Introdução: A epilepsia do lobo temporal mesial (ELTM) é a forma mais comum de epilepsia parcial e com índices significativos de resistência aos tratamentos farmacológicos. Um dos grandes desafios para a neurociência na atualidade é o desenvolvimento de biomarcadores que facilitem o diagnóstico e prognóstico para a epilepsia. Recentemente, algumas pesquisas demonstraram que microRNAs circulantes no sangue são biomarcadores sensíveis e específicos para várias doenças, incluindo do SNC, podendo ser obtido de forma não invasiva e representando um método de detecção eficiente e de baixo custo. Portanto, nosso trabalho teve como objetivo analisar o perfil de expressão dos microRNAs miR-145, miR-181c, miR199a e miR-1183 no hipocampo e fração leucocitária do sangue de pacientes com ELTM e investigar se os mesmos podem auxiliar como biomarcadores de diagnóstico e prognóstico para a epilepsia. Pacientes e métodos: Foram utilizadas amostras de hipocampo e sangue de 20 pacientes com ELTM, sendo 10 com boa evolução pós-operatória (Engel I) e 10 com evolução pósoperatória insatisfatória (Engel III e IV), e para controle foram utilizados hipocampos de necropsias e sangue de indivíduos saudáveis. A análise de expressão dos miRNAs foi feita utilizando a técnica de RQ-PCR. Resultados e Conclusões: As expressões dos miRNAs se comportaram de forma diferente no hipocampo e sangue de pacientes com ELTM quando comparados aos grupos controles. O miR- 145 apresentou-se hipoexpresso no hipocampo e hiperexpresso no sangue. Os miRNAs miR-145, miR-181c, miR199a e miR-1183 apresentaram-se hiperexpressos na fração leucocitária do sangue de pacientes com ELTM, podendo estes contribuir como possíveis biomarcadores para auxiliarem no diagnóstico. Ainda, não encontramos diferenças estatística desses miRNAs no sangue e hipocampo de pacientes Engel I versus Engel III e IV, no entanto, o miR-1183 apresentou-se progressivamente mais expresso no sangue dos pacientes nos grupos controle, no grupo Engel I e no grupo Engel III e IV respectivamente. / Introduction: The mesial temporal lobe epilepsy (MTLE) is the most common form of partial epilepsy and also presents significant resistance to pharmacological treatment. A major challenge for neuroscience today is the development of biomarkers that facilitate the diagnosis and prognosis for epilepsy. Recently, research has shown that microRNAs circulating in the blood are sensitive and specific biomarkers for various diseases, including CNS, they can be obtained noninvasively and represent a low cost detection method. Thus, our study aimed to analyze the expression profile of microRNAs miR-145, miR-181c, miR199a and miR- 1183 in the hippocampus and leukocyte fraction of the blood of patients with MTLE and investigate whether they can assist as diagnostic and prognosis biomarkers for epilepsy. Patients and Methods: Hippocampus and blood samples of 20 patients with MTLE were used; ten with good postoperative outcome (Engel I) and ten with poor postoperative outcome (Engel III and IV), and for the control group hippocampus of necropsy and blood of healthy individuals were used. The analysis of the expression of miRNAs was performed using RQ-PCR. Results and Conclusions: The expression of miRNAs behaved differently in the hippocampus and blood of patients with MTLE when compared with the control groups. This different behavior was most evident in miR-145, which was hypo-expressed in the hippocampus and hyper-expressed in the blood. MiRNAs miR-145, miR-181c, miR199a and miR-1183 were hyper-expressed in the leukocyte fraction from the blood of patients with MTLE, and these can be potential biomarkers to aid in the diagnosis. We found no statistical differences of these miRNAs in blood and hippocampus of patients Engel I versus Engel III and IV, however, miR-1183 was progressively expressed in the blood of patients in the control groups, Engel I and Engel III and IV respectively.

