As células-tronco processam e são sensíveis a múltiplos sinais dentro de seu microambiente, os quais podem exercer influências que regulam seu destino e sua função de forma espaço temporal. Neste contexto, células podem exercer seu papel biológico por transferir informação genética e alterar expressão gênica de alvos celulares através de vesículas extracelulares (VEs). MicroRNAs (miRNAs), uma classe de pequenos RNAs não codificantes, podem ser encontrados nestas vesículas e são considerados moléculas efetivas no controle do neurodesenvolvimento por regular genes chaves em tempo controlado. Pouco se sabe sobre como a diferenciação influencia o conteúdo de miRNAs liberados via VEs revelando o papel dos mesmos no microambiente de cada etapa do comprometimento neural. Assim, a proposta deste estudo foi analisar o perfil de miRNAs intracelulares e presentes em VEs envolvidos na diferenciação neural dopaminérgica de células-tronco pluripotentes e identificar os possíveis alvos regulados pelos mesmos como mecanismo de estabelecimento de um destino neural específico. / Stem cells sense and process multiple signals in their microenvironment, which can exert influences that regulate cell fate and function in a time spatial manner. In this context, the stem cells can exert their biological role transferring genetic information and altering the genetic expression of target cells through extracellular vesicles (EVs). MicroRNAs (miRNAs), a class of small non coding RNAs, can be found in those EVs and are considered effective molecules in the control of neurodevelopment and differentiation by regulating key genes in a time specific manner. However, little is known about how the cell differentiation influences the miRNAs content released through EVs, and how these molecules function in the microenvironment of each phase of neural commitment. Thus, the purpose of this study was to analyze the intracellular and EVs miRNAs profiles involved in the dopaminergic differentiation of pluripotent stem cells in attempt to identify possible targets regulated by miRNAs as a mechanism of specific neural fate decision.

MiRNAs são pequenas moléculas de RNAs endógenos não codificantes com aproximadamente 23nt que atuam na regulação da expressão gênica. A sua função é inibir a tradução de genes transcritos através de um mecanismo que viabiliza a ligação do miRNA com o mRNA alvo levando à inibição da tradução ou a degradação do RNA mensageiro. Estudos evidenciam a relação dos miRNAs com diversos processos biológicos como proliferação celular, diferenciação, desenvolvimento e doenças. Uma vez que estão envolvidos na regulação gênica, também alteram as vias metabólicas. Atualmente, as ferramentas computacionais disponíveis para o estudo dos miRNAs são o miRBase, microCosm, o miRGen e o miRNAmap. Elas possuem informações sobre as sequências dos miRNAs, genes alvos e sobre elementos que estão próximos à região dos miRNAs. Embora o avanço até o momento, não existia que relacionasse os miRNAs com as vias metabólicas, para isso foi construída a plataforma miRNApath que auxilia no estudo da função dos miRNAs por meio da análise do seus alvos dentro vias metabólicas. De modo semelhante, também não existia uma abordagem que relacione dados de expressão miRNAs e seus alvos dentro de um mesmo experimento. Para tanto, neste trabalho foi feita uma abordagem utilizando bibliotecas de SAGE (Serial Analysis of Gene Expression) que será incorporada no miRNApath. O miRNApath encontra-se disponível em http://lgmb.fmrp.usp.br/mirnapath. / MiRNAs are small molecules of endogenous non-coding RNAs with approximately 23nt in length that acts over gene expression regulation. Its function is inhibit the translation of gene transcripts through a mechanism that links the miRNA with its mRNA target leading to a translational repression or degradation. Studies show the relation of RNAs in many biological processes like cell proliferation, dierentiation and development of diseases. Since they are involved in gene regulation, they also change the metabolic pathways. Currently, the available computational tools for the study of miRNAs are miRBase, microCosm, miRGen and miRNAmap. They have information about miRNAs sequences, targets and features. Despite the the advances, until now, there is no tool that correlates the miRNAs with metabolic pathways, therefore we developed the miRNApath platform that helps in the analysis of miRNAs function through the study of its targets that are into the metabolic pathway. In the same way, there is no approach that put together information of expression of miRNAs and its targets in the same experiment. In this work we develop an approach with SAGE (Serial Analysis of Gene Expression ) libraries that will be integrated to miRNApath. The plataform is avaible at http://lgmb.fmrp.usp.br/mirnapath.

