MicroRNAs (miRNAs) sind eine Klasse von ca. 22 nt langen, nicht-kodierenden RNAs, welche mittels Basenpaarung die Translationsrate und Stabilität von mRNAs herabsetzen. Die vorliegende Studie untersucht mittels Hochdurchsatz-Sequenzierung Expressionsveränderungen von miRNAs nach Infektion von kultivierten Wirtszellen mit dem mikrobiellen Modellpathogen Salmonella enterica serovar Typhimurium. In Makrophagen, welche eine Schlüsselfunktion in der Orchestrierung der angeborenen Immunität spielen, wurde im Zuge der Infektion eine Induktion der miRNAs miR-21, miR-146 und miR-155 beobachtet. Darüberhinaus stellten sich alle Mitglieder der evolutionär konservierten let-7 miRNA Familie in infizierten Makrophagen als herab reguliert heraus. Es konnte gezeigt werden, dass let-7 miRNAs die zentralen Makrophagen-Zytokine IL6 und IL10 post-transkriptional reprimieren. Konsequenterweise bewirkt eine Reduktion der let-7 Expression in mikrobiell aktivierten Makrophagen eine Erhöhung der IL6 und IL10 Produktion. Weiterhin konnten den miRNAs miR-146 und miR-155 wichtige Funktionen in der Steuerung der Sensitivität und Aktivität von Makrophagen gegenüber mikrobiellen Stimuli nachgewiesen werden: während miR-146 primär die Aktivität des plasmamembranständigen Lipopolysaccharid-Rezeptors TLR4 herabsetzte und damit einer vorzeitigen inflammatorischen Makrophagenantwort vorbeugte, blieb miR-155 strikt an letztere gekoppelt, um die Aktivität diverser pro-inflammatorischer Signalwege zu begrenzen. Es konnte gezeigt werden, dass eine Stimulation des cytosolischen Immunrezeptors NOD2 eine inflammatorische Makrophagenantwort und die resultierende miR-155 Induktion begünstigt und der negativen Kontrolle durch miR-146 entzieht. Dies verhindert möglicherweise eine Hyposensitivität gegenüber zellinvasiven Pathogenen. Zusammenfassend legen diese Beobachtungen nahe, dass miRNAs zentrale Funktionen in der post-transkriptionalen Steuerung der Wirtszellantwort auf mikrobielle Pathogene ausüben. / MicroRNAs (miRNAs) are a class of approx. 22 nt long non-coding RNAs that interfere with mRNA translation and stability. Using high-throughput sequencing the present study investigated miRNA expression changes after infection of cultured host cells with the microbial model pathogen Salmonella enterica serovar Typhimurium. In macrophages, which play a key role in the orchestration of innate immunity, infection caused the induction of miRNAs miR-21, miR-146 and miR-155. Moreover, all members of the evolutionarily conserved let-7 miRNA family were down-regulated in infected macrophages. This work reports let-7 miRNAs to function in the macrophage inflammatory response by repressing the major cytokines IL6 and IL10 post-transcriptionally. Consequently a reduction of let-7 expression in microbially activated macrophages results in a specific increase in IL6 and IL10 production. Furthermore, miR-146 and miR-155 could be assigned important functions in the control of the sensitivity and activity of macrophages to microbial stimuli: while miR-146 primarily reduced the activity of the plasma membrane associated lipopolysaccharide receptor TLR4, thereby preventing a premature macrophage inflammatory response, miR-155 stayed strictly coupled to inflammation in order to limit the activity of various pro-inflammatory signaling pathways. Interestingly, it could be shown that stimulation of the cytosolic immune receptor NOD2 favors the macrophage inflammatory response and the concomitant induction of miR-155, while bypassing the negative control by miR-146. This may prevent hyposensitivity to cell-invasive pathogens. In summary, these observations suggest that miRNAs exert key functions in the post-transcriptional control of the host cell response to microbial pathogens.

