京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22330号 / 医博第4571号 / 新制||医||1041(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 小西 靖彦, 教授 妹尾 浩, 教授 湊谷 謙司 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Nous voulons mettre en évidence et analyser les réponses de systèmes biologiques à l’introduction de perturbations et de modulations spatio-temporelles. Plus précisément, afin de développer des stratégies innovantes pour l’étude des systèmes biologiques, nous proposons d’utiliser des outils microfluidiques. Nous concevons des microsystèmes adaptés qui peuvent influer localement sur les comportements biologiques, ceci afin qu’un experimentateur macroscopique puisse contrôler l’environnement externe des objects biologiques dont l’échelle est microscopique. Cette stratégie d’ingénierie est générique et multidisciplinaire. Au cours de cette thèse, elle a été mise en œuvre dans le cadre de deux projets collaboratifs, d’une part à l’échelle de la bactérie E.coli, et d’autre part à celle de l’embryon de souris à un stade post-implantation précoce. Les objets d’étude choisis sont caractéristiques à bien des égards des champs biologiques concernés : taille, représentativité, complexité.Nous avons mis nos compétences de spécialistes en conception et en fabrication de dispositifs fluidiques au service de la ligne DISCO du synchrotron SOLEIL et de l’équipe d’embryologie de la souris de l’IRIBHM. Le nœud de mon travail a été de concevoir et fabriquer les outils microfluidiques réutilisables pour des recherches génériques, qui permettent aux biologistes de se dispenser de l’utilisation d’une salle blanche.Plus précisément, le projet de microbiologie à SOLEIL avait pour object l’étude de l’influx et l’efflux de molécules antibiotiques dans des bactéries. Pour ce faire, nous avons developpé un dispositif réutilisable pour immobiliser les microorganisms et changer leur environnement chimique pendant l’imagerie en microscopie d’epifluorescence dans l’UV. Cette étude s’effectue en utilisant deux partenaires typiques : la bactérie Escherichia coli et un médicament de la famille des fluoroquinolones. Le projet d’embryologie a reposé sur l’électroporation localisée d’acides nucléiques au sein d’embryons de souris et le suivi des migrations cellulaires. Au cours de cette thèse, nous avons développé non seulement des microdispositifs réutilisables mais aussi des protocoles expérimentaux adaptés à l’utilisation de ces instruments miniaturisés.Plus précisément, le projet de microbiologie à SOLEIL avait pour object l’étude de l’influx et lantibiotiques dans des bactéries. Pour ce faire, nous avons developpé un dispositif réutilisable pour immobiliser lesmicroorganismes et changer leur environnement chimique pendant l’imagerie en microscopie d’épifluorescence dans l’UV.Cette étude s’effectue en utilisant deux partenaires typiques : la bactérie E. coli et un médicament de la famille desfluoroquinolones. Le projet d’embryologie a reposé sur l’électroporation localisée d’acides nucléiques des embryons desouris et le suivi des migrations cellulaires. / We want to analyze the responses of biological systems to the introduction of perturbations and spatio-temporal modulations. More specifically, in order to develop innovative strategies for the study of biological systems, we propose to use microfluidic tools. We design adapted microsystems that can locally influence biological behaviors, so that a macroscopic experimenter can control the external environment of biological objects whose scale is microscopic. This engineering strategy is generic and multidisciplinary. In this thesis, it has been implemented in two collaborative projects, on one hand, on the scale of the E.coli bacterium and on the other hand on that of the embryo of mouse at an early stage post-implantation. The selected study objects are characteristic in many respects of the biological fields concerned: size, representativeness, complexity.We extended our expertise in fluidic device design and manufacturing to the service of the DISCO beamline of the synchrotron SOLEIL and the IRIBHM mouse embryology team. The key point of my work has been to design and manufacture reusable microfluidic tools for generic research, which allow biologists to dispense with the use of a clean room.More precisely, the project of microbiology at SOLEIL had for object the study of the influx and the efflux of antibiotic molecules in bacteria. To do this, we have developed a reusable device for immobilizing microorganisms and changing their chemical environment during UV imaging on epifluorescence microscopy. This study is carried out using two typical partners: the Escherichia coli bacterium and a drug from the fluoroquinolone family. The embryology project relied on the localized electroporation of nucleic acids within mouse embryos and the monitoring of cellular migrations.In this thesis, we have developed not only reusable micro-devices but also experimental protocols adapted to the use of these miniaturized instruments.More precisely, the microbiology project at SOLEIL focused on the influx and the efflux of antibiWe have developed a reusable device for immobilizing those microorganisms and changing their chemical environmentduring UV imaging on an epifluorescence microscopy. This study was carried out using two typical partners: thebacterium and a drug from the fluoroquinolone family. The embryology project relied on the localized electroporation ofnucleic acids into mouse embryos and the monitoring of cell migrations.