Dentre outros fatores, a fertilidade depende da qualidade espermática (QE; características morfofuncionais) e dos componentes espermáticos moleculares. Devido à QE nem sempre estar associada à fertilidade, o estudo dos componentes moleculares ganha cada vez mais destaque. Os microRNAs, reguladores pós-transcricionais, estão presentes nos espermatozoides e desempenham funções importantes na espermatogênese, na maturação espermática e no desenvolvimento embrionário. Entretanto, os mecanismos pelos quais estes regulam a fertilidade masculina são pouco conhecidos. Portanto, o objetivo dos estudos aqui presentes foi investigar o papel dos miRNAs na determinação e regulação da fertilidade. No primeiro estudo, partidas de sêmen criopreservado de touros Aberdeen Angus (Bos taurus) com alta (54,3±1,0%; AF; n=3) e baixa fertilidade (41,5±2,3%; BF; n=3) foram submetidas às avaliações da QE; à produção in vitro e coleta de embriões com uma célula, duas células e blastocistos; e à avaliação do perfil de 380 miRNAs. Os genes-alvo dos miRNAs diferentemente abundantes nos espermatozoides e nos embriões dos touros AF e BF foram investigados. O diâmetro, número de células e taxa de proliferação dos blastocistos também foram analisados. Ainda, neste estudo, foi validado um novo modelo para provar que o espermatozoide é capaz de entregar miRNAs ao embrião. Para a análise estatística, foram empregadas as análises de variância (ANOVA) e de Qui-Quadrado. Quando não mencionado, o nível de significância foi de 5%. Dentre os miRNAs avaliados, o miR-216b foi menos abundante nos espermatozoides (P=0,08) e zigotos (P<0,05) dos touros AF. O gene-alvo deste miRNA, KRAS, associado à proliferação celular, foi mais abundante (P<0,05) nos embriões de duas células dos touros AF assim como a taxa de primeira clivagem e o número de células dos blastocistos que foram maiores neste grupo. Ainda, provou-se que o miR-216b é entregue pelo espermatozoide ao embrião. No segundo estudo, seis touros da raça Nelore (Bos indicus) foram utilizados, sendo três submetidos ao estresse térmico testicular (heat stress; HS). O sêmen foi coletado sete dias antes e 21 dias após o estresse. A QE foi avaliada seguida pela análise do perfil de 380 miRNAs espermáticos e exossomais. Os dados foram analisados por ANOVA. O nível de significância foi de 5%. HS apresentou 21 miRNAs espermáticos diferentemente abundantes, mas não apresentou diferença no conteúdo de miRNAs exossomais. Os miR-126-5p e -146a apresentaram menor abundância nos espermatozoides HS assim como apresentaram menor abundância nos espermatozoides dos touros BF do primeiro estudo. Já o miR-216b não apresentou diferença neste segundo estudo. Assim, com base nos dois estudos, podemos concluir que 1) os miRNAs são importantes para a fertilidade; 2) existem miRNAs importantes para a fertilidade que são alterados por injúrias à espermatogênese; e 3) o miR-216b, entregue pelo espermatozoide ao embrião, é importante para o desenvolvimento embrionário inicial regulando de maneira diferente embriões de touros de AF e BF; provando que miRNAs espermáticos possuem papel na regulação e determinação da fertilidade. Estes estudos possuem grande importância na geração de conhecimentos sobre os miRNAs espermáticos e apresentam resultados pioneiros que confirmam que o espermatozoide bovino contribui com mais que o DNA ao desenvolvimento embrionário. / Bulls fertility relies of many factors. In concern to sperm cells, it depends of sperm quality (SQ; morphofunctional features) and sperm molecular components. Due to the fact thatsperm quality is not always related with fertility, the effect of many molecular factors on fertility has been intensively studied. miRNAs, which have post transcriptional action, are present on sperm cells and are important to spermatogenesis, sperm maturation and embryo development. However, the mechanisms by which they regulate male fertility are not completely understood. Thus, the objective of the present studies was to investigate the role of miRNAs on fertility regulation. On the first study, frozen-thawed semen batches of high (54.3±1.0%; HF; n=3) and low fertility (41.5±2.3%; LF; n=3) Aberdeen Angus (Bos taurus) bulls were analyzed in concern to SQ; were used in embryo in vitro production to produce and collect embryos of one cell, two cells and blastocysts; and were analyzed to the profile of 380 sperm miRNAs. The target genes of the miRNAs that presented different abundance levels on sperm cells and embryos of HF and LF bulls were investigated. Blastocysts diameter, cell number and proliferation index were also