiocytes. Subsequently, we evaluated the role of miR-155, a miRNA widely described to be involved with the immune system. Hepatic expression of miR-155 was found to be overexpressed in AH patients, and also in Autoimmune Hepatitis (AIH) patients. Surprisingly, the expression of miR-155 in circulatory inflammatory cells was underexpressed. Next, we evaluated the deficiency of miR-155 in animal models. Concanavalina treatment, a model for AIH, was evaluated. miR-155 deficient animals showed high liver damage and overexpression of inflammatory mediators. Interestingly, these animals had a lower recruitment of CD4+CXCR3+ cells. Subsequently, we restore the expression of miR-155 in the inflammatory cells through bone marrow transplant. We achieved a reduction in the expression of inflammatory genes and hepatocellular damage. Altogether, these results show that restoration of miR-155 on inflammatory cells might alleviate liver damage. In conclusion, thesis demonstrates that there is a hepatic dysregulation of the miRNAs and that this deregulation is related to the pathophysiology of the disease, thus revealing that the miRNAs are potential therapeutic targets in the treatment of liver diseases in general and specifically in AH.
34
Foj, Capell Laura. "Nous biomarcadors per la detecció i pronòstic del càncer de pròstata." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/482236.
Full textAbstract:
INTRODUCCIÓ:
L'antigen prostàtic específic (PSA) segueix sent el biomarcador més utilitzat per a la detecció del càncer de pròstata (CaP), tot i els problemes relacionats amb resultats falsos positius i el sobrediagnòstic de tumors clínicament insignificants.
En els últims anys s'han proposat nous biomarcadors amb l'objectiu d'augmentar l'especificitat i distingir el càncer agressiu del no agressiu.
La hipòtesi principal dels estudis inclosos en aquesta tesi és que els biomarcadors de CaP estudiats: % [-2] proPSA, Prostate Health Index (PHI), PCA3 i miARNs són útils per millorar l'eficàcia en el diagnòstic del CaP, permetent així reduir el nombre de biòpsies negatives obtingudes amb l'algoritme utilitzat actualment. D'altra banda, aquests biomarcadors s'associen amb l'agressivitat del tumor, fet que permet distingir els CaP de baix risc dels d'alt risc de progressió.
METODOLOGIA:
1. Es va realitzar un primer estudi, que incloïa un total de 354 pacients amb biòpsia positiva o negativa. S’ analitzà [-2] proPSA en sèrum i es féu el càlcul de PHI. També es desenvolupà un model multivariat incloent %[-2] proPSA i PHI per predir el CaP en pacients amb PSA <10 μg/L. A més, s’estudià la influència del volum prostàtic en el rendiment diagnòstic d'aquests models. Les dades es publicaren en els articles "Clinical utility of %p2PSA and prostate health index in the detection of prostate cancer" i "The influence of prostate volume in prostate health index performance in patients with total PSA lower than 10 μg/L".
2. En un segon estudi, s’analitzà l'expressió de l'ARN no codificant del gen PCA3 en l’orina de 122 pacients sotmesos a biòpsia per PSA >4 μg/L. Les dades es publicaren a l'article "Real-time PCR PCA3 assay is a useful test Measured in Urine to improve prostate cancer detection".
3. En l'últim estudi, s’analitzaren 5 microARNs en exosomes i sediments de mostres d'orina post massatge prostàtic de 60 pacients amb càncer de pròstata i 10 voluntaris sans. S’estudià la seva expressió mitjançant PCR quantitativa. Les dades es publicaren a l'article "Exosomal and Non-Exosomal Urinary miRNAs in Prostate Cancer Detection and Prognosis".
CONCLUSIONS:
- % [-2] proPSA i PHI són útils en la detecció del CaP i superen el rendiment diagnòstic de PSA total i % PSA lliure.
- % [-2] proPSA i PHI mostren una associació significativa amb l'agressivitat del tumor.
- La implementació d'aquests biomarcadors a la clínica milloraria la precisió en la detecció del CaP, reduint el nombre de biòpsies innecessàries i millorant la predicció de l'agressivitat del tumor.
- La introducció de PHI i %[-2] proPSA millora el rendiment diagnòstic per detectar el CaP d'un model multivariat que inclou PSA total,% PSA lliure, edat del pacient i volum prostàtic.
- El volum prostàtic és un factor clau en la interpretació dels biomarcadors per detectar CaP.
- L'índex de PCA3 és útil en la detecció de CaP, superant el rendiment diagnòstic de PSA total i %PSA lliure.
- No es trobà associació significativa de l'índex de PCA3 amb l'agressivitat del tumor.
- MiR-21, miR-141, miR-214 i miR-375 en els sediments urinaris presenten diferències significatives comparant els grups de pacients amb CaP versus controls sans.
- MiR-21, miR-375 i let-7c en exosomes urinaris presenten diferències significatives comparant els grups de pacients amb CaP versus controls sans.
- MiR-21, miR-141 i miR-214 en sediments urinaris són capaços de distingir de manera significativa els grups de pacients amb CaP d'alt risc i intermedi versus els de baix risc i controls sans.
- MiR-21, miR-375 i let-7c en exosomes urinaris són capaços de distingir de manera significativa els grups de pacients amb CaP d'alt risc i intermedi versus els de baix risc i controls sans.PSA remains as the most used biomarker in the management of early prostate cancer (PCa), in spite of the problems related to false positive results and overdiagnosis. New biomarkers have been proposed in the last years with the aim to increase specificity and to distinguish aggressive from non-aggressive PCa. The main hypothesis of the studies included in this thesis is that several PCa biomarkers (%[-2] proPSA, PHI, PCA3 and miRNAs) are useful for improving the accuracy in the diagnosis of PCa, reducing the number of negative biopsies obtained with the current algorithm. Moreover, these biomarkers are associated with the aggressiveness of the tumor, distinguishing low-risk PCa from high-risk PCa patients. The results suggest that % [-2] proPSA i PHI are useful in the detection of PCa, with higher accuracy than PSA and % freePSA and that they are significantly associated with the tumor aggressiveness’. Therefore, the implementation of these biomarkers in routine will reduce the percentage of unnecessary biopsies. Besides, the inclusion of PHI and % [-2] proPSA in a multivariate model including total PSA, %free PSA, age and prostate volume improves the diagnostic accuracy for PCa. Available results about PCA3 score showed its usefulness to decide the repetition of biopsy in patients with a previous negative result, although its relationship with the aggressiveness of the tumor is controversial. According to our data, PCA3 score is useful in the detection of PCa, surpassing the diagnostic accuracy of total PSA and % free PSA. However, no significant association between PCA3 score and the aggressiveness of the tumor was found. More recently, aberrant microRNA expression in PCa has been reported by different authors. Our preliminary results suggest the utility of several exosomal and non-exosomal urinary microRNAs in the detection and prognosis of PCa. Although several of these new biomarkers have been recommended by different guidelines, large prospective and comparative studies are necessary to establish their value in PCa detection and prognosis.
35
Santasusagna, Canal Sandra. "Exosomes i microRNAs en biòpsia líquida com a biomarcadors pronòstic del càncer de colon." Doctoral thesis, Universitat de Barcelona, 2018. http://hdl.handle.net/10803/482165.
Full textAbstract:
El càncer de colon (CC) és el tercer tipus de càncer més freqüent a nivell mundial i representa la segona causa de mort per càncer en homes i la tercera en dones. La majoria dels CC tenen un origen esporàdic a partir d’un pòlip benigne que progressa a un adenocarcinoma maligne degut a l’acumulació successiva de determinats canvis genètics i epigenètics. El principal factor pronòstic per la recaiguda i la supervivència en el CC és l’estadiatge de la malaltia, essent l’estadi III el que està associat a major risc de recaiguda i pitjor supervivència. El tractament més adequat en funció de les característiques clinicopatològiques de cada pacient és una qüestió controvertida i en constant evolució, sobretot pels pacients en estadi II. Per això actualment està adquirint gran importància l’estudi de nous biomarcadors pronòstic, com els exosomes i els RNAs no codificants petits (microRNAs), mitjançant biòpsia líquida i obtinguts a partir de les venes més pròximes al tumor, per tal de dissenyar un tractament personalitzat més eficient.
Els exosomes són vesícules extracel·lulars (30-100nm) que poden afavorir el desenvolupament tumoral mitjançant la transferència de molècules empaquetades en el seu interior, com microRNAs i proteïnes, a les cèl·lules normals receptores. S’ha descrit que més del 40% del contingut exosomal correspon a microRNAs, l’expressió anòmala dels quals s’ha relacionat clàssicament amb el procés tumoral.
El treball presentat ens ha permès valorar la utilitat dels exosomes i els microRNAs com a biomarcadors pronòstic en el CC. En el primer estudi amb mostres de pacients de CC, s’ha obtingut que alts nivells de miR-21 en la vena que drena directament el tumor primari, la vena mesentèrica (VM), s’associen a un pitjor pronòstic en termes de supervivència lliure de malaltia.
Aquests resultats s’ampliaren al realitzar un array amb 754 microRNAs en VM i vena perifèrica (VP) de pacients quirúrgics de CC d’estadis I-III amb el qual s’obtingué un perfil de 4 microRNAs associats a valor pronòstic: let-7g, miR-15b, miR-155 i miR-328. Alts nivells de let-7g, miR-15b, miR-155 i miR-328, en VM s’associen a pitjor pronòstic en termes de temps a la recaiguda, mentre que, de forma interessant, s’obtenen resultats inversos a l’analitzar tals microRNAs en VP. Aquests resultats suggereixen que el tumor primari de colon allibera altes concentracions de microRNAs i exosomes a través de la VM i aquests nivells queden relativament diluïts en la circulació perifèrica. Aquest fet destaca la importància d’obtenir marcadors sanguinis abans de colonitzar l’òrgan diana. A continuació es van aïllar i caracteritzar exosomes de VM i VP dels pacients de la sèrie estudiada. Es va confirmar la presència de let-7g, miR-15b, miR-155 i miR-
328 en el seu interior mitjançant PCR quantitativa a temps real. El subgrup de pacients que va desenvolupar metàstasi presenta alts nivells de miR-328 exosomal en la VM.
