京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第18184号 / 医科博第49号 / 新制||医科||4(附属図書館) / 31042 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 萩原 正敏, 教授 松田 道行, 教授 岩井 一宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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© 2014, American Society for Microbiology. / 京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第19630号 / 医科博第68号 / 新制||医科||5(附属図書館) / 32666 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 松田 道行, 教授 竹内 理, 教授 杉田 昌彦 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Programmed cell death (PCD) is an essential biological process in animal development and tissue homeostasis that is necessary to ensure the physiological well-being of the organism. During PCD, phagocytes facilitate the selective removal of excess, damaged, and potentially deleterious cells, in a multi-step engulfment process. Genetic studies in Drosophila melanogaster, Caenorhabditis elegans, and mammals have identified two evolutionarily conserved signal transduction pathways that act redundantly to regulate engulfment: the CED-1/-6/-7 and CED-2/-5/-12 pathways. Of these cell death (CED) proteins, the ABC transporter CED-7 is the only protein reported to be required in both the engulfing cell and the dying cell. However, its function in the cell death process remains the most enigmatic and the ced-7 ortholog previously has not been identified in Drosophila. Homology searches revealed a family of putative ced-7 orthologs that encode transporters of the ABCA family in Drosophila. To determine which of these genes functions similarly to ced-7/ABCA1 in PCD, we analyzed their engulfment function in oogenesis, during which 15 germ cells in each egg chamber undergo programmed cell death and are removed by neighboring phagocytic follicle cells. It has been shown that genetically knocking down individual engulfment genes results in inefficient clearance of the germ cells, which then persist in late-stage egg chambers. Only two of the putative ced-7/ABCA1 genes are expressed significantly in the ovary, CG31731 and CG1718, and we have characterized these genes using transposon insertions, deficiencies, and RNAi knockdowns. Our genetic analysis reveals that CG31731 is necessary for germ cell clearance in the Drosophila ovary. Immunostaining shows that genetically knocking down CG31731 results in uncleared germ cells which persist in late-stage egg chambers. Altogether, our findings suggest that CED-7/ABCA1/CG31731 play evolutionarily conserved roles during engulfment.

Programmed cell death (PCD) and removal of cell corpses are important processes in animal development and homeostasis. Typically, engulfment of cell corpses is performed by professional phagocytes, such as macrophages. In tissues with limited accessibility to circulating cells, engulfment is carried out by neighboring non-professional phagocytes, such as epithelial cells. Compared to professional phagocytosis, the mechanisms that govern non-professional phagocytosis are not well characterized. The Drosophila ovary provides a powerful in vivo model for the study of PCD and engulfment by non-professional phagocytes. This dissertation identifies genetic pathways that govern non-professional phagocytosis during starvation-induced PCD and elucidates the major mechanism promoting non-apoptotic developmental PCD. During mid-oogenesis, germline nurse cells can be induced to die by starvation and their remnants are engulfed by surrounding epithelial follicle cells. We show that the engulfment receptor Draper is enriched and required in engulfing follicle cells. Additionally, we demonstrate that the JNK pathway is activated by Draper and required in engulfing follicle cells. Overexpression of Draper or the JNK pathway is sufficient to induce death of the germline, suggesting that there is coordination between the germline and follicular epithelium to promote cell death. Furthermore, activation of JNK bypasses the need for Draper in engulfment. These data demonstrate that JNK and Draper are crucial regulators of engulfment by non-professional phagocytes. During late oogenesis, nurse cells transfer their contents into the oocyte and undergo developmental PCD. Disruption of apoptosis or autophagy only partially inhibits PCD, indicating that other mechanisms contribute to the process. We demonstrate that the large-scale non-apoptotic developmental PCD in the Drosophila ovary occurs by a novel cell death program where follicle cells non-autonomously promote death of the germline. The phagocytic machinery of follicle cells, including Draper, JNK, and Ced-12, is essential for death and removal of nurse cells. Cell death events including acidification, nuclear envelope permeabilization, and DNA fragmentation are impaired when phagocytosis genes are inhibited. Moreover, elimination of a subset of follicle cells prevents nurse cell death and cytoplasmic dumping. Developmental PCD in the Drosophila ovary is an intriguing example of non-apoptotic, non-autonomous PCD, providing insight on the diversity of cell death mechanisms.

As an organism develops, multiple cellular processes need to occur in order to specify and organize tissue. One essential process is the establishment of cell polarity, which drives cell fate specification and stem cell differentiation. Another key process is programmed cell death, which is important for tissue remodeling and clearing damaged or diseased cells from the body. A loss in cell polarity can lead to defects in tissue organization and carcinogenesis. Defects in programmed cell death can lead to autoimmune diseases and cancer. However, hyperactive programmed cell death can lead to neurodegeneration. The Drosophila ovary, which is composed of germline and somatic cells, is an excellent model to study both cell polarity and cell death. In the germ cells, oocyte fate is specified and maintained through the asymmetric localization of cell cycle and cell polarity RNAs, proteins, and organelles, such as mitochondria, to and within the oocyte. Additionally the somatic follicle cells, which surround the germ cells, require a specific apical-basal polarity to function. During oogenesis, programmed cell death can be induced within the ovary to prevent oogenesis from maturing under low nutrient, high stress or crowded conditions. When this occurs, the germline is cleared from the ovary by a process known as engulfment. Somatic follicle cells surrounding the germline synchronously enlarge and engulf the corpses of the dying germline cells. It is unknown what triggers the enlargement of the follicle cells. Previous research has shown that the apical side of a follicle cell is heavily marked by cell polarity proteins, to specify the apical side away from the lateral and basal sides. Since many important genes regulating both cell polarity and engulfment are conserved between Drosophila and other eukaryotes, we can study the establishment and maintenance of cell polarity and its role during engulfment to obtain a better understanding of these processes in mammals and their relevance to diseases. This dissertation investigates the role of cell polarity in both the specification of oocyte cell fate, and the organization and enlargement of the follicle cells during engulfment in the ovary. / 2016-11-03T00:00:00Z

Some stars with similar properties to our sun (solar twins) have differ- ent chemical composition than the rest of the solar twins. One explanation might be planet engulfment. Therefore we did a large number of simu- lations where a disturbing star passed a sun and a planet at a distance closer than 100 AU to see how often the planet was engulfed. The result was that the planet in most cases was thrown out of the system, but it was engulfed in about 10 − 30% of the simulations when the planet was close to its star. The conclusion was that planet engulfment indeed can be a good explanation for the different chemical compositions of solar twins, at least in dense stellar clusters where such close passages should be quite common. / Vissa solliknande stja ̈rnor (s ̊a kallade soltvillingar) har en annorlunda kemisk sammansa ̈ttning ja ̈mfo ̈rt med resten av soltvillingarna. En mo ̈jlig fo ̈rklaring kan vara att dessa stja ̈rnor har slukat planeter. Da ̈rf ̈or gjorde vi ett stort antal simuleringar d ̈ar en sto ̈rande stja ̈rna passerade ett sys- tem best ̊aende av solen och en planet. Stj ̈arnan passerade p ̊a ett avst ̊and under 100 AU och vi observerade hur ofta planeten slukades. Resultatet var att planeten oftast slungades ut i rymden, men den slukades i 10-30% av simuleringarna da ̈r planeten kretsade na ̈ra sin stja ̈rna. Slutsatsen vara att planetslukning kan vara en rimlig f ̈orklaring till de annorlunda kemiska sammansa ̈ttningarna hos soltvillingar, ̊atminstone i ta ̈ta stja ̈rnhopar da ̈r na ̈ra stja ̈rnpassager bo ̈r vara ganska vanliga.

Microglia, the brain's resident immune cells and phagocytes, are emerging as critical regulators of developing synaptic circuits in the healthy brain after having long been thought to function primarily during central nervous system (CNS) injury or disease. Recent work indicates that microglia engulf synapses in the developing brain; however, how microglia know which synapses to target for removal remains a major open question. For my dissertation research, I studied microglia-mediated pruning in the retinogeniculate system and sought to identify the molecules regulating microglial engulfment of synaptic inputs. I discovered that "eat me" and "don't eat me" signals, immune molecules known for either promoting or inhibiting macrophage phagocytosis of cells or debris, localize to the dorsal lateral geniculate nucleus of the thalamus (dLGN) and direct retinogeniculate refinement. We found that "eat me" signal C3 and its microglial receptor, CR3, are required for normal engulfment, and that loss of either of these molecules leads to a reduction in phagocytosis and sustained deficits in refinement. These data suggest that microglia-mediated pruning may be analogous to the removal of non-self material by phagocytes in the immune system. To test this hypothesis, I examined whether protective signals are required to prevent excess microglial engulfment, as they prevent phagocytosis of self cells in the immune system. I found that protective "don't eat me" signal CD47 is required to prevent excess microglial engulfment and retinogeniculate pruning during development. Moreover, another "don't eat me signal", CD200, also prevents overpruning. Together, these findings indicate that immune molecules instruct microglia as to which synapses to engulf and present a model in which a balance of stimulatory and inhibitory cues is necessary to guide remodeling of immature synaptic circuits. These data shed new light on mechanisms regulating synaptic refinement and microglial function in the healthy, developing CNS, and may have implications for disorders characterized by immune dysregulation and circuit disconnectivity, such as autism spectrum disorder (ASD) and schizophrenia.