evaluated. Besides, in this study, we validated a new model to prove that some miRNAs are delivered by sperm cell to embryo. Data were analyzed by analyzes of variance (ANOVA) and Chi-Square. Signicante level was 5% unless otherwise stated. Among miRNAs evaluated, miR- 216b presented less abundance on HF sperm cells (P=0.08) and HF zygotes (P<0.05). miR- 216b target gene, KRAS, that is related with cell proliferation, presented high abundance (P<0.05) on HF two cells embryos such as the first clivage rate and the number of cells on blastocysts that were greater on this group. We also showed that miR-216b is delivered by sperm cells to embryos. On the second study, we used six Nelore (Bos indicus) bulls. Three bulls were submitted to testicular heat stress (HS group). Semen was collected three days before and 21 days after heat stress. Samples were analyzed in concern to SQ and to the profile of 380 miRNAs on sperm cells and on exossomes. Data were analyzed by ANOVA. Signicante level was 5%. HS presented 21 sperm miRNAs diferently abundant, but miRNAs from exossomes were not different between the HS and control groups. miR-126-5p and -146a presented less abundance on HS sperm cells and on BF bulls sperm cells of the first study. miR-216b was not different in the second study. Thus, we can conclude that 1) miRNAs are important to fertility; 2) there are miRNAs that are important to fertility and can be altered by spermatogenesis injuries; and 3) miR-216b is delivered by sperm cells to embryos and is important to initial embryo development regulating HF and LF embryos differently; showing that sperm miRNAs are important molecules for regulation and establishment of fertility. The studies here presented generate knowledge upon sperm miRNAs importance and present novel results that confirm that bull sperm cells contribute with more than DNA to embryo development.

MiRNAs são pequenas moléculas de RNAs endógenos não codificantes com aproximadamente 23nt que atuam na regulação da expressão gênica. A sua função é inibir a tradução de genes transcritos através de um mecanismo que viabiliza a ligação do miRNA com o mRNA alvo levando à inibição da tradução ou a degradação do RNA mensageiro. Estudos evidenciam a relação dos miRNAs com diversos processos biológicos como proliferação celular, diferenciação, desenvolvimento e doenças. Uma vez que estão envolvidos na regulação gênica, também alteram as vias metabólicas. Atualmente, as ferramentas computacionais disponíveis para o estudo dos miRNAs são o miRBase, microCosm, o miRGen e o miRNAmap. Elas possuem informações sobre as sequências dos miRNAs, genes alvos e sobre elementos que estão próximos à região dos miRNAs. Embora o avanço até o momento, não existia que relacionasse os miRNAs com as vias metabólicas, para isso foi construída a plataforma miRNApath que auxilia no estudo da função dos miRNAs por meio da análise do seus alvos dentro vias metabólicas. De modo semelhante, também não existia uma abordagem que relacione dados de expressão miRNAs e seus alvos dentro de um mesmo experimento. Para tanto, neste trabalho foi feita uma abordagem utilizando bibliotecas de SAGE (Serial Analysis of Gene Expression) que será incorporada no miRNApath. O miRNApath encontra-se disponível em http://lgmb.fmrp.usp.br/mirnapath. / MiRNAs are small molecules of endogenous non-coding RNAs with approximately 23nt in length that acts over gene expression regulation. Its function is inhibit the translation of gene transcripts through a mechanism that links the miRNA with its mRNA target leading to a translational repression or degradation. Studies show the relation of RNAs in many biological processes like cell proliferation, dierentiation and development of diseases. Since they are involved in gene regulation, they also change the metabolic pathways. Currently, the available computational tools for the study of miRNAs are miRBase, microCosm, miRGen and miRNAmap. They have information about miRNAs sequences, targets and features. Despite the the advances, until now, there is no tool that correlates the miRNAs with metabolic pathways, therefore we developed the miRNApath platform that helps in the analysis of miRNAs function through the study of its targets that are into the metabolic pathway. In the same way, there is no approach that put together information of expression of miRNAs and its targets in the same experiment. In this work we develop an approach with SAGE (Serial Analysis of Gene Expression ) libraries that will be integrated to miRNApath. The plataform is avaible at http://lgmb.fmrp.usp.br/mirnapath.

