Os princípios biológicos da regeneração óssea guiada (ROG) têm contribuído para o desenvolvimento de membranas que, em odontologia, são utilizadas em diversas situações como tratamentos com implantes dentários, aumento de rebordo alveolar e reparo de defeitos ósseos de origem traumática e patológica. Resultados de experimentos in vitro comparando a membrana obtida pela associação do polímero poli(fluoreto de vinilideno-trifluoretileno) e da cerâmica titanato de bário (P(VDFTrFE)/ BT) à membrana de politetrafluoretileno (PTFE) mostraram uma resposta favorável de osteoblastos, fibroblastos e queratinócitos à membrana de P(VDFTrFE)/ BT. O objetivo do presente estudo foi avaliar o efeito da membrana de P(VDFTrFE)/ BT sobre a formação óssea in vivo. Foram criados defeitos ósseos com 5 mm de diâmetro em calvárias de ratos machos Wistar (peso 200-250 g), distribuídos em três grupos com relação à utilização ou não de membranas nos defeitos ósseos: (1) membrana de P(VDF-TrFE)/BT; (2) membrana de PTFE; (3) nenhum tipo de membrana. Ao final de 4 e 8 semanas os animais foram eutanasiados e as amostras foram submetidas à: (1) análise por microtomografia computadorizada (micro-CT) para avaliar volume ósseo, superfície óssea, superfície óssea específica, número de trabéculas, separação trabecular e espessura trabecular, (2) análise histológica com base em cortes histológicos não descalcificados, (3) análise por reação em cadeia da polimerase em tempo real (PCR em tempo real) para avaliar a expressão gênica dos marcadores ósseos runt-related transcription factor 2 (RUNX2), fosfatase alcalina (ALP), sialoproteína óssea (BSP), osteocalcina (OC), osteoprotegerina (OPG) e receptor activator of nuclear factor-kappa B ligand (RANKL) e (4) análise da expressão de microRNAs (miRs) pela técnica de sequenciamento na plataforma Illumina. Os dados das análises morfométricas e da expressão gênica foram submetidos ao teste de Kruskal-Wallis, seguido pelo teste de Fischer quando apropriado, e para a análise da expressão de miRs foi utilizado o teste de Mann-Whitney para comparar a membrana de P(VDF-TrFE)/BT à de PTFE. Para todas as comparações o nível de significância adotado foi de 0,05. Os defeitos que não receberam a membrana tiveram uma formação óssea insignificante. Ambas as membranas favoreceram a formação óssea, sendo a superfície óssea maior sobre a membrana de P(VDF-TrFE)/BT comparada à de PTFE em 4 (p=0,01) e 8 (p=0,001) semanas e a separação trabecular maior sobre a membrana de PTFE comparada à de P(VDF-TrFE)/BT em 4 (p=0,05) e 8 (p=0,001) semanas. A expressão gênica de BSP (p=0,01), OC (p=0,001) e OPG (p=0,001) foi maior e de RANKL (p=0,001) foi menor sobre a membrana de P(VDFTrFE)/ BT comparada à de PTFE em 4 semanas. A expressão gênica de RUNX2 (p=0,001) e OC (p=0,05) foi maior e de ALP (p=0,05), RANKL (p=0,01) e OPG (p=0,001) foi menor sobre a membrana de P(VDF-TrFE)/BT comparada à de PTFE em 8 semanas. Quarenta e cinco e 13 miRs foram regulados positivamente (>2 vezes) em 4 semanas e 8 semanas, respectivamente, e 11 e 39 miRs foram regulados negativamente (>2 vezes) em 4 semanas e 8 semanas, respectivamente, pela membrana de P(VDF-TrFE)/BT comparada à de PTFE. Os resultados indicam que a membrana de