INTRODUÇÃO: O tratamento cirúrgico da endometriose profunda tem se mostrado benéfico para os resultados de Reprodução Assistida. O motivo que leva aos melhores resultados ainda é desconhecido, mas remete à etiologia multifatorial dessa doença. Observa-se, então, a oportunidade de investigar mecanismos moleculares que possam justificar este fato. Nesse contexto, os microRNAs podem desempenhar papel fundamental na medida em que alteram a expressão de diferentes genes por meio da inibição póstranscricional. OBJETIVO: Analisar o efeito da remoção cirúrgica das lesões de endometriose profunda na expressão dos microRNAs -21, -451 e -29c, além da expressão da proteína FKBP52. MÉTODOS: Trata-se de estudo clínico, prospectivo, longitudinal e comparativo no qual foram incluídas 26 pacientes que foram divididas em dois grupos, segundo o resultado da ultrassonografia transvaginal com preparo intestinal. As pacientes que não apresentaram alterações sugestivas de endometriose compuseram o grupo controle (n = 11) e foram submetidas à coleta de amostra de endométrio. As pacientes do grupo de estudo (n = 15) foram submetidas à amostragem endometrial antes e após o tratamento cirúrgico da endometriose, além de ter sido realizada a coleta de amostra da lesão profunda durante o ato operatório. Foi realizada a extração total de RNA dessas amostras e, posteriormente, PCR em tempo real para análise de expressão dos microRNAs -21, -451 e -29c, além da proteína FKBP52. RESULTADOS: A comparação da expressão relativa dos microRNAs -21 e -451 entre as amostras, de forma geral, não mostrou diferença estatisticamente significante. A expressão relativa do microRNA-29c foi maior no endométrio de pacientes com endometriose em relação ao grupo controle e ainda maior nas lesões de endometriose profunda. Após a cirurgia, a expressão do microRNA-29c no endométrio, foi equivalente à do grupo controle. A expressão da FKBP52 foi menor no endométrio das mulheres com endometriose e nas lesões da doença em comparação ao grupo controle. Após o tratamento cirúrgico houve aumento da expressão de FKBP52 nas amostras de endométrio nas pacientes com endometriose que passou a ser semelhante à do grupo controle. CONCLUSÕES: Os resultados sugerem que a presença das lesões de endometriose pode aumentar a expressão do microRNA-29c no endométrio e, por conseguinte, diminuir a expressão de um dos seus RNA mensageiros alvos que codifica a proteína FKBP52. Após o tratamento cirúrgico, com remoção das lesões de endometriose, a expressão do microRNA-29c e da FKBP52 se assemelhou à do grupo controle / INTRODUCTION: Surgical treatment of deeply infiltrating endometriosis appears to yield benefits to the affected women and also to the assisted reproduction treatments. Although the mechanisms involved on the improvement of the outcomes are still unknown; yet, they are similar to the multifactorial origin of the endometriosis. Considering that the microRNAs can modify different genes expression, it was investigated their role concerning the molecular pathways leading to endometriosis and the clinical betterment of the assisted reproductive procedures after the surgical treatment. OBJECTIVE: The aim of this study was to evaluate the effect of surgical treatment in women with endometriosis focusing the microRNAs -21, -451 and -29c expression, as well as the protein FKBP52 expression. METHODS: This is a clinical, prospective, longitudinal and comparative study which included 26 patients that were divided into two groups according to the findings of the transvaginal ultrasound with bowel preparation. Eleven women without evidence of DIE composed the control group and were submitted to eutopic endometrium sampling. Fifiteen women presented with evidence for DIE detected by pelvic ultrasound were also submitted to eutopic endometrium sampling before and after surgical treatment. Surgical procedures revealed the presence of relevant DIE. Total RNA extraction of all samples was performed and followed by real time PCR to evaluate microRNAs -21, -451, -29c and protein FKBP52 expression. RESULTS: MicroRNAs -21 and -451 expression analysis did not show statistically significant difference among samples. Nonetheless, expression of microRNA-29c was elevated in eutopic endometrium of women suffering from DIE in comparison to the control group. After surgery, the microRNA- 29c expression in eutopic endometrium became equivalent to the control group. The expression for FKBP52 was lower in women having DIE than those without endometriosis. The surgical treatment increased the expression of the protein FKBP52 in eutopic endometrial samples, turning it similar to the control group. CONCLUSIONS: The results of this study suggest that the presence of DIE might increase the expression of microRNA-29c in eutopic endometrium and consequently decrease the expression of one of its target messenger RNA that codifies the protein FKBP52. After surgical removal of endometriosis lesions, the microRNA-29c and the FKBP52 expression returned to a level similar to the control group