Une installation de mélange rapide entièrement automatisée permet d'étudier l'influence des conditions de mélange sur la co-précipitation d'oxydes mixtes de cérium et de zirconium. L'intensité du mélange est contrôlée par le débit d'entrée des solutions réactives. Un modèle d'engouffrement à iso-volume a permis d'estimer le temps de mélange à partir de la mesure d'un indice de ségrégation par le système de Villermaux Dushman pour trois mélangeurs Hartridge Roughton de géométries différentes. Pour une même puissance spécifique dissipée, le mélange est plus intense lorsqu'un rétrécissement est présent. L'intensification du mélange diminue la température maximale de réductibilité et augmente les contraintes du réseau cristallin des oxydes mixtes synthétisés et calcinés à 1100 °C. Cela est interprété par une meilleure homogénéité des particules. L'étude des particules directement en sortie du mélangeur rapide montre que pour les débits étudiés le mélange parfait avant précipitation n'est pas atteint. Une partie des particules se forme en milieu acide, incorporant des nitrates dans leur structure. Une modélisation simple des phénomènes de mélange couplée à une prise en compte des équilibres chimiques confirme ce résultat expérimental. / An automated experimental set-up with rapid mixers is used to study the influence of mixing conditions on the co-precipitation of cerium-zirconium mixed oxides. The intensity of mixing is controlled by the inlet flowrates of the reacting solutions. An engulfment model is used to estimate a mixing time from the measurement of a segregation index by the Villermaux-Dushman reaction system. Three geometries of Hartridge Roughton mixers are compared. Mixing performance is better when a separate mixing chamber upstream of a narrower outlet pipe is present. A better mixing decreases the maximal reducibility temperature of the material and increases the crystal strains of the particles calcined at 1100 °C. This is probably due to a better homogenization of the particles content. The important incorporation of nitrates in the particle at the outlet of the mixers shows precipitation occurs while the mixing process is not finished. This experimental result was confirmed by numerical simulation and an estimation of sursaturations during the mixing process.

Neurons in the olfactory epithelium that are responsible for detecting the odors we smell are constantly dying. However, the olfactory system has the unique ability to regenerate new neurons in order for the sense of smell to be maintained. After a new sensory neuron is born in the olfactory epithelium, it must extend a new axon that will travel to the olfactory bulb and make specific synaptic contact so that the odor information from the epithelium can be coded and sent to the higher cortical areas of the brain. The olfactory system’s ability to recover is also even more complex in that it is capable of regeneration after an injury in which a portion or even the entire olfactory epithelium is removed. A well established model for this type of injury in the olfactory epithelium is by inducing a chemical ablation by injection of the drug methimazole. A specific interest in the regenerative process after injury is the mechanism by which axonal debris from the dead neurons is removed. After ablation of the olfactory epithelium, the cell bodies of the neurons detach but their axons remain intact. The axonal debris must not only be removed, but must also be done so in a way that minimizes inflammation in order for new axons to be able to extend to the olfactory bulb. Axonal debris removal has been characterized both in vitro and during development. However, the mechanism of debris removal has yet to be characterized after an injury. Our lab has studied different engulfment proteins in the olfactory bulb after injury using RT-qPCR and found specific temporal expression profiles at 3, 14 and 21 days post injury. Our initial investigations involved some known engulfment proteins such as Jedi1, GULP, and Megf10. However, we found that these proteins are downregulated after an injury. Further investigation has shown that the proteins Cd11b and TLR2 are upregulated after injury. These changes in expression can begin to shed light on the mechanism of axonal debris removal after an injury and can further be used to study how inflammation is suppressed in order to allow for axon extension and synaptic contact to be reestablished.