P(VDF-TrFE)/BT favorece a formação óssea quando comparada à membrana de PTFE e, portanto, pode ser considerada um biomaterial promissor para ser utilizado em procedimentos de ROG. / Biological principles of guided bone regeneration (GBR) have contributed to development of membranes that, in dentistry, are used in several situations such as treatment with dental implants, alveolar bone augmentation and repair of traumatic and pathological bone defects. Results of in vitro experiments comparing membrane obtained by the combination of poly(vinylidene fluoride-trifluoroethylene) and barium titanate ceramics (P(VDF-TrFE)/BT) with polytetrafluoroethylene (PTFE) membrane showed a favorable response of osteoblasts, fibroblasts and keratinocytes to the P(VDF-TrFE)/BT membrane. The aim of this study was to evaluate the effect of P(VDFTrFE)/ BT membrane on in vivo bone formation. Bone defects with 5 mm in diameter were created in calvaria of male Wistar rats (weight 200-250 g), distributed into three groups regarding the use or not of membranes on defects: (1) P(VDF-TrFE)/BT membrane; (2) PTFE membrane; (3) no membrane. At the end of 4 and 8 weeks, the animals were euthanized and samples were subjected to: (1) computed microtomography analysis (micro-CT) to assess bone volume, bone surface, specific bone surface, trabecular number, trabecular thickness and trabecular separation; (2) histological analysis based on non-decalcified histological sections; (3) real time polymerase chain reaction (real time PCR) to evaluate gene expression of the bone markers runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), bone sialoprotein (BSP), osteocalcin (OC), osteoprotegerin (OPG) and receptor activator of nuclear factor-kappa B ligand (RANKL); (4) microRNAs (miRs) sequencing analysis at Illumina platform. Data from morphometric and gene expression analyses were submitted to the Kruskal-Wallis test followed by Fischer\'s test, when appropriate, and from miRs expression to the Mann-Whitney test to compare P(VDF-TrFE)/BT with PTFE membrane. For all comparisons, the significance level was 0.05. Defects without membrane exhibit a non significant bone formation. Both membranes favored osteogenesis with an increased bone formation on the P(VDF-TrFE)/BT membrane compared with PTFE at 4 (p=0.05) and 8 weeks (p=0.001). Trabecular separation was greater on PTFE membrane compared with the P(VDF-TrFE)/BT at 4 (p=0.05) and 8 weeks (p=0.001). At 4 weeks, the gene expression of BSP (p=0.01), OC (p=0.001) and OPG (p=0.001) were higher on P(VDF-TrFE)/BT membrane compared with PTFE, while RANKL (p=0.001) was lower. The gene expression of RUNX2 (p=0.001) and OC (p=0.05) were higher and ALP (p=0.05), RANKL (p=0.01) and OPG (p=0.001) were lower on the membrane of P(VDF- TrFE)/BT compared with PTFE at 8 weeks. Fortyfive and 13 miRs were up-regulated (> 2 fold) at 4 weeks and 8 weeks, respectively, and 11 and 39 miRs were negatively regulated (> 2 fold) at 4 weeks and 8 weeks, respectively, on the P(VDF-TrFE)/BT membrane compared with PTFE membrane. The results indicate that the P(VDF-TrFE)/BT membrane favors bone formation compared with PTFE membrane and, therefore, may be considered a promising biomaterial for using in GBR procedures.