O presente trabalho refere-se ao estudo do papel dos microRNAs no controle pós-transcricional das células T de camundongos Non Obese Diabetic (NOD) modelo que reproduz o diabetes mellitus do tipo 1 (DM-1). Durante o desenvolvimento do trabalho, procurou-se esclarecer a hipótese de que os microRNAs controlam os níveis de determinados RNAs mensageiros (mRNAs) das células T durante a indução ou perda de tolerância imunológica. Portanto, a expressão alterada dos microRNAs estaria contribuindo com o processo da autoimunidade. Sendo assim, o objetivo do estudo foi identificar os perfis de expressão e as redes de interação entre um conjunto de microRNAs e seus respectivos mRNAs alvos nos timócitos e nos linfócitos T CD3+ periféricos durante o desenvolvimento do diabetes mellitus do tipo 1 (DM-1) em camundongos NOD. Para avaliar a expressão de genes codificadores de mRNAs, sendo estes possíveis alvos de microRNAs, utilizou-se a tecnologia de microarrays. O uso de programas de análise e para a construção das redes foi imprescindível. Acreditase que fenômenos complexos como a regulação pós-transcricional de células T e seu envolvimento no processo de tolerância imunológica, bem como o surgimento de doenças autoimunes, podem ser melhor compreendidos por meio da genômica funcional. Os resultados encontrados evidenciam uma expressão diferenciada de mRNAs e microRNAs em timócitos e linfócitos T CD3+ periféricos durante o desenvolvimento do diabetes mellitus do tipo 1 (DM-1). As diferenças nos perfis transcricionais encontradas envolvem expressão de genes (mRNAs) relacionados diretamente ao sistema imune, a diferenciação e ativação de linfócitos T e a apoptose, bem como a outros processos relacionados a resposta imune. Além disso, as redes de interação microRNA-mRNA encontradas no presente trabalho evidenciam interações já conhecidas e apresentam novas interações, mostrando a participação de um grupo de microRNAs que estão atuando no controle pós-transcricional do diabetes do tipo 1 em camundongos NOD, contribuindo com a melhor compreensão do controle genético-molecular das doenças autoimunes, principalmente do diabetes do tipo 1. / This study refers to the role played by microRNAs in the post-transcriptional control of T cells from non obese diabetic (NOD) mice model which reproduces the type 1 diabetes (T1D). During the study, it was tried to clarify the hypothesis that microRNAs control certain messenger RNAs (mRNAs) levels of the T cells during the induction or loss of immunological tolerance. Therefore, the altered expression of microRNAs might be contributing to the process of autoimmunity. Thus, the study aim was to identify the expression profiles and interaction networks between a set of microRNAs and their mRNA targets in thymocytes and peripheral CD3+ T lymphocytes during the development of type 1diabetes (T1D) in NOD mice. The microarray technology was used to evaluate the expression of mRNAs as possible targets of microRNAs involved in this process. The use of bioinformatics software to reconstruct the networks was essential. It was realized that complex phenomena as post-transcriptional regulation in T cells and their involvement in the immune tolerance process, as well as the emergence of autoimmune diseases can be better understood only by means of functional genomics. The results show differential expression of mRNAs and microRNAs in thymocytes and peripheral CD3+ T lymphocytes during the development of type 1 diabetes (T1D). The differences found in the transcriptional profiles involve mRNAs related to the immune system, differentiation and activation of T lymphocytes and apoptosis as well as other processes related to immune response. In addition, the microRNA-mRNA interaction networks obtained in this study evidence the predicted interactions as well as new ones, showing the participation of a group of microRNAs that may be acting in post-transcriptional control of type 1 diabetes in NOD mice contributing to a better understanding of the molecular genetic control of autoimmune diseases in specially type 1 diabetes.