En base a aquests resultats previs, es procedeix a indagar l'efecte de miR-328 sobre els seus gens diana putatius per aprofundir en el mecanisme molecular que exerceix miR-328 durant el procés carcinogènic del CC. S'ha pogut comprovar que miR-328 inhibeix directament SLC2A1 / GLUT1 en dues línies cel·lulars de CC, LOVO i SW480, suggerint que miR-328 podria ser un element clau per modular les característiques metabòliques del microambient i afavorir la formació del nínxol pre-metastàsic, probablement participant en el desenvolupament de l'efecte Warburg.
Finalment es va realitzar un anàlisi proteòmic dels exosomes de VM i VP dels pacients de la sèrie estudiada. Els resultats obtinguts mostren que els pacients recaiguts presenten alts nivells de la proteïna ECM1 exosomal en la VM i aquests s’associen a valor pronòstic.Colon cancer (CC) is the third most common cancer worldwide. The main prognostic factor for relapse and survival is disease stage, and patients with stage III disease have a higher risk of relapse than those with stage II. Surgery is the standard treatment for stage I to III, and adjuvant treatment has been shown to be effective in stage III but less so in stage II. Consequently, the study of new prognostic and predictive biomarkers, such as exosomes and small non-coding RNAs, such as microRNAs (miRNAs), obtained from the tumor-draining vein can provide a useful tool for selecting treatment and improving outcome in these patients. In the present study, we have examined the role of exosomes and miRNAs as prognostic markers in CC. In a previous study, we examined the expression levels of the common oncogenic miRNA miR-21 in plasma samples from the tumor-draining mesenteric vein (MV) of CC patients and found that those with high expression had worse prognosis than those with low expression. In order to further explore this finding, we profiled the expression of 754 miRNAs in plasma samples taken from the MV and peripheral vein (PV) of surgically resected stage I-III CC patients. We found that let-7g, miR-15b, miR-155 and miR-328 in MV plasma were associated with shorter time to relapse. Interestingly, inverse results were obtained when analyzing the same miRNAs in PV plasma, highlighting the importance of obtaining blood biomarkers before they colonize the target organ. Next, we isolated and characterized exosomes from MV and PV and confirmed the presence of let-7g, miR-15b, miR-155 and miR-328 in exosomes. The subgroup of patients who developed metastases had high levels of exosomal miR-328 in MV. Based on these results, we proceeded to investigate the effect of miR-328 on its putative target genes during the development of CC. We found that miR-328 directly inhibits SLC2A1/GLUT1 in the LOVO and SW480 CC cell lines, suggesting that miR-328 could play a key role in modulating the metabolic characteristics of the microenvironment to induce the formation of the premetastatic niche. Finally, we performed a high throughput proteomic analysis in exosomes isolated from MV and PV from the same cohort of patients and found that relapsed patients showed high levels of ECM1 exosomal protein in the MV, which was associated with poor prognosis.
36
Vila, Casadesús Maria. "Design of bioinformatic tools for integrative analysis of microRNA-mRNA interactome applied to digestive cancers." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/663087.
Full textAbstract:
En esta tesis se han desarrollado e implementado distintas herramientas bioinformáticas que permiten el estudio de las interacciones miRNA-mRNA en contextos celulares específicos. oncretamente se ha creado un paquete de R (miRComb) que calcula las interacciones miRNA-mRNA partiendo de expresión de miRNAs y mRNAs, y predicciones bloinformáticas de bases de datos preexistentes. Las interacciones miRNA-mRNA finales son aquellas que muestran una correlación negativa y han estado predichas por al meno una base de datos. Como valor añadido, el paquete miRComb realiza un resumen en pdf con los resultados básicos del análisis (número de interacciones, número de mRNAs target por miRNA, análisis funcional, etc.), que permite comparar los datos de distintos estudios.
Hemos aplicado esta metodología en el contexto de cánceres digestivos. En un primer estudio hemos utilizado datos públicos de 5 cánceres digestivos (colon, recto, esófago, stómago e hígado) y hemos determinado las interacciones miRNA-mRNA comunes entre ellos y específicas de cada uno.
En un segundo estudio, hemos utilizado la misma metodología para analizar datos de IRNA-mRNA en biopsias de pacientes del Hospital Clínic de Barcelona con cáncer de páncreas. En este estudio hemos descrito interacciones miRNA-mRNA en el contexto de cáncer pancreático y hemos podido validar dos de ellas a nivel experimental.
En resumen, podemos concluir que el paquete miRComb es una herramienta útil para el estudio del interactoma de miRNA-mRNA, y que ha servido para establecer hipótesis biológicas que luego se han podido comprobar en el laboratorio.
37
Di, Domenico Tuany. "Avaliação de miRNAs como biomarcadores não invasivos de rejeição aguda em transplante renal." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2014. http://hdl.handle.net/10183/97171.
Full textAbstract:
Introdução: o transplante renal é o tratamento de escolha para uma significativa porção dos pacientes com perda crônica terminal da função renal. A rejeição aguda é uma importante complicação pós-transplante e entre outras disfunções agudas tem na biópsia do enxerto o padrão ouro para o seu diagnóstico. No entanto as biópsias apresentam uma série de limitações e riscos sendo necessário que se desenvolva biomarcadores não invasivos capazes de identificar disfunções do enxerto. Objetivos: analisar e quantificar a expressão dos microRNAs miR-142-3p, miR-155 e miR-210 em amostras de sangue periférico, urina e tecido renal coletadas de pacientes que submetidos à transplante renal que desenvolveram disfunção do enxerto. Métodos: estudo com delineamento transversal e executado no Laboratório de Biologia Molecular aplicado à Nefrologia (LABMAN), do Centro de Pesquisa Experimental do Hospital de Clínicas de Porto Alegre. As amostras são de pacientes submetidos a transplante renal que necessitaram de biópsia, por critério clínico. A expressão dos miRNAs miR-142-3p, miR-155 e miR-210 nos materiais biológicos (tecido renal, sangue periférico e células do sedimento urinário) foi avaliada através da técnica de reação em cadeia da polimerase quantitativo em tempo real. Resultados: foi encontrada, no sangue periférico uma diminuição estatisticamente significativa na expressão do miR-142-3p no grupo de pacientes com rejeição aguda (n=23) quando comparado ao grupo com outras causas de disfunção do enxerto (n=68) (P = 0,01). Não houve diferença entre os grupos na expressão do miR-155 e do miR-210, tampouco para o miR142-3p nos demais compartimentos. Conclusão: miR-142-3p mostra uma expressão diferenciada de rejeição aguda de enxertos renais, há um envolvimento deste marcador no grupo de biomarcadores moleculares em potencial para a disfunção do enxerto renal.Background: kidney transplantation is the treatment of choice for a significant portion of patients with end-stage kidney disease. Acute rejection is a major post-transplant complication among other acute disorders and has on graft biopsy the gold standard for diagnosis. Biopsy, however it is an invasive and potentially harmful procedure so it is desirable to develop new noninvasive markers for diagnosing graft dysfunction. Objective: to analyze and quantify the expression of microRNAs miR-142-3p, miR-155 and miR-210 in the peripheral blood, urinary sediment and kidney tissue obtained from patients who developed graft dysfunction after kidney transplantation. Methods: crosssectional study performed at the Laboratory of Molecular Biology applied to Nephrology (Labman), Center of Experimental Research from Hospital de Clinicas de Porto Alegre. The samples are from kidney transplant patients who undertook indication biopsies as a part of investigation of graft dysfunction. Micro-RNAs expression was evaluated by quantitative real-time polymerase chain reaction. Results: it was found that in peripheral blood, a significant decrease in the expression of miR-142-3p occurred in patients with acute rejection (n = 23) as compared to the group of patients with other causes of graft dysfunction (n = 68), (P = 0.01). No other significant differences were found in gene expression of miR-155 and miR-210, neither for miR142-3p in the other urine or kidney tissue. Conclusion: miR-142-3p presents differential expression in the peripheral blood of patients with rejecting kidney grafts. The role of miRNAs as biomarkers for kidney graft dysfunction is worth be further explored.
38
Martinez, Palacios Paulina. "Réponse des agents non codants du génome – éléments transposables et petits ARN – à un événement d'allopolyploïdie : le génome du colza (Brassica napus) comme modèle d'étude." Thesis, Paris 11, 2014. http://www.theses.fr/2014PA112055/document.
