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La plasticité du système nerveux central face aux contraintes environnementales ou morphologiques est un processus fondamental mis en place afin de permettre à l’animal de maintenir des comportements adaptés. Parce que le comportement locomoteur est essentiel à la survie de l'animal, les mécanismes neuronaux permettant sa genèse doivent s’adapter aux modifications morphologiques de l’organisme pendant son développement. Pour aborder cette question, nous avons développé un nouveau modèle expérimental pour lequel les modifications morphologiques au cours du développement sont extrêmes et impliquent des reconfigurations à long terme du système nerveux. L'amphibien Xenopus laevis lors de sa métamorphose est, en effet, un modèle pertinent pour étudier (par des approches comportementales, neuroanatomiques, électro-physiologiques et pharmacologiques), les mécanismes impliqués dans la réorganisation des réseaux neuronaux locomoteurs de la moelle épinière face à des modifications extrêmes du schéma corporel. En effet, pendant sa métamorphose, l'animal passe d'un mode de locomotion ondulatoire mettant en jeu sa musculature axiale, à un mode de locomotion appendiculaire grâce aux membres néo-formés. Il existe de plus des stades intermédiaires où les deux modes de locomotion coexistent et expriment des relations fonctionnelles variables. Nos expériences d’électrophysiologie extracellulaire nous ont permis de dégager la dynamique temporelle de l’émergence du réseau de neurones commandant la locomotion appendiculaire adulte et de ses relations fonctionnelles avec le réseau locomoteur commandant la nage larvaire lorsque ces deux réseaux coexistent. D’après les résultats présentés, il apparaît un changement de l’équilibre fonctionnel et des interactions entre les commandes locomotrices ondulatoire et appendiculaire, faisant des stades intermédiaires de la métamorphose les témoins privilégiés du passage de relais progressif entre les deux systèmes locomoteurs. Nos travaux ont également démontré que l’activité de chaque réseau ainsi que leurs relations fonctionnelles sont sujettes à modulation glutamatergique et aminergique destinées à adapter la locomotion aux besoins de l'animal. Nous montrons que certains modulateurs (tels que le glutamate, la sérotonine et la noradrénaline) exercent des effets opposés sur les réseaux locomoteurs larvaires et adultes, alors qu'à l'inverse, la dopamine conserve les mêmes propriétés modulatrices sur ces réseaux malgré les profonds bouleversements subis pendant le développement. Outre leur rôle modulateur, nos résultats suggèrent aussi un rôle des afférences aminergiques dans la maturation des réseaux locomoteurs et ouvrent de nombreuses interrogations quant aux mécanismes impliqués dans la plasticité des afférences neuromodulatrices elles-mêmes au cours de la métamorphose. L’apparition et la disparition de neurones sérotoninergiques intraspinaux concomitantes avec la croissance des membres postérieurs, et précédant la régression de l'appendice caudal laissent envisager un rôle de la sérotonine dans la maturation du réseau locomoteur appendiculaire ou dans la chronologie de la régression du réseau axial. / Plasticity of the central nervous system is fundamental to an animal's capacity to adapt to continually changing biomechanical and environmental demands. Although the neuronal mechanisms underlying such essential behaviours as locomotion must adapt to an organism's morphological modifications during growth and development, the associated changes that occur in central nervous function remain poorly understood. To address this issue, we have developed a new experimental model - the amphibian Xenopus laevis during its metamorphosis - in which the extreme biomechanical modifications occurring during this critical period necessitate a correspondingly extensive and long-term reorganisation of locomotor neural circuitry within the animal's spinal cord. During metamorphosis, the locomotory strategy of Xenopus shifts from undulatory swimming involving axial tail-based movements, to appendicular propulsion that uses the newly formed limbs. At intermediate metamorphic stages, moreover, the two locomotor strategies coexist within the same animal as the secondary limb-based motor circuitry is progressively replaces the primary axial network as the limbs are added and the tail regresses. By making extracellular recordings of spontaneous "fictive" locomotor patterns generated by isolated brainstem/spinal cord preparations, we have charted the temporal dynamics of the emergence of the appendicular neuronal network and determined its functional relationship with larval axial locomotor circuitry through the metamorphic period. Our results have shown that the limb circuitry is initially present but not functional, functional but subordinate to the embryonic axial network, functionally independent from the axial network, and ultimately alone after axial circuitry disappears with tail resorption. Furthermore, the use of pharmacological approaches established that during the metamorphic transition, the coexisting spinal locomotory networks and their functional interactions are subject to glutamatergic and aminergic modulation in order to adapt locomotory performance to the immediate behavioural needs of the animal. Interestingly, the neuromodulators glutamate, serotonin and noradrenaline exert directly opposing influences on the larval and adult locomotor networks, while dopamine preserves a similar modulatory action on the two circuits in spite of their profound remodelling during metamorphic development. Finally, in addition to a short-term modulatory role, our immunocytochemical evidence suggested that descending aminergic systems may contribute to the long-term maturation of spinal locomotor circuitry during metamorphosis in parallel with their own developmental reconfiguration. Specifically, the appearance and disappearance of a population of intraspinal serotonergic neurons concomitant with hindlimb growth and preceding tail regression suggested a role of serotonin in the maturation of the appendicular locomotor network and/or in the chronology of axial network regression.

