京都大学 / 新制・課程博士 / 博士(医学) / 甲第23410号 / 医博第4755号 / 新制||医||1052(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 村川 泰裕, 教授 長尾 美紀, 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

Cryptococcus laurentii and Cryptococcus flavescens are nonfermenting yeasts forming extracellular polysaccharide capsule. Both species are mainly saprofytic but Cr. laurentii is also known to be an opportunistic pathogen in immunocompromised patients. Cr. flavescens used to be considered a synonym of Cr. laurentii but nowadays it is classified as a separate species that belongs to the phylogenetic group I of the Cr. laurentii group. In the experimental part 28 strains of species Cr. laurentii, Cr. flavescens a Cr. victoriae were biotyped using MALDI-TOF MS. The yeasts were cultivated on three different media (Sabouraud, YPD and potato agar) and three methods were used for the protein extraction. The impact of growth medium composition from which the strains were inoculated on the quality of spectra was studied together with the suitability of individual methods for use on different media. Then the impact of growth medium composition on the quality of acquired spectra was evaluated. Finally, all strains were compared mutually and with the type strain of Cr. laurentii CCY 17 3-2. The composition of the medium cells were inoculated from was found to have little impact on the spectra quality. The same result was determined for the composition of the actual growth medium cells were cultivated on. Crucial for the quality of mass spectrum is the method of cells preparation. Best results were acquired when cultivating cells on YPD agar, washing the cells with ethanol and using mix of sinapinic and ferulic acid as a matrix. Potato agar was found not suitable for cultivating yeasts of the Cryptococcus genus due to significant production of extracellular polysaccharides which complicate the protein isolation process. All strains were compared to Cr. laurentii type strain CCY 17-3-2 and MSP dendrograms were created based on the spectra similarity. In the MSP dendrograms all strains were successfully divided into relevant species on all tested media. Finally sequences of D1/D2 domain of LSU gene were compared and phylogenetic tree was created. This tree was then compared to the MSP dendrograms.

Hepcidin is cysteine-rich cationic peptide produced by hepatocytes, secreted into blood plasma, and excreted in urine. Hepcidin is proposed to be the key regulator of iron metabolism and an evaluation of changes in the hepcidin level is important for diagnosis of several diseases. However, methods used for the hepcidin detection and determination in urine and serum have certain limitations. At present time MALDI-TOF MS based approaches have been applied for final analysis of urinary and/or serum hepcidin levels. Before MS analysis, separation of hepcidin from analyzed samples is an important and necessary step. The aim of this study was to compare the ability of several magnetic sorbents with different coating matrix and/or different terminal functionalized groups to adsorb hepcidin prior MS analysis. Either commercial magnetic sorbents containing -COOH groups or magnetic hydrophilic IDA-modified polymethacrylate microparticles P(HEMA-co-GMA)-IDA with immobilized metal ions were use for this purpose. Hepcidin was adsorbed to magnetic sorbents containing linked carboxyl groups (i.e. to weak cation exchange magnetic particles) at pH 6.8 independently on a nature of magnetic particle coating layer. Magnetic particles P(HEMA-co- GMA)-IDA with immobilized Cu(II) ions were found to adsorb hepcidin in a...

The system of mixed function oxygenase (MFO system) participates in significant roles in the metabolism of endogenous compounds and xenobiotics. This system contains cytochrome P450, NADPH:cytochrome P450 reductase, and also there are assigned NADH: cytochrome b5 reductase and cytochrome b5. It was proved that cytochrome b5 can stimulate or inhibit cytochrome P450 (CYP)-dependent reactions and even change the ratio of resulting metabolites. The mechanism of cytochrome b5 action has not been fully elucidated yet. Elucidation of protein-protein interactions in MFO system and determination of topology of this system could explain the mechanism of cyt b5 action. The covalent cross- linking technique is suitable method for identifying protein-protein interactions within the membrane. Cytochrome b5 contains 3 methionines and in 2 cases the methionines are localized in a short hydrophobic C-terminal membrane anchor. Interactions with cytochrome P450 in the membrane environment can be identified by substitution of two methionine for photoactivatable analogue of methionine (photo-methionine) and subsequent photoactivation. This work is focused on expression and isolation of photo-cytochrome b5 (photo- cyt b5), cytochrome b5 analogue with incorporated photo-methionine. Conditions for photo-methionine...

Among the heavy metal-binding ligands in cells the phytochelatins (PCs) and metallothioneins (MTs) are the best characterized. PCs and MTs are different classes of cysteine-rich, heavy metal-binding molecules. PCs are enzymatically synthesized peptides, whereas MTs are gene-encoded polypeptides. Several analytical methods have been used to analyze PCs, MTs and complexes of metal in different studies. Today, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDITOF MS) is renowned for its extreme ease of operation and requirement of inexpensive matrices for preparation of sample; more importantly, the instrumentation is capable to be fully automated. In this study, the optimal condition for the characterization of PC2 and Cd-PC2 complex by MALDI-TOF MS was determined. The human MT2A and MT3 genes were expressed in heterologous organisms (Escherichia coli) by cloning. Further, MT2 protein was isolated from rabbit liver. MTs were purified by fast protein liquid chromatography (FPLC). The present study demonstrates analytical approaches of employing MALDI-TOF MS for characterization MTs. Other electrochemical methods were used for confirmation of thiols complexes with metal. Moreover, easy-touse instruments for MTs isolation, coupled with simple analytical detection method in fully automatic mode, providing prototype for the construction of sensor was designed

L’identification de biomarqueurs demeure un véritable défi pour les sciences analytiques et un enjeu majeur pour la recherche clinique. Les glycosaminoglycanes (GAGs) ont été identifiés comme biomarqueurs potentiels de mucopolysaccharidoses (MPS), maladies génétiques rares et très souvent mortelles. Ces pathologies sont dues à une déficience en une des enzymes impliquées dans le catabolisme des GAGs. Le défaut enzymatique conduit à une accumulation de GAGs partiellement dégradés, et entraîne une neurodégénérescence pour les formes sévères de la pathologie. Les GAGs sont des polysaccharides polyanioniques complexes impliqués dans de nombreux processus physiologiques chez les mammifères. Leur étude demeure difficile en raison de leur hétérogénéité structurale, de leur faible biodisponibilité et du manque d'outils dédiés à leur analyse. Notre objectif a été de détecter et de quantifier ces composés à partir de fluides biologiques tels que l’urine et le liquide céphalo-rachidien, puis d’en élucider la structure par spectrométrie de masse. L’étude s’est focalisée sur la caractérisation d’oligosaccharides de type héparane sulfate (HS), biomarqueurs spécifiques de MPS à composante neurologique (MPS de type I, IIIB et IIIC) et responsables des atteintes du système nerveux central. Une stratégie expérimentale permettant l’extraction d’oligosaccharides de HS issus de fluides biologiques a été développée. Ainsi, la structure d’oligosaccharides sulfatés de HS urinaires, candidats biomarqueurs de MPS IIIB et IIIC, a pu être identifiée. Ces composés pourraient s’avérer utiles pour le diagnostic et le suivi de patients, notamment lors d’essais thérapeutiques. Des expériences in vitro d’exposition de différents types cellulaires du cerveau ont été menées afin d’établir la relation entre la structure des oligosaccharides accumulés et leurs effets neuropathologiques. Elles ont permis de mettre en évidence des processus cellulaires qui pourraient impliqués dans la neurodégénérescence et constituer de nouvelles cibles thérapeutiques. / The identification of biomarkers remains one of the main challenges for analytical sciences and a major stake for clinical research. Glycosaminoglycans (GAGs) have been identified as potential biomarkers of mucopolysaccharidoses (MPS) belonging to rare genetic diseases with often a deadly issue. These pathologies are due to a deficiency in one of the enzymes responsible for GAGs catabolism. This enzymatic defect results in the accumulation of partially catabolized GAGs in organism and leads to neurodegeneration for the most severe forms of the disease. GAGs are complex polyanionic polysaccharides involved in numerous physiological processes in mammals. Their study remains a challenging task because of their high structural heterogeneity and their low biodisponibility, besides the lack of dedicated analytical tools. Our aim was to detect and quantify these compounds in biologic fluids such as urine and cerebrospinal fluid, and to elucidate their structures by mass spectrometry. This study focused on heparan sulfate (HS) oligosaccharides, as potential biomarkers of MPS featured by neurological manifestations (MPS I, IIIB and IIIC), and possibly responsible of lesions in the central nervous system. An experimental strategy allowing the extraction of HS oligosaccharides from biological fluids was implemented, thereby the structures of urinary heparan sulfate oligosaccharides were deciphered, leading to possible biomarkers candidates of MPS IIIB and IIIC. These compounds could be useful for diagnostic and patient follow-up that are currently lacking for the monitoring of therapeutic assays. In vitro exposition of different cerebral cell types to HS oligosaccharides was carried out to establish the relation between the structure of oligosaccharides and neuropathological effects. These studies highlighted several cellular processes that could be involved in neurodegeneration and constitute new therapeutic targets.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第19576号 / 医博第4083号 / 新制||医||1013(附属図書館) / 32612 / 京都大学大学院医学研究科医学専攻 / (主査)教授 中川 一路, 教授 岩田 想, 教授 西渕 光昭 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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In this project, separation of trypsin digested transferrin (Tf) has been studied, using a RP HPLC- UV system equipped with a C18 column. 0.1% TFA/MQ-water and 90% MeOH were used as mobile phase A and mobile phase B, respectively. For economic reasons, the protein cytochrome c (cyt-C) was used to optimize the digestion procedure and LC system, before analysis of Tf. Four digestion methods were applied for analyzing cyt-C and Tf. The first method was digestion with no denaturing, reducing or alkylating agent. The other digestion methods used urea or heating as a denaturing agent, and lastly dithiothreitol (DTT) and iodoacetamide (IAA) as reducing and alkylating agent, respectively. The results from HPLC-UV showed that a gradient elution with a high concentration of organic solvent is favorable for the separation of cyt-C peptides. MALDI-MS was used to identify peptides, and the outcomes showed that denaturation by heat before digestion gave the best results. / I detta projekt har separation av trypsin-klyvt transferrin (Tf) studerats, med användning av ett RP HPLC-UV system, som bestod av en C18 kolonn. 0,1% TFA/MQ-vatten och 90% MeOH användes som mobilfas A respektive mobilfas B. Av ekonomiska skäl användes proteinet cytokrom c (cyt-C) före analys av Tf för att optimera klyvningsprocessen och LC systemet. Fyra klyvningsmetoder studerades för analysering av cyt-C och Tf. Den första metoden innehöll inget denaturerande, reducerande eller alkylerande medel. De andra klyvningsmetoderna innehöll urea eller värme som denaturerande medel, och slutligen ditiotreitol (DTT) och jodacetamid (IAA) som reducerande respektive alkylerande medel. Resultaten från HPLC-UV visade att en gradienteluering med en hög koncentration av den organiska lösningen är gynnsam för separationen av peptiderna från cyt-C. MALDI-MS användes för att identifiera peptiderna, och resultaten visade att denaturering med värme före klyvning gav bäst resultat.

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