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Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 134-148).

Thesis (Ph. D.)--University of Washington, 1998. / Vita. Includes bibliographical references (leaves [132]-134).

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite and an important signaling molecule that plays a significant role in fibrosis. S1P synthesis is catalyzed by sphingosine kinases (SphKs), which phosphorylate sphingosine into S1P. The present study tested the hypothesis that SphK1-S1P signaling pathway participates in the kidney damage in unilateral ureteral obstruction (UUO) model. Wild type and SphK1 knockout mice were subjected to UUO for 7 days or 14 days and then four groups of kidneys were collected: wild type control group (WT-C), wild type UUO group (WT-UUO), SphK1-/- control group (KO-C) and SphK1-/- UUO group (KO-UUO). The mRNA level of SphK1 in WT-UUO was increased by 6.1 folds compared to WT-C. The fibrotic markers α-smooth muscle actin (α-SMA) and collagen I were both upregulated in UUO groups, whereas the levels of these two markers were significant lower in KO-UUO than that in WT-UUO. The immunohistochemistry analyses showed that the distribution of α-SMA and collagen was located in the interstitial space and that the infiltration of immune cells was more in UUO groups than that in control groups, but there was no significant difference between KO-UUO and WT-UUO, suggesting a direct effect of SphK1 deletion on renal fibrotic markers independent of immune regulation. Further, the morphological examination showed that UUO-induced tubular injury and glomerular damage were significantly reduced in KO-UUO compared with WT-UUO. Our study suggests that SphK1-S1P signaling pathway mediates kidney damage in UUO mice. Manipulating SphK1-S1P signaling pathway may be used as a therapeutic strategy in renal interstitial fibrosis.

Intracellular recordings were made from isolated frog sciatic-sartorius nerve-muscle preparations, and the effects of sphingosine 1-phosphate (S1-P) on miniature endplate potentials (MEPPs) were studied. Extracellular application of S1-P (1 and 30 μM) had no significant effects on the frequency and amplitude of MEPPs. Delivery into nerve terminals by liposomes containing 10-5, 10-4 or 10-3 M S1-P was associated with a concentration-dependent increase in MEPP frequency of 37, 63 and 86%. The per cent of median MEPP amplitude was not significantly changed, but there was an increase in the number of 'giant' MEPPs. Pre-exposure of the preparations to S1-P 10-5 but not 10-8 M entrapped in liposomes for 15 min blocked the effects of subsequent superfusion of S1-P (10-4 M)-filled liposomes on MEPP frequency. Thus, intracellular S1-P receptors seem to undergo 'desensitization' to higher concentrations of S1-P. The result provides the first evidence that S1-P acting intracellularly but not extracellularly enhances spontaneous transmitter release at the frog neuromuscular junction.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20616号 / 医博第4265号 / 新制||医||1023(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 長船 健二, 教授 渡邊 直樹, 教授 岩田 想 / 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・論文博士 / 博士(医学) / 乙第13167号 / 論医博第2154号 / 新制||医||1029(附属図書館) / (主査)教授 宮本 享, 教授 小泉 昭夫, 教授 柳田 素子 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

