Orientadores: Michel Georges Albert Vincentz, Fabio Tebaldi Silveira Nogueira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-13T11:23:48Z (GMT). No. of bitstreams: 1 Zanca_AlmirSamuel_M.pdf: 11034884 bytes, checksum: 0545b58df6802e07009aef761dda3003 (MD5) Previous issue date: 2009 / Resumo: RNAs não-codificantes de 20-27 nucleotídeos (nt) regulam transcricionalmente ou pós-transcricionalmente a expressão de genes endógenos, modelando o transcriptoma e a produção de proteínas. Dentre estes, microRNAs (miRNAs) desempenliam papel chave no desenvolvimento vegetal, observação comprovada pela avaliação fenotípica e molecular de plantas transgênicas e de mutantes defectivos na produção de tais RNAs. MiRNAs são produzidos a partir de precursores longos (pri-miRNAs), os quais são posteriormente processados por enzimas específicas, gerando o miRNA maduro (20-22 nt). O miRNA maduro, por sua vez, guia a clivagem do mRNA de genes-alvo e bloqueia a tradução de proteínas, afetando diversos aspectos do desenvolvimento. O sequênciamento de populações de RNAs regulatórios possibilitou a identificação de miRNAs conservados e específicos em diferentes espécies vegetais, embora estudos em plantas de importância econômica sejam ainda incipientes. Atualmente, existem diversos bancos públicos de sequências ESTs disponíveis. Esses bancos possuem um grande número de sequências não-codíficantes, dentre as quais podem estar presentes pri-miRNAs, os quais são também são moléculas poliadeniladas similares a mRNAs codifícantes. O banco público de ESTs de cana-de-açúcar TIGR Gene Index foi usado como base para uma busca de miRNAs. O processo criado possibilitou a identificação de 20 precursores de miRNAs, agrupados em 15 famílias distintas. No presente trabalho desenvolveu-se também ferramenta para predição de potenciais alvos para os miRNAs encontrados. As famílias de miRNAs de cana-de-áçucar e a ferramenta de predição de genes-alvo estão integrados em banco de dados que estará disponível brevemente. Análise de expressão gênica demonstrou que precursors de miRNAs de cana-de-açúcar acumulam em níveis variáveis em distintos tecidos/órgãos. Além disso, tanto o acúmulo do miRNA maduro quanto a degradação do mRNA-alvo foram avaliados para alguns casos estudados. A caracterização de um miRNA específico de monocotiledôneas (miR528) e a confirmação de seu alvo, um gene comum em angiospermas, predito pela primeira vez neste trabalho, gera um interessante questionamento sobre a regulação desse gene via miRNA apenas em monocotiledôneas / Abstract: No-coding RNAs of 20-27 nucleotides (nt) transcriptional or posttranscriptionally regulate endogenous gene expression, affecting the cellular output of transcripts and proteins. Among these RNAs, microRNAs (miRNAs) play an important role in plant development as confirmed by phenotypic and molecular evaluation of transgenic plants and knockout mutants defective in miRNA biogenesis and function. miRNAs are produced from long precursors (pri-miRNAs), which are processed by specific enzymes into the mature miRNA (20-22 nt). The mature miRNA guides the cleavage of target genes as well as impairs protein translation, affecting several development processes. Deep sequencing of small RNAs identified conserved and species-specific miRNAs. Nevertheless, studies on crops are still in their infancy. Public ESTs databases are an important source of no-coding sequences, in which we can find miRNAs precursors, which are polyadenilated RNAs as messenger RNAs. In this work, the public sugarcane EST database TIGR Gene Index was used to search conserved miRNAs. The pipeline developed in this work made possible the identification of 20 miRNAs precursors, grouped into 15 families. It was also developed a search tool for potential miRNAs targets. Sugarcane miRNAs precursors displayed tissue/organ differential expression profiles. Moreover, a new identified miRNA target was confirmed experimentally. This new target is regulated by a monocot specific miRNA, miR528. Interestingly, this miRNA target is conserved in eudicots and monocots, even though its regulation by miRNA is not. This finding raises the question of why this gene has evolved in having a miRNA-mediated posttranscriptional regulation only in monocots / Mestrado / Bioquimica / Mestre em Biologia Funcional e Molecular

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MicroRNAs are short non-protein coding RNAs which negatively regulate gene expression by guiding the RNA-induced silencing complex to complementary target mRNAs. MicroRNA regulation is implicated in essentially all biological processes, and microRNAs have a prominent role in animal development. Several microRNA families are conserved between deuterostomes and protostomes, however the majority of microRNAs in animal species are a result of continuous de novo gene birth processes throughout natural history. The acquisition of novel microRNAs, and changes in existing microRNAs, has been suggested to play a role in animal evolution. However, the constraints on microRNA emergence, evolution and expression are not well understood. We have studied the interplay of microRNA developmental expression and evolutionary dynamics in model insects displaying different modes of embryogenesis. We first determined the evolutionary origins and rates of change of microRNAs in Drosophila melanogaster and Drosophila virilis, and analysed their temporal expression