The embryonic source and chemical nature of those factor/s directing the in vivo differentiation of melanocytes from crest cells are as yet unknown. To begin to address this issue, it is important to establish exactly when and where these signa/s first exert their effects. Therefore, in the present study, overtly differentiated melanocytes containing melanin were quantitated in developing Black Australorp X New Hampshire Red chick embryos. In contrast to previous studies, it was found that embryos synthesize melanin from as early as Day 5 of development, and that at this stage, the melanocytes are predominantly dermally located. Between 5 and 8 days, the numbers of both dermal and epidermal melanocytes increase, after which the dermal melanocyte population declines rapidly while the number of epidermal melanocytes continues to increase. These findings suggest that premelanocytes do not have to be epidermally located to initiate terminal differentiation and implicate the dermis as a possible source of melanocyte inducing factor/s. The next step was to examine stages of development prior to the onset of pigment production. For this reason, tyrosinase was purified for use as antigen in the production of a polyclonal antibody. The antibody was tested for specificity by western blotting, - immunocytochemistry and immunoinhibition procedures. Lack of specificity was demonstrated, rendering it unsuitable as an antibody marker for early melanocytes. Fowl melanocytes are thought to differentiate into either eumelanosome- or pheomelanosome synthesizing cells. To test the validity of this concept, embryonic skin of the red/black cross breed were screened for possible mixed type melanocytes by electron microscopy. The melanocytes contained melanosomes with a matrix of irregularly arranged filaments amongst typical eumelanogenic melanosomes. This suggests that these chick melanocytes may synthesize both eumelanosomes and pheomelanosomes in single cells. In a further study on pure breeding New Hampshire Reds, it was found that the melanocytes contained a mixture of typical and less typical pheomelanosomes. Outer membrane indentations in the latter melanosome type suggest that tyrosinase may enter these pheomelanosomes by a mechanism related to that proposed for the melanosomes of goldfish.

Multi-tissue pattern formation during development is a complex process in which extracellular communication events specify distinct cell types and regulate exquisite embryonic morphogenesis. The sea urchin embryo provides an excellent model for studying pattern formation due to relative genetic and morphological simplicity. The larval skeleton is secreted via biomineralization by the skeletogenic primary mesenchyme cells (PMCs). The PMCs undergo an epithelial-mesenchymal transition and migrate as individual cells within the blastocoel into stereotypic positions; this regulated PMC migration and positioning is required for normal skeletal patterning. Elegant PMC transplant experiments have demonstrated that PMC positioning, and thus skeletal patterning, is directed by ectodermal cues, and not by cues internal to the PMCs. In recent years, new efforts have been made to identify the ectodermal gene products that regulate skeletal development. The transcription factors Otp, Pax2/5/8 and Strim1, signaling by FGF, VEGF, and Wnt5a ligands have all been implicated in skeletal development in the sea urchin embryo; however, loss-of-function analysis for most of these gene products results in inhibition of skeletogenesis, suggesting that they regulate biomineralization and not PMC positioning and patterning. Notably, no skeletal patterning genes have previously been identified that pattern specific parts of the larval skeleton. This dissertation takes candidate-based and systems-level approaches to identify novel skeletal patterning genes that pattern distinct parts of the larval skeleton. We find that activation of the Alk4/5/7 receptor is required during gastrulation for anterior PMC positioning and skeletal patterning. We next test the function of the TGF-ß ligand Univin and find that it is necessary and sufficient for secondary skeletal patterning, indicating that Univin is a likely signaling ligand in anterior skeletal patterning. We also report a ventral accumulation of sulfated proteoglycans that requires function of the sulfate transporter, SLC26a2/7. This SLC-dependent sulfated proteoglycan accumulation is necessary and sufficient to attract PMCs to the ventral territory, and thereby pattern the ventral transverse skeletal elements. Finally, BMP5-8 function is required for left-side skeletal and serotonergic neuron development. Together, these studies reveal novel ectodermal genes that specifically regulate skeletal patterning across the anterior-posterior, dorsal-ventral, and left-right axes in Lytechinus variegatus embryos. / 2017-01-01T00:00:00Z