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Submitted by Mariângela Bueno Cordeiro Maldonado null (maribuenocordeiro@hotmail.com) on 2017-09-01T02:02:20Z No. of bitstreams: 1 TESE DE DOUTORADO 2017 - Mariangela Maldonado .pdf: 1446039 bytes, checksum: 2e08a41374d1ed14783e9142f58bdec8 (MD5) / Approved for entry into archive by LUIZA DE MENEZES ROMANETTO (luizamenezes@reitoria.unesp.br) on 2017-09-01T13:59:09Z (GMT) No. of bitstreams: 1 maldonado_mbc_dr_araca.pdf: 1446039 bytes, checksum: 2e08a41374d1ed14783e9142f58bdec8 (MD5) / Made available in DSpace on 2017-09-01T13:59:09Z (GMT). No. of bitstreams: 1 maldonado_mbc_dr_araca.pdf: 1446039 bytes, checksum: 2e08a41374d1ed14783e9142f58bdec8 (MD5) Previous issue date: 2017-08-22 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O objetivo desse estudo foi identificar polimorfismos de nucleotídeo único (SNPs) potencialmente sujeitos a controle epigenético exercido por metilação do DNA via seus envolvimentos na criação, remoção ou deslocamento de sítios CpG (meSNPs) e a partir de tal identificação criar um banco de dados para meSNPs, bem como determinar a possível associação desses marcadores com ilhas CpG (CGIs) e com o perfil metilacional de tecidos submetidos ao ensaio de recuperação de ilhas CpG metiladas combinado com plataformas de sequenciamento de nova geração (MIRA-seq) em bovinos. Usando as variantes anotadas para os SNPs identificados no Run5 do projeto 1000 Bull Genomes e a sequência genômica bovina de referência UMD3.1.1, identificamos e anotamos 12.836.763 meSNPs de acordo com o padrão de variação criado por cada SNP em um sítio CpG. Também analisamos a distribuição genômica desses meSNPs, sendo a maioria deles localizados em regiões intergênicas (68,00%) e intrônicas (26,32%). Globalmente, os meSNPs representam 22,53% dos 56.969.697 SNPs descritos na base de dados e 12,35% deles estão localizados em CGIs. Comparando o número observado com o número esperado de meSNPs nas CGIs e nos tecidos submetidos ao MIRA-seq, verificamos um enriquecimento médio (P<0,01) para meSNPs de 2,47 vezes em CGIs relaxadas e 1,90 vezes em CGIs rigorosas. Nos tecidos, o enriquecimento foi de 1,52 vezes em longissimus dorsi e 2,09 vezes em intestino delgado. Dez meSNPs com metilação diferencial, sendo 1 em longissimus dorsi e 9 em intestino delgado, causaram uma alteração na sequência genômica, a qual está associada ao fenótipo de eficiência alimentar em bovinos. / The aim of this study was to identify single nucleotide polymorphisms (SNPs) potentially subject to epigenetic control exerted by DNA methylation via their involvement in creating, removing or displacement CpG sites (meSNPs) and from this identification create a database for meSNPs, as well as to determine its possible association with CpG islands (CGIs) and the methylation profile of tissues submitted to the methylated-CpG island recovery assay combined with next generation sequencing platforms (MIRA-seq) in cattle. Using the variant annotations for SNPs identified in Run5 of the 1000 bull genomes project and the UMD3.1.1 bovine reference genome sequence assembly, we identified and classified 12,836,763 meSNPs according to the pattern of variation caused at the CpG site. We have also analyzed the genomic distribution of the meSNPs, with the majority being located in intergenic regions (68.00%) and then in introns (26.32%) and the remainder distributed among proximal promoters (3.93%), coding regions (1.27%), untranslated regions (UTRs) (0.29%), non-coding RNAs (0.11%) and splice regions (0.08%). Overall, meSNPs represent 22.53% of 56,969,697 SNPs described in the database of which 12.35% are located in CGIs. Comparing the observed number with the expected number of meSNPs in the CGIs and tissues submitted to the MIRAseq we found a mean enrichment (P<0.01) for meSNPs of 2.47 times in the relaxed CGIs and 1.90 times in the strict CGIs. In the tissues the enrichment was of 1.52 times in ribeye and 2.09 times in small intestine. Ten meSNPs, differing in methylation status, 1 in ribeye and 9 in small intestine, caused an alteration in the genomic sequence which is associated with a feed efficiency phenotype in cattle. / FAPESP: 2015/20557-5 / FAPESP: 2016/07584-6