Les monocytes sont des leucocytes dérivés de précurseurs de la moelle osseuse pouvant se différencier en macrophages, cellules dendritiques (CD), ou ostéoclastes (OC). Ils jouent un rôle critique dans la persistance de l'inflammation et la destruction des articulations par le biais de mécanismes encore mal connus. Les monocytes sont composés de deux grandes sous-populations chez la souris, discriminées sur la base du marqueur de surface Ly6C. Il a été suggéré que les OC pouvait être préférentiellement différenciés à partir de la sous-population monocytaire Ly6Chigh, dont l'activation excessive et prolongée est une signature clé de nombreuses pathologies inflammatoires. Parmi les acteurs moléculaires responsables de la régulation de l'expression des gènes, les miARNs jouent un rôle majeur dans de nombreux processus physiologiques, dont la réponse inflammatoire, en régulant finement les programmes génétiques au niveau post-transcriptionnel. Leur implication dans l'ostéoclastogénèse est encore mal connue. Par ailleurs, leur expression est perturbée dans de nombreuses pathologies, dont la polyarthrite rhumatoïde qui implique à la fois un dysfonctionnement de la réponse inflammatoire et de l'homéostasie osseuse. Mon projet de thèse vise à mieux comprendre l'implication des sous-populations monocytaires dans la persistance de l'inflammation et de l'activité des OC au travers de l'étude du rôle de miR-146a dans des conditions physiologiques et inflammatoires. J'ai montré que miR-146a est le miARN le plus différemment exprimé entre monocytes classiques Ly6Chigh et non-classiques Ly6Clow, et que son expression est diminuée dans les Ly6Chigh en conditions arthritiques. J'ai également montré que la perte de miR-146a augmente l'ostéoclastogénèse in vitro et l'érosion osseuse in vivo chez les souris arthritiques. Enfin, la surexpression artificielle de miR-146a dans les monocytes Ly6Chigh inhibe la différenciation osteoclastique et la perte osseuse dans l'arthrite expérimentale chez la souris. Mes résultats suggèrent que miR-146a contrôle l'hétérogénéité fonctionnelle des monocytes et qu'une diminution de son expression dans la sous-population Ly6Chigh serait responsable de l'augmentation de l'osteoclastogénèse et de l'érosion osseuse observées en conditions arthritiques. Pour finir, mes résultats montrent également qu'augmenter l'expression de miR-146a dans les monocytes Ly6Chigh présente un intérêt thérapeutique pour contrecarrer la perte osseuse associée à l'arthrite. / Introduction : Monocytes represent a prototypic cell type when investigating the interplay between immune and skeletal systems as they can give rise to different cell types including dendritic cells, macrophages and osteoclasts (OC), which play key roles in immunity and bone homeostasis. Circulating monocytes consist of at least two main functional subsets, Ly6Chigh and Ly6Clow monocytes. It has been suggested that OC might develop preferentially from the Ly6Chigh monocyte subset, which excessive and prolonged activation is a hallmark of many inflammatory diseases. Among key molecular rheostats of cell fate, micro(mi)RNAs are a class of regulatory RNAs that control basic biological functions and orchestrate inflammatory responses. Few miRNAs have been involved in osteoclastogenesis. The present study aimed at investigating the role of miRNAs in osteoclastogenesis in the context of monocyte subsets, under steady state and inflammatory conditions. Methods & Results : Using genome-wide miRNA expression study we have identified miRNAs and putative targeted pathways that are differentially expressed between Ly6Chigh and LyC6low FACS sorted mouse monocytes, and common to their human counter parts CD14+CD16- and CD14dimCD16+ monocytes, respectively. Among these, miR-146a showed higher expression in Ly6Clow monocytes when compared to Ly6Chigh monocytes. Under inflammatory arthritis conditions, expression of miR-146a in Ly6Chigh monocytes was down regulated as compared to healthy controls. Using mouse deficient for miR-146a, we showed that knockdown of miR-146a increased OC differentiation in vitro. While no bone phenotype was evidenced in miR-146a deficient mice, nor under steady state or ovariectomized conditions, arthritis-induced bone resorption and bone loss were increased in miR-146a knockout mice. Finally, using a liposomal formulation able to delivermiR-146a mimics to Ly6Chigh monocytes upon intravenous injection, we showed that enforced expression of miR-146a led to decreased number of TRAP positive cells within the synovium of arthritic mice, and efficiently reduced bone erosion in inflammatory arthritis. This effect was associated with decreased RelB expression in miR-146a-overexpressing Ly6Chigh osteoclast progenitors. Conclusion : Overall, our results show that specific over-expression of miR-146a in Ly6Chigh monocytes altered OC differentiation and decreased bone erosion in inflammatory arthritis. These data suggest a novel role for miR-146a in controlling osteoclast fate of Ly6Chigh monocyte progenitors and that reduced miR-146a expression in Ly6Chigh monocytes under arthritic conditions contributes to pathogenic bone loss. Finally, delivery of miR-146a mimics to Ly6Chigh monocytes may offer valuable therapeutic potential to interfere with pathological bone loss.