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Submitted by Izabel Franco (izabel-franco@ufscar.br) on 2016-10-11T17:36:54Z No. of bitstreams: 1 DissVAK.pdf: 4608619 bytes, checksum: 3022034b9035e4e8caf1195902d24581 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-21T13:03:21Z (GMT) No. of bitstreams: 1 DissVAK.pdf: 4608619 bytes, checksum: 3022034b9035e4e8caf1195902d24581 (MD5) / Approved for entry into archive by Marina Freitas (marinapf@ufscar.br) on 2016-10-21T13:03:27Z (GMT) No. of bitstreams: 1 DissVAK.pdf: 4608619 bytes, checksum: 3022034b9035e4e8caf1195902d24581 (MD5) / Made available in DSpace on 2016-10-21T13:03:34Z (GMT). No. of bitstreams: 1 DissVAK.pdf: 4608619 bytes, checksum: 3022034b9035e4e8caf1195902d24581 (MD5) Previous issue date: 2016-08-12 / Não recebi financiamento / Cluster analysis is the organization of a collection of patterns into clusters based on similarity which is determined by using properties of data. Clustering techniques can be useful in a variety of knowledge domains such as biotechnology, computer vision, document retrieval and many others. An interesting area of biology involves the concept of microRNAs (miRNAs) that are approximately 22 nucleotide-long non-coding RNA molecules that play important roles in gene regulation. Clustering miRNA sequences can help to understand and explore sequences belonging to the same cluster that has similar biological functions. This research work investigates and explores seven unsupervised clustering algorithms based on graphs that can be divided into three categories: algorithm based on region of influence, algorithm based on minimum spanning tree and spectral algorithm. To assess the contribution of the proposed algorithms, data from miRNA families stored in the online miRBase database were used in the conducted experiments. The results of these experiments were presented, analysed and evaluated using clustering validation indexes as well as visual analysis. / A análise de agrupamento é uma organização de coleção de padrões em grupos, baseando-se na similaridade das propriedades pertencentes aos dados. A técnica de agrupamento pode ser utilizado em muitas áreas de conhecimento como biotecnologia, visão computacional, recuperação de documentos, entre outras. Uma área interessante da biologia envolve o conceito de microRNAs (miRNAs), que são moléculas não-codificadas de RNA com aproximadamente 22 nucleotídeos e que desempenham um papel importante na regulação dos genes. O agrupamento de sequências de miRNA podem ajudar em sua exploração e entendimento, pois as sequências que pertencem ao mesmo grupo possuem uma função biológica similar. Esse trabalho explora e investiga sete algoritmos de agrupamentos não-supervisionados baseados em grafos que podem ser divididos em três categorias: algoritmos baseados em região de influência, algoritmos baseados em árvore spanning minimal e algoritmo espectral. Para avaliar a contribuição dos algoritmos propostos, os experimentos conduzidos utilizaram os dados das famílias de miRNAs disponíveis no banco de dados denominado miRBase. Os resultados dos experimentos foram apresentados, analisados e avaliados usando índices de validação de agrupamento e análise visual.