As células-tronco embrionárias (CTEs), extraídas da massa celular interna do blastocisto, tem como características principais a capacidade de auto-renovação e a pluripotência. Durante o desenvolvimento, as células perdem seu potencial de diferenciação e adquirem um perfil de expressão gênica mais restrito, modulado por mecanismos epigenéticos, assim como por microRNAs. Membros da família miR-29 têm como transcritos alvos enzimas responsáveis pela metilação da citosina em 5mC (DNMT3a e 3b) e também da desmetilação (TET1, 2 e 3) do DNA, pela hidroxilação de 5mC em 5hmC. Recentes trabalhos sugerem que a modulação do miR-29 sobre estes alvos teria um papel no início da diferenciação em CTEs de camundongos e no aumento de eficiência da geração de iPS em células humanas. No presente trabalho, buscou-se compreender o papel regulatório do miR-29a em seus alvos epigenéticos no contexto da pluripotência e no início da diferenciação com atRA. Para tanto, duas linhagens celulares pluripotentes humanas (H1 e NTera- 2) foram submetidas a indução de diferenciação com atRA e ao ganho de função do miR-29a durante quatro dias de cultivo para análises de expressão gênica. Ademais, em NT2, realizamos ensaios funcionais por microscopia de imunofluorescência quantitativa para avaliar os efeitos do ganho e perda de função do miR-29a, DNMT3b e TET1, sobre a expressão nuclear de OCT4 e os perfis globais de 5mC e 5hmC após 96 horas de transfecção. Neste ensaio, também avaliamos o papel específico da regulação pós-transcricional de DNMT3b e TET1 pelo miR-29a, utilizando moléculas bloqueadoras dos sítios alvo (TSB) do miR-29a nestes transcritos. Observamos que sob a indução do atRA, os níveis de expressão do miR- 29a e de seus genes alvos (com exceção de DNMT3b), assim como dos marcadores de endoderme e ectoderme, aumentaram, seguido da diminuição dos marcadores de pluripotência em ambas as linhagens. A transfecção de moléculas mímicas do miR-29a, reduziu os níveis de seus transcritos alvos após dois e quatro dias em NT2 e H1, além de reduzir os níveis nucleares de DNMT3b em NT2. Ainda, ocorreu um aumento na expressão de genes da endoderme, mesoderme e ectoderme em H1 e a queda da expressão gênica e nuclear de OCT4 em NT2. Com o uso de siRNA específicos, demonstramos que o knockdown dos níveis nucleares de DNMT3b foi acompanhado de uma queda nos níveis globais de 5mC e um aumento de OCT4 e de 5hmC. Já o knockdown de TET1, elevou os níveis de 5mC, mas também os níveis de 5hmC e OCT4 nuclear. As avaliações com o uso de TSB contra os sítios de ligação do miR-29a em seus transcritos alvo, TET1 e DNMT3b, demonstraram que em células NT2, o bloqueio da ligação do miR endógeno aos seus alvos resultam no aumento dos níveis globais de 5hmC, indicando que a regulação póstranscricional destes alvos pelo miR-29 teria um importante papel na regulação epigenética de células pluripotentes. / Embryonic stem cells (CTEs), extracted from the internal cell mass of the blastocyst, are main characterized by the capacity for self-renewal and pluripotency. During development, the cells lose their differentiation potential and acquire a restricter gene expression profile, modulated by epigenetic mechanisms, as microRNAs. Members of the miR-29 family have as target transcripts enzymes for cytosine methylation in 5mC (DNMT3a and 3b) and for DNA demethylation (TET1, 2 and 3), by hydroxylation of 5mC in 5hmC. Recent studies suggest that the modulation of miR-29 on these targets plays a role in early differentiation of mouse CTEs and in increasing human iPS cell generation efficiency. In the present study, we sought to understand the regulatory role of miR-29a in its epigenetic targets in the context of pluripotency and in early differentiation with atRA. For this, two human pluripotent cell lines (H1 and NTera-2) were submitted to differentiation induction with atRA and function gain of miR-29a during four days of culture for gene expression analysis. Furthermore, in NT2, we performed functional assays by quantitative immunofluorescence microscopy to evaluate the gain- and loss-of-function of miR-29a, DNMT3b and TET1 in the OCT4 nuclear expression and global profiles of 5mC and 5hC, 96 hours posttransfection. In this assay, we also evaluated the specific role of post-transcriptional regulation of DNMT3b and TET1 by miR-29a, using target site blocking molecules (TSB) of miR-29a. We observed that under the induction of atRA, the miR-29a expression levels and its target genes (except of DNMT3b), further the markers of endoderm and ectoderm, increased, followed by decreased pluripotency markers in both cell lines. Transfection of mimic molecules of miR-29a reduced the levels of their target transcripts after two and four days in NT2 and H1, and reduced nuclear levels of DNMT3b in NT2. In addition, the expression of endoderm, mesoderm and ectoderm genes increased in H1 and gene and nuclear expression of OCT4 decreased in NT2. With the use of specific siRNA, we demonstrated that the knockdown of nuclear levels of DNMT3b was accompanied by a drop in global 5mC levels and an increase of OCT4 and 5hmC. While, the knockdown of TET1 increased the levels of 5mC, 5hmC and nuclear OCT4. Evaluations using TSB against the miR- 29a binding sites in their target transcripts, TET1 and DNMT3b, show that in NT2 cells blocking the binding of endogenous miR to their targets results in an increase in global 5hmC levels, indicating that the post-transcriptional regulation of these targets by miR-29 would play an important role in the epigenetic regulation of pluripotent cells.

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.

A hipertensão arterial (HA) é uma síndrome multifatorial caracterizada por níveis elevados e sustentados de pressão arterial (PA). O treinamento físico (TF) aeróbio tem sido utilizado como um importante tratamento não farmacológico da HA, uma vez que ele corrige a rarefação microvascular e o perfil de fibras muscular e reduz a PA; entretanto, os mecanismos envolvidos são pouco conhecidos. Uma nova classe de pequenos RNAs, denominados de microRNAs (miRNAs), vem sendo muito estudados por regularem pós-transcricionalmente a expressão de seus genes alvo. Estudos avaliando o papel dos miRNAs na regulação do processo angiogênico e trofismo muscular tanto na HA quanto induzidos pelo TF são desconhecidos. Portanto, nosso objetivo é caracterizar o perfil de miRNAs e genes alvo do músculo esquelético envolvidos nos processos de rarefação microvascular e dano muscular na HA e verificar o papel terapêutico do TF de natação na correção desses parâmetros. Na primeira etapa do projeto, ratos espontaneamente hipertensos (SHR) com 3 meses de idade (HA recém estabelecida) e seus controles Wistar Kyoto (WKY) foram divididos em 4 grupos: SHR, SHR treinado (SHR-T), WKY e WKY treinando (WKY-T). O TF promoveu redução da PA em SHR e bradicardia de repouso acompanhando por um aumento VO2 nos animais treinados. O TF corrigiu a mudança na distribuição dos tipos de fibra muscular em SHR paralelamente a correção na expressão do miRNA-208b. A rarefação capilar foi prevenida em animais SHR-T concomitante a correção na expressão dos miRNAs-16, -21 e -126 envolvidos na promoção das vias angiogênicas e desativação de vias apoptóticas por alvejarem VEGF, Bcl-2 e PI3KR2. Na segunda etapa do projeto, SHR com 6 meses de idade (HA crônica) e seus controles WKY foram divididos nos mesmos 4 grupos da primeira etapa. Similares resultados foram observados nas respostas hemodinâmicas, mudança no perfil de fibras muscular e rarefação capilar. Em contraste, foi observado uma redução do VO2 acompanhado por uma atrofia e disfunção muscular em SHR. Além disso, estes animais apresentavam disfunção cardíaca e aumento na relação parede-lúmen da artéria femoral e arteríola muscular. O TF corrigiu estas alterações no vaso e no músculo. Curiosamente, o microarray de miRNAs muscular revelou um perfil de miRNAs envolvidos no remodelamento microvascular e muscular esquelético (miRNAs-96, -205, -182, -140, -328a, -665, -1, -499, -208b e -99b) possibilitando identificar genes alvo modificados pela HA e TF. Assim, alvos envolvidos na mudança do perfil de fibras, síntese protéica via IGF-I/ Akt/ mTOR e da via de sinalização angiogênica dependente e independente de VEGF foram prejudicadas na HA e corrigidos pelo TF. Juntos, os resultados apoiam a hipótese de que as alterações estruturais e funcionais advindas da progressão da HA possa ser regulada por um conjunto de miRNAs e genes alvo; e que o TF participa no restabelecimento muscular e da rede microvascular. Assim, há perspectiva do potencial terapêutico do uso de miRNAs no tratamento da HA / Hypertension is a multifactorial syndrome characterized by sustained high levels of blood pressure (BP). Aerobic exercise training (ET) has been used as an important non-pharmacological treatment of hypertension, since it corrects microvascular rarefaction, muscle fiber type profile and reduces BP; however, the mechanisms involved are poorly understood. A new class of small RNAs, called microRNAs (miRNAs), has been extensively studied because they regulate their target gene expression at the post-transcriptional level. Studies evaluating the role of miRNAs in the angiogenic process and muscle trophism both in hypertension and exercise training are unknown. Therefore, our goal is to characterize the skeletal muscle miRNA profiles and their target genes involved in microvascular rarefaction and muscle damage in hypertension and check the therapeutic role of swimming ET in correcting these parameters. In the first stage of the project, spontaneously hypertensive rats (SHR) aged 3 months (hypertension newly established) and their controls Wistar Kyoto (WKY) were divided into 4 groups: SHR, trained SHR (SHR-T), WKY and WKY training (WKY-T). ET promoted reduction in BP in SHR and resting bradycardia with subsequent increase in VO2in trained animals. ET corrected the change in proportion of muscle fiber types in SHR parallel with the correction in miRNA-208b expression. Capillary rarefaction was prevented in SHR-T group concomitant with the correction in the expression of miRNAs-16, -21 and -126 involved in promoting angiogenesis and deactivation of apoptotic pathways by targeting VEGF, Bcl-2 and PI3KR2. In the second stage of the project, 6-month-old SHR (chronic hypertension) and their controls WKY were divided into the same four groups as those in the first stage. Similar results were observed in hemodynamic responses, muscle fiber profile and capillary rarefaction. In contrast, we observed a reduction in VO2 accompanied by muscle atrophy and dysfunction in SHR. Moreover, these animals showed cardiac dysfunction and an increase in wall:lumen ratio of the femoral artery and muscle arteriole. ET corrected these changes in the vessel and muscle. Interestingly, the muscle miRNA microarray revealed a profile of miRNAs involved in microvascular and muscle remodeling (miRNAs-96, -205, -182, -140, - 328a, -665, -1, -499, -208b and -99b) making it possible to identify target genes modified by hypertension and ET. Thus, targets involved in changing the fiber profile, protein synthesis by the IGF-I/ Akt /mTOR pathway, and the VEGF-dependent and independent angiogenic signaling pathway, were impaired in hypertension and corrected by ET. Together, these results support the hypothesis that the structural and functional changes arising from the progression of hypertension may be regulated by a set of miRNAs and target genes; and ET participates in restoring the muscle and microvascular network. Thus, there are perspectives for the potential therapeutic use of miRNAs in the treatment of hypertension