Full textAbstract:
Le succès évolutif de la polyploïdie, notamment de l’allopolyploïdie (où la duplication de génome complet est associée à une hybridation entre génomes différenciés) est en partie lié au fait que cet événement s’accompagne de nombreux changements dans l'organisation du génome et la régulation de l'expression des gènes. On parle du « choc génomique » de l’hybridation interspécifique et de l’allopolyploïdie. Ces sources de diversité génétique, à la fois structurale et fonctionnelle, apparaissent utiles et nécessaires à l'adaptation et l’évolution des espèces. Alors que de nombreuses études portant sur la compréhension des mécanismes moléculaires à l’origine du succès des allopolyploïdes ont concerné les modifications de l’expression des gènes, mes travaux de thèse ont porté sur les agents non codants du génome que sont les éléments transposables et les petits ARN non codants. Le modèle d'étude est le colza (Brassica napus, AACC), espèce allotétraploïde issue de l'hybridation entre les espèces diploïdes navette (B. rapa, AA) et chou (B. oleracea, CC). Nous disposions de colzas néo-synthétisés, étudiés à différentes générations d’autofécondation, permettant de caractériser les changements génomiques accompagnant la formation puis l’évolution du génome néo-allopolyploïde. Une étude a tout d’abord été menée sur un élément transposable (ET) spécifique du génome C, Bot1, en vue d’identifier de nouvelles transpositions survenant chez les colzas néo-synthétisés par rapport aux parents diploïdes, par une approche SSAP. Quelques rares événements de transposition ont été identifiés. Ces résultats, confrontés à ceux obtenus sur deux autres ET, ont permis de mettre en évidence un impact modéré de l’allopolyploïdie sur la transposition de ces différents ET. Par contre, il est apparu que des changements de méthylation auraient accompagné cette allopolyploïdisation, sans doute à l’origine de la réactivation et la transposition de quelques copies de Bot1. Les petits ARN non codants ont été suggérés comme impliqués dans les différents événements génomiques accompagnant la formation d’un génome allopolyploïde. Pour étudier la dynamique d’expression des petits ARN chez des colzas néo-synthétisés pris à deux générations d’autofécondation (S1, S5) en comparaison de leurs parents diploïdes, j’ai exploité des données de séquençage haut débit obtenues pour 11 banques construites à partir des tiges de ces différents génotypes. J’ai ainsi démontré, qu’à une échelle globale, les petits ARN présentaient une réponse immédiate mais transitoire à l’événement d’allopolyploïdie. Les fractions particulièrement affectées par l’allopolyploïdie se sont révélées correspondre (1) à des petits ARN interférents dérivés d’éléments transposables avec une baisse de leur abondance en génération précoce S1, et (2) à des populations de petits ARN de 21 nucléotides exprimées uniquement de manière très précoce, de l’hybride F1 à la génération S1. Nous avons notamment identifié des transcrits de type viral correspondant à ces petits ARN de 21-nt, et présentant les mêmes profils d’expression (de l’hybride F1 à la génération S1), suggérant une réactivation d’éléments viraux endogènes (EVE) en réponse à l’hybridation et l’allopolyploïdie. L’ensemble de mon étude a démontré la mise en place d’une succession des voies de régulation par petits ARN où ET et EVE, réactivés au niveau transcriptionnel, sont immédiatement soumis à une répression post-transcriptionnelle (PTGS), renforcée ensuite par une répression de leur transcription (TGS). L’hypothèse d’une absence de cette régulation par petits ARN lors des phénomènes de nécrose et létalité hybride, amène à envisager ces populations de petits ARN comme les clés de la réussite de la formation d’un génome hybride, où la répression immédiate et efficace des ET et autres endovirus, réactivés suite au choc génomique, se révèle être une nécessitéThe evolutionary success of polyploid species is partly due to the dynamic changes in genome organization and gene expression patterns that occur at the onset of the polyploid formation. These changes are promoted by the merging of divergent genomes into a single nucleus (i.e. allopolyploidy) that causes a “genomic shock”; they are thought to provide a rich source of new genetic material upon which selection can act to promote adaptation and evolution. Many studies have thus aimed to uncover molecular mechanisms that are responsible for the evolutionary success of allopolyploid species, most of them focusing on gene expression changes. In the present PhD thesis, my interest has been concentrated on the non-coding components of the genome: transposable elements and small non-coding RNAs. My study involves oilseed rape (Brassica napus, AACC), a relatively young allopolyploid species that originated from hybridizations between B. rapa (AA) and B. oleracea (CC). Specifically, I have used resynthesized B. napus polyploids advanced by self-pollination of single plants for several generations; I have analyzed these plants at different generations for genomic changes accompanying polyploid formation and subsequent evolution. In a first part, sequence-specific amplification polymorphism (SSAP) targeting the C genome-specific transposable element Bot1, was used to evaluate transposition rate of Bot1 in resynthesized B. napus in comparison with the diploid parents. Only a few transposition events were identified. When combined with the results obtained for two other TEs, this work suggests that allopolyploidy has only a moderate impact on TE transposition and restructuring. The changes observed in SSAP profiles led us to hypothesize that some of them resulted from changes in DNA methylation, resulting in rare but highly specific TE activation and transposition. In a second part, I have concentrated on small non-coding RNAs (sRNAs), which are thought to mediate different aspects of the response to the “genomic shock” induced by allopolyploid formation. Comprehensive analyses of sRNA expression in resynthesized B. napus allopolyploids have been carried out by deep sequencing sRNAs from 11 libraries prepared from stems of three allotetraploids (surveyed at the two generations S1 and S5) and the two diploid parents. Characterization of sRNA distributions in these plants indicates that sRNAs show an immediate but transient response to allopolyploidy. The sRNAs derived from transposable elements (down-regulated in the S1) or targeting unknown sequences (no Blast hit against any available public database) were particularly affected. The use of B. napus mRNAseq data revealed that these latest unknown candidates, which are 21-nt long and over-expressed in the earliest generations (F1, S0, S1) were derived from endogenous viral elements (EVE). We confirmed that these EVEs showed the same expression patterns as the 21-nt long sRNAs that specifically target them (over-expression in the F1, S0 and S1). These results suggest that (at least) some EVEs might be reactivated as a response to the merging of divergent genomes (in interspecific hybrids and newly formed allopolyploids). Altogether, our results have demonstrated a succession of sRNA pathways that counteract the reactivation of some specific TEs and/or EVEs at the onset of polyploid formation; reactivated TEs and/or EVEs being immediately repressed at the post-transcriptional level (PTGS), and then fully repressed by transcriptional gene silencing (TGS) in the subsequent generations. Such data lead to hypothesize that sRNAs are essential to overcome interspecific hybrid incompatibilities due to the uncontrolled and deleterious reactivation of TEs / EVEs. Therefore, sRNAs should be considered as the guardians of genome integrity even in newly-formed allopolyploids
39
Laugier, Laurie. "Identification de marqueurs de susceptibilité dans les formes chroniques de la maladie de Chagas." Thesis, Aix-Marseille, 2017. http://www.theses.fr/2017AIXM0226.
Full textAbstract:
La maladie de Chagas est une maladie parasitaire causée par le protozoaire Trypanosoma cruzi et transmise par des insectes hématophages . Elle est composée de 2 phases : la phase aiguë et la phase chronique. Parmi les individus infectés, 30 % développent la forme chronique de la maladie. Les patients présentent des atteintes cardiaques, digestives (œsophage, côlon) et cardiodigestives. Notre étude a été focalisée sur les patients atteints de cardiomyopathie chagasique (CCC). Notre objectif est d’identifier des gènes de susceptibilité pouvant être impliqués dans le développement des formes chroniques. Notre étude a permis de mettre en évidence une variation d’expression de certains gènes entre les CCC et les contrôles. Nous nous sommes également intéressés aux processus épigénétiques pouvant réguler l’expression des gènes. Une étude de la méthylation de l’ADN croisée avec l’étude du transcriptome nous ont permis d’identifier des gènes présentant à la fois des variations d’expression et de méthylation. Pour certains de ces gènes, nous avons démontré que la méthylation est responsable de la variation d’expression observée. Enfin, nous avons étudié un ARN long non-codant, MIAT. Nous avons démontré qu’il est surexprimé chez les CCC par rapport aux contrôles et dans un modèle murin infecté par T. cruzi. De plus, l’analyse de l’expression de micro-ARNs couplée à une analyse de transcriptome nous a permis d'identifier plusieurs micro-ARNs indispensables à la régulation de l’expression des gènes. Enfin, une étude protéomique nous a permis de mettre en évidence une augmentation de la production de protéine pour certains gènes, en lien avec l’augmentation de l’expression observéeChagas disease is a parasitic disease caused by the protozoan Trypanosoma cruzi and transmitted by the hematophagous insects. The disease is composed by acute and chronic phases. Among the infected individuals, 30 % develop chronic form. They suffer from heart, digestive (esophagus, colon) and cardiodigestives injury. Our study was focused on patients with dilated chagasic cardiomyopathy (CCC). Our goal is to identify susceptibility genes that may be involved in the development of chronic forms. Our study revealed a variation in the expression of certain genes between CCC group and controls. We are also interested in epigenetic processes that can regulate the expression of genes. A study of the DNA methylation crossed with the transcriptome allowed us to identify genes presenting both variations in expression and methylation. For some of these genes we demonstrated that methylation is responsible for the expression variation observed. Finally, we studied a long non-coding RNA called MIAT. Our study demonstrated that it is overexpressed in CCC compared to controls and in a murine model infected by T. cruzi. Furthermore, the analysis of the expression of micro-RNAs crossed with transcriptome analysis allowed us to identify several micro-RNAs whose functions are essential in the regulation of gene expression. Finally, a proteomic study allowed us to demonstrate an increase in the production of protein for certain genes, correlated with the increase in expression levels observed
40
Sánchez-Cid, Pérez Lourdes. "Análisis de los perfiles de expresión de microRNAs en cáncer de mama y de la implicación de la familia miR-200 en metástasis." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/402827.
Full textAbstract:
Esta tesis consiste en el estudio de la regulación por microRNAs del proceso de metástasis de carcinomas ductales invasivos de mama. Hemos encontrado una expresión alterada de varios microRNAs en muestras tumorales, incluyendo las familia miR-200 y miR-181, miR-210, miR-101 y miR-10b, a lo largo de la progresión desde tumores primarios a metástasis regionales y a distancia. miR-7, miR-30, miR-148 y miembros de la familia let-7 se encontraron diferencialmente expresados entre tumores primarios no metastáticos vs primarios metastáticos a ganglios linfáticos. Además, los miembros del cluster 1 de la familia miR-200 se econtraban sobreexpresados en las metástasis ganglionares, las cuales mantenían o tenían una expresión más elevada de E-cadherina respecto de sus correspondientes tumores primarios. Adicionalmente y de acuerdo con la tendencia observada en los tejidos tumorales, observamos niveles elevados de miR-200b y miR-7 en la sangre de pacientes con metástasis regional o a distancia en comparación con pacientes con tumores primarios no metastáticos en el momento del diagnóstico.
Centrándonos en la significación biológica de la mayor expresión de los miembros de la familia miR-200 en asociación con la progresión metastática, utilizamos el modelo celular MCF10CA1h para inducir la expresión de miR-200. Observamos que miR-200 promovía una transición mesénquima-epitelio que conllevaba la inducción de un marcado programa epitelial. Éste era acompañado de un fuerte incremento de la actividad aldehido deshidrogenasa (ALDH), mayor capacidad de crecimiento en mamoesferas y la transición de un immunofenotipo CD44+ CD24- a CD44+ CD24+, lo cual era indicativo de la adquisición de características de progenitor luminal por parte de células que expresaban miR-200. Además, las células MCF10CA1h que expresaban miR-200 desarrollaron la capacidad de diferenciarse y organizarse en estructuras tubuloalveolares ramificadas similares a las de la mama normal. Se observó además una mayor capacidad de crecimiento tumoral y de colonización metastática in vivo. Además de la expresión de marcadores epiteliales, la expresión de miR-200 resultó en la inducción de la expresión de marcadores basales y de diferenciación luminal in vitro y de forma más prominente in vivo. Todo ello refueza la idea de que miR-200 induce características de progenitor luminal así como de agresividad en células tumorales mamarias. A nivel mecanístico encontramos que la mayor capacidad de autorrenovación promovida por miR-200 era debida en parte a la inhibición de su ya conocidad diana ZEB2 así como de una activación de la ruta de señalización PI3K-AKT.