MicroRNAs (miRNAs) sind kurze nicht-kodierende RNAs, die auf posttranskriptionaler Ebene die Genexpression hemmen. Dafür bilden miRNAs Ribonukleoprotein-Komplexe, deren Kernbestandteile aller Bilateria Argonaute (AGO) und GW182 /TNRC6 Proteine sind. GW182 / TNRC6-Proteine rekrutieren CCR4-NOT-Deadenylasen über kurze Tryptophan-reiche Motive (W-Motive), welche additiv wirken und fördern so die translationale Repression und den Abbau von Ziel-mRNAs. Um mehr über die Mechanismen der miRNA-abhängigen Genrepression zu erfahren, habe ich W-Motiv-abhängige Interaktionspartner humaner TNRC6C Proteine bestimmt. Hierzu habe ich, mithilfe von quantitativer Massenspektrometrie, das Interaktom von wildtyp TNRC6C Proteinen mit dem von TNRC6C Proteinen, deren W-Motive mutiert wurden, verglichen. Neben bekannten Interaktionspartnern, wie Untereinheiten des CCR4-NOT Komplexes, habe ich Komponenten von Clathrin-Vesikeln (CCVs), Stoffwechsel assoziierte Enzyme, mitochondriale Proteine, RNA Helikasen, Kinasen und Phosphatasen mit potentiellen Funktionen in der miRNA-assoziierten Repression identifiziert. Die im ersten Teil dieser Studie vorgestellten Ergebnisse legen nahe, dass CCVs die Speicherung oder das Recycling von TNRC6 und AGO Proteinen vermitteln können und somit das miRNA-Silencing modulieren. Der zweite Teil dieser Studie befasst sich mit der Konservierung von miRNA vermitteltem Gen-Silencing in Cnidaria (Nematostella vectensis), welche sich vor 600 Millionen Jahren von der Ahnenreihe der Metazoa abspalteten. Hier zeige ich anhand humaner Zellen, dass Nematostella GW182, ähnlich wie in Bilateria, von AGO rekrutiert wird und nachfolgend in der Repression der mRNA fungiert, was darauf hinweist, dass dieser Mechanismus der miRNA-vermittelten Geninhibition bereits in den letzten gemeinsamen Vorfahren von Cnidaria und Bilateria aktiv war. / MicroRNAs (miRNAs) are short non-coding RNAs that act as post-transcriptional repressors of gene expression. To function miRNAs are assembled in ribonucleoprotein complexes, whose core components in bilaterian animals are Argonaute (AGO) and GW182/TNRC6 proteins. GW182/TNRC6 proteins additively recruit CCR4-NOT deadenylases via short tryptophan-containing motifs (W-motifs), thereby promoting translational repression and the decay of target mRNAs. To gain deeper insights into the mechanisms of miRNA silencing I determined the W-motif-specific interactome of human TNRC6C proteins. Using Stable Isotope Labeling by Amino acids in Cell Culture (SILAC) coupled to affinity purification and Mass Spectrometry (MS) I identified proteins enriched with wild type TNRC6C as compared to two mutants with disrupted W-motifs. Besides known functional interactors, such as subunits of the CCR4-NOT complex, I identified several components of clathrin-coated vesicles (CCVs), metabolic enzymes, mitochondrial proteins, RNA helicases, kinases, and phosphatases with potential functional roles in miRNA-mediated repression. The results presented in the first part of this thesis indicate that CCVs may mediate the storage or recycling of TNRC6 and AGO proteins, thus modulating miRNA silencing. The second part of the thesis addressed the conservation of the mechanisms of miRNA silencing via W-motifs in the cnidarian Nematostella vectensis, separated by 600 million years from other Metazoa. Using cultured human cells, I showed that similarly to bilaterians, GW182 in Nematostella is recruited to the miRNA repression complex via interaction with AGO proteins, and functions downstream to repress mRNA, indicating that this mechanism of miRNA-mediated silencing was already active in the last common ancestor of Cnidaria and Bilateria.