MicroRNA (miRNA) are a class of small endogenous non-coding RNA that are mainly negative transcriptional and post-transcriptional regulators in both plants and animals. Recent studies have shown that miRNA are involved in different types of cancer and other incurable diseases such as autism and Alzheimer’s. Functional miRNAs are excised from hairpin-like sequences that are known as miRNA genes. There are about 21,000 known miRNA genes, most of which have been determined using experimental methods. miRNA genes are classified into different groups (miRNA families). This study reports about 19,000 unknown miRNA genes in nine species whereby approximately 15,300 predictions were computationally validated to contain at least one experimentally verified functional miRNA product. The predictions are based on a novel computational strategy which relies on miRNA family groupings and exploits the physics and geometry of miRNA genes to unveil the hidden palindromic signals and symmetries in miRNA gene sequences. Unlike conventional computational miRNA gene discovery methods, the algorithm developed here is species-independent: it allows prediction at higher accuracy and resolution from arbitrary RNA/DNA sequences in any species and thus enables examination of repeat-prone genomic regions which are thought to be non-informative or ’junk’ sequences. The information non-redundancy of uni-directional RNA sequences compared to information redundancy of bi-directional DNA is demonstrated, a fact that is overlooked by most pattern discovery algorithms. A novel method for computing upstream and downstream miRNA gene boundaries based on mathematical/statistical functions is suggested, as well as cutoffs for annotation of miRNA genes in different miRNA families. Another tool is proposed to allow hypotheses generation and visualization of data matrices, intra- and inter-species chromosomal distribution of miRNA genes or miRNA families. Our results indicate that: miRNA and miRNA genes are not only species-specific but may also be DNA strand-specific and chromosome-specific; the genomic distribution of miRNA genes is conserved at the chromosomal level across species; miRNA are conserved; More than one miRNA with different regulatory targets can be excised from one miRNA gene; Repeat-related miRNA and miRNA genes with palindromic sequences may be the largest subclass of miRNA class that have eluded detection by most computational and experimental methods.

Submitted by Leonardo Cavalcante (leo.ocavalcante@gmail.com) on 2018-04-25T12:06:32Z No. of bitstreams: 1 Arquivototal.pdf: 2117961 bytes, checksum: 458d516added97ab516b1c8ae4ec5ddd (MD5) / Made available in DSpace on 2018-04-25T12:06:32Z (GMT). No. of bitstreams: 1 Arquivototal.pdf: 2117961 bytes, checksum: 458d516added97ab516b1c8ae4ec5ddd (MD5) Previous issue date: 2017-10-27 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The microRNAs are of great interest in studies on viral infections. In the context of the Epstein-Barr virus (EBV), they act as important regulators of the expression of viral and cellular genes, facilitating viral persistence and oncogenesis. The present work aims to identify viral microRNAs, their target mRNAs and the essential target proteins specifically for Human gammaherpesvirus 4 tipo 2. First, we used the StructRNAfinder and IntaRNA programs to perform the identification tasks of miRNAs and target mRNAs, respectively. Subsequently, we created a network of proteins from the data obtained from the tool online virusmentha and visualized in the program cytoscape. In addition, we performed a topological analysis of the protein interaction network with the cytoscape tool. We found 55 viral miRNAs and 46 mRNAs and identified 10 proteins that were prominent in the protein interaction network. The BTRF1 and BSFR1 proteins exert a greater control in the protein network, although they are proteins of the integument with unknown functions. Thus, we suggest that the BTRF1 and BSRF1 proteins are potential targets for understanding and controlling EBV infections. / Os microRNAs são de grande interesse em estudos sobre infecções virais. No contexto do vírus Epstein-Barr (EBV), eles atuam como importantes reguladores da expressão de genes virais e celulares, facilitando a persistência viral e a oncogênese. O presente trabalho objetiva identificar microRNAs virais, seus mRNAs alvos e as proteínas alvos essenciais específicas para o Human gammaherpesvirus 4 tipo 2. Primeiramente, utilizamos os programas StructRNAfinder e o IntaRNA para realizar as tarefas de idenficação de miRNAs e mRNAs alvos, respectivamente. Posteriormente, criamos uma rede de proteínas a partir dos dados obtidos da ferramenta online virusmentha e visualizamos no programa cytoscape. Além disso, realizamos uma análise topológica da rede de interação de proteínas com a ferramenta cytoscape. Encontramos 55 miRNAs virais e 46 mRNAs e identificamos 10 proteínas que se destacavam na rede de interação de proteínas. As proteínas BTRF1 e BSFR1 exercem um maior controle na rede de proteínas, embora sejam proteínas do tegumento com funções desconhecidas. Assim, sugerimos que as proteínas BTRF1 e BSRF1 sejam alvos potenciais para compreender e controlar as infecções por EBV.

Introdução: A epilepsia do lobo temporal mesial (ELTM) é a forma mais comum de epilepsia parcial e com índices significativos de resistência aos tratamentos farmacológicos. Um dos grandes desafios para a neurociência na atualidade é o desenvolvimento de biomarcadores que facilitem o diagnóstico e prognóstico para a epilepsia. Recentemente, algumas pesquisas demonstraram que microRNAs circulantes no sangue são biomarcadores sensíveis e específicos para várias doenças, incluindo do SNC, podendo ser obtido de forma não invasiva e representando um método de detecção eficiente e de baixo custo. Portanto, nosso trabalho teve como objetivo analisar o perfil de expressão dos microRNAs miR-145, miR-181c, miR199a e miR-1183 no hipocampo e fração leucocitária do sangue de pacientes com ELTM e investigar se os mesmos podem auxiliar como biomarcadores de diagnóstico e prognóstico para a epilepsia. Pacientes e métodos: Foram utilizadas amostras de hipocampo e sangue de 20 pacientes com ELTM, sendo 10 com boa evolução pós-operatória (Engel I) e 10 com evolução pósoperatória insatisfatória (Engel III e IV), e para controle foram utilizados hipocampos de necropsias e sangue de indivíduos saudáveis. A análise de expressão dos miRNAs foi feita utilizando a técnica de RQ-PCR. Resultados e Conclusões: As expressões dos miRNAs se comportaram de forma diferente no hipocampo e sangue de pacientes com ELTM quando comparados aos grupos controles. O miR- 145 apresentou-se hipoexpresso no hipocampo e hiperexpresso no sangue. Os miRNAs miR-145, miR-181c, miR199a e miR-1183 apresentaram-se hiperexpressos na fração leucocitária do sangue de pacientes com ELTM, podendo estes contribuir como possíveis biomarcadores para auxiliarem no diagnóstico. Ainda, não encontramos diferenças estatística desses miRNAs no sangue e hipocampo de pacientes Engel I versus Engel III e IV, no entanto, o miR-1183 apresentou-se progressivamente mais expresso no sangue dos pacientes nos grupos controle, no grupo Engel I e no grupo Engel III e IV respectivamente. / Introduction: The mesial temporal lobe epilepsy (MTLE) is the most common form of partial epilepsy and also presents significant resistance to pharmacological treatment. A major challenge for neuroscience today is the development of biomarkers that facilitate the diagnosis and prognosis for epilepsy. Recently, research has shown that microRNAs circulating in the blood are sensitive and specific biomarkers for various diseases, including CNS, they can be obtained noninvasively and represent a low cost detection method. Thus, our study aimed to analyze the expression profile of microRNAs miR-145, miR-181c, miR199a and miR- 1183 in the hippocampus and leukocyte fraction of the blood of patients with MTLE and investigate whether they can assist as diagnostic and prognosis biomarkers for epilepsy. Patients and Methods: Hippocampus and blood samples of 20 patients with MTLE were used; ten with good postoperative outcome (Engel I) and ten with poor postoperative outcome (Engel III and IV), and for the control group hippocampus of necropsy and blood of healthy individuals were used. The analysis of the expression of miRNAs was performed using RQ-PCR. Results and Conclusions: The expression of miRNAs behaved differently in the hippocampus and blood of patients with MTLE when compared with the control groups. This different behavior was most evident in miR-145, which was hypo-expressed in the hippocampus and hyper-expressed in the blood. MiRNAs miR-145, miR-181c, miR199a and miR-1183 were hyper-expressed in the leukocyte fraction from the blood of patients with MTLE, and these can be potential biomarkers to aid in the diagnosis. We found no statistical differences of these miRNAs in blood and hippocampus of patients Engel I versus Engel III and IV, however, miR-1183 was progressively expressed in the blood of patients in the control groups, Engel I and Engel III and IV respectively.

