Skeletal development requires stringent control of programs for gene activation and suppression in response to physiological cues. There has been a principal focus on the identification of the mechanisms by which a particular cell phenotype is activated. MicroRNAs (miRNAs, miRs) have emerged as key negative regulators of diverse biological and pathological processes, including developmental timing, organogenesis, apoptosis, cell proliferation and differentiation; how they regulate osteoblast specific gene expression, is poorly understood. miRNAs are small 22 nucleotides (nt) endogenous non-coding RNAs (ncRNAs) that anneal to 3’ untranslated region (3’UTR) of target messenger RNA (mRNA) to mediate inhibition of translation and lower protein level. It remains to be established how specific miRNAs contribute to regulate the onset of a tissue-specific phenotype. One previously identified important player in the activation of skeletal-related genes that control formation of bone tissue is Wnt (wingless) signaling. The Wnts are regulating the differentiation of multiple cell types but also are driving embryonic stem cells (ESCs) into specific lineages, for example they support osteoblastogenesis. By attaching to the membrane, Wnts direct a signaling cascade for accumulation of β-catenin (CatnB), which in turn activates osteoblast-essential genes. The contribution of global mechanisms is equally important for understanding tissue development and diseases. The aim of this study was to identify miRNAs that are differentially expressed in osteogenically differentiated ESCs. In addition, functional characterization of these miRNAs was performed to further unravel the molecular mechanisms underlying osteogenesis. Finally, an important goal was to identify the mRNA targets of these miRNAs, which are required for differentiation of ESCs into osteoblasts with a primary focus on mRNAs associated with the Wnt signaling pathway. miRNA expression profiling reveals an overall down-regulation of miRNAs during osteogenic differentiation of ESCs To identify miRNAs that are potentially involved in osteogenesis ESCs were differentiated into osteoblasts and compared to undifferentiated ESCs using a miRNA microarray. miRNA profiling during the initial stages of osteoblast differentiation showed 25 miRNAs significantly differentially expressed. Differential expression of 4 miRNAs tested was confirmed using quantitative real-time PCR (RT-qPCR). Many miRNAs were expressed at low levels in differentiated ESCs. Indeed, down-regulation of miRNAs appeared to be common during differentiation. Furthermore, related miRNAs encoded on the same chromosome showed similar expression profiles. In summary, though several miRNAs were identified that can significantly distinguish between undifferentiated and osteogenically differentiated ESCs, 11 were chosen for further functional analysis. Functional studies show that miR-127, miR-183, miR-291b-5p, miR-293, miR-361, miR-467b and miR-665 affect osteogenesis of ESCs Undifferentiated and differentiated ESCs were used for functional studies of 11 miRNAs (miR-22, miR-127, miR-130a, miR-183, miR-291b-5p, miR-293, miR-300, miR-361, miR-467b, miR-665 and miR-690), which were down-regulated during osteogenic differentiation. To asses the function of these miRNAs, gain- and loss-of-function experiments were performed. Overexpressing and knocking down these miRNAs caused changes in cell survival, cell morphology, and osteogenic differentiation capacity as measured with calcium deposition, ALP activity and expression of osteogenic markers. Particularly, overexpression of miR-361 and knockdown of miR-665 significantly enhanced mineralization and expression levels of osteogenic markers. Thus, both miRNAs might regulate osteogenic differentiation in the early stages of lineage specification and commitment. miRNAs are modulators of osteogenic differentiation To identify miRNA target candidates that may account for the observed effects on cell survival and osteogenic differentiation of ESCs, a combined approach of bioinformatic predictions, mRNA expression analysis, and TurboGFP reduction upon miRNA overexpression coupled with the search of known literature was performed to identify cellular events that the identified miRNAs might be involved in. Target identification suggested that the candidate miRNAs may interfere with the Wnt pathway as many target candidates were detected that were known to be Wnt signaling-associated. To confirm that miR-183, miR-293, miR-361, miR-665 and miR-690 regulated osteoblast differentiation, target mRNA/miRNA interaction was studied using RT-qPCR. Overexpression of these miRNAs reduced the levels of the key factors involved in Wnt signaling; particularly Wnt inhibitor factor 1 (WIF-1) levels were decreased by miR-293, nuclear factor of activated T cells 3 (NFATc-3) and Prickle-1 by miR-361, Dishevelled 1 (Dvl-1) by miR-665 and for forkhead box O 3 (FoxO-3), Ras homolog gene family, member A (RhoA) and CatnB-1 by miR-690. Thus, to address the hypothesis that miR-361 activates osteoblast differentiation by targeting Prickle-1 and NFATc-3, the p2FP-RNAi vector system was applied. It was shown that expression of miR-361 down-regulates Prickle-1 levels, which to our knowledge have not been described so far. As it was found previously, Prickle-1 reduced Dvl-3 levels by promoting its ubiquitination, resulting in inhibition of Wnt canonical signaling in liver cancer. Since Dvls are positive regulators of osteogenesis by elevating CatnB levels and stimulating lymphoid enhancer factor/T cell factor proteins (LEF/TCF) -dependent transcription in the canonical Wnt pathway, Prickle-1 might be a negative regulator of osteogenic differentiation by eliminating Dvls from the complex. This interaction offers a novel mechanism of Wnt signaling activation in osteogenesis and can be explored to identify key components in the Wnt signaling pathway. In summary, we suggested that miR-361 acts as an activator in osteogenic differentiation of ESCs. / Die Embryonalentwicklung des Skelettsystems ist in Bezug auf programmierte Genaktivierung in Antwort auf physiologische Schlüsselreize strikten Kontrollen unterworfen. Studien zur Untersuchung solcher Kontrollelemente haben sich dabei vor allem auf die Identifikation von Mechanismen fokussiert, die einen bestimmten zellulären Phänotyp aktivieren. Zum Vorschein kamen microRNAs (miRNAs), die als negative Schlüsselregulatoren diverser biologischer und pathologischer Prozesse wirken, wie zum Beispiel der zeitlichen Regulation von Entwicklung, der Organogenese, Apoptose, zellulärer Proliferation und Differenzierung. Wie sie allerdings die Osteogenese, den Prozess der Knochenbildung, regulieren ist weitestgehend unbekannt. MiRNAs sind kurze 22 Nukleotid lange endogene nicht-kodierende RNAs (ncRNAs), die an die 3\'' nicht translatierte Region (3\''UTR) einer Ziel mRNA binden und somit die Inhibition der Translation vermitteln, was letzten Endes zu einer Erniedrigung des Proteinlevels führt. Es bleibt allerdings zu etablieren, wie spezifische miRNAs zur Spezifikation in einen bestimmten Zell- oder Gewebephänotyps beitragen. Einer der bisher identifizierten Akteure, der die Aktivierung von skelettalen Genen kontrolliert, ist der Wnt (wingless) Signalweg. Wnt Moleküle regulieren die Differenzierung vieler unterschiedlicher Zelltypen, aber lenken auch die Differenzierung von embryonalen Stammzellen (ESCs) in spezifische Richtungen, so z.B. in die Richtung von Knochenzellen, den Osteoblasten. Indem sie an die Zellmembran andocken, dirigieren Wnts eine Signalkaskade, die die Akkumulation von beta-catenin (CatnB) im Zellkern nach sich zieht, wodurch knochenspezifische Gene aktiviert werden. Obwohl die Wnt Signalkaskade weitestgehend beschrieben ist, ist der Beitrag globalerer Regulationsmechanismen, wie die der miRNAs, an der Osteogenese jedoch gleichfalls für das Verständnis von Gewebeentwicklung und -fehlfunktion von Bedeutung. Das Ziel dieser Arbeit war es deshalb bestimmte miRNAs zu identifizieren, die differentiell in ESCs exprimiert werden, die zu Knochenzellen ausdifferenzieren. Desweiteren sollten diese miRNAs funktionell charakterisiert werden, um die molekularen Mechanismen, die der Osteogenese unterliegen, aufzudecken. Letztendlich war es ein weiteres wichtiges Ziel die Ziel mRNAs der knochenspezifischen miRNAs zu identifizieren und deren Bezug zum Wnt Signalweg zu charakterisieren. miRNA Expression ist während der osteogenen Differenzierung herunter reguliert Um solche miRNAs zu identifizieren, die potentiell in die Osteogenese eingreifen, wurden ESCs zu Osteoblasten differenziert und mit undifferenzierten ESCs mit Hilfe eines miRNA Microarrays verglichen. Das so durchgeführte miRNA Profiling zeigte, dass 25 miRNAs während der initialen Phase der osteogenen Differenzierung signifikant unterschiedlich exprimiert wurden. Die differentielle Expression von 4 getesteten miRNAs wurde in einem nächsten Schritt über quantitative real-time PCR (RT-qPCR) beispielhaft bestätigt. Generell zeigte sich, dass differenzierende ESCs viele miRNAs auf geringem Niveau exprimieren. Tatsächlich schien die Herunterregulation der miRNA Expression mit der Differenzierung der Zellen einherzugehen. Desweiteren zeigten miRNAs, die auf dem gleichen Chromosom kodiert sind, ähnliche Expressionsmuster. Zusammenfassend fanden sich etliche miRNAs, die in undifferenzierten Zellen im Vergleich zu differenzierenden Zellen unterschiedlich exprimiert werden, von denen schlussendlich 11 für weitere Analysen ausgewählt wurden (miR-22, miR-127, miR-130a, miR-183, miR-291b-5p, miR-293, miR-300, miR-361, miR-467b, miR-665 and miR-690). miR-127, miR-183, miR-291b-5p, miR-293, miR-361, miR-467b und miR-665 beeinflussen die Osteogenese In einem nächsten Schritt wurden undifferenzierte und differenzierende ESCs für funktionelle Studien dieser 11 herrunterregulierten miRNAs herangezogen. Um die Funktion dieser miRNAs aufzudecken, wurden sogenannte Gain-of-function und Loss-of-function Studien durchgeführt. Die experimentelle Überexpression und der Knock-down dieser miRNAs führten zu Änderungen in der zellulären Morphologie, der Viabilität und der osteogenen Differenzierungskapazität wie durch einen Kalziumdepositionsassay, einen ALP Aktivitätsassay und die Expression knochenspezifischer Markergene gezeigt werden konnte. Im Besonderen erhöhte die Überexpression der miR-361 und der Knock-down der miR-665 den Mineralisierungsgrad der Zellen und die Expressionniveaus knochenspezifischer Gene. Daher ist zu schließen, dass beide miRNAs das Potential besitzen, die Osteogenese - besonders in den frühen Stadien der Keimbahnspezifikation - zu regulieren. miRNAs als Modulatoren der Osteogenese Um miRNA Zielkandidaten zu identifizieren, die die beobachteten Effekte auf die Zellviabilität und auf die osteogene Differenzierungen bedingen könnten, wurde ein kombinierter Ansatz aus Bioinformatischer Sequenz- und Prädiktionsanalyse, mRNA Expressionsanalyse und TurboGFP Reduktion nach miRNA Überexpression gewählt. Gepaart mit einer Literatursuche deutete diese Zielkandidatenanalyse darauf hin, dass die identifizierten miRNAs tatsächlich den Aktivierungsstatus des Wnt Signalwegs manipulieren könnten, da viele der prädiktierten Target mRNAs bekannt dafür sind, mit dem Wnt Signalweg zu interagieren. Um zu bestätigen, dass miR-183, miR-293, miR-361, miR-665 und miR-690 die Osteogenese regulieren, wurde die mRNA/miRNA Interaktion indirekt mittels RT-qPCR studiert. Die Überexpression dieser miRNAs führte zu einer Erniedrigung des mRNA Expressionsspiegels von WIF-1 (Wnt inhibitory factor 1) durch miR-293, NFATc-3 (nuclear factor of activated T cells 3) und Prickle-1 durch miR-361, Dishevelled 1 (Dvl-1) durch miR-665, sowie forkhead box O3 (FoxO-3), Ras homolog gene family, member A (RhoA) und CatnB durch miR-690. In einem nächsten Schritt konnte durch Nutzung eines speziellen Reportersystems (TurboGFP) eine direkte Interaktion zwischen miR-361 und Prickle-1 nachgewiesen werden. Wie bereits in anderen Studien gezeigt, ist Prickle-1 in der Lage, die Spiegel an Dvl-3 durch Ubiquitinierung des Proteins zu reduzieren, was zur Inhibierung des kanonischen Wnt Signalweges führt. Da Dvls als positive Regulatoren der Osteogenese bekannt sind, indem sie den CatnB Spiegel erhöhen und die lymphoid enhancer factor/T cell factor protein (LEF/TCF) abhängige Transkription stimulieren, könnte Prickle-1 als negativer Regulator fungieren, indem es Dvls von diesem Transkriptionskomplex entfernt. Abschließend lässt sich zusammenfassen, dass miR-361 in dieser Arbeit als neuartiger Aktivator der osteogenen Differenzierung vorgeschlagen wird. Die molekulare Interaktion zwischen miR-361, Prickle-1 und Dvls bietet einen neuartigen Mechanismus der Wnt Signalaktivierung während der Osteogenese und kann für weitere Untersuchungen zur Identifizierung von Schlüsselkomponenten des Wnt Signalweges herangezogen werden.

Yes / Increased cardiovascular morbidity and mortality in individuals with type 2 diabetes (T2DM) is a significant clinical problem. Despite advancements in achieving good glycaemic control, this patient population remains susceptible to macrovascular complications. We previously discovered that vascular smooth muscle cells (SMC) cultured from T2DM patients exhibit persistent phenotypic aberrancies distinct from those of individuals without a diagnosis of T2DM. Notably, persistently elevated expression levels of microRNA-145 co-exist with characteristics consistent with aging, DNA damage and senescence. We hypothesised that increased expression of microRNA-145 plays a functional role in DNA damage signalling and subsequent cellular senescence specifically in SMC cultured from the vasculature of T2DM patients. In this study, markers of DNA damage and senescence were unambiguously and permanently elevated in native T2DM versus non-diabetic (ND)-SMC. Exposure of ND cells to the DNA-damaging agent etoposide inflicted a senescent phenotype, increased expression of apical kinases of the DNA damage pathway and elevated expression levels of microRNA-145. Overexpression of microRNA-145 in ND-SMC revealed evidence of functional links between them; notably increased secretion of senescence-associated cytokines and chronic activation of stress-activated intracellular signalling pathways, particularly the mitogen-activated protein kinase, p38a. Exposure to conditioned media from microRNA-145 overexpressing cells resulted in chronic p38a signalling in naïve cells, evidencing a paracrine induction and reinforcement of cell senescence. We conclude that targeting of microRNA-145 may provide a route to novel interventions to eliminate DNA-damaged and senescent cells in the vasculature and to this end further detailed studies are warranted.

The aim of this review is to discuss current findings regarding the roles of miRNAs in periodontal diseases and the potential use of saliva as a diagnostic medium for corresponding miRNA investigations. For periodontal disease, investigations have been restricted to tissue samples and five miRNAs, that is, miR-142-3p, miR-146a, miR-155, miR-203, and miR-223, were repeatedly validated in vivo and in vitro by different validation methods. Particularly noticeable are the small sample sizes, different internal controls, and different case definitions of periodontitis in in vivo studies. Beside of that, the validated miRNAs are associated with inflammation and therefore with various diseases. Furthermore, several studies successfully explored the use of salivary miRNA species for the diagnosis of oral cancer. Different cancer types were investigated and heterogeneous methodology was used; moreover, no overlap of resultswas found. In conclusion, fivemiRNAs have consistently been reported for periodontitis; however, their disease specificity, detectability, and expression in saliva and their importance as noninvasive markers are questionable. In principle, a salivary miRNA diagnostic method seems feasible.However, standardized criteria and protocols for preanalytics, measurements, and analysis should be established to obtain comparable results across different studies.

Metabolic reprogramming in cancer cells provides energy and important metabolites required to sustain tumour proliferation. Hypoxia represents a hostile environment that can encourage these transformations and other adaptive responses that contribute to poor prognosis and resistance to radiation and chemotherapy. Hypoxic signatures associated with worse prognosis were previously derived in different cancer types, and led to the selection of two candidates with potential metabolic implications, namely the mir210-putuative target iron-sulfur scaffold protein ISCU and glycogen phosphorylase (PYGL). Firstly, it was verified that the hypoxia-induced miR-210 targets ISCU. Iron-sulfur clusters represent cofactors for key enzymes involved in Krebs cycle and electron transport chain. Downregulation of ISCU was associated with the induction of reactive oxygen species (ROS) and reduced mitochondrial complex I and aconitase activity, caused a shift to glycolysis in normoxia and enhanced cell survival. This indicates that the induction of a single microRNA, miR-210, can mediate a new mechanism of adaptation to hypoxia, by regulating mitochondrial function via iron-sulfur cluster metabolism and free radical generation. Secondly, it was found that changes in PYGL expression reflect a characteristic upregulation of glycogen metabolism in hypoxia in both tumour xenografts and in cancer cell lines. More specifically, hypoxia stimulates glycogen accumulation and its utilisation, as well as the concurrent upregulation of several glycogen-metabolizing enzymes such as glycogen synthase (GYS1) and PYGL. PYGL depletion led to glycogen accumulation in hypoxic cells, increased intracellular levels of ROS, and a reduction in proliferation due to a p53-dependent induction of senescence. Furthermore, depletion of PYGL was associated with markedly impaired tumorigenesis in vivo. Finally, metabolic analyses indicated that glycogen degradation by PYGL is important for the optimal functioning of the pentose phosphate pathway. Collectively, this study shows the contribution of two important pathways to the metabolic adaptations induced by hypoxia.

NOD2 is an intracellular innate immune receptor expressed in dendritic cells and gastrointestinal epithelial cells. Polymorphisms in the NOD2 gene convey a strong predisposition to Crohn’s disease (CD), a form of inflammatory bowel disease. Understanding the function of NOD2, and in what way it is aberrant in the presence of NOD2 polymorphisms, would confer a valuable paradigm for understanding Crohn’s pathogenesis. CD is thought to arise both from defects in the gut mucosal barrier and from a dysregulated Th17 immune response to commensal gut flora. Aberrant expression of IL-23 is present in both human CD and in murine models of colitis. Wild-type NOD2 contributes to NFκB activation and pro-inflammatory cytokine production on recognition of its cognate ligand, a function that is lost in CD-associated mutations. How the predominantly loss-of-function CD-NOD2 contributes to the pro-inflammatory response present in Crohn’s is not yet understood. In this thesis a set of experiments is described that aim to shed light on the function of NOD2, firstly through identification of negative regulators of immune activation that are dependent on NOD2 for their expression. This work identifies the microRNAs that are expressed following NOD2 triggering in human dendritic cells. Specifically, up-regulation of the miR-29 family was found to be dependent on wild-type NOD2 function. A number of novel miR-29 targets and their functional consequences are presented, including the cytokine subunits IL-12p40 and IL-23p19, directly linking NOD2 polymorphisms and aberrant IL-23 expression. Secondly, a project aiming to identify components of the NOD2 signalling complex (or signalosome) is described. To this end I employed a model system that involved tagging NOD2, and stable expression in a human cell line. These clones were validated for expression and function before an immunoprecipitation protocol was optimised. Mass spectrometry analysis of these samples identified the known NOD2-interacting protein Erbin.