L’hybridation est une technique qui consiste à interconnecter un réseau de neurones biologique et un réseau de neurones artificiel, utilisée dans la recherche en neuroscience et à des fins thérapeutiques. Durant ces trois années de doctorat, ce travail de thèse s’est focalisé sur l’hybridation dans un plan rapproché (communication directe bi-directionnelle entre l’artificiel et le vivant) et dans un plan plus élargies (interopérabilité des systèmes neuromorphiques). Au début des années 2000, cette technique a permis de connecter un système neuromorphique analogique avec le vivant. Ce travail est dans un premier temps, centré autour de la conception d’un réseau de neurones numérique, en vue d’hybridation, dans deux projets multi-disciplinaires en cours dans l’équipe AS2N de l’IMS, présentés dans ce document : HYRENE (ANR 2010-Blan-031601), ayant pour but le développement d’un système hybride de restauration de l’activité motrice dans le cas d’une lésion de la moelle épinière, BRAINBOW (European project FP7-ICT-2011-C), ayant pour objectif l’élaboration de neuro-prothèses innovantes capables de restaurer la communication autour de lésions cérébrales.Possédant une architecture configurable, un réseau de neurones numérique a été réalisé pour ces deux projets. Pour le premier projet, le réseau de neurones artificiel permet d’émuler l’activitéde CPGs (Central Pattern Generator), à l’origine de la locomotion dans le règne animale. Cette activité permet de déclencher une série de stimulations dans la moelle épinière lésée in vitro et de recréer ainsi la locomotion précédemment perdue. Dans le second projet, la topologie du réseau de neurones sera issue de l’analyse et le décryptage des signaux biologiques issues de groupes de neurones cultivés sur des électrodes, ainsi que de modélisations et simulations réalisées par nos partenaires. Le réseau de neurones sera alors capable de réparer le réseau de neurones lésé. Ces travaux de thèse présentent la démarche de conception des deux différents réseaux et des résultats préliminaires obtenus au sein des deux projets. Dans un second temps, ces travaux élargissent l’hybridation à l’interopérabilité des systèmes neuromorphiques. Au travers d’un protocole de communication utilisant Ethernet, il est possible d’interconnecter des réseaux de neurones électroniques, informatiques et biologiques. Dans un futur proche, il permettra d’augmenter la complexité et la taille des réseaux. / HYBRID experiments allow to connect a biological neural network with an artificial one,used in neuroscience research and therapeutic purposes. During these three yearsof PhD, this thesis focused on hybridization in a close-up view (bi-diretionnal direct communication between the artificial and the living) and in a broader view (interoperability ofneuromorphic systems). In the early 2000s, an analog neuromorphic system has been connected to a biological neural network. This work is firstly, about the design of a digital neural network, for hybrid experimentsin two multi-disciplinary projects underway in AS2N team of IMS presented in this document : HYRENE (ANR 2010-Blan-031601), aiming the development of a hybrid system for therestoration of motor activity in the case of a spinal cord lesion,BRAINBOW (European project FP7-ICT-2011-C), aiming the development of innovativeneuro-prostheses that can restore communication around cortical lesions. Having a configurable architecture, a digital neural network was designed for these twoprojects. For the first project, the artificial neural network emulates the activity of CPGs (Central Pattern Generator), causing the locomotion in the animal kingdom. This activity will trigger aseries of stimuli in the injured spinal cord textit in vitro and recreating locomotion previously lost. In the second project, the neural network topology will be determined by the analysis anddecryption of biological signals from groups of neurons grown on electrodes, as well as modeling and simulations performed by our partners. The neural network will be able to repair the injuredneural network. This work show the two different networks design approach and preliminary results obtained in the two projects.Secondly, this work hybridization to extend the interoperability of neuromorphic systems. Through a communication protocol using Ethernet, it is possible to interconnect electronic neuralnetworks, computer and biological. In the near future, it will increase the complexity and size of networks.

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Les protéines à « Methyl-CpG-binding domain » (MBD) jouent un rôle important dans l’interprétationde la méthylation de l’ADN conduisant à la répression transcriptionnelle via le recrutement decomplexes remodelant la chromatine. Dans les cancers, MBD2 jouerait un rôle essentiel dans la perted’expression des gènes hyperméthylés. Ainsi, MBD2 serait une cible potentielle pour rétablir, enpartie au moins, leur expression. Caractériser, à l’échelle du génome, la distribution de MBD2 et sesconséquences sur la répression transcriptionnelle au cours de la cancérogenèse est donc une étapeincontournable. (1) L’impact sur l’expression génique de l’inhibition de MBD2 par interférence àl’ARN, a été étudié en utilisant des puces, dans des cellules normales MRC5. La perte de MBD2n’induit pas de surexpression génique globale et la densité en CpG des promoteurs méthylés sembleêtre une composante importante dans la force de répression par MBD2. (2) Les profils de méthylationde l’ADN, de liaisons de MBD2 et de l’ARN polymérase II dans les cellules HeLa ont été analysés parChIP-on-chip avec des puces promoteurs. Ces mêmes approches couplées à l’analyse de l’acétylationdes histones H3 ont été réalisées dans un modèle cellulaire syngénique de progression tumoralemammaire humain. Dans les modèles étudiés, une forte proportion de gènes silencieux et méthylés estliée par MBD2. Les comparaisons entre cellules immortalisées et transformées ne montrent pas dechangements majeurs de la méthylation de l’ADN ou de la répression transcriptionnelle, par contreune redistribution de MBD2 parmi ces sites est observée, suggérant une redondance entre les protéinesliant l’ADN méthylé. / The Methyl-CpG-Binding Domain (MBD) proteins represent key molecules in the interpretation ofDNA methylation signals leading to gene silencing through recruitment of chromatin remodelingcomplexes. In cancer, a member of this protein family, MBD2, seems to play an important role in theloss of expression of aberrantly methylated genes. Thus, MBD2 may be a potential target toreestablish their expression. Mapping of MBD2 binding sites and the relationship between MBD2binding and transcriptional activity was, therefore, a crucial step. (1) We investigated the impact ofMBD2 inhibition by RNA interference on gene expression, using microarray analysis, in a normalhuman fibroblastic cell line, MRC-5. MBD2 depletion did not induce global gene overexpression andCpG density of the methylated promoters seems to be an important parameter in the strength of thetranscriptional repression mediated by MBD2. (2) Global profiling for different layers of epigeneticmodifications (DNA methylation, MBD2 association) and RNA polymerase II binding sites in HeLacells was analyzed by a ChIP-chip method using human promoter arrays. This approach, combinedwith an analysis of H3 histone acetylation patterns, was performed in a syngenic model of breastcancer progression. In the models analyzed MBD2 appeared to be a true methylation-dependenttranscriptional repressor. Furthermore, MBD2 binds to a high proportion of methylated silent genes.Comparisons between immortalized and transformed cells did not indicate major changes of DNAmethylation or gene silencing, while a redistribution of MBD2 among these sites was observed,suggesting a redundancy between methylated binding proteins.