O objetivo desse estudo foi investigar a participação de integrina &alpha;1&beta;1 e microRNAs (miRs) no potencial osteogênico de superfícies de titânio (Ti) com nanotopografia. Discos de Ti previamente polidos foram tratados quimicamente com H2SO4/H2O2 para obtenção de nanotopografia, que foi observada por microscopia eletrônica de varredura. Para o estudo da participação da integrina &alpha;1&beta;1, células-tronco mesenquimais (CTMs) de ratos foram cultivadas em condições osteogênicas e não osteogênicas sobre superfícies de Ti com nanotopografia e sem tratamento químico (controle). O resultados mostraram que a nanotopografia de Ti aumentou a proliferação celular, a atividade de fosfatase alcalina (Alp) e regulou positivamente a expressão gênica de marcadores da diferenciação osteoblástica em CTMs cultivadas tanto em condições osteogênicas quanto em condições não osteogênicas. Além disso, uma maior expressão gênica para as integrinas &alpha;1 e &beta;1 foi observada em culturas crescidas sobre nanotopografia em condições não osteogênicas em relação ao Ti controle. O uso de obtustatina, um inibidor de integrina &alpha;1&beta;1, reduziu os efeitos da nanotopografia sobre os marcadores osteoblásticos, indicando a participação da via de sinalização dessa integrina nos efeitos da nanotopografia sobre CTMs. Para investigar a participação de miRs no efeito osseoindutor da nanotopografia de Ti, foram utilizadas CTMs humanas e células préosteoblásticas de camundongos da linhagem MC3T3-E1. A análise em larga escala da expressão de miRs revelou que 60 miRs foram regulados positivamente (no mínimo, 2x maior), enquanto 58 miRs foram regulados negativamente (no mínimo, 2x menor) em CTMs crescidas sobre a nanotopografia. Três desses miRs, miR-4448, -4708 e -4773, cuja expressão foi significativamente reduzida pela nanotopografia de Ti (no mínimo, 5x menor), afetaram a diferenciação osteoblástica de CTMs. Esses miRs atuam diretamente sobre SMAD1 e SMAD4, proteínas transdutoras da sinalização da proteína óssea morfogenética 2 (Bmp-2), conhecida por sua capacidade osseoindutora. Além disso, verificou-se que a sobreexpressão de miR-4448, -4708 e -4773 em células pré-osteoblásticas MC3T3-E1 inibiu a expressão gênica e proteica de SMAD1 e SMAD4 e, consequentemente, a expressão gênica de marcadores ósseos. Esses dados sugerem a influência do circuito miR-SMAD-Bmp-2 sobre o efeito osseoindutor da nanotopografia. Conjuntamente, os achados do presente estudo mostraram que o efeito da nanotopografia de Ti sobre a diferenciação osteoblástica resulta de um mecanismo regulatório complexo, do qual fazem parte as vias de sinalização da integrina &alpha;1&beta;1 e da Bmp-2, com a participação de miRs. Esses resultados podem representar um avanço para o desenvolvimento de novas modificações de superfície, com o objetivo de acelerar e/ou melhorar o processo de osseointegração. / The aim of this study was to investigate the role of the &alpha;1&beta;1 integrin and microRNAs (miRs) on the osteogenic potential of titanium (Ti) with nanotopography. Polished Ti discs were chemically treated with H2SO4/H2O2 to generate nanotopography, which was observed under scanning electron microscopy. For the study related to the &alpha;1&beta;1 integrin, rat mesenchymal stem cells (MSCs) were cultured under osteogenic and non-osteogenic conditions on Ti with nanotopography and non-treated Ti discs (control). Nanotopography increased cell proliferation and alkaline phosphatase (Alp) activity and upregulated the gene expression of bone markers in cells cultured under osteogenic and non-osteogenic conditions. Furthermore, the gene expression of &alpha;1 and &beta;1 integrins was higher in cells cultured on nanotopography under non-osteogenic conditions compared with control. Obtustatin, an inhibitor of &alpha;1&beta;1 integrin, reduced the higher gene expression of the bone markers induced by nanotopography. These results indicate that &alpha;1&beta;1 integrin signaling pathway determines the osteoinductive effect of nanotopography on MSCs. The role of miRs in the osteogenic potential of Ti with nanotopography was evaluated using human MSCs and MC3T3-E1 mouse pre-osteoblastic cells. The miR sequencing analysis revealed that 60 miRs were upregulated (> 2 fold), while 58 miRs were downregulated (< 2 fold) in MSCs grown on nanotopography. Three miRs, miR-4448, -4708 and -4773, which were significantly downregulated (< 5 fold) by nanotopography, affected the osteoblast differentiation of MSCs. These miRs directly target SMAD1 and SMAD4, both key transducers of the bone morphogenetic protein 2 (Bmp-2) osteogenic signal, which were upregulated by nanotopography. Overexpression of miR-4448 - 4708 and 4773 in MC3T3-E1 cells noticeably inhibited gene and protein expression of SMAD1 and SMAD4 and by targeting them, these miRs repressed gene expression of key bone markers. These results suggest that a miR-SMAD-Bmp-2 circuit acts in the Ti nanotopography-mediated osteoblast differentiation. Taken together, our data showed that the osteoblast differentiation induced by Ti with nanotopography is governed by a complex regulatory network involving a crosstalk between &alpha;1&beta;1 integrin and Bmp-2 signaling pathways with participation of miRs.