Em ouriços, os esferulócitos vermelhos são responsáveis pela biossíntese do equinocromo, um pigmento naftaquinônico considerado antioxidante e bactericida, no entanto seu papel na resposta imune permanece pouco elucidado. O presente trabalho avaliou a reposta imune inata de ouriços-do-mar Lytechinus variegatus, através da atividade fagocítica frente a diferentes concentrações de equinocromo (50 e 100 µg/ml). Para tanto, o equinocromo foi extraído e purificado por RP-HPLC. Nossos resultados demonstraram que o equinocromo em ambas as concentrações modula positivamente a fagocitose, aumentando a quantidade de células fagocitando. A concentração de 50 µg/ml foi capaz de ativar os amebócitos fagocíticos (AF), e aumentar a quantidade de AF com quatro ou mais leveduras fagocitadas. Já na concentração de 100 µg/ml, além da ativação dos AF, aumentou também, a quantidade de AF com uma, duas, quatro ou mais leveduras fagocitadas, sugerindo uma atuação dose-dependente. Desta forma, os dados apresentados demonstram que o equinocromo exerce um importante papel na resposta imune. / The biosynthesis of echinochrome is mediated by red sphere cell. This naphthoquinonic e pigment presents antioxidant and bactericidal characteristics. However, the echinochrome role in immune response remains unclear. In this study, we evaluated the innate immune response of the sea urchin Lytechinus variegatus. To this purpose, the echinochrome was extracted and purified by RP-HPLC. Finally, phagocytic amoebocytes were exposed to different concentrations of echinochrome (50 and 100 mg/ml), when phagocytic activity was analysed. Here, we showed that echinochrome positively modulate phagocytosis, increasing the number of phagocytizing cells. The concentration of 50 mg/ml activated phagocytic amoebocytes (AF), and increased the number of AF containing four or more phagocytosed yeasts. For the other hand, at 100 mg/ml exposure, the activation of AF also increased the number of AF with one, two, four or more yeast phagocytosed, suggesting a dose-dependent activity. Thus, the data presented demonstrated that echinochrome plays an important role in the immune response.

In the adult nervous system, neurotransmitters mediate cellular communication within neuronal circuits. In developing tissues and primitive organisms, neurotransmitters subserve growth regulatory and morphogenetic functions. Accumulated evidence suggests that acetylcholine, (ACh), released from growing axons, regulates growth, differentiation, and plasticity of developing central nervous system neurons. In addition to intrinsic cholinergic neurons, the cerebral cortex and hippocampus receive extensive innervation from cholinergic neurons in the basal forebrain, beginning prenatally and continuing throughout the period of active growth and synaptogenesis. Acute exposure to ethanol in early gestation (which prevents formation of basal forebrain cholinergic neurons) or neonatal lesioning of basal forebrain cholinergic neurons, significantly compromises cortical development and produces persistent impairment of cognitive functions. Neonatal visual deprivation alters developmental expression of muscarinic acetylcholine receptors (mAChR) in visual cortex, whereas local infusion of mAChR antagonists impairs plasticity of visual cortical neurons. These findings raise the possibility that exposure to environmental neurotoxins that affect cholinergic systems may seriously compromise brain development and have long-lasting morphologic, neurochemical, and functional consequences.

The analysis of Gene Regulatory Networks (GRNs) is essential to understanding the complete process of embryo development. Elucidating every gene regulatory circuit from maternal regulatory inputs all the way to the activation of differentiation gene batteries is an important step in increasing our understanding of developmental biology. In this work I study the cis-regulatory architecture of a pigment cell differentiation gene, polyketide synthase (SpPks) in the sea urchin Strongylocentrotus purpuratus. SpPks encodes an enzyme that is responsible for the biosynthesis of the sea urchin pigment echinochrome in larval pigment cells. The analysis of the promoter of a differentiation gene will lead to identifying the direct upstream regulators and ultimately to elucidating the structure of the upstream gene regulatory network, which is mostly uncharacterized. From previous studies the transcription factors SpGcm and SpGatae are predicted to be positive regulators of SpPks. Here, I identify a minimal 1kb promoter region containing putative DNA-binding sites for both GCM and GATAE that is able to recapitulate the expression of SpPks. I further show by mutagenesis that a putative DNA-binding site for GCM located 1,179 base pairs upstream of the start of transcription is a direct target for the positive cis-regulation of SpPks. Quantitative analysis of the transcriptional regulatory function of the GCM-mutagenized construct suggests that GCM is not necessary for the start of SpPks transcription but is required for its maintenance. Several GATA E binding sites have been identified within the minimal promoter for SpPks by means of consensus sequence. My analysis suggests that GATA E may be a direct positive regulator and could potentially be required for the onset of transcription of SpPks, though further experimentation will be necessary to characterize the exact regulatory function of GATA E.

