1. The role of calmodulin (CaM) in transmitter release was investigated using liposomes to deliver CaM and monoclonal antibodies against CaM (antiCaM) directly into the frog motor nerve terminal. 2. Miniature endplate potentials (MEPPs) were recorded in a high K+ solution, and effects on transmitter release were monitored using estimates of the quantal release parameters m (number of quanta released), n (number of functional transmitter release sites), p (mean probability of release), and vars p (spatial variance in p). 3. Administration of CaM, but not heat-inactivated CaM, encapsulated in liposomes (1000 units ml-1) produced an increase in m (25%) that was due to an increase in n. MEPP amplitude was not altered by CaM. 4. Administration of antiCaM, but not heat-inactivated antiCaM, in liposomes (50 μl ml-1) produced a progressive decrease in m (40%) that was associated with decreases in n and p. MEPP amplitude was decreased (15%) after a 25 min lag time, suggesting a separation in time between the decreases in quantal release and quantal size. 5. Bath application of the membrane-permeable CaM antagonist W7 (28 μM) produced a gradual decrease in m (25%) that was associated with a decrease in n. W7 also produced a decrease in MEPP amplitude that paralleled the decrease in m. The decreases in MEPP size and m produced by W7 were both reversed by addition of CaM. 6. Our results suggest that CaM increases transmitter release by mobilizing synaptic vesicles at the frog motor nerve terminal.

The work presented in this thesis provides insights into the Drosophila smooth septate junction complex Ssk-Mesh that regulates ISC proliferation and tissue homeostasis in addition to the well-known barrier function in the epithelial integrity. With CRISPR-generated tag knockin alleles of Ssk and Mesh, I characterized the intracellular expression pattern of Ssk and Mesh. Ssk and Mesh had low but detectable expression in punctate format in the cytoplasm of enteroblasts (EBs). The protein expression profile of Ssk and Mesh correlated with their ability to regulate the ISC proliferation even though the septate junctions in EBs had not fully formed. Along with further differentiation into mature enterocytes (ECs), Ssk and Mesh gradually localized to the epithelial apical domain, where they coordinated with other junction proteins, such as Tsp2A and Coracle, to form the septate junction. RNAi-conducted genetic assays and mutant clonal analyses by knockout mutant alleles of Ssk and mesh further revealed that Ssk and Mesh restricted the activity of the transcription coactivator Yki, which governs the production of the cytokine Upd3 along the EB-EC differentiation lineage in adult midgut. Loss of Ssk or Mesh activated Yki to elevate the upd3 expression and thereby to induce the robust ISC proliferation non-autonomously. Although the total number of EBs in midgut is much fewer than that of ECs, surprisingly, knockdown Ssk or mesh in EBs resulted in a comparable upd3 upregulation and ISC proliferation as knockdown their expression in ECs. Leaky midgut caused by knockdown of Ssk or mesh in ECs activated the stress-responding mechanisms to repair the damaged intestinal epithelium, and was eventually associated with death of animals. The reduction of Ssk and Mesh in EBs displayed much milder gut leakage and lower lethality further confirmed that Ssk and Mesh in the two distinct cell types had their own roles in governing ISC proliferation.

Emerging evidence in both vertebrates and invertebrates is redefining glia as active and mobile players in synapse formation, maturation and function. However, the molecular mechanisms through which neurons and glia interact with each other to regulate these processes is not well known. My thesis work begins to understand how glia use secreted factors to modulate synaptic function. We use Drosophila melanogaster, a simple and genetically tractable model system, to understand the molecular mechanisms by which glia communicate with neurons at glutamatergic neuromuscular junctions (NMJs). We previously showed that a specific subtype of glia, subperineurial peripheral glia cells (SPGs), establish dynamic transient interactions with synaptic boutons of the NMJ and is required for synaptic growth. I identified a number of potential functional targets of the glial transcription factor, reverse polarity (repo) using ChIP-chip. I found that one novel target of Repo, Wg, is expressed in SPGs and is regulated by repo in vivo. Wnt/Wg signaling plays a pivotal role during synapse development and plasticity, including the coordinated development of the molecular architecture of the synapse. While previous studies demonstrated that Wg is secreted by motor neurons, herein I provide evidence that a significant amount of Wg at the NMJ is additionally provided by glia. I found that Wg derived from SPGs is required for proper GluR distribution and electrophysiological responses at the NMJ. In summary, my results show that Wg expression is regulated by Repo in SPGs and that glial-derived Wg, together with motor neuron-derived Wg, orchestrate different aspects of synapse development. My thesis work identifies synapse stabilization and/or assembly as a new role for SPGs and demonstrates that glial secreted factors such as Wg regulate synaptic function at the Drosophila NMJ.