A interação entre fragmentos de bicamada (BF) de brometo de dioctadecildimetilamônio (DODAB) e um mononucleotídeo-modelo (deoxiadenosina monofosfato, dAMP) ou um oligodeoxinucleotídeo-modelo (5\'- AAAAAAAAAA-3\', poli(dA)) ou um oligodeoxinucleotídeo terapêutico (5\'- TTGACGTTCG -3\', CpG) foi investigada por turbidimetria, espalhamento de luz dinâmico, espectroscopia de dicroísmo circular e de fluorescência e calorimetria diferencial de varredura (DSC). Respostas imunológicas foram caracterizadas com ensaio de hipersensibilidade tardia por inchamento de coxim patelar de camundongo, dosagem de anticorpos IgG1 e IgG2a e de citocinas secretadas por células de linfonodo em cultura. Poli(dA), em contraste com dAMP, induziu fusão máxima de DODAB BF a partir da neutralização de cargas, quando houve obtenção de um tamanho máximo e um potencial-zeta igual a zero para os arranjos. Para [poli(dA)] maiores do que aquela correspondente à neutralização de cargas, houve recuperação da estabilidade coloidal com reversão do potencial-zeta e com obtenção de tamanhos que foram aproximadamente o dobro daqueles determinados inicialmente para DODAB BF. A proporção molar de neutralização poli(dA): DODAB foi 1:10 para DODAB BF e 1:20 para vesículas grandes (LV) de DODAB, de acordo com as estruturas de bicamada aberta e fechada dessas duas dispersões de bicamada de DODAB. A fusão de DODAB BF induzida por poli(dA) foi extensiva aumentando o grau de empacotamento das bicamadas formadas conforme inferido a partir dos termogramas de DSC. Em condições de equivalencia de cargas, nucleotídeo não causou fusão de DODAB BF, mostrando a importância do caráter de polieletrólito do poli(dA) para induzir fusão. O sal divalente Na2HPO4 causou fusão e aumentou o empacotamento da bicamada graças à blindagem eficiente de cargas. Reestabilização coloidal como aquela induzida por poli(dA) não ocorreu em presença de Na2HPO4, NaCl ou nucleotídeo. Para complexos DODAB BF/CpG em presença de ovalbumina (OVA) como antígenomodelo, a neutralização de cargas de DODAB BF/OVA por CpG reduziu a estabilidade coloidal, enquanto que supercompensação de cargas levou à reestabilização por repulsão eletrostática, como observado para a interação DODAB BF/poli(dA). Diferenças no tamanho e nas proporções de neutralização por CpG indicaram que os fragmentos são capazes de carregar mais moléculas de OVA do que de BSA. Na região de supercompensação de cargas com potenciais-zeta negativos, arranjos Al(OH)3/ OVA/ CpG são coloidalmente bem mais instáveis que DODAB BF/ OVA ou DODAB BF / OVA/ CpG. O complexo negativamente carregado DODAB (0,1 mM) / OVA (0,1mg/mL)/ CpG (0,020 mM) potencializou a resposta Th1 obtida com DODAB (0,1 mM)/ OVA (0,1 mg/mL). Houve um aumento de 25 % no inchamento do coxim patelar, de 36 % na produção de IFN-&#947;, de 60 % de IL-12 e produção sustentada de IgG2a ao longo de 35 dias pós-imunização, todos indícios fortes de potencialização da resposta Th1 por CpG. Arranjos negativamente carregados de oligonucleotídeos em fragmentos de bicamada de DODAB possuem excelente potencial para terapias baseadas em oligonucleotídeos e para produção de vacinas para diferentes antígenos de interesse. / The interaction between bilayer fragments (BF) of dioctadecyldimethylammonium bromide (DODAB) and a model nucleotide (deoxyadenosine monophosphate, dAMP) or a model oligodeoxynucleotide (5\'- AAAAAAAAAA-3\', poly(dA)) or a therapeutic oligodeoxynucleotide (5\'- TTGACGTTCG -3\', CpG) was investigated by means of turbidimetry, dynamic light scattering, circular dichroism and fluorescence spectroscopies and differential scanning calorimetry. Immune responses were characterized using footpad swelling delayed type hipersensitivity assay and antibody and cytokine measurements. In contrast to dAMP, poly(dA) induced maximal DODAB BF fusion from charge neutralization, where assemblies presented maximal size and zero zeta-potential. Above charge neutralization colloid stability was recovered with negative zeta-potentials and sizes that were about the double of those initially determined for DODAB BF. The poly(dA):DODAB molar ratio for neutralization was 1:10 for DODAB BF and 1:20 for DODAB LV, in agreement with the open and closed bilayer structures of these two DODAB bilayer dispersions. The poly(dA)-induced DODAB BF fusion was extensive and increased the packing of the formed bilayers, as inferred from DSC thermograms. In conditions of charge equivalence, nucleotide did not cause DODAB BF fusion, highlighting the importance of poly(dA)\'s polyelectrolyte character to induce fusion. Divalent Na2HPO4 salt caused fusion and increased bilayer packing due to efficient BF charge shielding. Colloid restabilization as induced by poly(dA) was not observed in presence of Na2HPO4, NaCl and nucleotide. For DODAB BF/CpG complexes in presence of the ovalbumin (OVA) model antigen, the charge neutralization of DODAB BF/OVA by CpG reduced colloid stability, while charge overcompensation led to restabilization due to electrostatic repulsion, as observed for DODAB BF/poly(dA) interaction. Differences in size and neutralization proportions by CpG indicate that BF are able to load more OVA than BSA molecules. In the charge overcompensation region with negative zeta-potentials, Al(OH)3/OVA/CpG assemblies are colloidally less stable than DODAB BF/OVA or DODAB BF/OVA/CpG. The negatively charged DODAB (0.1mM)/OVA (0.1mg/ml)/CpG (0.020mM) assembly enhanced the Th1 response obtained with DODAB (0.1mM)/OVA (0.1mg/ml). There was a 25% increase in footpad sweeling, a 36% and 60% increase in the production of IFN-&#947; and IL-12 and sustained IgG2a production for the 35-day period after immunization, all indicative of strong Th1 response enhancement by CpG. Negatively charged assemblies of oligonucleotides in DODAB bilayer fragments have excellent potential in oligonucleotidebased therapies and in vaccine production for different antigens of interest.

A atividade de neurônios em rajadas de disparo (bursts) é onipresente em sistemas nervosos. No entanto, o papel funcional dos bursts na codificação de informação ainda não é completamente compreendido. A dinâmica de burst tem sido intensivamente estudada em centros geradores de padrões (CPGs) que são exemplos clássicos de sistema nervoso autônomo em que a atividade em bursts está diretamente associada ao controle motor. Estudos recentes investigaram pequenas perturbações na dinâmica aparentemente periódica de neurônios com atividade em burst (bursters): padrões sutis nos tempos de disparo (spikes) dentro de um mesmo burst. Padrões de spikes intraburst (PSIBs) são tradicionalmente negligenciados por falta de relação com a função motora do CPG, no entanto, esses estudos mostraram que PSIBs são específicos para cada tipo de célula do CPG estomatogástrico de crustáceos e além disso, são capazes de refletir mudanças na conectividade da rede, indicando um possível papel na codificação de informação. Nesse trabalho, abordamos esse assunto investigando como um neurônio motor com atividade em bursts expressa informação a respeito de outros neurônios da rede através dos PSIBs. Realizamos experimentos registrando a atividade de neurônios pilóricos do gânglio estomatogástrico tanto na rede intacta como em uma rede híbrida na qual um neurônio pilórico interage em tempo real com um neurônio modelo através de uma sinapse artificial. Para inferir a sensibilidade dos PSIBs pós sinápticos aos pré-sinápticos desenvolvemos uma ferramenta de análise baseada em Teoria da Informação para encontrar padrões de máxima informação (ou seja, encontrar o bin que produz PSIBs de máxima entropia) e calcular informação mútua média entre eles (IMM) ao longo do burst de cada neurônio. Esta ferramenta também é potencialmente útil à análise de outros tipos de processos puntuais por fornecer um método de revelar informação oculta em padrões de eventos. Encontramos que um único neurônio motor é capaz de expressar no início de seu burst informação contida nos PSIBs do início do burst anterior do neurônio pré-sináptico. Esse fenômeno é observado em diferentes espécimes e espécies, o que sugere um mecanismo geral de codificação de informação. Além disso, esse efeito foi reproduzido em experimentos com uma rede híbrida, onde os estímulos pré-sinápticos são totalmente controlados, livre de qualquer influência de outros elementos no circuito. Esses resultados sugerem que a microestrutura dos padrões de disparo pré-sinápticos são codificadas se dá através de uma única sinapse, de maneira não linear e não homogênea nos PSIBs pós sinápticos. Dessa forma, neurônios motores são capazes de usar escalas de tempo diferentes para expressar dois tipos de informação: o ritmo de burst (associado à taxa de disparo) carrega informação sobre a contratação motora, enquanto que em uma escala de tempo muito menor, os PSIBs (associados à codificação temporal) expressam informação sobre o comportamento de outros neurônios do CPG. Além disso, encontramos que a informação dos PSIBs de um neurônio pilórico é codificada na estrutura temporal de disparo das unidades ativas registradas em um nervo do sistema estomatogástrico que projeta em áreas sensoriais do cérebro do animal. Assim, o mecanismo de codificação descrito pode ser parte de uma via de transmissão de informação previamente desconhecida, sugerindo que a codificação de informação através dos PSIBs poderia ser aproveitada para a regulação dos padrões de disparo em circuitos remotos pelo sistema nervoso central. / Burst firing is ubiquitous in nervous systems. However, the functional role of burst firing in information coding is mostly unknown. Bursting dynamics have been intensively studied in Central Pattern Generators (CPGs), classical examples of autonomous nervous circuits in which the most conspicuous bursting activity is clearly associated to motor function. Recent studies have investigated small perturbations embedded in the otherwise seemingly periodic bursting: the subtle intra-burst spike timing patterns (IBSPs), traditionally neglected for their lack of relation to the CPG motor function. Moreover, IBSPs were found to be cell-type specific and able to reflect changes in CPG connectivity, indicating a potential role in information coding. Here we addressed this matter by investigating how a bursting motor neuron expresses information about other neurons in the network. We performed experiments on the crustacean stomatogastric pyloric CPG, both in control conditions and when interacting in real-time with computer model neurons. The sensitivity of post- to pre-synaptic IBSPs was inferred by computing their average mutual information along each neuron burst. We found that a single motor neuron is able to express, at the beginning of its burst, information about the IBSPs of the beginning of the pre synaptic neuron\'s burst. This phenomenon is observed in different specimens and species, sugesting a genera information coding mechanism. Moreover, this effect was reproduced in a hybrid circuit, in which the presynaptic stimuli are completely controled by the experimenter free of any influence of other elements in the circuit. These results suggest that the presynaptic spiking microstructure are non-linearly and in homogeneously encoded through a single synapse in the post synaptic IBSPs. This way, motor neurons are able to use different time scales to express two types of information simultaneously: muscle contraction (related to bursting rate), and the behavior of other CPG neurons (in a much smaller timescale by using IBSPs as information carriers). Therefore, the coding mechanism described takes part in a previously unsuspected information pathway, providing evidence of the general physiological role of information coding through IBSPs in the regulation of neuronal _ring patterns in remote circuits by the central nervous system.

Esta tese é estruturada em dois capítulos. O primeiro capítulo explora os retrotransposons com LTR (LTR-RT) em cana-de-açúcar e grande parte de seus resultados foram publicados no artigo \'\'Analysis of plant LTR-retrotransposons at the fine-scale family level reveals individual molecular patterns\'\'. Nossos resultados mostraram que as diferentes famílias de LTR-RT em cana-de-açúcar possuem estruturas e regulação distintas. O segundo capítulo desta tese visa responder a perguntas que surgiram durante a primeira metade deste trabalho, mas ao invés de focar no genoma de uma planta optamos por trabalhar com linhagem Del de LTR-RT em dez genomas de angiospermas sequenciados. Os resultados desta parte do trabalho foram submetidos para publicação no artigo intitulado \'\'Virus-like attachment sites and plastic CpG islands: landmarks of diversity in plant Del retrotransposons\'\'. Os resultados mostraram que a LTR é uma região dinâmica e importante para a evolução dos LTR-RTs. Nós especulamos que mudanças nas LTR atuem como gatilhos para a diversificação dos LTR-RTs. / This doctoral thesis is structured in two chapters. In the first chapter we explore the LTRretrotransposons (LTR-RT) in sugarcane, these results were published in an article entitled \'\'Analysis of plant LTR-etrotransposons at the fine-scale family level reveals individual molecular patterns\'\'. In this paper we show that different sugarcane LTR-RT families have distinct structure and are differentially regulated. In the second chapter we try to find answers to questions that came up in the first half of this work, but instead of focusing in one plant genome we chose to work with the Del lineage of LTR-RT in tem angiosperm sequenced genomes. These results are submitted to publication as an article entitled \'\'Virus-like attachment sites and plastic CpG islands: landmarks of diversity in plant Del retrotransposons\'\'. Our results indicate that the LTR region is dynamic and important in the evolution of LTR-retrotransposons, we speculate that it is a trigger for retrotransposon diversification.