The amount of solar ultraviolet radiation (UVR, 290-400 nm) reaching Earth’s surface is increasing due to ozone depletion and global climate change. Embryos of the purple sea urchin, Strongylocentrotus purpuratus, provide an ideal system for examining how UVR affects developing marine organisms and cells in general. To model the protein-mediated cell cycle response to UV-irradiation, six batches of S. purpuratus embryos were exposed to UVR, monitored for delays in the first mitotic division and examined for global proteomic changes. Embryos from each batch were exposed to or protected from artificial UVR for 25 or 60 min. Embryos treated with UVR for 60 min cleaved an average of 23.24 min (±1.92 s.e.m) later than the UV-protected embryos. Protein expression of UV-protected and UV-treated embryos was examined at 30 and 90 min post-fertilization using two dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (2D SDS-PAGE) and mass spectrometry (MS). Proteins were isoelectrically focused (pH 4-7) and separated by molecular weight using SDS-PAGE. At least 1,306 protein spots were detected in all gels. A total of 171 protein spots (13% of the detected proteome) migrated differently in UV-treated embryos at 30 min post-fertilization and 187 spots (14%) at 90 min post-fertilization (2-way ANOVA, P= 0.03, n=6). Our results identify the differential migration of proteins from multiple cellular pathways and are the first to indicate that the mechanisms involved in the protein mediated UV-induced developmental delay are integrated among pathways for cellular stress, protein turnover and translation, signal transduction, general metabolism and involve the cytoskeleton.

Ion channels and pumps play critical roles during sea urchin development including mediating the blocks to polyspermy, regulating left-right and dorsal-ventral axis specification, directing ventral PMC migration, and controlling biomineralization of the larval skeleton. We performed a screen of pharmacological ion channel inhibitors, and we chose two inhibitors to investigate further. First, we found that tricaine, a potent inhibitor of voltage-gated sodium channels (VGSCs), induces aberrant skeletal patterning in Lytechinus variegatus larvae. The larval skeleton is secreted by the primary mesenchyme cells (PMCs), which migrate within the blastocoel into a stereotypical pattern. We show that VGSC activity is required for normal PMC migration and skeletal patterning. Timed inhibitor studies identified VGSC activity as specifically required from early gastrula to the onset of late gastrula for normal skeletal patterning. Tricaine inhibits the voltage-gated sodium channel LvScn5a which is strongly expressed in the developing nervous system in pluteus larvae. We found that exogenous expression of an anesthetic-insensitive version of LvScn5a is sufficient to rescue hallmark tricaine-mediated skeletal patterning defects, demonstrating the specificity of the inhibitor. LvScn5a exhibits a ventrolateral ectodermal expression domain in gastrulating embryos that is spatiotemporally congruent with triradiate formation in the ventrolateral PMC clusters at the onset of skeletogenesis. This ectodermal territory normally expresses the patterning cue Wnt5, and we find that the expression of Wnt5 is dramatically spatially expanded by tricaine treatment. We also observe ectopic PMC clusters in tricaine-treated embryos. We found that knockdown of Wnt5 expression is sufficient to rescue tricaine-mediated skeletal patterning defects. These results are consistent with a model in which LvScn5a activity in the ventrolateral ectoderm functions to spatially restrict the expression of the ectodermal patterning cue Wnt5 that in turn induces PMC cluster formation. Together, these findings show that spatially restricted sodium channel activity regulates ectodermal cue expression that, in turn, regulates PMC differentiation and skeletal morphogenesis. Second, we show that V-type H⁺ ATPase (VHA) activity is required for specification of the dorsal-ventral (DV) axis. DV specification is controlled by the TGF-β signal Nodal that specifies the ventral territory and indirectly activates dorsal specification via induction of BMP 2/4 expression. Nodal expression occurs downstream of p38 MAPK, which is transiently, asymmetrically inactive on the presumptive dorsal side of the blastula embryo. VHA activity is required for that transient inactivation of p38 MAPK, and it is required for the subsequent spatial restriction of Nodal expression. We show that VHA inhibition is sufficient to induce global Nodal expression during the blastula stage, resulting in ventralization of the embryo. We show that this phenotype can be rescued by experimentally imposing asymmetric Nodal expression at the 4-cell stage. We discover a VHA-dependent voltage gradient across the DV axis and find that VHA activity is required for hypoxia inducible factor (HIF) activation. We show that neither hyperpolarization nor HIF activation is sufficient to perturb DV specification, which implicates a third unknown pathway connecting VHA activity and p38 MAPK symmetry breaking. These results are consistent with a model in which dorsal VHA activity is required to inhibit Nodal expression and signaling, potentially via dorsal p38 MAPK inhibition. Together, these studies demonstrate that ion channels are required for both DV specification and for normal skeletal patterning.

Continuous efforts have been directed towards controlled calcium carbonate biomineral synthesis in recent years. Compared to their inorganic counterparts, biominerals are more tensile in industrial applications, biocompatible with scientific designs, and sustainable for the environment. Most current approaches for synthetic biomineral production rely heavily on sophisticated engineering techniques to constrain the physical property of their crystals, which limits the adaptability of these products. Here, we proposed a novel approach to synthesize calcium carbonate biominerals by reproducing skeletogenesis of the sea urchin larva in vitro using common cellular and molecular methods. Skeleton formation in Lytechinus variegatus sea urchin embryos is a highly coordinated event, where ectodermal cells in different domains express distinct patterning cues that are received by adjacent primary mesenchyme cells (PMCs), which in turn secrete the skeleton. Our group and others have identified a range of skeletal patterning cues, and based on our current understanding of the mechanism, we envisioned a synthetic ectoderm culture using defined ectodermal lineages that, when combined with PMCs, will direct the synthetic production of skeletal structures. Here we have developed a detailed protocol for establishing such as ectoderm culture and have begun initial experiments towards this goal. Future deployment of this protocol will provide invaluable insights into the mechanism of skeletal patterning in sea urchins, as well as an unprecedented system for customized synthetic calcium carbonate biomineral production. Finally, improving our mechanistic understanding of skeletal patterning in echinoderms has the potential to shed light on analogous biomineralization processes in other species as well. / 2024-10-03T00:00:00Z

Made available in DSpace on 2015-05-07T14:49:31Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 3590758 bytes, checksum: bf7818f89b1ec249bca8891f256b8f1a (MD5) Previous issue date: 2013-04-19 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / This thesis called Sustainability in Suape Bay: between the Suape Port and Industrial Complex and the Sea Urchin Festival seek to understand the relationship into the realization of the Sea Urchin Festival as a cultural and folk religious manifestation of Suape region. The relationships maintained by the population that inhabits the Suape Bay for generations is a complexity composed by relevant elements as historical, economic, social, cultural or environmental which are analyzed in this study. Through a quantitative and qualitative perspective, we seek to analyze the mode of production and reproduction of theirs lives, which are strongly attached with elements of the local nature, as well the marine territory category and with their cultural manifestations. They are facing the growing social changes resulted by the pressures coming from the enterprises and the development promises of the most emblematic economic policy megaproject of Pernambuco State, the Suape Port and Industrial Complex. We can say that the contemporary scenario showed to the Suape population, who are rooted into a rustic culture and traditional modal of life based on their small scale fishing practice, is a turning point, once some of them realize they are living one social and environmental conflict against the economic development machine, in the same time that others see each other in an uprooting process with their own territory. So there are some of the Sea Urchin Festival expressions that now just belong to the social and folk memory sphere, as well other ones get the drift, getting an uncertain way. / Esta dissertação intitulada Sustentabilidade na Baía de Suape: entre o Complexo Industrial Portuário de Suape e a Festa da Ouriçada busca compreender as relações que transpassam a realização da Festa da Ouriçada, enquanto manifestação da cultura e da religiosidade popular suapense. Estas relações protagonizadas pela população que habita a Baía de Suape há gerações são compostas pela complexidade de sua pertinência seja histórica, econômica, social, cultural ou ambiental, as quais são analisadas na presente pesquisa. Através de uma abordagem quantitativa e qualitativa, buscou-se a análise dos fortes vínculos de seu modo de produção e reprodução de vida com os elementos naturais locais, com a categoria territorialidade marinha e com suas manifestações culturais face às constantes transformações sociais provenientes das pressões exercidas por empreendimentos e pelas promessas de desenvolvimento do megaprojeto mais emblemático da política econômica pernambucana, o Complexo Industrial Portuário Governador Eraldo Gueiros de Suape (Cips). Pode-se considerar que o cenário posto na contemporaneidade para a população suapense enraizada no modelo de cultura rústica e tradicional de sua prática da pesca artesanal é um ponto de mutação, já que alguns se percebem vivendo uma situação de conflito socioambiental diante dos avanços das máquinas do desenvolvimento econômico, enquanto outros se veem em processo de desenlace com sua territorialidade. Na mesma direção, algumas manifestações da Festa da Ouriçada agora só pertencem à memória social de seu povo, enquanto sua prática fica à deriva, tomando um caminho, no mínimo, incerto.