<p>Funding Agency:</p><p>Health Research Council in the South-East of Sweden</p>

Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever (MCF) in susceptible ruminants. Through an unknown mechanism, presence of the virus leads to proliferation of NK-like T cells that are not targetrestricted by the MHC class molecules. These host cells cause the symptoms found in MCF; fever, swollen lymph nodes, and necrotic lesions of the nasal, conjunctival, and oral mucosa, which usually leads to death of the host. MicroRNAs (miRNAs) are ~22 nt RNA molecules expressed by eukaryotes and viruses that regulate genes post-transcriptionally. Viral miRNAs have been found to regulate cellular genes to control the cell cycle and have a role in pathogenesis. It was hypothesised that OvHV-2 expresses miRNAs and these play a role in MCF pathogenesis. The aim of this project was to determine if OvHV-2 encodes miRNAs. Bioinformatic analysis was conducted on deep sequencing data from RNA of OvHV-2- immortalised T cells. Candidate miRNAs were selected if they adhered to miRNA secondary structure. 46 candidate miRNAs were found, with three clusters on the minus strand; one at the 5’ end and the other two in a 9.3 kb region that contains no predicted open reading frames. The 8 most abundant candidates were successfully validated by northern hybridisation for small RNAs. The majority of the predicted targets for the 8 validated OvHV-2 miRNAs were from the OvHV-2 genome. This study adds OvHV-2 to the list of herpesviruses that encode miRNAs and provides another tool for studying the pathogenesis of MCF.

THÈME DE RECHERCHE : Le phénotype callipyge est une hypertrophie musculaire généralisée post-natale décrite chez le mouton (introduit dans le Chapitre 1). Son mode de transmission non mendélien, qualifié de surdominance polaire, est unique : seuls les hétérozygotes ayant reçu la mutation callipyge (CLPG) de leur père expriment le phénotype. La mutation CLPG, localisée dans un domaine soumis à l'empreinte parentale, est une mutation ponctuelle (SNP) détruisant un élément qui contrôle, en cis, le taux dexpression musculaire des gènes voisins. L'hypertrophie musculaire, sans doute causée par l'expression ectopique de la protéine DLK1 chez les animaux +mat/Cpat, n'est pas observée chez les animaux Cmat/Cpat dont l'allèle maternel apparaît trans-inactiver la traduction du messager DLK1. Cette thèse a pour objectif d'approfondir notre compréhension de la surdominance polaire, aussi bien au travers de l'étude des mécanismes impliqués dans l'effet de contrôle à longue distance en cis de la mutation que de la caractérisation des inhibitions en trans induites par les transcrits maternels sur les messagers paternels. RÉSULTATS : L'étude de l'effet en cis (Chapitre 2) a démontré que la mutation CLPG se différenciait de l'allèle sauvage par au moins trois marques épigénétiques distinctes : (i) l'hypométhylation de l'ADN à proximité du SNPCLPG, (ii) la création d'un site d'hypersensibilité à la DNase, et (iii) l'activation de la transcription bidirectionnelle de la région autour de la mutation. En démontrant que la mutation inactivait bel et bien un élément de contrôle à longue distance, ces données nous ont permis d'élaborer un modèle plus précis de la surdominance polaire. L'étude de la trans-inhibition des transcrits protéiques à expression paternelle par les transcrits non codants maternels (Chapitres 3 et 4) se fondait sur l'hypothèse d'une implication des nombreux microARNs (miRNAs) du domaine DLK1-GTL2. Nous avons ainsi pu montrer que les cinq miRNAs abrités par anti-PEG11 étaient capables, par leur parfaite complémentarité de séquence, de cliver le messager PEG11 (Chapitre 3). Bien que le rôle de la protéine PEG11 dans le phénotype callipyge soit inconnu, cette étude a néanmoins démontré l'existence d'une trans-inhibition entre les deux