The current study focuses on finding differential expression between circulating microRNAs in plasma from patients with abdominal aortic aneurysms compared to un-diseased individuals by using a qPCR-based array. In addition, we evaluated the expression of deregulated microRNAs in human tissue samples as well as microarray data from two independent mouse models of aneurysm development. Fifteen miRNAs were found to be significantly differentially expressed, with four of them surviving multiple testing. Interestingly all four of them were substantially different in murine aneurysm development.

<p>As a major component of the adaptive immune system, CD4+ T cells play a vital role in host defense and immune tolerance. The potency and accuracy of CD4+ T cell-mediated protection lie in their ability to differentiate into distinct subsets that could carry out unique duties. In this dissertation, we dissected the roles and interplays between two emerging mechanisms, miRNAs and epigenetic processes, in regulating CD4+ T cell-mediated responses. Using both gain- and loss-of-function genetic tools, we demonstrated that a miRNA cluster, miR-17-92, is critical to promote Th1 responses and suppress inducible Treg differentiation. Mechanistically, we found that through targeting Pten, miR-17-92 promotes PI3K activation. Strong TCR-PI3K activation leads to the accumulation of DNMT1, elevated CpG methylation in the foxp3 promoter, and suppression of foxp3 transcription. Furthermore, we demonstrated that an epigenetic regulator, methyl CpG binding protein 2 (MeCP2), is critical to sustain Foxp3 expression in Tregs, and to support Th1 and Th17 differentiation in conventional CD4+ T cells (Tcons). In Tregs, MeCP2 directly binds to the CNS2 region of foxp3 locus to promote its local histone H3 acetylation; while in Tcons, MeCP2 enhances the locus accessibility and transcription of miR-124, which negatively controls SOCS5 translation to support STAT1, STAT3 activation and Th1, Th17 differentiation. Overall, miRNAs and epigenetic processes may crosstalk to control CD4+ T cell differentiation and function.</p> / Dissertation

Dicer endoribonuclease catalyzes the maturation of microRNAs (miRNAs) from double stranded precursors. Studies conditionally inactivating Dicer in the mouse embryonic forebrain continue to shed light on the spectrum of biological processes subject to miRNA regulation. This study looked at defects of brain development following a widespread ablation of Dicer in the early forebrain. The neuroepithelial stem cells failed to specify the radial glia appropriately around the time when the first postmitotic neurons begin to be generated in the neuroepithelium. Ablation of Dicer in only a subset of radial glia was not accompanied by the early apoptosis observed in all other models of Dicer ablation in the cortex. This allowed the study of the role of miRNAs in regulating cell numbers in the cortex. The study revealed that generation of cortical cells is increased during postnatal development. Finally, the study identified a miRNA which is able to negatively regulate the development of neuronal precursor cells of the developing cortex by targeting Tbox transcription factor 2. Together the results presented in this Thesis contribute to the understanding of the roles of endogenous RNA interference in the development of the brain.

No description available.

<p>The cellular microRNA (miRNA) pathway has emerged as an important regulator of host-virus interactions. While miRNAs of viral and cellular origin have been demonstrated to modulate viral gene expression and host immune responses, reports detailing these activities in the context of mammalian retroviruses have been controversial. Using modern, high-throughput small RNA sequencing we provide evidence that the spumaretrovirus bovine foamy virus expresses high levels of viral miRNAs via noncanonical biogenesis mechanisms. In contrast, the lentivirus human immunodeficiency virus type 1 (HIV-1) does not express any viral miRNAs in a number of cellular contexts. Comprehensive analysis of miRNA binding sites in HIV-1 infected cells yielded several viral sequences that can be targeted by cellular miRNAs. However, this analysis indicated that HIV-1 transcripts are largely refractory to binding and inhibition by cellular miRNAs. In addition, we demonstrate that HIV-1 exerts minimal perturbations on cellular miRNA profiles and that viral replication is not affected by the ablation of mature cellular miRNAs. Together, these data demonstrate that the ability of retroviruses to encode miRNAs is not broadly conserved and that lentiviruses, particularly HIV-1, have evolved to avoid targeting by cellular miRNAs.</p> / Dissertation