O aquecimento global é uma realidade e seus efeitos são bastante estudados atualmente. No entanto, pouca atenção tem-se dado para as alterações que ocorrem com invertebrados marinhos em decorrência dessa alteração climática. Sabe-se que uma das alterações que ocorrem em consequência do aumento da temperatura da água do mar, é o aumento do aparecimento de doenças no ambiente marinho. Para tanto, é importante que se avalie sua resposta imune inata frente a esse fator estressor. Os ouriços-do-mar foram escolhidos como modelo por serem considerados bons biondicadores ambientais, além de serem filogeneticamente próximos aos cordados, partilhando com estes, amplo repertório de genes e receptores envolvidos no sistema imune inato. O presente trabalho avaliou a resposta imune inata de ouriços-do-mar antárticos (S. neumayeri) e tropicais (L. variegatus e E. lucunter) frente ao estresse térmico por diferentes temperaturas e períodos de exposição. No que se refere aos ouriços-do-mar tropicais, foram estudadas duas espécies, uma que habita regiões menos submetidas a variações de marés (L. variegatus) e outra constantemente exposta a variações de maré (E. lucunter). Constatou-se uma diferença entre a resposta ao estresse térmico nas três espécies estudadas. A espécie antártica demonstrou alterações mais significativas de aumento na porcentagem de esferulócitos vermelhos (EV) e da capacidade fagocítica no período agudo de exposição (24h) quando submetidos a temperaturas intermediárias. Por outro lado, a espécie tropical E. lucunter apresentou alteração somente na porcentagem de EV no período de exposição crônico (7 e 14 dias) a temperaturas altas, enquanto que L. variegatus apresentou alteração neste tipo celular em todas as temperaturas experimentais e períodos avaliados, além de uma importante redução da capacidade fagocítica nos mesmos períodos que foi diretamente proporcional ao aumento da temperatura. Buscando compreender tais diferenças entre animais que habitam uma mesma região, verificou-se que a espécie L. variegatus apresentou alteração na adesão e espraiamento celular, no citoesqueleto de actina dos amebócitos fagocíticos (AF), na migração celular sem adição de substância quimiotática e na resposta quimiotática frente a leveduras S. cerevisiae, além de diminuição do estresse oxidativo em elevadas temperaturas. Constatou-se também que os AF desta espécie apresentaram alterações morfométricas importantes, como diminuição na circunferência celular e alteração na área de espraiamento. Por outro lado, a espécie E. lucunter não apresentou alteração em nenhum parâmetro analisado. Conclui-se, assim, que o estresse térmico atua de maneiras diferentes em espécies polares e tropicais de ouriços-do-mar. No entanto, os mecanismos moleculares pelos quais a espécie E. lucunter apresenta maior termotolerância em comparação à espécie L. variegatus permanecem imcompreendidos e merecem ser estudados. / Global warming is a reality and its effects are widely studied today. However, little attention has been given to the changes that occur to marine invertebrates due to this climate change. It is known that one of the changes that occur as a result of the seawater temperature rise is the increased occurrence of diseases in the marine environment. Therefore, it is important to evaluate the innate immune response against this stressor. The sea urchins were chosen as a model for being considered good environmental bioindicators, and are phylogenetically close to chordates, sharing with them large repertoire of genes and receptors involved in the innate immune system. This study evaluated the innate immune response of Antarctic (S. neumayeri) and tropical (L. variegatus and E. lucunter) sea urchins against thermal stress by different temperatures and exposure periods. With regard to tropical sea urchins, two species, one that inhabits sites less subject to tidal variations (L. variegatus) and the other constantly exposed to tidal oscilations (E. lucunter) were studied. A difference between the response to thermal stress in the three species was observed. The Antarctic species showed more significant changes regarding increase of the percentage of red spherule cells and phagocytic capacity in acute exposure period (24 h) when subjected to mild temperatures. On the other hand, tropical species E. lucunter presented only change in the percentage of red spherule cells during chronic exposure (7 and 14 days) at extreme temperatures, while L. variegatus showed a change in this cell type in all experimental temperatures and exposure periods evaluated, as well as a significant reduction in phagocytic capacity in the same periods that was directly proportional to the temperature increase. Trying to understand the differences between animals that inhabit the same region , it was found that the species L. variegatus showed changes in cell adhesion and spreading, actin cytoskeleton of phagocytic amoebocytes, cell migration without the addition of chemotactic substance and chemotactic response against yeasts S. cerevisiae, besides a reduction of oxidative stress at elevated temperatures. It was also found that the phagocytic amoebocyte of this species showed significant morphological changes, such as reduction in cell circunference and change in the area of spreading. On the other hand, the species E. lucunter presented no change in any parameter analyzed. Thus, it is concluded that the thermal stress acts differently in polar and tropical species of sea urchins. However, the molecular mechanisms by which the species E. lucunter shows higher thermotolerance compared to the species L. variegatus remain obscure and deserve to be studied.

CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / Echinodermata are marine-exclusive animals found in various habitat types and depth.Sea urchins have great relevance in this group because of important control exerted on algal community and reef bioerosion. Population of sea urchins has been experiencing variances on its natural density, thus impacting the marine biodiversity and testing habitats resilience. Anthropogenic or natural aspects are the main drivers of this event once observed only in temperate areas, but now noticed worldwide. As a result of such rapid expansion and new migratory routes, the genetic characteristics of these species might undergo changes in heterozigosity levels as well as in gene flow rates amongst other subpopulations geographically distant. Molecular markers can provide the rates of genetic variability in animals, showing their patterns of diversification, origin and migratory movements. This project analyzed the genetic characteristics of the white sea urchin populations in Fernando de Noronha and Bahia using mtDNA citocrome oxidase 1 from 89 samples, aiming to evaluate the gene flow within Brazilian populations and those spread in the Atlantic ocean. The results showed that sea urchins from F. Noronha form a group genetically diverse and, regardless its recent population expansion haplotype diversity suggests these organisms are not the result of a massive population migration from other areas. The haplotype network well linked with the population from Bahia is a result of continuous gene flow between these groups, taking a continentisland direction and the likely migration rates through the North Equatorial undercurrent. The frequency of unique haplotypes in the island population demonstrates high heterozigosity, and low divergence rates in comparison with Bahia population. / Os Echinodermata sÃo animais exclusivamente marinhos encontrados nos mais diversos tipos de habitat e profundidades. Nesse grupo se destacam os ouriÃos-do-mar por seu papel fundamental no controle da comunidade de algas e bioerosÃo do substrato rÃgido. Suas populaÃÃes vem comumente sofrendo variaÃÃes em sua densidade natural, impactando a biodiversidade marinha e colocando em prova a resiliÃncia dos habitats afetados. Fatores antrÃpicos ou naturais sÃo os principais responsÃveis por esse fenÃmeno antes conhecido apenas em Ãreas temperadas, mas jà observado em latitudes tropicais. Com a expansÃo populacional repentina e novas rotas migratÃrias, as caracterÃsticas genÃticas da espÃcie podem sofrer variaÃÃes na heterozigosidade e no fluxo genÃtico entre subpopulaÃÃes geograficamente separadas. Marcadores moleculares podem revelar a variabilidade genÃtica entre populaÃÃes, demonstrando os padrÃes de diversificaÃÃo de espÃcies, ancestralidade e movimentos migratÃrios. Esse trabalho analisou as caracterÃsticas genÃticas da populaÃÃo de ouriÃo-branco em Fernando de Noronha e Bahia atravÃs seqÃenciamento parcial do mtDNA, gene da citocromo oxidase 1 de 89 indivÃduos, com a finalidade de avaliar o fluxo gÃnico entre as populaÃÃes brasileiras e suas relaÃÃes com as demais existentes no AtlÃntico. Os resultados obtidos mostraram que os ouriÃos-brancos do arquipÃlago formam um grupo com caracterÃsticas genÃticas especÃficas e, embora haja ocorrido expansÃo da populaÃÃo, as modificaÃÃes haplotÃpicas observadas revelam que esses animais nÃo sÃo provenientes de uma migraÃÃo em massa de uma populaÃÃo. O compartilhamento de haplÃtipos com ouriÃos da Bahia à resultado de um fluxo gÃnico contÃnuo, na direÃÃo continente-ilha, e que a possÃvel rota migratÃria se daria pela contracorrente Equatorial Norte. A freqÃÃncia de haplÃtipos especÃficos da ilha demonstra alta heterozigosidade da populaÃÃo e baixa divergÃncia com relaÃÃo à populaÃÃo da Bahia.

<p>Specification and differentiation of a cell is accomplished by changing its gene expression profiles. These processes require temporally and spatially regulated transcription factors (TFs), to induce the genes that are necessary to a specific cell type. In each cell a set of TFs interact with each other or activate their targets; as development progresses, transcription factors receive regulatory inputs from other TFs and a complex gene regulatory network (GRN) is generated. Adding complexity, each TF can be regulated not only at the transcriptional level, but also by translational, and post-translational mechanisms. Thus, understanding a developmental process requires understanding the interactions between TFs, signaling molecules and target genes which establish the GRN.</p><p>In this thesis, two genes, FoxN2/3, a TF and FGFR1, a component of the FGF signaling pathway are investigated. FoxN2/3 and FGFR1 have different mechanisms that function in sea urchin development; FoxN2/3 regulates gene expression and FGFR1 changes phosphorylation of target proteins. However, their ultimate goals are the same: changing the state of an earlier GRN into the next GRN state. </p><p>First, we characterize FoxN2/3 in the primary mesenchyme cell (PMC) GRN. Expression of foxN2/3 begins in the descendants of micromeres at the early blastula stage; and then is lost from PMCs at the mesenchyme blastula stage. foxN2/3 expression then shifts to the secondary mesenchyme cells (SMCs) and later to the endoderm. Here we show that, Pmar1, Ets1 and Tbr are necessary for activation of foxN2/3 in the descendants of micromeres. The later endomesoderm expression is independent of the earlier expression of FoxN2/3 in micromeres and independent of signals from PMCs. FoxN2/3 is necessary for several steps in the formation of larval skeleton. A number of proteins are necessary for skeletogenesis, and early expression of at least several of these is dependent on FoxN2/3. Furthermore, knockdown (KD) of FoxN2/3 inhibits normal PMC ingression. PMCs lacking FoxN2/3 protein are unable to join the skeletogenic syncytium and they fail to repress the transfating of SMCs into the skeletogenic lineage. Thus, FoxN2/3 must be present for the PMC GRN to control normal ingression, expression of skeletal matrix genes, prevention of transfating, and control fusion of the PMC syncytium.</p><p>Second, we show that the FGF-FGFR1 signaling is required for the oral-aboral axis formation in the sea urchin embryos. Without FGFR1, nodal is induced in all of the cells at the early blastula stage and this ectopic expression of nodal requires active p38 MAP kinase. The loss of oral restriction of nodal expression results in the abnormal organization of PMCs and the larval skeleton; it also induces ectopic expression of oral-specific genes and represses aboral-specific genes. The abnormal oral-aboral axis formation also affected fgf and vegf expression patterns; normally these factors are expressed in two restricted areas of the ectoderm between the oral and the aboral side, but when FGFR1 is knocked down, Nodal expands, and in response the expression of the FGF and VEGF ligands expands, and this in turn affects the abnormal organization of larval skeleton.</p> / Dissertation