Thompson (1967) defines three developmental types for Opisthobranchia with representative nudibranchs in each category. The types are: 1.) planktotrophic larvae which are obligatory plankton feeders prior to progressive metamorphosis, 2.) lecithotrophic larvae which may feed on the plankton, but do not need to do so in order to metamorphose, 3.) direct development which results in hatching of a post veliger, benthic juvenile. Tardy (1970) feels that Thompson's Type 3 development is artificial. Thus, he incorporates direct development into the lecithotrophic developmental type. Tardy then proposes a classification of metamorphic types based on larval shell type (Thompson, 1961) and on larval feeding behavior (ie. lecithotrophic vs. planktotrophic larvae). I agree with Thompson's (1967) distinction between direct and lecithotrophic developmental types in the sense that they represent ecologically diverse ontogenies. The major explicit assumption in the above definition of planktotrophic larvae is that feeding is a necessary prerequisite to progressive metamorphosis. However, both Thompson (1967) and Tardy (1970) appropriately note that metamorphic observations of planktotrophic larvae are fragmentary and circumstantial. Thus, the definition of planktotrophic larvae rests its credibility on larval morphology at hatching, and the implicit assumption that energy is required (ie. feeding) to develop the organs necessary to accommodate the functional transition to the adult mode of life. The present study describes the early embryology, larval development, veliger morphology and feeding behavior of Hermissenda crassicornis and Aeolidia papillosa. Veligers of the facelinid, Hermissenda crassicornis and the aeolid, Aeolidia papillosa have striated, Type 1 (coiled) larval shells and fall within Thompson's (1967) definition of planktotrophic larvae. Veligers of both species remain planktonic for two to five days after hatching. They subsequently become epibenthic swimmers and discard their larval shells. There is considerable variation in the amount of yolk reserves in the gut and diverticulae of recently hatched veligers. An individual egg mass yields larvae with and without yolk reserves. The results show that shell length frequency of hatching larvae is distributed bimodally. There is a larval dimorphism based on shell length at hatching, the presence of yolk reserves and feeding ability. Feeding larvae are found to differ with respect to diet; the difference being associated with shell length of the larvae in relation to food particle size. The results are discussed in a comparative review of larval development in the Eolidoidea. Secondly, the relative dependency of nudibranch larvae on feeding ability is discussed with respect to the morphological and developmental categorization of opisthobranch metamorphic responses. The functional and ecological considerations of feeding in gastropod and selected invertebrate larvae are discussed with respect to the evolution of larval strategy and life cycle phenomena.

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The development of Sacropodus opercularis Linnaeus, (Perciformes: Anabantidae), is described from cleavage to five days post hatching. For the most part M. opercularis follows the pattern of typical teleostean development except as noted below: 1. The heart develops under the left eye along the anterior margin of the yolk sac. This is similar to that reported by Ingesoll (1951) for the Blue Gourami, Trichogaster trichopterus. 2. The ear develps from the head mesenchyme alongside the brain rather than from a placode. 3. The gut develops first in the posterior region then differentiates cephalad. It was also learned that: 1. No true bone exists in the skeletal system up to five days of development post hatching at 80°F. 2. The hatching time was 35-37 (36) hours after fertilization at 80°F.

The purpose of this paper is to critically study and analyze all species belonging to the genus Pantosteus and to determine tbe validity of the characteristics that were used to separate the genus Notolepidomyzon from the genus Pantoateus. Special reference was made to the morphology of the fontanelle and the weberian ossicles. In order to more thoroughly comprehend the reasons for Cope describing Pantosteus as a new genus and Fowler describing Notolepidomyzon as a new sub-genus and then Snyder elevating it to the status of a genus as complete a historical sketch as possible was made using all literature known and available. A description has been made of the genus, the sub-genera, and all species concerned in this study. The two critical factors which separate the genus Notolepidomyzon from the genus Pantosteus received special attention. Illustrations showing the structure of the cranium have been made of representative specimens of all species except P. generosus. The fontanelle remained constant in some species, but in others it varied greatly. The weberian ossicles vere studied and illustrations made or species within both genera. No definite critical factor could be found with the ossicles that could be used to separate the genera. It is the proposal of the writer that since the characteristics that were used to distinguish these two genera are not consistent nor completely reliable, the genus Notolepidomyzon not be recognized as separate genus, but retained as a sub-genus distinguished from the sub-genus Pantosteus primarily on the thickness of the cranium and the open or obliterated fontanelle. The material and evidence needed to support this proposal is contained in this paper.