Over the last decade, a significant amount of research has been done on sphingosine-1-phosphate (S1P). S1P regulates survival, proliferation and migration of breast cancer cells45, as well as playing an important but still unknown role in ovarian46, prostate, and glioma47 cell carcinomas. S1P plays a significant role in the regulation, proliferation and angiogenesis, and is therefore an area of considerable interest for cancer research. Previous research in our group has shown that two stilbenoid sphingosine analogs (1 & 2) were effective at sphingosine kinase (SphK) inhibition in addition to showing down-regulation of Vascular Endothelial Growth Factor (VEGF), a novel and distinctly different mechanism of action compared to reported sphingosine kinase inhibitors. Both compounds showed novel toxicity toward the parasite Toxoplasma gondii. T. gondii causes toxoplasmosis; a disease carried by up to 1/3rd of the world’s population and one that has been implicated in schizophrenia. Unfortunately, use of 1 & 2 as fluorescent probes is limited; their emission wavelengths fall between 360 and 380 nm, within the range of background fluorescence. The synthesis of five fluorescent sphingosine analogs is described herein, three of which have emission wavelengths above 420 nm. These compounds show single digit µM cytotoxicity towards T. gondii. The second generation synthesis of Fingolimod (FTY720) is also described. The process was optimized for large-scale production. Improvements to the synthesis included increased atom economy, purification and overall reaction efficiency. Yield was increased to 36%, from 33% in the first generation synthesis. Lastly, a synthesis of p-divinylbenzene is described, using “green” solvents and easy sample work-up. The product contains no meta impurities, an issue that arises when p-divinylbenzene is purchased commercially. / Thesis / Candidate in Philosophy

京都大学 / 新制・論文博士 / 博士(医学) / 乙第13535号 / 論医博第2275号 / 新制||医||1065(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 伊達 洋至, 教授 湊谷 謙司, 教授 小林 恭 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM

The respiratory system is amongst the most important compartments in the human body. Due to its connection to the external environment, it is one of the most common portals of pathogen entry. Airborne pathogens like measles virus (MV) carried in liquid droplets exhaled from the infected individuals via a cough or sneeze enter the body from the upper respiratory tract and travel down to the lower respiratory tract and reach the alveoli. There, pathogens are captured by the resident dendritic cells (DCs) or macrophages and brought to the lymph node where immune responses or, as in case of MV, dissemination via the hematopoietic cell compartment are initiated. Basic mechanisms governing MV exit from the respiratory tract, especially virus transmission from infected immune cells to the epithelial cells have not been fully addressed before. Considering the importance of these factors in the viral spread, a complex close-to-in-vivo 3D human respiratory tract model was generated. This model was established using de-cellularized porcine intestine tissue as a biological scaffold and H358 cells as targets for infection. The scaffold was embedded with fibroblast cells, and later on, an endothelial cell layer seeded at the basolateral side. This provided an environment resembling the respiratory tract where MV infected DCs had to transmigrate through the collagen scaffold and transmit the virus to epithelial cells in a Nectin-4 dependent manner. For viral transmission, the access of infected DCs to the recipient epithelial cells is an essential prerequisite and therefore, this important factor which is reflected by cell migration was analyzed in this 3D system. The enhanced motility of specifically MV-infected DCs in the 3D models was observed, which occurred independently of factors released from the other cell types in the models. Enhanced motility of infected DCs in 3D collagen matrices suggested infection-induced cytoskeletal remodeling, as also verified by detection of cytoskeletal polarization, uropod formation. This enforced migration was sensitive to ROCK inhibition revealing that MV infection induces an amoeboid migration mode in DCs. In support of this, the formation of podosome structures and filopodia, as well as their activity, were reduced in infected DCs and retained in their uninfected siblings. Differential migration modes of uninfected and infected DCs did not cause differential maturation, which was found to be identical for both populations. As an underlying mechanism driving this enforced migration, the role of sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) was studied in MV-exposed cultures. It was shown in this thesis that MV-infection increased S1P production, and this was identified as a contributing factor as inhibition sphingosine kinase activity abolished enforced migration of MV-infected DCs. These findings revealed that MV infection induces a fast push-and-squeeze amoeboid mode of migration, which is supported by SphK/S1P axis. However, this push-and-squeeze amoeboid migration mode did not prevent the transendothelial migration of MV-infected DCs. Altogether, this 3D system has been proven to be a suitable model to study specific parameters of mechanisms involved in infections in an in vivo-like conditions. / Die respiratorische System ist ein wesentlicher physiologischer Bestandteil. Durch die direkte und konstante Verbindung der Atemwege mit der äußeren Umgebung sind sie einer der häufigsten Pfade für den Eintritt von Krankheitserregern in den Körper. Luftübertragene Krankheitserreger wie das Masern-Virus (MV), das in Flüssigkeitströpfchen mitgeführt und von Patienten durch Husten oder Niesen ausgeatmet wird, können über die oberen Atemwege in den Körper gelangen und sich bis in die unteren Atemwege und bis zu den Alveolen ausbreiten. Dort werden diese Krankheitserreger von den dort residenten dendritischen Zellen (DC) oder Makrophagen erworben und zu sekundären lymphatischen Organen transportiert, in denen sowohl virus-spezifische Immunantworten, aber auch – wie im Falle von MV – die hämatogene Dissemination initiiert wird. Der Austrittsmechanismus des MV aus den Atemwegen, insbesondere dessen Übertragung von infizierten Immunzellen auf die Epithelzellen und die Faktoren, die diesen Ablauf bestimmen, wurden jedoch bisher unzureichend untersucht. In Anbetracht der Bedeutung dieser Faktoren für die Virusausbreitung wurde ein komplexes, realitätsnahes in-vivo 3D-Modell der menschlichen Atemwege erstellt. Dieses Modell wurde unter Verwendung von de-zellularisiertem Schweinedarmgewebe als biologischem Gerüst und H358 Epithelzellen als Empfänger etabliert. Dieses Grundgerüst wurde mit Fibroblastenzellen eingebettet. Später wurde auf der basolateralen Seite der Modelle eine Endothelzellschicht eingebracht, um eine Umgebung zu schaffen, die der der Atemwege ähnelt. Somit mussten die Virus-Donoren, MV-infizierte DC durch das Kollagengerüst wandern und das Virus auf Epithelzellen in einer Nektin-4 abhängigen Weise übertragen. Für die Virusübertragung ist der Zugang infizierter DC zu den Empfänger-Epithelzellen eine wesentliche Voraussetzung, weshalb dieser wichtige Faktor, der sich in der Zellmigration widerspiegelt, in diesem 3D-System analysiert wurde. Eine erhöhte Beweglichkeit spezifisch MV-infizierter DCs wurde in den 3D-Modellen beobachtet. Dies erwies sich als unabhängig von löslichen Faktoren der anderen Zelltypen in den Modellen. Erhöhte Beweglichkeit infizierten DCs wurde auch in 3D-Kollagenmatrizes gesehen, was auf einen infektionsvermittelten zytoskelettalen Umbau hindeutete, der auch anhand von Zytoskelettpolarisation und Uropodbildung bestätigt wurde. Die MV-Infektion induzierte einen schnellen amöboiden Migrationsmodus in den DCs, der sich als sensitiv gegenüber ROCK-Hemmung erwies. Im Gegensatz zu uninfizierten DCs gleichen Reifungsstadiums waren in infizierten DCs Podosomenstrukturen und Filopodien sowie deren Aktivität stark reduziert. Als potentiell zur verstärkten Motilität infizierter DCs beitragender Faktor wurde die Rolle der Sphingosinkinase (SphK) und des Sphingosin-1-phosphats (S1P) in MV-exponierten Kulturen untersucht. In dieser Arbeit wurde gezeigt, dass die S1P-Produktion durch eine MV-Infektion erhöht wurde, und in der Tat zur für infizierte DCs beobachteten erhöhten Geschwindigkeit beitrug, da diese sensitiv gegenüber Hemmung der Sphingosinkinase-Aktivität war. Diese Ergebnisse zeigen, dass die MV-Infektion einen schnellen amöboid-artigen Migrationsmodus induziert, der von der SphK/S1P-Achse unterstützt wird. Dieser Push-and-Squeeze-Amoeboid-Migrationsmodus verhinderte jedoch nicht die transendotheliale Migration von MV-infizierten DCs. Insgesamt hat sich dieses 3D-System als geeignetes Modell erwiesen, um die spezifische Parameter von Mechanismen von Infektionen in einem in-vivo-ähnlichen Zustand zu untersuchen.