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18145号 / 医博第3865号 / 新制||医||1002(附属図書館) / 31003 / 京都大学大学院医学研究科医学専攻 / (主査)教授 萩原 正敏, 教授 柳田 素子, 教授 稲垣 暢也 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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MiRNR molekulės yra 21-24 nukleotidų ilgio dvigrandės RNR molekulės. Jos pačios nieko nekoduoja. Viena svarbiausių jų funkcijų yra genų raiškos valdymas potranskripciniame lygmenyje. Jas koduoja daugelis organizmų. miRNR brendimas skiriasi priklausomai nuo ląstelės tipo (gyvūninė ar augalinė). Augalų ląstelėse yra papildomas etapas, kuriu metu dvigrandė miRNR yra metilinama HEN1 baltymo. HEN1 metiltransferazė yra neseniai augaluose identifikuotas baltymas. HEN1 baltymas yra didelis (106,6 kDa), daugiadomeninis baltymas, kurio savybės mažai ištirtos. Jis vienintelis sugeba modifikuoti mažąsias RNR molekules pernešdama metilo grupę nuo kofaktoriaus S-adenozil-L-metionino ant 3′ galinio nt 2′ -OH grupės. HEN1 baltymas yra labai specifiškas savo substratui, kurį atpažįsta ne pagal nt seką, o pagal jo antrinę struktūrą ir ilgį. (Chen, 2005) Pagal homologiją su kitais baltymais HEN1 buvo nustatyti konservatyvūs motyvai. (Č. Venclovas) N-galinėje dalyje nustatyti du RNR prijungiantys (dvRNR-PD ir La- tipo) motyvai. Naudojant sutrumpintus baltymus ir išanalizavus juos elektroforetinio judrumo poslinkio PAA gelyje metodu buvo nustatyta, kad dvRNR-PD atlieka RNR prijungimo funkciją. dvRNR-PD buvo sumodeliuota erdvinė struktūra. Išanalizavus ją buvo nustatytos keturios aminorūgščių liekanos (12K, 22K, 69K ir 70K), kurios gali būti svarbios formuojantis baltymo ir RNR kompleksui. Ištyrus mutantinius baltymus, turinčius šių lizinų pakaitas buvo nustatyta, kad 12-as lizinas... [toliau žr. visą tekstą] / MiRNAs are 21-24 nt length double-stranded RNA molecules which do not encode anything. They play mayor role in post-transcriptional gene regulation level. These molecules are encoded by various organisms. miRNA maturation depends on the type of the cell (animal or plant). There is an additional step in plants when double stranded miRNA is methylated by HEN1 protein. HEN1 methyltransferase is a novel protein identified in plants. HEN1 protein has high molecular weight (106,6 kDa) and it is multistructural. Its features are not yet investigated. This is the only protein which modifies small RNA molecules by transferring methyl group from cofactor S-adenosil-L-methyonin to the 2′ -OH group of the last 3′ nt. HEN1 proteins is very specific to its substrate and recognizes it not by the nt sequence but by the molecule length and structure. After analysis of homologous proteins HEN1 conservative motives were identified. (Č. Venclovas) Two RNA binding motives (dsRNA-BM and La-type) were identified in the N-terminal region. By using shortened proteins and analyzing those by electrophoretic gel mobility in PAA gel shift it was find out that dsRNA-BM is responsible for binding miRNA. Theoretical structure model was made for dsRNA-BM. After analyzing it four amino acid residues (12K, 22K, 69K, and 70K) which can be important for the formation of protein and RNA complex were identified. After examination of mutant proteins with changed lysines it was determined that 12th... [to full text]

Lung diseases are one of the leading causes of mortality and morbidity worldwide. Understanding these diseases at a molecular level remains a critical component to developing effective therapeutics. Previous work has shown that gene expression alterations play an important role in disease initiation, maintenance, and progression as well as serve as diagnostic tools in disease. However, much remains to be uncovered regarding the role that microRNAs play in both healthy and diseased lung tissue. This thesis seeks to utilize methods of bioinformatics, cell biology, and molecular biology to examine the effect of miR-4423 on lung epithelial cell differentiation (Aim 1), miR-424 on never smoker derived lung adenocarcinoma (Aim 2), and miR-34c isomiRs in interstitial lung disease (ILD) (Aim 3). First, we examined the role of miR-4423 in lung mucociliary epithelium by employing the use of an air-liquid interface culture system, finding miR-4423 has an effect in ciliated cell differentiation and that a loss of miR-4423 is associated with cancer progression. These findings suggest that miR-4423’s actions in airway