allèles du locus, tout en identifiant le premier cas de dégradation miRNA/cible impliquant des gènes soumis à l'empreinte chez les mammifères. L'étude de la trans-inhibition appliquée au messager DLK1 (Chapitre 4) a quant à elle nécessité la génération par séquençage haut débit d'un catalogue exhaustif des miRNAs du muscle squelettique ovin. Ce travail nous a permis de démontrer que les miRNAs du domaine DLK1-GTL2 étaient bien exprimés maternellement et eux aussi soumis à l'effet en cis de la mutation. Si aucun miRNA capable d'inhiber le messager DLK1 n'a été identifié sans ambiguïté, nos analyses d'affinité ont toutefois révélé un effet significatif de miR-376c sur sa 3'UTR, ainsi que de l'ensemble des miRNAs du locus DLK1-GTL2 sur sa région codante. Notons que nous avons également considéré comme potentiels candidats à la trans-inhibition les snoRNAs du locus, de même que les miRNAs du locus soumis à l'édition ARN. CONCLUSIONS ET PERSPECTIVES : À l'issue de cette thèse, nous avons donc contribué à la compréhension du phénomène de surdominance polaire au locus DLK1-GTL2 du mouton callipyge. Si de nombreuses questions restent en suspend, elles devraient néanmoins trouver réponse grâce aux nouveaux outils d'analyse en cours de développement. Ainsi, deux lignées de souris transgéniques développées dans notre laboratoire, respectivement porteuses de la mutation CLPG ou surexprimant PEG11 dans le muscle, éclaireront les mécanismes moléculaires de l'effet en cis de la mutation ainsi que le rôle de PEG11 dans l'établissement du phénotype callipyge. Par ailleurs, pour confirmer in vivo l'effet inhibiteur sur DLK1 des miRNAs identifiés dans notre étude, les progrès apportés par le séquençage haut débit (notamment le HITS-CLIP) et la transfection en modèle cellulaire se révéleront sans doute d'une grande aide. / RESEARCH OVERVIEW: The callipyge phenotype is a generalized muscular hypertrophy described in sheep (see Chapter 1). It features a non-mendelian mode of inheritance termed polar overdominance: only heterozygous animals having inherited the mutation from their father display the phenotype. The callipyge mutation (CLPG), which falls in an imprinted domain, is a single-nucleotide polymorphism (SNP) that disrupts a putative long-range control element affecting, in cis, the expression level of neighboring imprinted genes in skeletal muscle. The callipyge phenotype is thought to be caused by ectopic expression of DLK1 protein in +mat/Cpat animals. In contrast, Cmat/Cpat animals exhibit a wild-type phenotype that is certainly due to a trans-inhibition from the maternal allele on DLK1 translation. The objective of our thesis was to improve our knowledge of polar overdominance, both by studying mechanisms of long-range cis regulation and by characterizing the trans effect of maternal noncoding transcripts on the paternally-expressed messenger RNAs. RESULTS: Our study of the cis effect (Chapter 2) showed that the CLPG mutation differs from the wild-type allele by at least three distinct epigenetic marks: (i) DNA hypomethylation in the vicinity of the SNPCLPG, (ii) creation of a DNase-hypersensitive site, and (iii) activation of a bidirectional transcription start site centered on the mutation. Altogether, our data provided strong evidence for the SNPCLPG inactivating a long-range control element and allowed us to refine our model of polar overdominance. Our working hypothesis for the trans-inhibition of paternally-expressed genes by maternal noncoding transcripts (Chapters 3 and 4) involved microRNAs (miRNAs) from the DLK1-GTL2 domain. In this respect, we showed that five miRNAs from anti-PEG11 were able to cleave PEG11 transcript, owing to perfect sequence complementarity (Chapter 3). This study was the first demonstration of miRNA-mediated RNAi involving imprinted genes in mammals. Furthermore, it allowed us