A key process underlying synapse development and plasticity is stimulus-dependent translation of localized mRNAs. This process entails RNA packaging into translationally silent granules and exporting them over long distances from the nucleus to the synapse. Little is know about (a) where ribonucleoprotein (RNP) complexes are assembled, and if in the nucleus, how do they exit the nucleus; (b) how RNPs are transported to specific synaptic sites. At the Drosophila neuromuscular junction (NMJ), we uncovered a novel RNA export pathway for large RNP (megaRNP) granules assembled in the nucleus, which exit the nucleus by budding through the nuclear envelope. In this process, megaRNPs are enveloped by the inner nuclear membrane (INM), travel through the perinuclear space as membrane-bound granules, and are deenveloped at the outer nuclear membrane. We identified Torsin (an AAA-ATPase that in humans is linked to dystonia), as mediator of INM scission. In torsin mutants, megaRNPs accumulate within the perinuclear space, and the mRNAs fail to localize to postsynaptic sites leading to abnormal NMJ development. We also found that nuclear envelope budding is additionally used for RNP export during Drosophila oogenesis. Our studies also suggested that the nuclear envelope-associated protein, Nesprin1, forms striated F-actin-based filaments or ‘‘railroad tracks,’’ that span from muscle nuclei to postsynaptic sites at the NMJ. Nesprin1 railroad tracks wrap aoround the postsynaptic regions of immature synaptic boutons, and serve to direct RNPs to sites of new synaptic bouton formation. These studies elucidate novel cell biological mechanisms for nuclear RNP export and trafficking during synapse development.

XPA (xeroderma pigmentosum group A) protein is an essential factor for NER (nucleotide excision repair) which is believed to be involved in DNA damage recognition/verification, NER factor recruiting and stabilization of repair intermediates. Past studies on the structure of XPA have focused primarily on XPA interaction with damaged DNA. However, how XPA interacts with other DNA structures remains unknown though recent evidence suggest that these structures could be important for its roles in both NER and non-NER activities. Previously, we reported that XPA recognizes undamaged DNA ds/ssDNA (double-strand/single-strandDNA) junctions with a binding affinity much higher than its ability to bind bulky DNA damage. To understand how this interaction occurs biochemically we implemented a structural determination of the interaction using a MS-based protein footprinting method and limited proteolysis. By monitoring surface accessibility of XPA lysines to NHS-biotin modification in the free protein and the DNA junction-bound complex we show that XPA physically interacts with the DNA junctions via two lysines, K168 and K179, located in the previously known XPA(98-219) DBD (DNA-binding domain). Importantly, we also uncovered new lysine residues, outside of the known DBD, involved in the binding. We found that residues K221, K222, K224 and K236 in the C-terminal domain are involved in DNA binding. Limited proteolysis analysis of XPA-DNA interactions further confirmed this observation. Structural modelling with these data suggests a clamp-like DBD for the XPA binding to ds/ssDNA junctions. Our results provide a novel structure-function view of XPA-DNA junction interactions.

This study integrates detailed mapping of Tertiary deposits along the divide between the lower Bear River basin and the Cache Valley basin with several other techniques to generate a depositional model, define extension-related structures, and compile a geologic history for this part of the northeastern Basin and Range province. The study area is situated along the topographic divide between Box Elder and Cache Counties, Utah, from the Wellsville Mountains north almost to Clarkston Mountain. These ranges are cored by folded and thrusted Paleozoic rocks. They are bound on the west by normal faults of the Wasatch fault zone and on the east by the West Cache fault zone. Between these two fault zones, poorly consolidated Tertiary deposits of the Wasatch Formation and Salt Lake Formation overlie Paleozoic rocks in the foothills and low divide between the north-trending ranges. The Miocene to Pliocene Salt Lake Formation accumulated above non-tuffaceous conglomerates of the Paleocene to Eocene Wasatch Formation, up to 0.5 km thick in the Wellsville Mountains, but thin or absent northward. The Salt Lake Formation in the study area consists of an apparently non-tuffaceous lower conglomerate member, up to 0.5 km thick in the Wellsville Mountains, and a widespread younger tuffaceous and lacustrine member, at least 1 km thick. The traditional names of Collinston Conglomerate and Cache Valley Member were used for these two lithologies. The Cache Valley Member was further subdivided into a local tuffaceous basal conglomerate, a widespread tuffaceous subunit, and an overlying oolitic subunit. Normal faults in the study area comprise three groups. North-striking normal faults are the youngest, and include major range-bounding faults. East-striking normal faults are less numerous, and are cut by the north-striking faults. The southwest-dipping low-to moderate-angle Beaver Dam fault separates the Cache Butte Divide and Junction Hills from the Wellsville Mountains. It may be unique within the area of study, and may comprise a newly identified segment of the Wasatch fault zone. Most of its displacement appears to pre-date the late Miocene, at the time that previous authors have suggested for the onset of Basin-and-Range normal faulting.