microRNA (miRNA) är korta icke-kodande RNA sekvenser som har fått ökande uppmärksamhet då de kan binda till mRNA och styra vårt genuttryck. Omkring trettio procent av våra proteinkodande gener styrs av miRNA. miRNA binder till mRNA vilket hindrar dess translation eller leder till nedbrytning av mRNA. Studier visar att miRNA är involverade i olika cancersjukdomar där miRNAs kan ha olika roller. De kan antingen hämma cancern (tumörsupressor) eller agera som en onkogen. Idag pågår mycket forskning kring miRNAs påverkan på cancer och hur eventuellt miRNA kan användas som läkemedel i framtiden. Trots pågående forskning saknar vi avgörande insikter om både strukturer och biofysiska egenskaperna hos miRNA: mRNA-interaktionen som bestämmer deras funktionella resultat. Att förstå detta är viktigt för att förstå miRNA fulla terapeutiska potential. För att bidra med kunskap inom detta område var rapportens första fokus att studera bindningen mellan mRNA HNF4α och miRNA-34a med hjälp av en metod som kallas SHAPE MaP. Genom att studera bindningen är det möjligt att få mer kunskap om dess reaktiva områden och därmed öka förståelsen för den inre strukturen. SHAPE MaP är baserad på en metod som utför massiv parallell sekvensering för att identifiera mutationer introducerade med ett SHAPE-reagens. Efter detta utförs kartläggning av dessa mutationer med hjälp av ett datorprogram för att få fram en struktur. Strukturen är avgörande för att förstå de biofysiska egenskaperna såsom stabilitet, vikningsdynamik och interaktioner med andra biomolekyler. Under arbetets gång flyttades fokus till att optimera SHAPE MaP-experimentet. En serie experiment utfördes för att bestämma den optimala tiden mellan transfektion av miRNA och modifiering av SHAPEreagens med qPCR. Detta tydde på att den optimala tiden är 100 minuter, men ytterligare studier behövs dock för validering. Sammanfattningsvis beskriver denna rapport ett optimeringsexperiment för SHAPE MaP-applikationen. / microRNA (miRNA) are short non-coding RNA sequences that are important as they can bind to mRNA and control our gene expression. miRNAs control around thirty percent of our protein-coding genes. They bind to mRNAs and induce cleavage, transcriptional inhibition, or degradation. Studies show that miRNAs are involved in various cancers, where they can have different roles under different conditions. They can either act as a tumor suppressor or act as an oncogene. Today, there is extensive research into the impact of miRNAs on cancer and how miRNAs can potentially be used as drugs in the future. Nevertheless, we lack crucial insights into their intrinsic structural and biophysical properties and interactions with mRNAs that determine their functional outcomes. Addressing this knowledge gap is essential for unlocking their full therapeutic potential. To contribute knowledge in this area, the first focus of this report was studying the binding between the mRNA HNF4α and miRNA-34a using a method called SHAPE MaP. By studying the binding, it is possible to get more knowledge about their reactive sites, and by knowing this, it is possible to investigate the intrinsic structure. SHAPE MaP is based on a method that performs massively parallel sequencing to detect mutations made by a SHAPE reagent. These mutations are then mapped using a computer program to extract a structure. The structure is crucial for understanding the biophysical properties such as stability, folding dynamics, and interactions with other biomolecules. During the work, however, the focus shifted towards optimizing the SHAPE MaP experiment. A series of experiments were conducted to determine the optimal time between transfection of miRNA and modification of SHAPE reagent with qPCR. This indicated that the optimal time is 100 minutes, but further studies are needed for validation. In conclusion, this report describes an optimization experiment for the SHAPE MaP application.

Pancreatic cancer (PC) is extremely severe malignant disease with a five-year survival of less than 5%. Currently there is no reliable tool for the diagnosis of PC in its early stages. At the time of clinical symptoms most patients are in an advanced stage of the disease and the treatment does not usually have a significant effect. For these reasons emphasis is gradually shifting to the search for the suitable molecular markers for improvement of the diagnosis and assessment of the survival prognosis with respect to a possibility of surgical treatment. MiRNA represent one of the most promising markers, although, their examination in pancreatic tissue is a complicated process. One of the reasons is the very small amount of the source material coming from a fine needle biopsy. A second cause of problems is the subtle character of the pancreatic tissue resulting in significantly lower yields of molecular genetic analysis when compared to other epithelial tissues. An additional negative factor is heterogeneity of the tissue resulting in disproportionate representation of tumor cells within the sample. A suitable choice of procedures for isolation of nucleic acids (NA) and subsequent analysis including quantification of tumor cells is critical for accurate evaluation of the miRNA levels. This work is...