to confirm the existence of a trans-inhibition between both alleles in the domain, albeit the role of PEG11 protein in the callipyge phenotype is still unknown. To study the trans-inhibition of DLK1 messenger (Chapter 4), we used high-throughput sequencing to build an exhaustive catalogue of skeletal muscle-specific miRNAs in sheep. Our analyses showed that miRNAs from the DLK1-GTL2 domain are maternally expressed and affected by the cis effect of the CLPG mutation. Even if we could not find miRNAs unambiguously able to repress DLK1 transcript, affinity analyses revealed a significant effect on its 3' UTR for miR-376c, as well as on its coding region for all miRNAs considered as team. Of note, we also investigated snoRNAs and miRNAs subjected to RNA editing in the DLK1-GTL2 locus. CONCLUSIONS AND PERSPECTIVES: During the course of this research, we contributed to improve the understanding of polar overdominance in the sheep DLK1-GTL2 locus. Although many questions remain, most will eventually be answered thanks to upcoming analysis tools. Hence, our lab has already generated two transgenic mouse lines, either carrying the CLPG mutation or over-expressing PEG11 in skeletal muscle. These mice should respectively shed light on the molecular mechanisms underlying the cis effect of the CLPG mutation and on the role of PEG11 in the callipyge phenotype. Finally, to confirm inhibiting effects of miRNAs identified in our study, technological improvements granted by high-throughput sequencing (such as HITS-CLIP) and miRNAs transfection in cell model systems will both prove to be very useful.

Amyotrophic lateral sclerosis (ALS) is a progressive adult onset neurodegenerative disease characterized by selective death of the upper and lower motor neurons of the brain and spinal cord. Neuromuscular synapses are lost leading to paralysis and ultimately death. Non-neuronal cells, such as astrocytes, oligodendrocytes, and microglia, have been shown to contribute to ALS disease progression in mouse models. Microglia, the innate immune cells of the central nervous system, have been shown to be activated in ALS and contribute to disease progression. Hundreds of mRNAs have shown to be dysregulated in a variety of ALS cell types and tissues, including total spinal cord, acutely isolated microglia, and in vitro differentiated motor neurons. These mRNAs can be regulated post-transcriptionally by microRNAs (miRNAs), which are small endogenous non-coding RNAs with important regulatory roles in a wide range of cellular processes. This dissertation examines the contribution of miRNAs to ALS disease progression in microglia. I acutely isolated primary microglia from the spinal cords of transgenic mice overexpressing human wild type (WT) SOD1 and human G93A SOD1. I used small RNA sequencing to profile the miRNAs that are expressed during disease progression, and identified miRNAs that are differentially expressed. I confirmed these results by quantitative PCR and examined the expression changes of predicted targets in a microglia RNA-seq dataset. Here I show that miRNAs are dysregulated in acutely isolated microglia from SOD1 G93A transgenic mice, and that miR-155, a pro-inflammatory miRNA, and miR-210, a hypoxia-inducible miRNA, are significantly upregulated during disease progression. In addition, miR-1198-5p, miR-182, miR-503, and miR-668 are also dysregulated, and predicted mRNA targets of all six of these miRNAs are differentially expressed during disease progression. To my knowledge, this is the first analysis of miRNA expression in microglia during ALS disease progression. This work contributes to the understanding of the contribution of a non-neuronal cell type to ALS disease progression and serves as a paradigm for studies in other non-neuronal cell types, such as astrocytes and oligodendrocytes, and other ALS mouse models.