epithelium differentiation may potentially provide a therapeutic role in lung cancer. Next, we validated transcriptomic differences between lung tumor tissues resected from never and ever smokers. Specifically, miR-424, a predicted regulator of a large number of gene expression changes in never smoker lung adenocarcinoma, was found to regulate cell migration, potentially identifying a novel target and/or pathway for therapeutic action. Lastly, the function of microRNA isomiRs is relatively unknown. We validated miR-34c as upregulated in ILD and modulated both miR-34c and a miR-34c 5’ isomiR in lung relevant cell lines to explore their differing biological roles. We found that they are capable of targeting differing mRNA, indicating an independent role for isomiRs in disease. The studies contained in this dissertation offer valuable insight into the biology of microRNAs in the lung and how they might be employed as therapeutic targets for a number of common lung diseases. In addition, biological insights into the complexity of microRNAs in the lung highlight the need to better understand diseases influenced by microRNA expression and microRNA variants in regards to actionable therapeutics. / 2017-12-01T00:00:00Z

MicroRNAs are short RNA molecules that are central to the regulation of many cellular and developmental pathways. They are processed in several stages from structured precursors in the nucleus, into mature microRNAs in the cytoplasm where they direct protein complexes to regulate gene expression through, often imperfect base-pairing with target messenger RNAs. The broad aim of this project is to better understand how polymorphisms and new mutations can disrupt microRNA processing and targeting, and ultimately define their contributions to human disease. I have taken two approaches towards this. The first approach is to comprehensively identify the microRNA targets by developing and applying a novel computational pipeline to identify microRNA binding events genome-wide in RNA-RNA interaction datasets. I use this to examine the transcriptomic diversity of microRNA binding, finding microRNA binding events along the full length of protein coding transcripts and with a variety of non-coding RNAs. This reveals enrichment for non-canonical microRNA binding at promoters and intronic regions around splice sites, and identifies highly spatially clustered binding sites within transcripts that may be acting as competitive endogenous RNAs to compete for microRNAs, effectively sequestering them. Using statistical models and new cell fractionated RNA-seq data, I rank the features of microRNAs and their binding sites which contribute to the strength and specificity of their interaction to provide a better understanding of the major determinants of microRNA targeting. The second approach is to directly identify DNA sequence changes in microRNA precursors that alter processing efficiency affecting mature microRNA abundance which are routinely overlooked in the search for disease or trait associated causal variants. I have systematically screened public datasets for both rare and common polymorphisms that overlap microRNA precursors and are correlated with mature microRNA levels as measured in short RNA sequencing. I use these eQTL SNPs to examine the most important microRNA precursor regions and sequence motifs. Several of these SNPs have been observed as risk factors in cancer or other clinically relevant traits, and correlated with microRNA processing efficiency. I demonstrate that a specific DNA change which is known to be important in the development of some cancers, is located in a microRNA precursor and affects the balance of its two products, miR-146a-3p and miR-146a-5p, that can be produced from that single precursor providing new insights into the mechanisms of microRNA production and the aspects of genetic mis-regulation that result in cancer. I find further examples of common human polymorphisms that appear to affect microRNA production from their precursors, several of these variants are independently implicated in human immune disease, cancer susceptibility and associated with other complex traits. As they exhibit a molecular phenotype and immediately lead to mechanistic hypotheses of trait causality that can be tested, these variants could provide a route into the frequently intractable problem of mechanistically linking non-coding genetic variation to human phenotypes. Applying similar studies to patient DNA has revealed rare and unique DNA changes that are now candidates for causing human disease that are being subject to follow-up experimental studies. Collectively this work has started to define which sequences changes in microRNAs are likely to disrupt their function and provides a paradigm for the analysis of microRNA sequence variants in human genetic disease.

Pancreatic cancer is the fourth leading cause of cancer death in the United States, with a five-year survival rate under 5%. Given the disease’s deadliness, increasing our understanding of the molecular nature of the pancreatic cancer is key to developing more effective preventive measures and treatments. Dietary energy restriction (DER) has been shown to have potent anticancer effects in pancreatic cancer, but the mechanism of action has yet to be completely elucidated. Here we investigate the potential of altered microRNA expression as a mechanism by which DER exerts its anticancer effect. Using the Exiqon microRNA Array, we identified several microRNAs of interest for further study. This includes microRNA (mir) 669c, a known regulator of glutathione-S transferases (linked to carcinogen metabolism and oxidative stress) that increases with age. To our knowledge, this is the first exploration of the effects of DER (which is known to suppress oxidative stress and other processes associated with aging and cancer) on microRNA expression. These findings may provide the initial steps towards identifying novel targets for pancreatic cancer prevention or treatment. / text

<p>The development and function of the nervous system relies on complex regulation of gene expression programs. MicroRNAs (miRNAs) are small RNAs that have diverse functions in mammalian development and disease. In concert with the RNA-induced silencing complex, miRNAs repress translation by binding to target mRNAs. The nervous system contains the largest proportion of miRNAs, yet few have been functionally characterized <italic>in vivo</italic>. </p><p>miR-133b is a highly conserved miRNA embedded in the sequence of 7H4, a noncoding RNA that is enriched at the neuromuscular junction (NMJ), a large synapse that is essential for eliciting muscle contraction and movement. I have found that, like 7H4, miR-133b expression is enriched at the NMJ and upregulated postnatally, coinciding with important events in synaptic maturation, including synaptic growth and elimination. Knockdown of miR-133b in postnatal muscle by electroporation of modified antisense oligonucleotides gave rise to abnormally large synapses, indicating a role for miR-133b in synaptic maturation. To specifically remove miR-133b <italic>in vivo</italic>, I generated a mouse containing a targeted deletion of the miR-133b stemloop. NMJ maturation and synapse elimination proceeded normally in miR-133b knockout mice, suggesting that miR-133b may have other functions at the synapse. The expression of 7H4 and miR-133b is upregulated following nerve transection, consistent with a role in synaptic regeneration. Indeed, NMJ reinnervation is delayed in miR-133b KO mice following nerve crush, but not nerve cut. These data suggest that miR-133b may have a specific protective function at the synapse that could be relevant to disease states, including amyotrophic lateral sclerosis (ALS), where NMJ denervation occurs following motor neuron cell death. However, loss of miR-133b did not affect survival or disease progression in the SOD1(G93A) mouse model, differentiating its role from that of miR-206, another miRNA found in 7H4.</p><p>miR-133b has recently been proposed to regulate the development and maintenance of midbrain dopaminergic (mDA) neurons. mDA neurons have critical functions in the control of movement and emotion, and their degeneration leads to motor and cognitive defects in Parkinson's disease. miR-133b is enriched in the midbrain and regulates mDA neuron differentiation <italic>in vitro</italic> by targeting Pitx3, a transcription factor required for appropriate development of substantia nigra DA neurons. However, the function of miR-133b in the intact midbrain has not been determined. miR-133b KO mice have normal numbers of midbrain dopaminergic neurons during development and aging. Moreover, dopamine neurotransmitter levels are unchanged in the striatum and other brain regions, while expression of dopaminergic genes including Pitx3 is also unaffected. Finally, miR-133b null mice display normal motor coordination and activity, suggesting that miR-133b does not play a significant role in the development or maintenance of the mDA neuron population.</p> / Dissertation