Finalmente, la morfología de los tumores formados in vivo por células que expresaban miR-200 recordaba a la de los carcinomas metaplásicos mamarios, por lo que decidimos estudiarlos. De hecho, observamos que el componente epitelial de tumores metaplásicos expresaban niveles significativamente superiores de miR-200 que el componente mesenquimal y además, mostraban un perfil de marcadores compatible con el de células luminales progenitoras.
En vista de los resultados, proponemos que los microRNAs de la familia miR-200 inducen características de células progenitoras luminales, las cuales asociadas a un fenotipo epitelial, promueven la capacidad metastática de las células tumorales.The maintenance of an epithelial gene program is essential for the metastatic growth of epithelial cancers, including breast cancer. However, little is known of the molecular and cellular mechanisms leading to the enhanced proliferative and survival properties of metastatic epithelial cells. We report here that forced expression of miR-200s in MCF10CA1h mammary cells induced not only a strong epithelial program but also aldehyde dehydrogenase (ALDH) activity, mammosphere growth and ability to form branched tubuloalveolar structures while promoting orthotopic tumor growth and lung colonization in vivo. This was accompanied with the expression of luminal differentiation markers in vitro and in vivo, the overall phenotype being compatible with a promotion of luminal progenitor traits. Interestingly, the morphology of tumors formed in vivo by MCF10CA1h cells expressing miR-200s was reminiscent of metaplastic breast cancer (MBC) and the epithelial components of MBC samples expressed significantly higher levels of miR-200s than their mesenchymal components and displayed a marker profile compatible with luminal progenitor cells. Additionally, the miR-200 family showed enhanced expression along progression of invasive ductal breast cancer (IDC) from primary tumors to distant metastases, further reflected in higher blood levels of miR-200b in patients with regional or distant metastases relative to patients with primary node-negative tumors. We propose that the expression of epithelial gene programs through miR-200 family microRNAs promote traits of highly proliferative mammary luminal progenitor cells, thereby exacerbating the growth and metastatic properties of transformed mammary epithelial cells.
41
Rodríguez, Rius Alba. "Study of the Regulatory Mechanisms of Gene Expression in Venous Thromboembolic Disease: microRNAs." Doctoral thesis, Universitat de Barcelona, 2020. http://hdl.handle.net/10803/671757.
Full textAbstract:
Venous Thrombosis (VT) is a frequent complex disease that involves a disruption of the balance of the hemostatic system. microRNAs (miRNAs) are small non-coding RNAs that regulate gene expression. Promising findings in other complex diseases encourage their use as clinical biomarkers. However, up to now, the implication of miRNAs in VT has not been studied in-depth.
The main objective of this Thesis was to study the regulatory mechanisms of the gene expression through miRNAs in VT. The specific objectives were (1) to identify a plasma miRNA profile associated with VT and to analyze its suitability as biomarker, (2) to dissect the interactions between biological layers in the biological context of VT and (3) to identify factors affecting either expression or quantification of miRNAs in plasma.
In the first article, the differential expression of miRNAs in plasma was analyzed in the ‘Genetic Analysis of Idiopathic Thrombophilia’ 2 (GAIT-2) Project, which involves 935 individuals belonging to 35 extended Spanish families with idiopathic thrombophilia. First, we conducted a discovery phase, in which 752 miRNAs were measured in plasma by quantitative Polymerase Chain Reaction in 104 individuals of GAIT-2 (52 VT cases and 52 controls). Sixteen miRNAs were selected, which were measured in the entire GAIT-2 (n=935). Four of the miRNAs were significantly associated with VT (false discovery rate
<0.1): hsa-miR-885-5p, hsa-miR-126-3p, hsa-miR-192-5p and hsa-miR-194-5p. All the four miRNAs returned significant odds ratio for VT, in the range 1.3-2.12. The discriminatory ability of the profile including the four miRNAs, age and sex retuned an area under the curve (AUC) of 0.7. In addition, significant correlations were found between the miRNAs and clinical VT phenotypes.
In the second article, the four miRNAs identified above were integrated with the gene expression levels of 260 genes of the blood coagulation pathway and 14 clinical VT phenotypes. 51 VT cases and 51 controls of the GAIT-2 Project were included. Feature selection was conducted by the building of linear models for VT discrimination, which were then optimized using penalized regression. We obtained three models with AUC >0.7. The first model (VT ~ GATA2 + von Willebrand Factor) showed that the expression of GATA2 in blood was inversely correlated with the blood levels of von Willebrand Factor, and that the disruption of this relationship represents a prothrombotic phenotype. The second model (VT ~ Factor IX, ANXA2 + ENTPD1 + ILK + PDPK1 + PRKAR1A + STXBP3 +hsa-miR-
885-5p + hsa-miR-192-5p) represented an interaction between the fibrinolytic system and platelet activation through the αIIbβ3 signaling pathway. The third model (VT ~CSRP1+ LYN + hsa-miR-192-5p+ hsa-miR-885-5p) revealed the interaction between two group of genes involved in platelet activation, correlated with Protein S, and two miRNAs, in the biological context of VT.
In the third article, we used the miRNA expression data of the discovery phase (103 miRNAs in 104 subjects) to analyze the effect of biological and technical factors in their expression. First, we found that the hemolysis marker represented ~10% of the shared variability of miRNA expression. Therefore, using this value as a continuous covariate, beyond as a categorical control, could help to increase consistency across miRNA studies. Second, we found that the expression of miRNAs in plasma was not biased by any blood cell count. Then, we identified 1,323 genetic variants associated with the expression of 16 miRNA genes, that represent 158 independent loci. Finally, we found that these loci were enriched in promoter regions from several tissues, though not in blood tissue. This finding is in agreement with the results regarding blood cell counts, and encourages the role of circulating miRNAs as biomarkers of tissue specific conditions.El objetivo general de esta Tesis fue estudiar los mecanismos de regulación de la expresión génica mediante microRNAs (miRNAs) en la trombosis venosa (VT). En el primer artículo, se explora la expresión diferencial de miRNAs en VT utilizando la población del proyecto ‘Genetic Analysis of Idiopathic Thrombophilia’ 2 (GAIT-2), compuesta por 935 individuos de 35 familias con trombosis idiopática. El diseño experimental implicó una fase de descubrimiento en la que 752 miRNAs plasmáticos se cuantificaron en 104 individuos del GAIT2 (52 casos y 52 controles) y se seleccionaron 16 miRNAs para la fase de validación, en la que se cuantificaron en toda la población GAIT-2 (n=935). Cuatro de los miRNAs se asociaron significativamente con la enfermedad (false discovery rate <0.1): hsa-miR-885-5p, hsa-miR-126-3p, hsa-miR-192-5p y hsa-miR-194-5p. El segundo artículo supone la integración de los cuatro miRNAs identificados con los niveles de expresión en sangre de 260 genes de la vía de la coagulación y con 14 fenotipos clínicos de VT. Se incluyeron 102 individuos del GAIT-2 y se llevó a cabo mediante la construcción de modelos lineales para la discriminación de VT. El primer modelo reveló que los niveles de expresión de GATA2 están inversamente correlacionados con los niveles de Factor von Willebrand y que la disrupción de dicha relación representa un fenotipo protrombótico. El segundo modelo representó una interacción entre el sistema fibrinolítico y la activación plaquetaria mediante la vía de señalización αIIbβ3. El último modelo identificó dos grupos de genes implicados en la activación plaquetaria, correlacionados con la Proteína S, y dos miRNAs. En el tercer artículo, se analiza el efecto de variables biológicas y técnicas en la expresión de miRNAs en plasma. Primero, identificamos que utilizar el marcador de hemolisis como covariable continua podría ayudar a mejorar la concordancia entre estudios de miRNAs. Segundo, de nuestros datos se desprende que los conteos celulares no sesgan sistemáticamente la expresión de miRNAs en plasma. Por último, identificamos 1,323 variantes genéticas asociadas con la expresión de 16 genes de miRNAs, que suponen 158 loci independientes, enriquecidos en promotores de diversos tejidos, aunque no de sangre.
42
Boces, Pascual Clara. "Implicació del miRNorma tumoral en l´expressió del transportador concentratiu de nucleòsids hCNT1." Doctoral thesis, Universitat de Barcelona, 2020. http://hdl.handle.net/10803/672712.
Full textAbstract:
Human Concentrative Nucleoside Transporters (hCNTs) are encoded by SLC28 gene family. It has been demonstrated that hCNT1 is lost in some cancers. Furthermore, hCNTs have the ability of translocating selected nucleoside- derivatives, currently used in anticancer therapies, for that reason, a change in their expression profile could affect drug bioavailability and drug chemoresistance. Recent studies demonstrate that hCNT1 has also additional functions, being a transceptor and it is likely to be relevant in tumor biology and during the carcinogenic process. Although mechanisms responsible for hCNT1 loss during carcinogenesis are unknown, it seems likely that epigenetic modifications might be able to affect hCNT1 expression and functional activity. miRNAs are small, non-codding transcripts which play a key role during the carcinogenic process with a discriminatory expression profiles significantly different to healthy individuals. miRNAs pair to mRNA by complementarity to 3’UTR extreme of the target gene, modulating its expression. The objective of this work aims to elucidate miRNAs associated with hCNT1 loss of expression in CRC, PDAC and HCC. The analysis of paired clinical samples of CRC, PDAC and HCC confirm the decrease of hCNT1 compared to the adjacent non-tumoral tissue. Also, increased expression of miR-106a, miR-17 and miR- 18a is correlated with hCNT1 loss of expression in the same clinical samples. The luciferase assay validates hCNT1 as a direct target of miR-106a and miR-17. Furthermore, the modulation of these miRNAs could affect hCNT1 mRNA and protein levels. A new 3D cell culture model (spheroids) is presented to increase basal levels of endogenous hCNT1 compared to monolayer cell culture. This new model allows to evaluate hCNT1 modulation by miRNAs as an easy and complete way. The results obtained in spheroid culture confirm the ability of miR-106a and miR-17 to regulate hCNT1 expression. Moreover, miRNAs silencing increase gemcitabine effect in spheroids. All these results propose miR-106a and miR-17 as possible new therapeutic targets to improve nucleoside analogs treatment by increasing hCNT1 and also the possibility of using them as biomarkers.