La résistance aux anti-aromatases (AAs) constitue un obstacle thérapeutique majeur dans le traitement des cancers du sein RE+. Les objectifs de ce travail étaient : (i) de caractériser les événements moléculaires associés à la résistance acquise aux AAs ; (ii) d’identifier de manière globale de nouveaux profils de miRNAs spécifiquement associés à la résistance aux AAs. Notre étude a mis en évidence le rôle central de la voie Akt/mTOR dans la résistance acquise et de novo aux AAs dans des modèles cellulaires, mais également dans des échantillons de patientes ayant récidivé sous anastrozole. La combinaison d’un AA avec le MK-2206, inhibiteur d’Akt ou avec la rapamycine, inhibiteur de mTOR, augmente la sensibilité à l’AA dans les cellules contrôles et est suffisante pour surmonter la résistance et restaurer la sensibilité à l'hormonothérapie dans les cellules résistantes. Notre travail propose également un modèle de résistance acquise aux AAs basé sur la sélection de cellules « cancer-initiating-like » dotées de propriétés d'auto-renouvellement, d’une résistance intrinsèque aux AAs et d’une sensibilité au MK-2206. Notre étude à grande échelle des miRNAs a identifié la voie Akt/mTOR comme une des cibles privilégiées de ces miRNAs. Nous avons identifié et validé trois miRNAs dérégulés capables de moduler le statut d’activation de la voie Akt/mTOR, qui représentent des cibles potentielles. En conclusion, notre projet a mis en évidence de nouvelles voies de signalisations ciblées par ces miRNAs et de nouveaux évènements moléculaires, qui représentent des candidats potentiels dans la résistance aux AAs / Resistance to aromatase inhibitors (AIs) remains a major drawback in the treatment of ER+ breast cancers. Our objectives were (i) to characterize molecular events associated with acquired AI resistance (ii) to capture a global view of the miRNA expression profiles associated with AI resistance. Our results showed the major role of the Akt/mTOR pathway in both de novo and acquired resistance to AI in cellular models and also in breast tumors of patients who relapsed under anastrozole. Combining AI with the Akt inhibitor MK-2206 or with the mTOR inhibitor rapamycin increased sensitivity to this AI in the control cells and was sufficient to overcome resistance and restore sensitivity to endocrine therapy in the resistant cells. Our findings propose a model of AI-acquired resistance based on the selection of cancer-initiating-like cells possessing self-renewing properties, intrinsic resistance to AI and sensitivity to MK-2206. Our large-scale study identified the Akt/mTOR pathway as one of the main targets of the deregulated miRNAs. We identified and validated three miRNAs able to modulate the Akt/mTOR activation status, suggesting these miRNAs as potential targets. To conclude, our project identified new miRNA-targeted signaling pathways and new molecular events, representing strong candidates in the mediation of AI resistance

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Micro (mi)RNAs have recently been found to play an important role in cancer biology. In order to further understand how miRNAs affect prostate tumour progression, we evaluated miRNA expression in two invasive prostate tumour lines, PC3 and DU145. We then focused our evaluation on a novel miRNA, miR-105, whose levels were significantly decreased in both tumour cell lines as compared to normal prostate epithelial cells. As miR-105 levels were reduced in prostate tumour cell lines, we restored its expression following transfection of cells with mimic constructs to over-express miR-105 in both cell lines, in order to determine its effect on various tumourigenic properties. Over-expression caused decreased tumour cell proliferation, anchorage-independent growth and invasion in vitro and inhibited tumour growth in vivo. We further identified CDK6 as a putative target of miR-105, which likely contributed to its inhibition of tumour cell growth. Our results suggest that miR-105 inhibits tumour cell proliferation and may be an interesting target to regulate tumour growth or potentially used as a biomarker to differentiate between less and more aggressive tumours in patients.