Spinal cord injury (SCI) results in a number of devastating consequences, including loss of motor function, paralysis, and neuropathic pain. Concomitant peripheral tissue injury below the lesion site can result in uncontrollable nociception that sensitizes spinal neurons and promotes chronic pain. Additionally, drugs like morphine, though critical for pain management, elicit pro-inflammatory effects that exacerbate chronic pain symptoms. Currently, there is a lack of effective therapeutic mechanisms to promote regeneration at the lesion site, and a limited understanding of regulatory mechanisms that can be utilized to therapeutically manipulate spinal cord plasticity. MicroRNAs (miRNAs) constitute novel targets for therapeutic intervention to both promote repair and regeneration, and mitigate maladaptive plasticity that leads to neuropathic pain. Microarray and qRT-PCR comparisons of contused and sham rat spinal cords at 4 and 14 days following SCI indicated that a total of 35 miRNAs were dysregulated, with miR1, miR124, and miR129 exhibiting significant down-regulation after SCI, and both miR21 and miR146a being transiently induced. Localized expression of miRNAs and cellular markers indicated that changes in miRNA regulation favor the emergence of neural stem cell niches and reversion of surviving neurons to a pre-neuronal phenotype. Additionally, both uncontrollable nociception and morphine administration resulted in further dysregulation of SCI-sensitive miRNAs, along with their mRNA targets. Morphine administration significantly induced expression of both miR21 and IL6R expression, indicating that morphine-induced miRNA dysregulation is involved in the promotion of neuroinflammation that drives increased pain-sensitivity. Similarly, uncontrollable nociception significantly modulates expression of miR124, miR129, and miR146a, which inhibit cell cycle proteins and microglial activation, and dysregulation of these miRNAs, along with BDNF and IGF-1, likely contributes towards promotion of hypersensitivity in spinal neurons that underlies neuropathic pain. Consequently, SCI- sensitive miRNAs may constitute therapeutic targets for modulation of neuroinflammation and microglial activation in order to mitigate secondary injury, promote regeneration, and prevent maladaptive plasticity that drives neuropathic pain and exacerbation of chronic pain symptoms by morphine administration.

Submitted by LÍVIA CARVALHO FERREIRA null (livinha_ferreira@hotmail.com) on 2017-12-07T17:35:22Z No. of bitstreams: 1 TESE FINAL.pdf: 2973167 bytes, checksum: a3584a471121034b22fc9cfcef852a73 (MD5) / Approved for entry into archive by Elza Mitiko Sato null (elzasato@ibilce.unesp.br) on 2017-12-07T18:59:29Z (GMT) No. of bitstreams: 1 ferreira_lc_dr_sjrp.pdf: 2973167 bytes, checksum: a3584a471121034b22fc9cfcef852a73 (MD5) / Made available in DSpace on 2017-12-07T18:59:29Z (GMT). No. of bitstreams: 1 ferreira_lc_dr_sjrp.pdf: 2973167 bytes, checksum: a3584a471121034b22fc9cfcef852a73 (MD5) Previous issue date: 2017-11-10 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O câncer de mama apresenta altas taxas de incidência e também de mortalidade, sendo a neoplasia mais comum entre as mulheres. MicroRNAs (miRNAs) são pequenas moléculas de RNAs não codificantes que desempenham papel fundamental na regulação gênica. Estudos recentes têm demonstrado que miRNAs estão diretamente envolvidos na iniciação e progressão de vários tipos tumorais, incluindo o câncer de mama. Diversos miRNAs têm sido descritos como promotores ou supressores tumorais, podendo estar associados ao crescimento do tumor e metástase. Atualmente, tem sido demonstrado que a administração exógena da melatonina, um hormônio naturalmente secretado pela glândula pineal, apresenta diversos efeitos oncostáticos em diferentes tipos tumorais. Assim, o objetivo do presente estudo foi avaliar o papel da melatonina em uma possível via metastática envolvendo a regulação de miRNAs em células da linhagem de câncer de mama metastática e triplo-negativa MDA-MB-231. Inicialmente, a expressão de 384 miRNAs foi avaliada utilizando placas “Taqman