43
Ruiz, Martinez Marc. "Estudio del gen YKT6 y miRNAs asociados en la liberación de exosomas en pacientes quirúrgicos de cáncer de pulmón." Doctoral thesis, Universitat de Barcelona, 2016. http://hdl.handle.net/10803/401865.
Full textAbstract:
En los últimos años los exosomas han emergido como importantes mediadores en el desarrollo de cáncer, demostrándose su utilidad como biomarcadores para diagnóstico y pronóstico de pacientes oncológicos. Los exosomas son vesículas extracelulares de entre 30-100nm de diámetro de origen endosomal que pueden favorecer diferentes procesos importantes en el progreso tumoral, como la modulación del microambiente tumoral, inmunosupresión y preparación del nicho premetastático. El estudio de la biogénesis y liberación de los exosomas se ha centrado principalmente en los mecanismos ESCRT y de proteínas Rab, postulándose también la importancia de las proteínas SNARE en estos procesos. YKT6 es una proteína SNARE que se ha descrito como necesaria para la liberación de exosomas en diferentes tipos celulares. Sin embargo el papel que podría tener en la liberación de exosomas en cáncer de pulmón no se había investigado. El trabajo realizado nos ha permitido investigar la importancia de YKT6 en el proceso de liberación de exosomas en líneas celulares, sus mecanismos de regulación mediante microRNAs (miRNAs), así como también evaluar el posible papel pronóstico en pacientes de cáncer de pulmón de célula no pequeña. En el estudio in vitro con la línea celular A549 de cáncer de pulmón observamos que la inhibición de YKT6 producía una disminución drástica en la liberación de exosomas. Además, identificamos y validamos miR- 34a, miR-134, miR-135a y miR-135b como miRNAs capaces de regular la expresión del ARNm de YKT6. El efecto de la sobreexpresión de miR-134 y miR-135b, los miRNAs que más efectivamente disminuían la expresión de YKT6 también provocaron una disminución en la liberación de exosomas en las células A549. En los pacientes de cáncer de pulmón de célula no pequeña observamos que la expresión de YKT6 estaba infraexpresada y que en contraste la expresión de los miRNAs miR-134 y miR-135b se encontraban sobrexpresados en tejido tumoral respecto al tejido normal. Aunque en el tejido tumoral no se pudo apreciar ninguna correlación negativa entre los miRNAs y la expresión de YKT6, en el tejido normal si que observamos la correlación negativa significativa entre miR-135b y la expresión de YKT6. El análisis de la expresión de YKT6 en las muestras tumorales de los pacientes resultó en la clasificación de los pacientes según niveles de expresión alto o bajo de YKT6, observándose un peor pronóstico en el grupo de pacientes con alta expresión en términos de una menor supervivencia global y supervivencia libre de enfermedad. Estos resultados indican que el estudio de los procesos de liberación de exosomas y sus posibles mecanismos de liberación, en este caso YKT6 a través de miRNAs, pueden ser de utilidad en cáncer.Cancer-derived exosomes are involved in metastasis. YKT6 is a SNARE protein that participates in the regulation of exosome production and release, but its role in non- small cell lung cancer (NSCLC) has not been examined. This study has allowed us to investigate the importance of YKT6 in the process of releasing exosomes in cell lines, its mechanisms of regulation by microRNAs (miRNAs), as well as to evaluate the possible prognostic role in patients with non-small cell lung cancer . In the in vitro study with the lung cancer cell line A549, we observed that the inhibition of YKT6 produced a drastic decrease in the release of exosomes. In addition, we identified and validated miR-34a, miR-134, miR-135a and miR-135b as miRNAs capable of regulating the expression of YKT6 mRNA. The effect of overexpression of miR-134 and miR-135b, miRNAs that most effectively decreased YKT6 expression also led to a decrease in the release of exosomes in A549 cells. In non-small cell lung cancer patients we observed that the expression of YKT6 was underexpressed and that in contrast the expression of miRNAs miR-134 and miR-135b were overexpressed in tumor tissue compared to normal tissue. Although no negative correlation between miRNAs and YKT6 expression in the tumor tissue was observed, in the normal tissue, we observed a significant negative correlation between miR-135b and YKT6 expression. Analysis of YKT6 expression in patient tumor samples resulted in the classification of patients according to high or low expression levels of YKT6, with a worse prognosis in the group of patients with high expression in terms of lower overall survival and disease-free survival. These results indicate that the study of the exosome release processes and their possible release mechanisms, in this case YKT6 through miRNAs, may be useful in cancer.
44
Guasch, i. Casany Eduard. "Estudi de la Fibril·lació Auricular: de la fisiopatologia al tractament." Doctoral thesis, Universitat de Barcelona, 2012. http://hdl.handle.net/10803/127299.
Full textAbstract:
La fibril.lació auricular és l’arítmia cardiaca més freqüent, amb una elevada morbimortalitat i impacte econòmic. Augmentant-ne la importància epidemiològica, s’espera que la seva incidència s’incrementi de forma molt marcada com a conseqüència de l’envelliment de la població. En aquesta tesi doctoral, estudio la fisiopatologia i el tractament de la fibril.lació auricular a través d’un abordatge experimental i clínic.
L’exercici físic de molt elevada intensitat és una de les causes emergents de fibril.lació auricular, especialment en individus joves i de mitjana edat. La fisiopatologia n’és desconeguda. En aquesta tesi, en un model exeperimental d’exercici físic en rata, es demostra mitjançant tècniques in vivo i in vitro que la hipertonia vagal característica dels atletes és un factor clau en el desenvolupament de la fibril.lació auricular de l’atleta. El substrat aritmogènic induit per l’exercici és reversible, especialment la hipertonia vagal. Per contra, el remodelat estructural (dilatació auricular i fibrosi) reverteixen de forma molt més lenta o incompleta.
Els microRNA són seqüències curtes de RNA, recentment descrites, que participen en la regulació post-transcripcional de l’expressió génica. Els microRNA participen en gran quantitat de processos fisiològics i patològics, entre els quals el desenvolupament de fibrosi en diferents teixits. Tanmateix, es desconeix la seva participació en el desenvolupament del substrat de la fibril.lació auricular. En aquesta tesis, s’estudia la participació de miR-21 en el remodelat auricular post-infart de miocardi ventricular. S’usa un model en rata en el que objectivem una elevada inducibilitat de fibril.lació auricular. Es demostra que miR-21 participa en el desenvolupament de fibrosi miocàrdica auricular, i que la seva inhibició és capaç de prevenir-ne el desenvolupament i la inducibilitat de fibril.lació auricular. Es postul.la miR-21 com a possible diana terapéutica en el tractament de la fibril.lació auricular.
El tractament anticoagulant constitueix la base de la prevenció del risc embòlic en la fibril.lació auricular. Ocasionalment, els pacients diagnosticats de fibril.lació auricular pateixen malaltia coronària com a comorbilitat, i requereixen tractament antiplaquetari amb aspirina i tienopiridines. El risc hemorràgic i trombòtic de la triple combinació d’anticoagulació, aspirina i tienopiridina roman desconeguda. En aquesta tesi estudiem aquest problema en pacients sotmesos a intervencionisme coronari i mostrem que es presenta en un de cada vuit pacients. En el seguiment, els pacients als que se’ls prescriu la triple combinació antitrombòtica presenten una incidència significativament menor d’esdeveniments trombòtics que els que no la van rebre, amb una major incidència d’hemorràgies no greus com a contrapartida.Atrial fibrilliation is the most common cardiac arrhythmia, with a high morbidity, mortality and economic burden. An aging population in the upcoming years will likely increase its incidence and intensificate its epidemiological importante. This doctoral thesis approaches the pathology and therapy of atrial fibrillation through clinical and experimental studies. Very high intensity exercise is an emerging cause of atrial fibrillation in young and middle-aged individuals. However, its pathophysiology is unknown, yet. Here, a rat model of endurance, very high intensity exercise is used to study its mechanisms. By mean of in vivo and in vitro techniques, vagal enhancement is shown to be key factor in the development of exercise-induced atrial fibrillation. Moreover, exercise-induced arrhythmogenic atrial substrate is reversible, particularly concerning vagal enhancement. In contrast, structural remodeling (atrial fibrosis and dilatation) slowly or uncompletely reversed. MicroRNAs are recently discovered short RNA sequences that regulate post-transcriptional gene expression. MicroRNAs are involved in a large number of physiological and pathological processes, including fibrosis development. However, it is unknown whehter they participate in atrial fibrillation substrate. In this thesis, miR-21 involvement in post-left ventricle myocardial infarction atrial remodeling is explored. A left ventricle myocardial infarction rat model with a high burden of atrial fibrillation inducibility is used. We show that miR-21 is involved in atrial fibrosis pathology, and its inhibition may prevent its development and atrial fibrillation inducibility. Mir-21 is postulated as a potential therapeutic target in the treatment of atrial fibrillation. Anticoagulant therapy is on the basis of thrombo-embolic risk prevention in atrial fibrillation patients. Occasionally, patients diagnosed with atrial fibrillation are also diagnosed of ischemic cardiomyopathy and require antiplatelet therapy with aspirin and tienopiridines. The hemorrhagic risk of triple thrombotic combination of anticoagulation, aspirin and thienopiridines is unknown. In this thesis we study this problem in patients undergoing coronary intervention and show that this occurs in one out of eight patients in the cath lab. At follow-up patients who were prescribed triple combination antithrombotic had a significantly lower incidence of thrombotic events than those who did not receive it. A higher risk of non-serious bleeding balanced its benefits.