profiles throughout development by deep sequencing. We found a good correlation between microRNA conservation and abundance at most stages except for the early embryo, where fast-evolving microRNAs are highly expressed. We further showed that the temporal expression of orthologous microRNAs is highly similar between species, and the global microRNA profiles across development display an hourglass-like conservation pattern, previously observed for protein-coding genes. We next extended our analyses to the red flour beetle Tribolium castaneum, which develops via the short germband mode of embryogenesis. This developmental mode is ancestral and widespread in arthropods, yet the microRNA complement of a representative species has not been previously characterized. We find a number of conserved features between Drosophila and Tribolium, including microRNA maternal loading and modifications, and microRNA-mediated targeting of maternally deposited transcripts. We also describe an abundant pool of maternally loaded and zygotically expressed piRNAs, which appear to be turned on in response to active transposons. In contrast to the previously observed piRNA profile in flies, beetle piRNAs are abundant throughout the entire embryogenesis. A majority of the newly identified microRNAs in the flour beetle are expressed during a discrete period in the early blastoderm, and also target maternally provided transcripts. The observation that the early embryos of both Drosophila and Tribolium are uniquely characterized with high levels of novel and dynamically evolving microRNAs suggests a conserved phenomenon where the blastoderm stage is a highly permissive environment for microRNA innovations.

Os Transtornos do Espectro do Autismo (TEA) reúnem um conjunto de alterações no desenvolvimento, caracterizado por dificuldades nos níveis de interação social, linguagem, comunicação, processo imaginativo e no repertório restrito de interesses e atividades. O autismo ainda possui etiologia desconhecida e até o momento nenhum tratamento ou marcador clínico para diagnóstico foi identificado. Apesar de alta herdabilidade, há uma alta heterogeneidade na sua arquitetura genética. Fatores ambientais podem contribuir com o aumento do risco do desenvolvimento do autismo, incluindo a exposição pré-natal a teratógenos como o ácido valpróico (VPA). MicroRNA são pequenos RNA não codificadores com aproximadamente 19-25 nucleotídeos que atuam como reguladores da expressão gênica, podendo agir no controle de diversos processos biológicos. O objetivo deste estudo foi avaliar os perfis de expressão de alguns miRNA no sangue total do modelo animal de autismo induzido por exposição pré-natal ao ácido valpróico (VPA). Além disso, as funções potenciais dessas moléculas foram avaliadas através da correlação com seus genes alvos, os quais poderiam, por sua vez, estar associados com os fundamentos da patofisiologia do autismo. Mudanças nos níveis de miRNA induzidas pelo VPA podem contribuir para algumas características relacionadas ao autismo. Os alvos preditos e validados dos miRNA que se mostraram alterados incluem alguns genes que estão envolvidos na síntese de proteínas, na resposta imune, inflamatória e à hipóxia, no desenvolvimento dos linfócitos e do sistema nervoso entérico e na transmissão sináptica. O presente estudo sugere que a avaliação da expressão de miRNA pode ser utilizada para a identificação potencial de rotas alteradas no autismo e podem constituir marcadores para a detecção e diagnóstico, além de auxiliar nas investigações sobre mecanismos de ação molecular algumas em vias de sinalização supostamente alterados no autismo. / The Autism Spectrum Disorders (ASD) assemble a group of developmental disorders characterized by difficulties in levels of social interaction, language, communication, imaginative process and restricted repertoire of activities and interests. The etiology of autism has still unknown and no treatment or clinical markers for diagnosis were identified. Despite its high heritability, there is a high heterogeneity in its genetic architecture. Environmental factors may contribute to the increased risk of developing autism, including prenatal exposure to teratogens such as valproic acid (VPA). MicroRNA are small noncoding RNA with approximately 19-25 nucleotides that act as regulators of gene expression and may act in the control of many biological processes. The aim of this study was to evaluate the expression profile of miRNA levels in whole blood samples from animal model of autism by prenatal exposure to VPA. In addition, some potential roles of these miRNA were analyzed through correlation with their target genes, which could, in turn, be associated with the basis of the pathophysiology of autism. Changes in the miRNA levels induced by VPA may contribute to some features related to autism. The predicted and validated targets of the altered miRNA included some genes that are involved in protein synthesis, immune, inflammatory and hypoxia responses, lymphocytes and enteric nervous system development and synaptic transmission. The present study suggests that the assessment of the expression of miRNA may be used to perform the identification of potential pathways altered in autism and may constitute markers for the detection and diagnostics, in addition to studies related therapies and mechanistic investigations on signaling pathways putatively altered in autism.

La Macroglobulinémie de Waldenström (MW) est un type de lymphome à cellules B de faible grade dont la pathogénèse est caractérisée par différents stades de progression. La MW asymptomatique n’occasionne pas de symptômes sévères chez les patients qui en sont atteints et nécessite une observation régulière mais pas de traitement. Elle peut évoluer en une forme symptomatique aux effets nocifs qui requiert un traitement par chimiothérapie. Le développement de la MW est généralement précédé par l’apparition d’un syndrome précurseur appelé Gammapathie Monoclonale de Signification Indéterminée à Immunoglobuline M (GMSI à IgM). Bien que les conséquences cliniques des formes asymptomatique et symptomatique de la maladie soient dramatiquement différentes pour les patients, au niveau cellulaire peu de différences permettant d’expliquer ce contraste ont été identifiées. Les mécanismes moléculaires de progression de la maladie restent donc majoritairement à élucider.Les exosomes sont des vésicules d’une taille de 40 à 100 nanomètres sécrétées par les cellules. Ils assurent le transport de protéines, de lipides et de molécules d’ARN ou d’ADN entre les cellules de l'organisme et constituent ainsi un moyen de communication intercellulaire. Ces propriétés permettent aux exosomes de conditionner le microenvironnement tumoral afin de le rendre favorable à la survie et la dissémination des cellules tumorales.Le but de cette étude est d’analyser le contenu des exosomes circulants chez les patients atteints de MW afin d’identifier des microARNs exprimés différentiellement en fonction du stade de progression de la maladie.Pour cela, les exosomes présents dans le sang circulant de patients à différents stades de la maladie (GMSI à IgM, MW asymptomatique et MW symptomatique) et d’individus sains ont été isolés. Le contenu en microARNs de ces exosomes a été analysé dans un premier temps par qRT-PCR puis par une technique utilisant la cytométrie de flux. Ces analyses ont permis d’identifier un groupe de microARNs dont l’expression corrèle directement ou inversement avec la progression de la maladie.En parallèle, l’ADN contenu dans les exosomes de certains de ces échantillons a également été extrait afin de détecter la fraction de variants mutés pour MYD88, un gène muté chez environ 90% des patients atteints de MW. Cette analyse permettra d’identifier une potentielle corrélation entre la progression de la maladie, la fraction de variants mutés de MYD88 et l’expression de certains microARNs dans les exosomes circulants. / Waldenström Macroglobulinemia (WM) is a low- grade B-cell lymphoma with a heterogenous clinical presentation. In patients with the asymptomatic form of the disease, no severe symptoms are observed and only monitoring is recommended. However, it can evolve into symptomatic WM in which case the patients will require treatment with chemotherapy .In many patients, the diagnosis is preceded with an asymptomatic precursor state of IgM monoclonal gammopathy of undetermined significance (MGUS). To date, the molecular mechanisms involved in the progression from asymptomatic WM to symptomatic WM remain to be elucidated.Exosomes are small vesicles secreted by cells with a size ranging from 40 to 100 nanometers that mediate the transfer of nucleic acids, proteins and lipids between distant cells in the organism. Exosomes enable communication between cells and the conditioning of distant tissues throughout the body.The goal of this study is to analyze the microRNA content of circulating exosomes in patients at different stages of WM progression to identify microRNAs which expression correlates with disease progression.Exosomes were isolated from the peripheral blood of healthy individuals and patients with WM at different stages (IgM MGUS, asymptomatic WM and symptomatic WM). The microRNA content of these exosomes was analyzed first by qRT-PCR and then by a technique involving flow cytometry. These analyses revealed a group of microRNAs which expression correlated directly or inversely with disease progression.In parallel, the DNA content of some of the exosomes was extracted in order to detect the fraction of mutated MYD88, a gene mutated in approximately 90% of patients with WM. This could potentially identify a correlation between disease progression, the fraction of mutated MYD88 and the expression of specific microRNAs in circulating exosomes.

Orientador: Debora Aparecida Pires de Campos Zuccari / Banca: Sonia Maria Oliani / Banca: Flávia Cristina Rodrigues Lisoni / Banca: Dorotéia Rossi Silva Souza / Banca: Marina Gobbe Moschetta / Resumo: O câncer de mama apresenta alta incidência e alta taxa de mortalidade devido a rápida progressão tumoral e disseminação metastática que ocorrem com o incentivo da angiogênese. MicroRNAs (miRNAs) são pequenas moléculas de RNA mensageiro (RNAm) não codificantes que desempenham papel fundamental na regulação gênica. A desregulação dessas moléculas está relacionada com a iniciação e progressão de diferentes tipos tumorais humanos, incluindo o câncer de mama. Existem fortes evidências de miRNAs atuando como oncogenes e supressores tumorais, regulando o processo de angiogênese, crescimento tumoral e metástase. É neste cenário que a melatonina, hormônio secretado pela glândula pineal, vem ganhando destaque como potencial tratamento contra o câncer de mama por apresentar efeitos oncostáticos, antiangiogênicos, além de atuar na regulação da expressão de miRNAs. Assim, o objetivo deste estudo foi avaliar o potencial valor terapêutico da melatonina na regulação do supressor tumoral miR148a-3p e relação com a progressão tumoral e angiogênese. Inicialmente, células tumorais de mama MDA-MB-231 foram cultivadas nas condições experimentais controle e tratamento com melatonina (1 mM). O PCR Array foi realizado para análise da expressão de 84 miRNAs relacionados com o câncer de mama e, após análise em banco de dados e literatura, o miR-148a-3p foi selecionado para validação. As células MDA-MB-231 foram cultivadas em quatro grupos experimentais: controle, tratamento com melatonina (1 mM),... / Abstract: Breast cancer has a high incidence and high mortality rate due to rapid tumor progression and metastatic dissemination that occur with the encouragement of angiogenesis. MicroRNAs (miRNAs) are small non-coding messenger RNA (mRNA) molecules that play a key role in gene regulation. Deregulation of these molecules is related to the initiation and progression of different human tumor types, including breast cancer. There is strong evidence for miRNAs acting as oncogenes and tumor suppressors, regulating the process of angiogenesis, tumor growth and metastasis. It is in this scenario that melatonin, a hormone secreted by the pineal gland, has been gaining prominence as a potential treatment against breast cancer for having oncostanic and antiangiogenic effects, in addition to regulating the expression of miRNAs. Thus, the objective of this study was to evaluate the potential therapeutic value of melatonin in the regulation of the tumor suppressor miR-148a-3p and its relation to tumor progression and angiogenesis. Initially, MDA-MB-231 breast tumor cells were cultured under the experimental control and treatment conditions with melatonin (1 mM). The PCR Array was performed to analyze the expression of 84 miRNAs related to breast cancer and, after analysis in the database and literature, miR-148a-3p was selected for validation. MDA-MB-231 cells were cultured in four experimental groups: control, treatment with melatonin (1 mM), overexpression of miR-148a-3p and control of the ... / Doutor

Recent work has demonstrated that microRNAs (miRNAs) are involved in critical biological processes by suppressing the translation of coding genes. This work develops an integrated database, miRNAMap, to store the known miRNA genes, the putative miRNA genes, the known miRNA targets and the putative miRNAtargets. The knownmiRNAgenes in fourmammalian genomes such as human, mouse, rat and dog are obtained from miRBase, and experimentally validated miRNA targets are identified in a survey of the literature. Putative miRNA precursors were identified by RNAz, which is a non-coding RNA prediction tool based oncomparative sequence analysis. The mature miRNA of the putative miRNA genes is accurately determined using a machine learning approach, mmiRNA. Then, miRanda was applied to predict the miRNAtargets within the conserved regions in 30-UTR of the genes in the four mammalian genomes. The miRNAMap also provides the expression profiles of the known miRNAs, cross-species comparisons, gene annotations and cross-links to other biological databases. Both textual and graphical web interface are provided to facilitate the retrieval of data from the miRNAMap.

Background: MicroRNAs have been identified as crucial regulators in both animals and plants.Here we report on a comprehensive comparative study of all known miRNA families in animals.We expand the MicroRNA Registry 6.0 by more than 1000 new homologs of miRNA precursorswhose expression has been verified in at least one species. Using this uniform data basis we analyzetheir evolutionary history in terms of individual gene phylogenies and in terms of preservation ofgenomic nearness across species. This allows us to reliably identify microRNA clusters that arederived from a common transcript. Results: We identify three episodes of microRNA innovation that correspond to majordevelopmental innovations: A class of about 20 miRNAs is common to protostomes anddeuterostomes and might be related to the advent of bilaterians. A second large wave ofinnovations maps to the branch leading to the vertebrates. The third significant outburst of miRNAinnovation coincides with placental (eutherian) mammals. In addition, we observe the expectedexpansion of the microRNA inventory due to genome duplications in early vertebrates and in anancestral teleost. The non-local duplications in the vertebrate ancestor are predated by local(tandem) duplications leading to the formation of about a dozen ancient microRNA clusters. Conclusion: Our results suggest that microRNA innovation is an ongoing process. Majorexpansions of the metazoan miRNA repertoire coincide with the advent of bilaterians, vertebrates,and (placental) mammals.

Proper development and activation of cells of the myeloid lineage are critical for supporting innate immunity. This myelopoiesis is orchestrated by interdependent interactions between cytokine receptors, transcription factors and, as recently described, microRNAs (miRNAs). miRNAs contribute to normal and dysregulated myelopoiesis. Alterations in myelopoiesis underlie myeloid-derived suppressor cell (MDSC) expansion, a poorly understood heterogeneous population of immature and suppressive myeloid cells that expand in nearly all diseases where inflammation exists. MDSCs associated with inflammation often have immunosuppressive properties, but molecular mechanisms responsible for MDSC expansion are unclear. Emerging data implicate miRNAs in MDSC expansion. This review focuses on miRNAs that contribute to myeloid lineage differentiation and maturation under physiological conditions, and introduces the concept that altered miRNA expression my underlie expansion and accumulation of MDSCs. We divide our miRNAs into those with potential to promote MDSC expansion and two with known direct links to MDSC expansion, miR-223 and miR-494.

Multiple Sclerosis (MS) is a chronic, inflammatory disease of the central nervous system. MS is caused by the immune-mediated destruction of myelin and oligodendrocytes, resulting in demyelination and neurodegeneration. The microRNA miR-145-5p has been demonstrated to be upregulated in MS lesions. Our lab has previously shown that dysregulation of miR-145-5p can interfere with oligodendrocyte differentiation in mice and that knockout of miR-145-5p protects mice from experimental autoimmune encephalomyelitis (EAE), a model for MS. The objective of this study is to determine if inhibition of miR-145-5p with an antisense oligonucleotide (ASO) is sufficient to protect mice from EAE. Female mice were induced with EAE and then treated with a control or miR-145 ASO at the onset of disease. We evaluated disease progression by monitoring clinical severity, and evaluating molecular and structural characteristics of EAE by RT-qPCR, histology, immunohistochemistry and electron microscopy. We have shown that the miR-145 ASO reduced miR-145-5p expression in the lumbar spinal cord, spleen and thymus following EAE induction. Treatment with the miR-145-5p ASO resulted in improved clinical severity of EAE, reduced neuroinflammation and increased myelination. Inhibition of miR-145-5p may represent a novel treatment for MS.