La Dysplasie Epithéliale Intestinale (DEI) est une maladie intestinale rare chez l'homme qui a récemment été liée à des mutations des gènes EPCAM et SPINT2. Peu de données sont disponibles pour ces deux gènes, principalement étudiés comme marqueurs du cancer. Le but de ma Thèse était d'identifier les défauts cellulaires induits par l'inactivation de ces gènes et de comprendre les fonctions d’EpCAM et de Spint2dans les entérocytes. Matériel et méthode : Nous avons utilisé comme modèle des cultures cellulaires de cellules Caco2‐BBe stablement inactivées pour EpCAM ou pour Spint2, en utilisant la stratégie shRNA, en comparaison avec les biopsies intestinales de patients DEI, mutés pour l'un ou l'autre gène. J'ai analysé par microscopie électronique,microscopie confocale ou vidéo microscopie, différents marqueurs, et étudié ces cellules sur des modèles 3Dtrès novateurs développés dans notre laboratoire.Résultats: Notre étude a mis en évidence que EpCAM est un acteur clé dans le maintien de l'organisation entérocytaire apico‐basale. Son inactivation conduit à une hyperactivation majeure de l'actomyosine focalisée sur les sommets tricellulaires, entraînant une profonde perturbation de l'organisation cellulaire globale. Les inhibiteurs de la myosine II restaurent l'organisation cellulaire normale. L'étude de Spint2 vient de commencer,mais il semble que l'inactivation de Spint2 conduit à la perturbation de l'adhésion cellulaire en relation avec desdéfauts dans la fin de la division cellulaire à l'étape de la cytocinèse. Cette hypothèse nécessite des analyses complémentaires afin de confirmer ces résultats, les compléter et préciser le mécanisme impliqué.Conclusion: Au cours de ma thèse, j'ai dévoilé de nouvelles fonctions d'EpCAM, et en soupçonne d'autres pour Spint2. Nos résultats devraient aider la recherche de traitements pour ces malades. Les données devraient non seulement servir la cause de la DEI mais également aider les biologistes cellulaires de l'intestin et la recherche sur le cancer. / Congenital Tufting Enteropathy is a rare human intestinal disease that has recently been ascribed tomutations in EPCAM and SPINT2. Few data were available for both, mainly studied as cancer markers. Thepurpose of my thesis was to identify cell defects induced by the inactivation of these genes and to understand EpCAM and Spint2 functions in enterocytes. Materiel & Method : The model used was cell culture of Caco2‐BBe cells stably inactivated for EpCAM or for Spint2 using shRNA strategy, in comparison with intestinal biopsies from patients mutated for either one or theother gene. I analysed by electron microscopy, confocal, or video microscopy different markers. Moreover, Istudied the cells on very novative 3D patterns developped in the laboratory. Results : My studies highlighted that EpCAM is a key player in maintenance of enterocyte apico‐basalorganization. Its inactivation leads to major actomyosin hyperactivation focused in tricellular vertices resulting in tremendous perturbation of the global cell organization. Inhibitors of myosin‐II rescued the normal cellorganization. The Spint2 study has just started, but it seems that Spint2 inactivation leads to cell adhesionperturabtion in relationship with defects in finishing cell division at the cytokinesis step. This hypothesis needs further analyses to be confirmed and to precise the involved mechanism. Conclusion : During my thesis I unraveled new functions of EpCAM, suspected others for Spint2, and have progressed in the seeking of treatments. The data should not only serve the cause of CTE, but also should help members of intestinal biology and cancer research.

The aim of this thesis is the relocation of roads II/431 and III/4317 near Kojátky village. This is due to the unsuitable layout of current routes which were poorly arranged because of too many horizontal and vertical curves with small radius. Another reason was a bad technical state of bridge structures, one of them was in serious disrepair. The project deals with the relocation of current roads to more suitable routes including the option allowing access to the nearby estates and the connection to the existing cart tracks. The project also addresses two bridge structures across the streams.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18483号 / 生博第312号 / 新制||生||41(附属図書館) / 31361 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 上村 匡, 教授 垣塚 彰, 教授 藤田 尚志 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第19312号 / 工博第4109号 / 新制||工||1633(附属図書館) / 32314 / 京都大学大学院工学研究科電子工学専攻 / (主査)教授 木本 恒暢, 教授 引原 隆士, 准教授 船戸 充 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM