京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第17896号 / 農博第2019号 / 新制||農||1017(附属図書館) / 学位論文||H25||N4792(農学部図書室) / 30716 / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 菅原 達也, 教授 左子 芳彦, 教授 澤山 茂樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第18596号 / 工博第3957号 / 新制||工||1608(附属図書館) / 31496 / 京都大学大学院工学研究科化学工学専攻 / (主査)教授 山本 量一, 教授 秋吉 一成, 准教授 谷口 貴志, 教授 大嶋 正裕 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12880号 / 論農博第2807号 / 新制||農||1028(附属図書館) / 学位論文||H26||N4879(農学部図書室) / 31598 / (主査)教授 河田 照雄, 教授 金本 龍平, 教授 谷 史人 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19580号 / 医博第4087号 / 新制||医||1013(附属図書館) / 32616 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妹尾 浩, 教授 木村 剛, 教授 柳田 素子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21372号 / 工博第4531号 / 新制||工||1706(附属図書館) / 京都大学大学院工学研究科合成・生物化学専攻 / (主査)教授 梅田 眞郷, 教授 浜地 格, 教授 秋吉 一成 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

No description available.

Background: Tissue non-specific alkaline phosphatase (TNAP) is an enzyme which functions within the body to catalyze the hydrolysis of pyrophosphate to phosphate, and is a well-known mediator of bone mineralization. It has also been identified as a positive mediator of intracellular lipid accumulation (ICLA) in both murine and human preadipocytes as well as in the hepatocellular cell line HepG2. However, the mechanism through which TNAP functions to control ICLA is not known. Both osteoblasts and adipocytes are both of mesenchymal origin and thus may share conserved mechanisms through which TNAP functions. Within bone, TNAP converts pyrophosphate (which inhibits mineralization) to phosphate. This phosphate is essential to the mineralization process through binding to hydroxyapatite crystals, and it also activates the transcription of genes whose products function in osteoblast differentiation, including NRF2. This thesis therefore aimed to determine the role of both pyrophosphate and TNAP-generated phosphate in ICLA. In addition, it is possible that TNAP may interact with other proteins, as it is known that TNAP is able to dephosphorylate proteins such as tau. This thesis therefore aimed to determine whether TNAP binds to other proteins in the context of ICLA. Lipids are not only stored within hepatocytes and adipocytes, but are also found in cells of the adrenal cortex, and TNAP is known to be expressed within such cells. Therefore, this thesis also aimed to determine whether TNAP is involved in the accumulation of cholesterol esters within lipid droplets in the adrenal cortex. Methods: To determine the effect of high intracellular pyrophosphate levels on ICLA, 3T3-L1 cells (a preadipocyte cell line) were cultured in the presence and absence of probenecid, an inhibitor of the pyrophosphate transporter ANK, and induced to accumulate lipids. Lipid accumulation was monitored through Oil red O staining. The effect of probenecid treatment on TNAP activity and intracellular pyrophosphate levels was also analysed. To determine whether TNAP functions in ICLA by producing phosphate for gene induction, 3T3-L1 cells were stimulated to undergo ICLA in the presence and absence of the TNAP inhibitor levamisole, which in turn blocks ICLA. Levamisole treated cells were also incubated with phosphate to see if this would overcome the inhibitory effect of levamisole on ICLA. The ability of phosphate to induce gene expression of NRF2 was determined through real-time PCR. In addition, an NRF2 expressing plasmid was transfected into cells treated with the TNAP inhibitor levamisole to determine if this would also overcome the block on ICLA caused by TNAP inhibition. In silico analysis identified TRAF2 as a potential binder of TNAP. The expression of TRAF2 during ICLA was determined through real time PCR, and the effect of overexpression of TRAF2 on intracellular lipid accumulation was determined through the transfection of a TRAF2 expressing plasmid in cells induced to undergo ICLA. To determine whether TNAP modulates lipid accumulation in cells of the adrenal cortex, the Y1 murine adrenocortical cell line was cultured in the presence and absence of TNAP inhibitor levamisole, and ICLA measured by Oil Red O staining. The location of TNAP within Y1 cells was identified by histochemical staining. Results: Cells treated with probenecid showed increased pyrophosphate levels (expressed as a % of levels observed at baseline) when compared to untreated controls (155.5 ± 15.1 % vs 51.1 ± 18.9 %; p=0.001) after 24 hours of culture. Increased pyrophosphate levels resulted in ICLA within 3T3-L1 cells surpassing levels seen in untreated controls (507.4 ± 30.4 % vs 337.6 ± 16.17 %; p=0.004). This increase in pyrophosphate was coupled to an increase in TNAP activity within the initial 24 hours (291.5 ± 72.8 % vs baseline of 100%; p=0.038) compared to that seen in control experiments (103.43 ± 24.3 % vs baseline of 100%; p=0.848). Cells treated with levamisole showed minimal ICLA and when exogenous phosphate was added, lipid levels were reconstituted to levels similar to that seen in cells induced to accumulate lipids in the absence of levamisole (284.01 ± 62.52% vs 275.86 ± 35.52%; p= 0.83). In the presence of levamisole plus exogenous phosphate, NRF2 expression was upregulated within 1 hour of treatment to levels greater than that seen in the absence of phosphate but presence of levamisole (216.64 ± 19.24% vs 98.28 ± 3.79%; p=0.004). Expression of NRF2 (through transfection with an NRF2 expression plasmid) in cells deficient in TNAP activity (via levamisole treatment), and induced to accumulate lipids, was not able to completely reconstitute ICLA when compared to cells not treated with levamisole (193.72 ± 16.51 vs 326.46 ± 47.64; p = 0.019), but ICLA was still greater than that observed at baseline. In silico analysis predicted that TNAP would bind to TRAF2, yet neither band shift assays nor immune co-precipitation showed evidence of this. However, TRAF2 mRNA was down regulated within 3T3-L1 cells during adipogenesis, reaching levels of 15.27 ± 10.27% (p= 0.014) of baseline (levels prior to induction of intracellular lipid accumulation) by day 4 of lipid accumulation. Overexpression of TRAF2 during adipogenesis markedly reduced intracellular lipid accumulation (147.88 ± 11.28% vs 326.46 ± 47.64%; p=0.028 (after 8 days of culture)). In Y1 cells TNAP activity is upregulated during ICLA, reaching 233 ± 37.56% (p=0.019 vs. baseline) of baseline levels within the initial 24 hours. Inhibition of TNAP activity through levamisole treatment resulted in a decrease in ICLA when compared to cells not treated with levamisole. Histochemical analysis showed that TNAP activity was localised to the lipid droplet. Discussion and Conclusions: Within 3T3-L1 cells TNAP mediates intracellular lipid accumulation through the generation of phosphate. The phosphate is able to increase the expression of NRF2, however it is likely that NRF2 is not the only gene whose expression is regulated by TNAP-generated phosphate. It was found that TNAP and TRAF2 do not bind to each other in the context of ICLA; however TRAF2 is a negative mediator of ICLA through a TNAP-independent mechanism. Functional TNAP is necessary for the accumulation of cholesterol esters within the Y1 cell line, suggesting that TNAP is essential for lipid accumulation in cell types that store lipids in intracellular membrane-bound droplets in the form of triglycerides or cholesterol esters. / GR2018

The development of cellular life on earth is coupled to the formation of lipid-based biological membranes. Although many tools to analyze their biophysical properties already exist, their variety and number is still relatively small compared to the field of protein studies. One reason for this, is their small size and complex assembly into an asymmetric tightly packed lipid bilayer showing characteristics of a two-dimensional heterogenous fluid. Since membranes are capable to form dynamic, nanoscopic domains, enriched in sphingolipids and cholesterol, their detailed investigation is limited to techniques which access information below the diffraction limit of light. In this work, I aimed to extend, optimize and compare three different labeling approaches for sphingolipids and their subsequent analysis by the single-molecule localization microscopy (SMLM) technique direct stochastic optical reconstruction microscopy (dSTORM). First, I applied classical immunofluorescence by immunoglobulin G (IgG) antibody labeling to detect and quantify sphingolipid nanodomains in the plasma membrane of eukaryotic cells. I was able to identify and characterize ceramide-rich platforms (CRPs) with a size of ~ 75nm on the basal and apical membrane of different cell lines. Next, I used click-chemistry to characterize sphingolipid analogs in living and fixed cells. By using a combination of fluorescence microscopy and anisotropy experiments, I analyzed their accessibility and configuration in the plasma membrane, respectively. Azide-modified, short fatty acid side chains, were accessible to membrane impermeable dyes and localized outside the hydrophobic membrane core. In contrast, azide moieties at the end of longer fatty acid side chains were less accessible and conjugated dyes localized deeper within the plasma membrane. By introducing photo-crosslinkable diazirine groups or chemically addressable amine groups, I developed methods to improve their immobilization required for dSTORM. Finally, I harnessed the specific binding characteristics of non-toxic shiga toxin B subunits (STxBs) and cholera toxin B subunits (CTxBs) to label and quantify glycosphingolipid nanodomains in the context of Neisseria meningitidis infection. Under pyhsiological conditions, these glycosphingolipids were distributed homogenously in the plasma membrane but upon bacterial infection CTxB detectable gangliosides accumulated around invasive Neisseria meningitidis. I was able to highlight the importance of cell cycle dependent glycosphingolipid expression for the invasion process. Blocking membrane accessible sugar headgroups by pretreatment with CTxB significantly reduced the number of invasive bacteria which confirmed the importance of gangliosides for bacterial uptake into cells. Based on my results, it can be concluded that labeling of sphingolipids should be carefully optimized depending on the research question and applied microscopy technique. In particular, I was able to develop new tools and protocols which enable the characterization of sphingolipid nanodomains by dSTORM for all three labeling approaches. / Die Entwicklung von zellulären Lebensformen auf der Erde basiert auf der Entstehung biologischer Lipid-Membranen. Obwohl viele Techniken zur Verfügung stehen, welche es erlauben deren biophysikalische Eigenschaften zu untersuchen, sind die Möglichkeiten, verglichen mit der Analyse von Proteinen, eher eingeschränkt. Ein Grund hierfür, ist die geringe Größe von Lipiden und deren komplexe Zusammenlagerung in eine asymmetrische dicht gepackte Lipiddoppelschicht, welche sich wie eine heterogene zweidimensionale Flüssigkeit verhält. Durch die lokale Anreicherung von Sphingolipiden und Cholesterol sind Membranen in der Lage dynamische, nanoskopische Domänen auszubilden, welche lediglich mit Techniken, welche die optische Auflösungsgrenze umgehen, detailliert untersucht werden können. Ein wesentliches Ziel meiner Arbeit war es, drei Färbeverfahren für Sphingolipide zu vergleichen, erweitern und optimieren, um eine anschliessende Untersuchung mit Hilfe der einzelmolekülsensitiven Technik dSTORM (direct stochastic optical reconstruction microscopy) zu ermöglichen. Zunächst verwendete ich das klassische Färbeverfahren der Immunfluoreszenz, um Sphingolipid-Nanodomänen auf eukaryotischen Zellen mit Hilfe von Farbstoff-gekoppelten Antikörpern zu detektieren und quantifizieren. Dieses Vorgehen ermöglichte es mir, Ceramid-angereicherte Plattformen mit einer Größe von ~ 75nm auf der basalen und apikalen Membran verschiedener Zell-Linien zu identifizieren und charakterisieren. Als nächstes Verfahren verwendete ich die Klick-Chemie, um Sphingolipid-Analoge in lebenden und fixierten Zellen zu untersuchen. Eine Kombination aus Fluoreszenz-Mikroskopie und Anisotropie-Messungen erlaubte es mir Rückschlüsse über deren Zugänglichkeit und Konfiguration innerhalb der Plasmamembran zu ziehen. Hierbei lokalisierten Azid-Gruppen am Ende kurzkettiger Fettsäurereste außerhalb des hydrophoben Membrankerns, wodurch sie mittels membran-undurchlässige Farbstoffe angeklickt werden konnten. Im Gegensatz dazu, waren Azide an längeren Fettsäureresten weniger zugänglich und konjugierte Farbstoffe tauchten tiefer in die Plasmamembran ein. Durch die Einführung photoreaktiver Diazirin-Gruppen oder chemisch modifzierbarer Amin-Gruppen wurden Wege geschaffen, welche eine Immobilisierung und anschließende Analyse mit Hilfe von dSTORM ermöglichen. Schließlich nutzte ich das spezifische Bindeverhalten der nicht toxischen B Untereinheiten von Shiga- (STxB) und Cholera-Toxin (CTxB) aus, um Glycosphingolipid Nanodomänen im Kontext einer Neisseria meningitidis Infektion zu untersuchen. Unter physiologischen Bedingungen waren diese homogen in der Plasmamembran verteilt, jedoch reicherten sich CTxB-detektierbare Ganglioside um eindringende Bakterien an. Darüber hinaus konnte ich einen Zusammenhang zwischen der zellzyklusabhängigen Expression von Glycosphingolipiden und dem Eindringen der Bakterien herstellen. Eine Absättigung der Zucker an der äußeren Membran durch CTxB-Vorbehandlung reduzierte die Anzahl von invasiven Bakterien signifikant und bestätigte die Schlüsselrolle von Gangliosiden bei der Aufnahme von Bakterien. Meine Ergebnisse legen Nahe, dass das Färbeverfahren für Sphingolipide an die jeweilige Fragestellung und Mikroskopietechnik angepasst werden sollte. Im Rahmen dieser Arbeit konnten neue Werkzeuge und Protokolle geschaffen werden, die die Charakterisierung von Sphingolipid-Nanodomänen mittels dSTORM für alle drei Färbeverfahren ermöglichen.

In the 19th century, halothane hepatitis became a sensitive and well-known subject in human anesthesiology due to the production of a noxious metabolite further discovered, trifluoroacetic acid. Subsequently, isoflurane, enflurane, and desflurane were also investigated for potentially causing hepatitis through the same metabolite. Sevoflurane, however, does not generate trifluoroacetic acid and is quickly conjugated and excreted. For more than four decades these anesthetics have been experimentally developed for intravenous injection by having added either a lipid or fluorocarbon-based carrier to produce general anesthesia with less drug and faster onset of action. The use of intravenous emulsified halogenated anesthetics as an alternative to inhalation brought contradictory findings, therefore they are still not utilized in the clinical settings of veterinary and human anesthesia. The high solubility of these anesthetic emulsions increases their tissue uptake, volume of distribution, and potency. By this means, the amount of anesthetic necessary to establish general anesthesia could be significantly reduced but would still carry the risk of causing hepatic toxicity. On the other hand, because the emulsified anesthetics have a higher tissue uptake and are liposoluble, they remain for longer periods in the cellular membrane providing cellular pre- and postconditioning effects by minimizing cellular deleterious responses to a critical environment. Emulsified isoflurane and sevoflurane are the most investigated anesthetics for this purposein the heart, brain, kidneys, liver, and central nervous system of laboratory animals and human volunteers. The focus of this study is to evaluate the cellular effects of the loaded-lipid emulsified isoflurane and sevoflurane at different concentrations on cultured primary canine hepatocytes considering their viability and apoptosis response. Specifically, the overall objective is to establish a basis for in vitro metabolism of these emulsified anesthetics on canine hepatocytes under normal oxygen tension and on canine hepatocytes exposed to extreme hypoxia (1% O2). Thus, this study is sectioned into three major chapters followed by conclusions and future studies to determine the safety and indication of these anesthetic formulations in canine hepatocytes to be further explored in the clinical setting with live animals.

Obesity is linked to nonalcoholic steatohepatitis. Peroxisome proliferator-activated receptor-a (PPARa) regulates lipid metabolism. Cytochrome P-450 2A5 (CYP2A5) is a potential antioxidant and CYP2A5 induction by ethanol is CYP2E1 dependent. High-fat diet (HFD)-induced obesity and steatosis are more severe in CYP2A5 knockout (cyp2a5 -/- ) mice than in wild-type mice although PPARa is elevated in cyp2a5 -/- mice. To examine why the upregulated PPARa failed to prevent the enhanced steatosis in cyp2a5 -/- mice, we abrogate the upregulated PPARa in cyp2a5 -/- mice by cross-breeding cyp2a5 -/- mice with PPARa knockout (ppara-/- ) mice to create ppara-/- /cyp2a5 -/- mice. The ppara-/- /cyp2a5 -/- mice, ppara-/- mice, and cyp2a5 -/- mice were fed HFD to induce steatosis. After HFD feeding, more severe steatosis was developed in ppara-/- /cyp2a5 -/- mice than in ppara-/- mice and cyp2a5 -/- mice. The ppara-/- /cyp2a5 -/- mice and ppara-/- mice exhibited comparable and impaired lipid metabolism. Elevated serum alanine transaminase and liver interleukin-1β, liver inflammatory cell infiltration, and foci of hepatocellular ballooning were observed in ppara-/- /cyp2a5 -/- mice but not in ppara-/- mice and cyp2a5 -/- mice. In ppara-/- /cyp2a5 -/- mice, although redox-sensitive transcription factor nuclear factor erythroid 2-related factor 2 and its target antioxidant genes were upregulated as a compensation, thioredoxin was suppressed, and phosphorylation of JNK and formation of nitrotyrosine adduct were increased. Liver glutathione was decreased, and lipid peroxidation was increased. Interestingly, inflammation and fibrosis were all observed within the clusters of lipid droplets, and these lipid droplet clusters were all located inside the area with CYP2E1-positive staining. These results suggest that HFD-induced fibrosis in ppara-/- /cyp2a5 -/- mice is associated with steatosis, and CYP2A5 interacts with PPARa to participate in regulating steatohepatitis-associated fibrosis.