45
Raimondi, Giulia. "Broadening Adenoviral Oncolysis in PDAC: Interrogation of Patient-Derived Organoids for personalized virotherapy and modulation of miRNA content to boost adenoviral potency." Doctoral thesis, Universitat de Barcelona, 2020. http://hdl.handle.net/10803/671205.
Full textAbstract:
The general goal of this thesis has been to progress oncolytic adenovirus therapy for PDAC, by the incorporation of novel preclinical models to test for patient-specific responses and the generation of oncolytic adenoviruses with enhanced therapeutic index.
The two main objectives have been the following:
i) Evaluate patients-derived organoids (PDOs) technology as a platform to screen for personalized virotherapy in vitro
1) Establishment of a battery of PDOs from PDAC and normal pancreatic tissues, and evaluation of their applicability in the study of adenoviral infection;
2) Screening of a battery of PDOs to identify individual sensitivities to virotherapies, and the effects derived from the combination with chemotherapy;
3) Study virotherapy-responses in metastasis originated from
PDOs xenografted in mice;
(ii) Improve oncolytic adenovirus potency by modulation of miRNAs deregulated in PDAC
4) Screening of aberrantly expressed miRNAs sensitizing viral oncolysis in PDAC via CRISPR/Cas9 system;
5) Generation of a miRNA sponge-adenovirus and evaluation of its oncolytic effects in vitro and in vivo;
6) Modulation of miRNA levels with the THZ1 transcriptional inhibitor, and assessment of the effects of its combination
with oncolytic adenoviruses.
46
Ventayol, Espinazo Marina. "Paper del miRNA en la diferenciació de les cèl.lules mare." Doctoral thesis, Universitat de Barcelona, 2013. http://hdl.handle.net/10803/109374.
Full textAbstract:
Les patologies renals s’han convertit en una problemàtica a nivell mundial, en l’actualitat poden afectar a una de cada 9 persones al llarg de la seva vida i porten associats elevats costos econòmics. Malgrat els últims avenços científics i millores en el tractament d’aquestes patologies, el transplantament renal i la diàlisi continuen sent les dues úniques opcions terapèutiques efectives en el tractament de la insuficiencia renal. La regeneració de l’epiteli renal és determinant en la recuperació del pacient ja que determina que es pugui restablir la funcionalitat d’aquest òrgan. L’obtenció de precursors renals a partir de cèl•lules mare que puguin integrar-se i regenerar el ronyó lesionat s’ha convertit en una àrea de recerca biomèdica molt important.
L’objecte d’estudi d’aquesta tesi va ser conèixer els mecanismes involucrats en la diferenciació de les cèl•lules mare embrionàries (ESCs) i les cèl•lules mare adiposes (ASCS) cap al llinatge epitelial renal ja que aquestes cèl•lules poden ser una potencial font d’aquests precursors renals amb capacitat regenerativa. Recentment s’ha descobert que els miRNAs, que tenen la funció de regular l’expressió gènica en l’etapa post-transcripcional, són essencials en la diferenciació de les cèl•lules mare i s’han trobat miRNAs específics que regulen la diferenciació a un llinatge cel•lular en concret. En aquest sentit, el nostre objectiu general en aquest treball va ser estudiar el paper dels miRNAs en la diferenciació de cèl•lules mare a progenitors epitelials renals.
Per això, primer de tot es van realitzar experiments de diferenciació en que els cossos embrionaris (EBs) induïts a partir de les cèl•lules mare embrionàries (ESCs) es van cultivar amb un medi de cultiu suplementat amb all-trans-retinoic acid (ATRA) i activina A, i les cèl•lules mare adiposes (ASCs) amb un medi amb una concentració fisiológica de glucosa suplementat amb ATRA. Amb aquests protocols de diferenciació, es van obtenir cèl•lules amb característiques de progenitors renals. Els EBs cultivats amb el protocol de diferenciació expressaven els marcadors de l’organogènesi renal inicial (Pax2, WT1, Wnt4, Notch2 i Wnt9b). Les ASCs cultivades amb ATRA varen canviar la seva morfologia a una morfologia epitelial i van expressar marcadors tant de l’organogènesi renal inicial (Pax2, WT1, Wnt4, Six2 i Megalina) com els marcadors epitelials (Citoqueratina 18, E-caderina).
Un anàlisis de miRNAs va demostrar que la família de miRNAs let-7 es sobreexpressava durant la diferenciació dels EBs i que el miRNA let-7e més concretament era essencial en l’expressió dels marcadors Pax2, Wt1i Wnt4 ja que la seva silenciació disminuïa l’expressió d’aquests gens.
En les ASCs, en canvi, es va demostrar que el miRNA let-7e també augmentava en la diferenciació i que a més tenia característiques d’inductor de la diferenciació, essent essencial en l’expressió tant dels marcadors de l’organogènesi renal (Pax2, Wt1, Wnt4, Megalina) com del marcador epitelial CK18.
Profunditzant en el paper del miRNA let-7e en la diferenciació, es va demostrar amb experiments de Western blot, que el miRNA let-7e modula els nivells de β-catenina a través d’un mecanisme que implica la inhibició de la PKCβ i la conseqüent disminució en la fosforilació de la GSK3β (Ser-9) i que això és imprescindible per la correcta diferenciació de les ESCs.
Per altra banda, en les ASCs es va demostrar utilitzant l’assaig del gen reporter de la luciderasa, que el miRNA let-7e inhibeix directament l’expressió de la metal•loproteinasa 9 i que d’aquesta manera modula la diferenciació de les ASCs al llinatge epitelial renal.Role of miRNA in stem cell differentiation Renal diseases have become a worldwide problem that can affect one in nine people throughout their life. Despite recent scientific advances, kidney transplantation and dialysis are still the only two effective therapeutic options in renal failure. The regeneration of the epithelium is critical for patient recovery as it determines the restoration of the kidney functionality. Cell precursors obtained from renal stem cells that can regenerate and integrate the injured kidney have become an important research area. The aim of our study was to determine the mechanisms involved in the differentiation of embryonic stem cells (ESCs) and adipose stem cells (ASCs) to renal epithelial lineage as these cells can be a source of these renal precursors with regenerative potential. It was recently discovered that miRNAs, which have the function of regulating gene expression in the post-transcriptional level, are essential in the differentiation of stem cells. In this sense, our main objective was to study the role of miRNAs in stem cells differentiation to renal epithelial progenitors. For this purpose, We have carried out experiments of stem cells differentiation. Embryoid bodies (EBs) from ESCs were cultured with activin A and ATRA and ASCs where cultured in a medium with physiological concentration of glucose supplemented with ATRA, obtaining progenitor cells with renal epithelial characteristics. EBs expressed the early kidney organogenesis markers (Pax2, WT1, Wnt4, Notch2 Wnt9b) and ASCs changed their morphology to a more epithelial one and expressed both markers of kidney organogenesis (Pax2, WT1, Wnt4, SIX2 i Megalin) and epithelial markers (cytokeratin-18 and E-cadherin). Furthermore, miRNA analysis and the subsequent overexpression and silencing of the miRNA let-7e in stem cells, demonstrates that this miRNA has characteristics of differentiation inductor and that is essential in the expression of both kidney organogenesis markers and epithelial markers. Furthermore, the ESCs, let-7e miRNA modulates β-catenin levels through a mechanism involving inhibition of PKCβ and the consequent decrease in the phosphorylation of GSK3 (Ser-9). Moreover, it was demonstrated using the luciferase assay, that the miRNA let-7e directly inhibits expression of gelatinase B (MMP9) in ASCs and thereby modulates its renal epithelial differentiation.
47
Farwati, Abduljalil. "Potencial diagnóstico de los miRNAs en patología inflamatoria vascular (preeclampsia e ictus)." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/457692.
Full textAbstract:
Durante las últimas décadas, una de las principales aspiraciones de la investigación biomédica ha constituido el diagnóstico precoz de diferentes enfermedades de alta tasa de mortalidad. La posibilidad de detección en fase temprana o, mejor dicho, en la etapa asintomática, del desarrollo de la enfermedad ha impulsado la búsqueda de nuevos biomarcadores. Los biomarcadores convencionales, por lo general, se han identificado a partir de mecanismos de acción ya conocidos. Por otro lado, las nuevas tecnologías “- ómicas” emergentes permiten el descubrimiento y caracterización no sesgada de las variaciones genéticas y epigenéticas asociadas con predisposición a la enfermedad.
Los microRNAs (miRNAs) juegan un importante papel en diferentes patologías gracias a su capacidad de influir tanto en procesos fisiológicos: diferenciación celular, proliferación, crecimiento, apoptosis, angiogénesis, e inmuno- inflamatorios, y en la comunicación celular, como en multitud de patologías: el cáncer, las enfermedades autoinmunes y las patologías de origen vascular, como el ictus y la preeclampsia (PE).
Según la OMS, el accidente cerebrovascular es la tercera causa más común de muerte en los países desarrollados, sólo superada por las enfermedades coronarias y el cáncer. A nivel mundial, cada año 15 millones de personas en todo el mundo sufren un accidente cerebrovascular. De éstos, 5,5 millones mueren y otros 5 millones quedan discapacitadas de forma permanente, lo cual supone una carga familiar y comunitaria. La PE es un trastorno multisistémico que complica 5%-8% de los embarazos en los países occidentales, y constituye una fuente importante de morbilidad y mortalidad en todo el mundo. Es responsable de aproximadamente 76.000 muertes maternas y 500.000 muertes infantiles por año en todo el mundo.
El objetivo principal del presente estudio ha sido la evaluación de los miRNAs circulantes como biomarcadores no-invasivos para el diagnóstico/pronóstico de dos patologías inflamatorias de origen vascular: PE e ictus.
Para evaluar la utilidad de los miRNAs circulantes como biomarcadores moleculares no invasivos para la predicción precoz de la PE, se realizó un análisis de perfilado molecular diferencial utilizando la plataforma OpenArray. El cribado de 754 miRNAs ha confirmado la presencia de 63 de ellos en el suero de mujeres gestantes de primer trimestre, aunque sólo 7 miRNAs parecieron estar diferencialmente, aunque modestamente (rango FC: 0.4-1.4), regulados cuando se compararon gestantes con PE y gestantes con embarazos no complicados. Sin embargo, no hemos podido confirmar la expresión diferencial de estos 7 miRNAs en muestras individuales mediante RT-qPCR TaqMan.
Los resultados obtenidos muestran que los miRNAs del suero materno durante el primer trimestre de la gestación no parecen tener ningún valor diagnóstico para PE temprana.
En el segundo estudio de esta Tesis se sugiere, según análisis bioinformáticos, que la expresión alterada de miR-638 podría modular la cascada de expresión de diferentes genes diana que desempeñan un papel importante en diferentes procesos y vías de señalización implicadas en la patogénesis de la aterosclerosis y la isquemia cerebral.
La inflamación juega un papel crucial en la iniciación y progresión de la aterosclerosis. Hemos verificado la presencia y la participación del miR-638 en el proceso inflamatorio en células endoteliales HUVEC inducidas por estímulos pro-inflamatorios (TNFα y IFNγ), observando una regulación que concuerda con los resultados obtenidos en suero humano
Por primera vez, hemos sugerido que los niveles séricos de miR-638 pueden constituir un biomarcador no invasivo prometedor asociado con placa aterosclerótica carotidea vulnerable e ictus isquémico, particularmente en individuos con elevado riesgo cardiovascular. Asimismo, el miR-638 sérico podría resultar útil como biomarcador pronóstico, para monitorizar la efectividad del tratamiento médico y/o la aparición de reestenosis en pacientes revascularizados.
48
Gómez, González Soledad. "Tumores del Desarrollo, Epigenética y miRNAs." Doctoral thesis, Universitat de Barcelona, 2017. http://hdl.handle.net/10803/457765.
Full textAbstract:
Evidencias recientes han mostrado que los tumores sólidos pediátricos, incluidos el neuroblastoma y el meduloblastoma, albergan una baja cantidad de mutaciones genéticas recurrentes, pero presentan una proporción significativa de alteraciones recurrentes en los mecanismos epigenéticos. Además, la epigenética es un mecanismo regulatorio fundamental durante el desarrollo en mamíferos.
En este proyecto hemos estudiado los cambios epigenéticos que afectan a dos tumores neuronales pediátricos prototípicos, el neuroblastoma y meduloblastoma. Mediante el estudio de los cambios de metilación del ADN, los miRNA y la expresión génica, hemos identificado modificaciones genéticas y epigenéticas que subyacen a la base molecular de la patogénesis y tienen implicación clínica en la evolución de estos tumores neurales. El objetivo principal de nuestro proyecto era identificar marcadores moleculares y posibles dianas de interés para el desarrollo de nuevas estrategias terapéuticas.
Este proyecto fue el primer estudio completo de metilación del ADN en neuroblastoma empleando microarrays de alta densidad. Detectamos por primera vez en neuroblastoma la presencia de metilación del ADN en citosinas no-CpG, asociada a tumores de bajo riesgo clínico. El estado de metilación detectado en el contexto no-CpG indica un papel potencial de esta metilación en la diferenciación y regulación transcripcional de genes clave el en desarrollo, como por ejemplo ALK. Observamos que la metilación del ADN en el neuroblastoma afecta no sólo a los promotores, sino también a las regiones intragénicas e intergénicas tanto en sitios CpG como no-CpG en dominios funcionales de la cromatina de genes implicados en desarrollo y cáncer.
Los cambios de metilación del ADN en sitos CpG y no-CpG tanto dentro como fuera de islas CpG (CGI) y localizados en el cuerpo del gen podian estar asociados con el comportamiento clínico del neuroblastoma. Detectamos cambios en contexto no-CpG que sugieren que el neuroblastoma puede ser epigenéticamente diferente a lo largo de la terapia. Además, analizando citosinas fuera de las regiones promotoras, en el cuerpo del gen y de las regiones intergénicas, en sitios CpG asociados a CGI y en sitios no-CpG dentro y fuera de CGIs observamos una asociación de la metilación con el comportamiento clínico en neuroblastoma.
La segunda parte del proyecto se centró en tratar de dar respuesta a la creciente necesidad de aplicar en la práctica clínica el sistema de clasificación del meduloblastoma en los subgrupos moleculares WNT, SHH, Grupo 3 y Grupo 4, recientemente identificados y propuestos mediante análisis (epi)genómicos. El reconocimiento de los subgrupos consenso ha cambiado la forma en que se estudia el meduloblastoma en el contexto de la investigación y cómo se diagnostica y trata en el contexto clínico. Estos subgrupos moleculares se definen actualmente mediante robustos sistemas de clasificación basados técnicas difícilmente incorporables a la rutina clínica de la mayoría de hospitales que tratan tumores cerebrales infantiles.
Hemos desarrollado una metodología clínicamente aplicable, robusta, rápida y reproducible para la predicción exacta de subgrupos de meduloblastoma de muestras individuales en base a dos paneles compuestos por conjuntos reducidos de biomarcadores epigenéticos, y por tanto, potencialmente incorporables en la rutina de diagnóstico. Los biomarcadores epigenéticos identificados podían aplicarse a muestras individuales de ADN de meduloblastoma obtenidas a partir de tejido congelado o FFPE. El patrón bimodal de los paneles permitía el análisis mediante diversas técnicas moleculares, tanto cuantitativas como cualitativas. Nuestro enfoque era técnicamente más simple, rápido y coste efectivo en comparación con las actuales técnicas estándar de clasificación de subgrupos de meduloblastoma.Recent evidence has shown that pediatric solid tumors harbor a paucity of recurrent genetic mutations as compared to other tumors from adults, suggesting that additional mechanisms such as epigenetic alterations may play an important role in the molecular pathogenesis of developmental tumors. So far the great majority of studies that have investigated DNA methylation in developmental tumors were biased towards CpG sites and promoter regions. Recent genome- wide studies are starting to reveal a role for DNA methylation outside such genomic contexts. Furthermore, it has recently been described that cytosine methylation also occurs in sites other than CpG dinucleotides (mCHG and mCHH) in embryonic stem cells and during brain development. However, DNA methylation at non-CpG contexts has rarely been described in cancer. We have analyzed the DNA methylome of two prototypical developmental neural tumors, neuroblastoma and medulloblastoma, using high-density microarrays with the aim of detecting epigenetic modifications at a genome-wide level that may have clinical relevance in the pathogenesis of these pediatric tumors, to identify molecular markers and potential targets of interest for the development of new therapeutic strategies. Our DNA methylation studies in neuroblastoma using high-density microarrays have defined the epigenetic landscape of this pediatric tumor and its potential clinicopathological impact. Our results reveal that: - DNA methylation changes in neuroblastoma affect not only promoters but also intragenic and intergenic regions at CpG sites and, for the first time in neuroblastoma, at non-CpG sites. - These epigenetic changes show a non-random distribution relative to functional chromatin states, and frequently target development and cancer-related genes relevant for neuroblastoma, such as CCND1 and ALK. - DNA methylation patterns in non-CpG sites provide new insights into the differentiation stage of high and low-risk neuroblastomas. - DNA methylation changes at CpG and non-CpG sites are strongly associated with clinicopathological features of neuroblastoma and with patient outcome. Furthermore, we have developed a simplified and reproducible approach to classify medulloblastoma tumors into clinically relevant subgroups applying epigenetic markers. Using this strategy, MB patients can be accurately classified into the three consensus subgroups WNT, SHH and non-WNT/non-SHH. In addition, we propose a similar approach for the specific classification of Group 3 and Group 4 medulloblastoma tumors. The proposed strategies allows for classification of single DNA samples from biopsies both frozen as well as FFPE of primary, metastasis or relapse compartments, using diverse molecular approaches. Our results show that the proposed strategy is robust, accurate, and cost-effective, making it adequate for molecular subgrouping of medulloblastoma in daily diagnostic practice of most centers treating children with brain tumors.
49
Romano, Antonietta. "Hepatocarcinoma in chronic viral hepatitis: from epidemiological data, through new pathophysiological findings, up to new possible clinical tools." Doctoral thesis, Università degli studi di Padova, 2019. http://hdl.handle.net/11577/3423317.
Full textAbstract:
Background and aim: Hepatocellular carcinoma (HCC) is the first cause of liver tumor and the leading cause of death among patients with liver cirrhosis. HCC is a major public health concern, with an incidence and mortality of HCC in increasing in North America and in some European regions1. In this contest, is strategically relevant to determine HCC epidemiology, physiopathological alterations and its potential early biomarkers. The aim of the three studies presented in this thesis is to define HCC incidence in HCV patients treated with direct acting antiviral drugs (DAAs), and investigate the role of novel targets and circulating biomarker in HCC related to chronic viral hepatitis. The main aim of study 1 is to evaluate incidence of newly diagnosed HCC in patients with advanced hepatitis C treated with DAAs. Aim of study 2 is to evaluate the expression of mi-RNAs in patients with HCC, compared with patients without HCC. The main aims of study 3 is to better define the role of OPN in HCC and elucidate the immunoregulatory functions of OPN in Hepatocellular Carcinoma
Methods: Study1. The study is based on the NAVIGATORE platform, a prospectively recording database of all patients with hepatitis C receiving DAAs in Veneto region (Italy). Inclusion criteria: fibrosis stage ≥ F3. Exclusion criteria: Child-Pugh C, liver transplantation before DAAs, history or presence of HCC, follow-up <4 weeks after starting DAAs. Study 2. A total of 68 patients with cirrhosis HCV related who achieved SVR after therapy with DAAs have bee enrolled. 20 patients with HCC within the 24 weeks after the end of therapy with DAAs and 48 patients without HCC after DAAs. In the two groups selected micro-RNAs have been analyzed at the start and at the end of DAAs therapy. Study 3. This study includes: measurement of Osteopontin (OPN) in the plasma of patients with HCC, analysis of OPN on supernatant of Peripheral blood mononuclear cells (PBMCs) culture in HCC patients and evaluation of the impact of recombinant OPN on modulating the phenotype of PBMCs in patients with HCC.
Results: Study 1. During the first year, HCC incidence was 0.46% (95% CI: 0.12-1.17) in F3, 1.49% (1.03-2.08) in Child-Pugh-A and 3.61% (1.86-6.31) in Child-Pugh-B cirrhotics. In the second year HCC incidences were: 0%, 0.2%, and 0.69%, respectively. Study 2. When we have compared mi-RNAs expression between the two groups (patients with HCC or without HCC after DAAs) at baseline, miR28-5p is resulted significantly increased (p=0.029) in HCC group compared to not HCC group. Study 3. In patients with HCC hepatitis B (HCC-CHB) related and with HCC non-viral related, the levels of OPN are higher than patients with chronic hepatitis without HCC (p=0.0016 and p=0.0063 respectively). In the analysis of OPN on supernatant of PBMCs cultured with Glypican 3, following PD1 blockade, OPN production was reduced (p=0.023) in HCC-non viral but not HCC-CHB or Healthy controls. We also observed a reduction in OPN in both HCC-non viral (p=0.0078) and HCC-CHB (p=0.0010) in PBMCs cultured with p53.
Conclusions: Study 1 These data, obtained in a large, prospective, population-based study, indicate that in patients with advanced hepatitis C receiving DAAs, the risk of "de novo" hepatocarcinoma during the first year is not higher, and might be lower, than that of untreated patients, and further declines thereafter. Study 2. In conclusion the expression of miR 28-5p is altered in patients treated with DAAs who develop HCC, suggesting its potential prognostic role in HCC related to HCV. Study 3. These results suggest its use as biomarker of HCC and reveal an immunomodulatory role for Osteopontin in HCC and warrant its further investigation as an immunotherapeutic target for HCC.
50
Gamez, Molina Beatriz. "Molecular mechanisms regulating osteoblast differentiation: miR-322/Tob2 and PI3K/SMAD networks." Doctoral thesis, Universitat de Barcelona, 2015. http://hdl.handle.net/10803/398891.
Full textAbstract:
We propose a molecular description of the mechanism whereby the osteogenic master gene Osx is controlled post-transcriptionally through a mechanism driven by miR-322/Tob2, strongly suggesting that control of specific mRNA decay is relevant in bone development and homeostasis. miR-322 has been previously studied together with miR-503 in myogenesis as promoters of cell cycle quiescence and differentiation by down-regulation of Cdc25A. Our results also show that, after differentiation by BM P-2, the miR-322 level progressively decreases in C2C12 and MC3T3-E1 cells and primary cultures of BM-MSCs. We have mentioned the up-regulated expression of osteogenic transcription factors during BMP-2 treatment. Then, miR-322, by means of Tob2 down-regulation, adjusts the expression levels of some of these factors, particularly Osx. This profile seems to allow the transcriptional up-regulation of the osteogenic transcription factors, whereas miR-322 may later exert a regulatory mechanism that allows fine-tuning of bone homeostasis.
Tobi and Tob2 proteins constitute a Tob subfamily and belong to the BTG/Tob antiproliferative factor protein family. Tob genes have emerged as key players in mediating post-transcriptional gene expression by regulating mRNA deadenylation and therefore cytoplasmic mRNA levels. Our results suggest that new target genes displaying compatible Tob-interacting secondary structures at their 3'-UTR could be subjected to specific mRNA repression by Tob family members, as we suggest here for Osx. These data are in agreement with previous research showing that, although Tobi knock-out mice are born without apparent phenotypic abnormalities, Tobi-deficient adult mice were shown to have higher bone mass compared with wild-type mice.
It has been shown that Tob proteins can interact with CPEB2-4 and specifically intensify the rapid decay of particular transcripts. Their RNA-binding domains recognize mRNAs with specific secondary structures containing U-rich loops and interact with single-stranded uridines as well as double-stranded stems present in the 3'-UTR of the target mRNA. Recent studies showed Tobi interaction with cytoplasmic CPEB2-4, which negatively regulate the expression of a target by tethering to specific mRNA and mediating recruitment of the deadenylase Cnot7, leading to specific mRNA decay. The Osx 3'-UTR contains secondary structures with a stem-loop structure similar to those bound by CPEB2-4. Our RNA pulldown analysis showed that these sequences are directly bound by CPEB proteins and Tob2. Thus, in view of our results, we hypothesize that Osx mRNA could be bound by Tob2 and CPEB2-4 and, as a consequence, specifically degraded. Future work is necessary to discern which CPEB-like proteins are involved in the interaction between Tob2 and stem-loop structures in the 3'-UTR of Osx and other osteogenic genes.
2. Class I PI-3-Kinase signaling is critical for bone formation through regulation of SMADi activity in osteoblasts
The present work reveals that PI3Ka and (3 isoforms are major regulators of osteoblast differentiation and survival. Deletion of either p110alpha and/or piio6 impairs osteoblast differentiation by decreasing the expression of key osteoblast-determining transcription factors and their transcriptional targets. More importantly, these results establish a network of molecular events that integrate distinct osteogenic inputs such as IGFi, Wnts and BMPs. Signals from these cytokines converge on GSK3 inhibition and higher SMADi levels, which confers a larger response to the osteogenic BMP action on osteoblasts.
Our results confirm that p110alpha is critical for early bone formation and development and further demonstrate this requirement for postnatal bone homeostasis. Deletion of p110alpha during early bone development causes an osteopenic phenotype in bones of both endochondral and intramembranous origin. We also took advantage of an inducible Cre system to delete p110alpha at 1-2 days of age, when bone architecture is already established and BMD and cortical thickness were approximately 4o-5o% of that of adult mice. Postnatal p110alpha deletion also led to a significant loss of either calvarial, cortical or trabecular bone at 12 weeks of age.
When p110alpha was deleted, although a slow proliferation rate and higher sensitivity to apoptosis was seen in cultured osteoblasts in vitro, there were no significant changes in the total number and proliferation rate of osteoblasts in bones of prim-deficient mice. These data mirror those previously obtained in Pten-deficient mice. Moreover, deletion of p1106 did not increase sensitivity to apoptosis in osteoblasts, but it also produced a strong bone phenotype. In our mouse model, lack of PI3K isoforms leads to similar changes in the expression of osteoblast-specific transcription factors in calvaria, long bone and osteoblast cultures. Whereas Runx2 expression was significantly reduced only after deletion of both p110alpha and piio6, Osx expression was strongly suppressed in all cases. OSX has been shown to transcriptionally regulate the expression of Cohal, Ibsp, Bglap and Fmod. Thus, lower levels of OSX could account for impaired osteoblast maturation and function, through decreased transcription of these genes..
GSK3 is a multifunctional kinase that is constitutively active and negatively regulated by numerous signaling pathways such as PI3K/AKT and canonical Wnt. Evidence suggests a negative role for GSK3 activity in osteogenesis. Our data identified GSK3 activity as a novel node of integration for multiple osteogenic signals. Previous studies have shown that MAPK and GSK3 pathways can interfere with BMP signaling. SMADi is sequentially phosphorylated on its linker region by MAPKs and GSK3. The latter modification primes for the recognition and polyubiquitination of SMAD1 by the SMURF1 and -2 E3 ubiquitin ligases. Thus, GSK3-regulated cellular levels of SMAD1 integrate signals from PI3K activators and Wnts with those of the BMPs to give a coordinated osteogenic readout. Our results conclude that genetic or pharmacological inhibition of PI3K blocks the inhibitory phosphorylation of GSK3 and regulates SMAD1 protein stability. These effects on SMADi levels were partially reversed by pharmacological inhibition of GSK3. The effects on osteoblast-specific gene expression could be reversed by ectopic expression of exogenous SMAD1. Further cooperation comes from the fact that GSK3 activity also governs nuclear levels of (3-catenin. Moreover, SMAD1 and 13-catenin transcriptionally cooperate in key osteogenic gene promoters. Thus, the present findings represent a molecular framework to understand the mounting evidence showing cooperative activation of osteoblast differentiation and function by IGFs, Wnts and BMPs.En esta tesis se han estudiado y demostrado distintos mecanismos moleculares involucrados en la diferenciación osea. Los microRNAs emergieron hace pocos años como reguladores post-transcripcionales presentes en todos los procesos biológicos. En esta tesis se describe el microRNA-322 como un miRNA reprimido durante el tratamiento con BMP-2 en células osteoblásticas y se detalla cómo éste actúa sobre el extremo 3'-UTR del mRNA de Tob2, produciendo su degradación. TOB2 es una proteína que forma parte de la familia de proteínas antiproliferativas TOB2/BTG. Tob2 se ha descrito como mediador de modificaciones post-traduccionales regulando la deadenilación general de los mRNAs. A demás, se ha visto que participa acelerando específicamente la degradación de determinados mRNAs al unirse a proteínas CPEBlike (CPEB2-4). En la presente tesis se describe el mecanismo por el cual el miR-322 degrada específicamente Tob2 y lo retiene de ejercer su función específica sobre Osterix. El uso de inhibidores específicos y los estudios con ratones mutantes existentes hasta el momento sugerían un papel importante para la proteína PI3-quinasa en el desarrollo y mantenimiento óseo. En esta tesis se describen los fenotipos derivados de la deleción específica en osteoblastos de las isoformas más abundantes en hueso (p110alpha, p 1 10beta y p 1 10alpha/beta). La ausencia de cualquiera de las subunidades catalíticas de PI3K se traduce en una reducción de masa ósea cuando los fémures de dichos ratones son analizados por microtomografia computerizada (micro-CT). El estudio in vitro de osteoblastos primarios obtenidos de los mismos ratones muestran una disminución de los principales genes osteogénicos a nivel de mRNA y proteína, concluyendo que la deficiencia de PI3K en la célula provoca una menor diferenciación osteoblástica. PI3K regula, a través de la fosforilación de AKT, un gran número de efectores. Entre ellos está GSK3, quinasa que se fosforila e inactiva en respuesta a PI3K. Estudios anteriores a esta tesis describen cómo GSK3 fosforila la región "linker" de Smadl (proteína efectora de la vía de BMPs), marcándola para degradación. En concordancia con dichos trabajos, tanto los ratones como los osteoblastos primarios defectivos en PI3K presentan menor cantidad de SMAD1 y de SMAD1 fosforilado, constituyendo así una mecanismo en el cual la falta de PI3K, a través de una menor inactivación de GSK3, aumentaría la degradación de Smads. Dicho mecanismo describe un eje PI3K-GSK3-SMAD en el que la deficiencia de PI3K conlleva a un desarrollo óseo deficitario.