Low-density Array” (TLDA). Para futuras validações, foram selecionados apenas miRNAs que apresentaram fold change >1,5 e <0,5. Os resultados demonstraram que a melatonina modulou a expressão de 17 miRNAs (11 superexpressos e 6 inibidos). Dentre os miRNAs modulados pela melatonina, miR-148b e miR-210 foram confirmados por qRT-PCR, selecionados e utilizados para investigações funcionais. As células MDAMB-231 foram então transfectadas para inibição de miR-148b e/ou miR-210 (permanente ou transiente, respectivamente), e avaliadas após tratamento com melatonina. Em seguida foram avaliadas a taxa de migração e expressão proteica de cMyc, possível gene alvo de miR-148b e miR-210. Os resultados demonstraram que a melatonina foi capaz de reduzir a expressão de c-Myc e afetar a taxa de migração das células modificadas ou não com miR-148b. No entanto, nenhum efeito sobre c-Myc ou migração foi observado em células modificadas para miR-148b, quando comparadas as células controle. No que se refere ao miR-210, a melatonina alterou a expressão de cMyc e migração celular, nas células tratadas com melatonina ou expressando anti-miR210. Em resumo, nossos resultados demonstraram que apesar da melatonina influenciar na modulação positiva do miR-148b e miR-210 em células tumorais de mama triplonegativas, esta regulação não foi necessariamente causativa para alterar a expressão de c-Myc ou diminuir a taxa de migração destas células. Nossos resultados confirmam o efeito da melatonina na inibição da proliferação e migração celular, especialmente em células triplo-negativas, sugerindo seu importante papel no controle da progressão tumoral. No entanto, ainda é necessário estabelecer uma ligação direta entre as modulações de certas proteínas ou miRNAs com os efeitos da melatonina. / Breast cancer has high rates of incidence and mortality, and it is the most common cancer among women. MicroRNAs (miRNAs) are small molecules of non-coding mRNA that play a key role in gene regulation. Recent studies have shown that miRNAs are directly involved in the initiation and progression of various tumor types, including breast cancer. Several miRNAs have been described as promoters or suppressors of metastasis and may be associated with tumor growth and metastasis. Exogenous administration of melatonin, a hormone secreted by the pineal gland, has been shown several oncostatics effects on different types of cancers. Herein, we investigated if the antimetastatic effects of melatonin were coordinated by miRNAs involved in tumor progression. The expression of 384 miRNAs was measured using Taqman Low-density Array (TLDA) cards. Considering the cut-off we imposed (fold change >1.5 and (fold change >1.5 and<0.5) were evidenced the modulation of 17 miRNAs (11 up and 6 down). Among all miRNAs modulated, the selected miR-210 and miR-148b were further confirmed by qRT-PCR and tested for functional investigations. First, we engineered cells for miR-210 or miR-148b overexpression or depletion (stable or transient), then we evaluated the effect of melatonin on c-Myc protein expression and migration. Melatonin reduced c-Myc expression and migration in cell depleted or not for miR-148b. However no effect on c-Myc or migration was observed for cells depleted for miR-148b when compared with control cells. c-Myc and migration were reduced in cells treated with melatonin or expressing anti-miR-210 (depleted). In summary, our results suggest that, even if melatonin alters miRNA expression, the modulations of the miRNAs we studied, miR-210 and miR-148b, are not essential for melatonin inhibition of cell migration and c-Myc expression. One of the hypotheses is that c-Myc is not a target of these miRNAs in MDA-MB-231 cells, and it is believed that melatonin effects on miRNAs could be just “epiphenomenon” due to general Dicer/Drosha deregulations. Nevertheless, melatonin remains a powerful molecule for metastatic traits inhibition, which modulates a set of protein-coding genes, such as c-Myc. However, a direct link between expression modulations of certain proteins or miRNAs and melatonin effects has still to be established. / 2013/24612-5