Neural plasticity is the process by which anatomical (structural) and physiological (functional) changes in the nervous system of an organism lead to alterations in behavior. This dissertation examines the structural and functional changes that occur during neural morphallaxis, a rare form of neural plasticity, in the annelid worm, Lumbriculus variegatus. Neural morphallaxis involves the reorganization of the animal's nervous system during segmental regeneration following injury. Here, I have examined neural morphallaxis of the giant fiber pathway, which mediates rapid escape reflex behaviors in Lumbriculus. Electrophysiological recording techniques, immunohistochemistry, and transmission electron microscopy were used to demonstrate that prior to injury and neural morphallactic regeneration, activation of the escape reflex neural circuitry is nonfunctional in specific regions of the worm's nervous system. Following body fragmentation, neural circuits underlying specific escape responses rapidly become functional. The speed of functional changes in sensory-to-giant interneuron physiology, less than 24 hours, did not coincide with significant anatomical changes to sensory afferent synapses, suggesting a role for the unsilencing of existing sensory synapses. Furthermore, I have discovered and described a sensory interneuron system that mediates sensory inputs via electrical synapses onto the giant interneuron pathway. This finding led to my hypothesis that the site of sensory plasticity during neural morphallaxis is not at the giant axon, but rather at the glutamatergic synapses between sensory neurons and their sensory interneuron targets. Results from this dissertation demonstrate that sensory inputs onto the giant interneuron pathway are functionally silent prior to neural morphallaxis and the awakening of ineffective synapses occurred rapidly, within hours, following injury. Neural morphallactic plasticity was determined to occur at glutamatergic synapses onto bilaterally paired sensory interneurons that were coupled to the giant interneuronal pathway. The early phase of morphallaxis is then followed by gradual structural and functional changes to enhance aspects of the escape response network. This research provides a foundation for future studies of the mechanisms underlying neural morphallactic regeneration in Lumbriculus variegatus and provides comparative insight into the evolution and plasticity of neural circuit underlying discrete animal behavior.

Submitted by LIERGE RAMOS null (lrg.ramos@hotmail.com) on 2017-09-06T20:31:40Z No. of bitstreams: 1 Lierge_Ramos_Dissertação_Biotecnologia.2017.pdf: 2068937 bytes, checksum: 107fae4a7bea607b2de9a6fd531de01f (MD5) / Approved for entry into archive by Monique Sasaki (sayumi_sasaki@hotmail.com) on 2017-09-11T19:41:07Z (GMT) No. of bitstreams: 1 ramos_l_me_araiq.pdf: 2068937 bytes, checksum: 107fae4a7bea607b2de9a6fd531de01f (MD5) / Made available in DSpace on 2017-09-11T19:41:07Z (GMT). No. of bitstreams: 1 ramos_l_me_araiq.pdf: 2068937 bytes, checksum: 107fae4a7bea607b2de9a6fd531de01f (MD5) Previous issue date: 2017-08-11 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / As hemoglobinas constituem um grupo de proteínas que desempenham um papel vital nos organismos. Suas propriedades intrínsecas, assim como a sua relação estrutura-atividade, envolvem fenômenos tais como a cooperatividade e afinidade por ligantes específicos, como o oxigênio, que estão associados a uma variedade de processos que viabilizam a vida. As hemoproteínas, em especial as hemoglobinas de anelídeos têm sido objeto de estudo de diferentes grupos de pesquisa, devido a sua alta estabilidade oligomérica, resistência à oxidação, alta cooperatividade e afinidade por ligantes específicos, apresentando um alto potencial em aplicações biotecnológicas como, por exemplo, substituto sanguíneo. Estudos sobre a caracterização estrutural e a determinação da estabilidade de hemoproteínas na presença de surfactantes, por meio de várias técnicas como absorção ótica, emissão de fluorescência, CD (Dicroísmo Circular) e espalhamento de luz podem trazer informações sobre esta classe de proteínas, principalmente sobre o mecanismo de oxidação, dissociação e desnaturação. Desta forma, no presente projeto de pesquisa objetivou realizar a caraterização biofísica da hemoglobina extraída de Amynthas gracilis (HbAg) na presença de surfactantes iônicos (SDS e CTAC) nos valores de pH 5,0 e 7,0. Os resultados nos mostram que ambos os surfactantes são capazes de interagir fortemente com a HbAg, sendo que o pH do meio influência diretamente na intensidade da interação proteína-surfactante. O SDS em pH 5,0 interage fortemente com a HbAg formando precipitados de complexo proteína-surfactante, podendo ser observados em baixas concentrações de SDS (0,01 – 0,2 mmolL-1). Enquanto que para o CTAC ocorre uma forte interação entre o surfactante e a HbAg em pH 7,0 em uma faixa de concentração de 0,01 – 0,07 mmolL-1. A formação de agregados nestes sistemas provavelmente ocorre em função do ponto isoelétrico (pI) da HbAg ser ácido (6,0 ±3), assim como o de outras Hb extracelulares, como resultado de uma forte interação eletrostática. As medidas espectroscópicas indicam que com o aumento da concentração dos surfactantes ocorre a ressolubilização dos agregados. Os resultados obtidos neste estudo demonstraram que o SDS e o CTAC promovem o processo de oxidação/dissociação da HbAg em baixas concentrações e que nas concentrações máximas de surfactantes utilizadas neste trabalho o processo de desnaturação da HbAg não é completo. / Hemoglobins are a group of proteins that play a vital role in organisms. Their intrinsic properties, as well as their structure-activity relationship, involve phenomena such as cooperativity and affinity for specific ligands, such as oxygen, which are associated with a variety of processes that make life possible. Hemoproteins, especially hemoglobins of annelids have been studied by different research groups, due to their high oligomeric stability, resistance to oxidation, high cooperativity and affinity for specific ligands, presenting a high potential in biotechnological applications, for example, a blood substitute. Studies on the structural characterization and determination of hemoprotein stability in the presence of surfactants by optical absorption, fluorescence emission, CD and light scattering can bring information about this class of proteins, mainly on the mechanism of dissociation and denaturation. Thus, in the present master's project the main objective was to perform biophysics characterization studies, with the hemoglobin extracted from the annelid of Amynthas gracilis (HbAg) in the presence of ionic surfactants (SDS and CTAC) at pH values 5,0 and 7,0. The results show that both surfactants are capable of interacting strongly with HbAg, and the pH of the medium directly influences the intensity of the protein-surfactant interaction. SDS at pH 5.0 strongly interacts with HbAg forming precipitates of protein-surfactant complex, can be observed with low concentrations of SDS (0.01 - 0.2 mmolL -1). While for CTAC a strong interaction between surfactant and HbAg occurs at pH 7.0 in a concentration range of 0.01-0.07 mmolL-1. The formation of aggregates in these systems probably occurs as a function of the isoelectric point (pI) of HbAg being acid (6.0 ± 3), as well as that of other extracellular Hb, as a result of a strong electrostatic interaction. This study showed that SDS and CTAC promote the oxidation/dissociation process of HbAg at low concentrations and that at the maximum concentrations of surfactants used in this work the denaturing process of HbAg is not complete.

Stylaria is a genus of globally distributed clitellates consisting of two recognized species with a difficult taxonomic history: Stylaria lacustris and Stylaria fossularis. The current species-level taxonomy of this genus is unclear due to variation in several morphological characters. To assess the amount of species-level diversity in Stylaria, an integrative approach using morphological investigations and genetic information was employed. I investigated the phylogenetics of this genus using three loci, two mitochondrial (cox1, and rrnL), and one nuclear (ITS) gene regions. Using several species delimitation tools, I was able to estimate the number of species in this group. Automatic barcode gap discovery, assemble species by automatic partitioning (ASAP), and the Geneious Prime Species delimitation package all delimited at least five species within Stylaria, Bayesian Poisson Tree Processes(bPTP) recovered at least six, and DELINEATE proposed only three species, all within S. fossularis. Morphologically, there seems to be no clear, consistent distinction between members of S. lacustris from Europe and S. lacustris from North America. Based on my morphological investigations and disagreement among species delimitation methods, I conservatively propose that the subclades discovered via phylogenetic analysis of mitochondrial and nuclear sequence data within S. lacustris represent population-level rather than species-level variation. However, there appear to be at least two previously unknown species in need of proper descriptions within the S. fossularis species complex. Within Stylaria, morphological criteria might be useless at the species level as there is a high level of intraspecific morphological variation. Analyses of sexual characteristics of this group could help further document any differences between populations of S. lacustris.

Ramisyllis multicaudata is a member of Syllidae (Annelida, Errantia, Phyllodocida) with a remarkable branching body plan. Using a next-generation sequencing approach, the complete mitochondrial genomes of R. multicaudata and Trypanobia sp. are sequenced and analysed, representing the first ones from Syllidae. The gene order in these two syllids does not follow the order proposed as the putative ground pattern in Errantia. The phylogenetic relationships of R. multicaudata are discerned using a phylogenetic approach with the nuclear 18S and the mitochondrial 16S and cox1 genes. Ramisyllis multicaudata is the sister group of a clade containing Trypanobia species. Both genera, Ramisyllis and Trypanobia, together with Parahaplosyllis, Trypanosyllis, Eurysyllis, and Xenosyllis are located in a long branched clade. The long branches are explained by an accelerated mutational rate in the 18S rRNA gene. Using a phylogenetic backbone, we propose a scenario in which the postembryonic addition of segments that occurs in most syllids, their huge diversity of reproductive modes, and their ability to regenerate lost parts, in combination, have provided an evolutionary basis to develop a new branching body pattern as realised in Ramisyllis.

Ramisyllis multicaudata is a member of Syllidae (Annelida, Errantia, Phyllodocida) with a remarkable branching body plan. Using a next-generation sequencing approach, the complete mitochondrial genomes of R. multicaudata and Trypanobia sp. are sequenced and analysed, representing the first ones from Syllidae. The gene order in these two syllids does not follow the order proposed as the putative ground pattern in Errantia. The phylogenetic relationships of R. multicaudata are discerned using a phylogenetic approach with the nuclear 18S and the mitochondrial 16S and cox1 genes. Ramisyllis multicaudata is the sister group of a clade containing Trypanobia species. Both genera, Ramisyllis and Trypanobia, together with Parahaplosyllis, Trypanosyllis, Eurysyllis, and Xenosyllis are located in a long branched clade. The long branches are explained by an accelerated mutational rate in the 18S rRNA gene. Using a phylogenetic backbone, we propose a scenario in which the postembryonic addition of segments that occurs in most syllids, their huge diversity of reproductive modes, and their ability to regenerate lost parts, in combination, have provided an evolutionary basis to develop a new branching body pattern as realised in Ramisyllis.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21830号 / 農博第2343号 / 新制||農||1068(附属図書館) / 学位論文||H31||N5202(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 佐藤 健司, 教授 澤山 茂樹, 准教授 豊原 治彦 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM