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...

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

No description available.

Les arthropodes vecteurs sont hématophages et peuvent assurer la transmission biologique active d'un agent pathogène responsable de maladies humaines ou animales. La lutte anti-vectorielle et la surveillance épidémiologique des vecteurs sont essentielles dans la stratégie de lutte contre les maladies vectorielles. Disposer d'outils d'identification précis, fiable et rapides des vecteurs et des pathogènes associés est indispensable. Ainsi dans ce projet nous avons évalué l'utilisation du MALDI-TOF MS pour identifier les arthropodes vecteurs ainsi que la détection des pathogènes associés. La première partie de notre travail consistait à utiliser MALDI TOF pour identifier les tiques, moustiques et les puces. Nous avons déterminé quelle partie du spécimen permettait d'obtenir une reproductibilité des spectres et une identification correcte par des tests à l'aveugle après création d'une base de données de référence. La deuxième partie consistait à utiliser le MALDI-TOF MS pour détecter des Rcikettsies associés aux tiques dont Rickettsia conorii et R. slovaca, deux pathogènes humains transmis respectivement par Rhipicephalus sanguineus et Dermacentor marginatus. Des variations spectrales étaient obtenues entre les spécimens infectés et non infectés, avec des masses spécifiques liés à l'infection des tiques par les rickettsies. La technique d'identification était validée par des tests à l'aveugle. Les résultats obtenus permettent de conclure que le MALDI TOF pourra être utilisé dans l'avenir pour identifier les tiques prélevées chez des patients, les arthropodes vecteurs lors des enquêtes entomologiques et préciser la prévalence d'infection de ces arthropodes. / Arthropods are vectors bloodsucking and can ensure the active biological transmission of a pathogen responsible of human or veterinary diseases. The vector control and vectors epidemiological surveillance are essential in the strategy against the vectors-borne diseases. Accurate, reliable and rapid identification of vectors and associated pathogens are essential. Thus, in this project we evaluated the use of MALDI-TOF MS for the arthropods vectors identification as well as for the detection of associated pathogens. This proteomics technology emerged since few years ago and is currently used in routine for bacteria identification in many microbiology laboratories. In the first part of our work, we used the MALDI TOF to identify the tick, mosquito and flea species. For each arthropod, we determined which part allowed obtaining reproducible spectra by MALDI TOF and correct identification by blind test, after reference database creation. The second part consisted to use the MALDI-TOF MS to detect the associated Rickettsia in ticks including Rickettsia conorii and R. slovaca, two human pathogens transmitted by Rhipicephalus sanguineus and respectively Dermacentors marginatus. The spectral variations were obtained between infected and non infected specimens with specific masses related to the tick infection by Rickettsia. The identification technique of not or infected ticks was validated by blind tests. The obtained results allowed concluding that the MALDI-TOF MS could be used in the future to identify the ticks removed from patient, the arthropods vectors and during entomological survey and determine the prevalence of infection of these arthropods.

A prolactina humana (hPRL) é um hormônio sintetizado pela hipófise com inúmeras funções tais como: lactação, reprodução e regulação osmótica. Este hormônio é frequentemente dosado em casos de problemas na lactação, infertilidade, além de estudos que elucidam sua ligação em alguns tipos de câncer (mama, próstata e útero). A hPRL é encontrada na forma não glicosilada (NG-hPRL) (23 kDa) e glicosilada (G-hPRL) (25 kDa), sendo a isoforma glicosilada um modelo ideal de análise de perfil de N-glicanos, já que possui um único sítio de glicosilação localizado na Asparagina 31. A glicosilação está relacionada diretamente à solubilidade, à estabilidade, ao enovelamento, à meia-vida e atividade biológica in vivo. As células de ovário de hamster chinês (CHO) e as células embrionárias de rim humano (HEK293) são os hospedeiros mais utilizados para expressão de proteínas recombinantes, já que podem ser cultivadas em altas densidades e por possuírem similaridade nas modificações pós-traducionais. O objetivo foi expressar, purificar e realizar uma caracterização físico-química e biológica da hPRL glicosilada de células HEK293, incluindo análise da estrutura de carboidratos. Para tanto, foi realizada uma transfecção em células HEK293T (aderidas) com o vetor pcDNA 3.4-TOPO. Foi obtida uma expressão de 21,26 ± 8,3 μg/mL de hPRL no meio condicionado sem soro. A hPRL foi purificada por cromatografia de afinidade a metais imobilizados (IMAC), eluindo 92% da hPRL em uma única fração que, analisada por HPSEC, apresentou pureza de 97%. O perfil de N-glicanos da amostra apresentou seis espécies, todas com terminação em ácido-siálico, do tipo complexo, sendo bi, tri e tetra-antenárias, com relativa predominância da espécie N2G2S1 (29,4%). A bioatividade in vitro da G-hPRL HEK293 demonstrou ser ≅ 16 vezes menor que a G-hPRL produzida em células CHO. / Human prolactin (hPRL) is a hormone synthesized by the pituitary gland with innumerable functions such as lactation, reproduction and osmotic regulation. This hormone is often determined in cases of lactation problems, infertility, and studies that elucidate its connection in some types of cancer (breast, prostate and uterus). The hPRL is found in the non-glycosylated (NG-hPRL) (23 kDa) and glycosylated (G-hPRL) (25 kDa) form, being the glycosylated isoform an ideal model for N-glycan profile analysis, since it has a single glycosylation site located in Asparagine 31. Glycosylation is directly related to solubility, stability, folding, half-life and biological activity in vivo. Chinese hamster ovary (CHO) cells and human embryonic kidney (HEK293) cells are the most widely used hosts for expression of recombinant proteins, since they can be grown at high densities and have similarity in post-translational modifications. The objective of this work was to express, purify and perform a physicochemical and biological characterization of the glycosylated hPRL from HEK293 cells, including analysis of the carbohydrate structure. For this purpose, a transfection was performed on HEK293T (adhered) cells with the 3.4-TOPO pcDNA vector. Expression of 21.26 ± 8.3 μg/mL hPRL in the serum free conditioned medium was obtained. The hPRL was purified by immobilized metal affinity chromatography (IMAC), eluting 92% of the hPRL in a single fraction which analyzed by HPSEC, showed 97% purity. The N-glycans profile of the sample showed six species, all with sialic acid termination, complex type, being bi, tri and tetra antennary, with a relative predominance of N2G2S1 (29.4%). In vitro bioactivity of G-hPRL HEK293 demonstrated to be ≅ 16-fold lower than G-hPRL produced in CHO cells.

Snabb och korrekt artidentifiering är avgörande för effektiv behandling av svampinfektioner, särskilt bland immunsupprimerade patienter. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) används rutinmässigt på kliniska laboratorier för identifiering av karaktäristiska proteinmönster hos bakterier och jästsvampar genom tolkning av proteinspektra i en masspektradatabas för korrekt artidentifiering. Mögelsvamparnas hårda cellvägg och heterogena växtsätt med varierande proteinuttryck beroende på mognadsstadie, försvårar identifiering med MALDI-TOF MS. Metodens tänkbara fördelar mot traditionella metoden mikroskopering är förkortade svarstider, säkrare artidentifiering av fler arter och mindre beroende av subjektiv morfologisk bedömning. Studiens syfte var att undersöka om MALDI-TOF MS kunde anpassas och användas för identifieringen av mögelsvamp i klinisk rutindiagnostik. Fyra referensstammar (Aspergillus niger, A. fumigatus, A.terreus, A.flavus) och ett kliniskt isolat (A.terreus) undersöktes. Preparationsmetoderna (I) fullständig myrsyraextraktion, (II) direktapplicering och (III) suspension i destillerat vatten användes för analys av sporer och frontmycel hos yngre och äldre mögelkulturer. Två olika masspektradatabaser för artidentifiering jämfördes; rutindatabasen BDAL och den specialiserade mögeldatabasen Filamentous Fungi Library. Även plocktekniken av mögelmaterial inför analys med MALDI-TOF MS utvärderades. Vid vissa tillfällen förbättrades artidentifieringen efter extraktion av mögelkulturerna, medan i andra fall var direktapplicering fullt tillräcklig. Mögelmaterial med mycket sporer tenderade ge något fler artidentifieringar i BDAL oavsett kulturernas ålder.  Filamentous Fungi Library tenderade i vissa fall ge bättre resultat jämfört med BDAL för yngre kulturer. Fler studier krävs för att utvärdera och optimera MALDI-TOF MS som metod för artidentifiering av mögelsvamp. / Rapid and accurate species identification is crucial for successful treatment of fungal infections, especially among immunosuppressed patients. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is used routinely at clinical laboratories to identify characteristic protein patterns of bacteria and yeast by the interpretation of protein spectra in a database for accurate species identification. The hard cell wall of the mold and the heterogeneous growth with varying protein expression due to maturation, complicates identification with MALDI-TOF MS. The potential benefits of this method compared to microscopy as traditional method are shortened turn-around times, safer species identification of more species that is independent on subjective morphological assessment. The purpose of the study was to investigate whether MALDI-TOF MS could be adapted and used for the identification of molds in clinical routine diagnostics. Four reference strains (Aspergillus niger, A.fumigatus, A.terreus, A.flavus) and a clinical isolate (A.terreus) were examined. The preparation methods (I) complete formic acid extraction, (II) direct application and (III) suspension in distilled water were used for analysis of spores and frontmycelium from younger and older mold cultures. Two different masspektradatabases for species identification were compared; routine database BDAL and the specialized mold database, Filamentous Fungi Library. Also the collecting technique of mold prior to analysis with MALDI-TOF MS was evaluated. Sometimes, the species identification improved after extraction of mold cultures, while in other cases direct application was sufficient. Cultures with a lot of spores tended to give slightly more species identifications in BDAL regardless of the age of cultures. Filamentous Fungi Library, in some cases, tended to improve the performance compared to BDAL for younger cultures. More studies are required to evaluate and optimize MALDI-TOF MS as a method of mold identification.

A prolactina humana (hPRL) é um hormônio sintetizado pela hipófise com inúmeras funções tais como: lactação, reprodução e regulação osmótica. Este hormônio é frequentemente dosado em casos de problemas na lactação, infertilidade, além de estudos que elucidam sua ligação em alguns tipos de câncer (mama, próstata e útero). A hPRL é encontrada na forma não glicosilada (NG-hPRL) (23 kDa) e glicosilada (G-hPRL) (25 kDa), sendo a isoforma glicosilada um modelo ideal de análise de perfil de N-glicanos, já que possui um único sítio de glicosilação localizado na Asparagina 31. A glicosilação está relacionada diretamente à solubilidade, à estabilidade, ao enovelamento, à meia-vida e atividade biológica in vivo. As células de ovário de hamster chinês (CHO) e as células embrionárias de rim humano (HEK293) são os hospedeiros mais utilizados para expressão de proteínas recombinantes, já que podem ser cultivadas em altas densidades e por possuírem similaridade nas modificações pós-traducionais. O objetivo foi expressar, purificar e realizar uma caracterização físico-química e biológica da hPRL glicosilada de células HEK293, incluindo análise da estrutura de carboidratos. Para tanto, foi realizada uma transfecção em células HEK293T (aderidas) com o vetor pcDNA 3.4-TOPO. Foi obtida uma expressão de 21,26 ± 8,3 μg/mL de hPRL no meio condicionado sem soro. A hPRL foi purificada por cromatografia de afinidade a metais imobilizados (IMAC), eluindo 92% da hPRL em uma única fração que, analisada por HPSEC, apresentou pureza de 97%. O perfil de N-glicanos da amostra apresentou seis espécies, todas com terminação em ácido-siálico, do tipo complexo, sendo bi, tri e tetra-antenárias, com relativa predominância da espécie N2G2S1 (29,4%). A bioatividade in vitro da G-hPRL HEK293 demonstrou ser ≅ 16 vezes menor que a G-hPRL produzida em células CHO. / Human prolactin (hPRL) is a hormone synthesized by the pituitary gland with innumerable functions such as lactation, reproduction and osmotic regulation. This hormone is often determined in cases of lactation problems, infertility, and studies that elucidate its connection in some types of cancer (breast, prostate and uterus). The hPRL is found in the non-glycosylated (NG-hPRL) (23 kDa) and glycosylated (G-hPRL) (25 kDa) form, being the glycosylated isoform an ideal model for N-glycan profile analysis, since it has a single glycosylation site located in Asparagine 31. Glycosylation is directly related to solubility, stability, folding, half-life and biological activity in vivo. Chinese hamster ovary (CHO) cells and human embryonic kidney (HEK293) cells are the most widely used hosts for expression of recombinant proteins, since they can be grown at high densities and have similarity in post-translational modifications. The objective of this work was to express, purify and perform a physicochemical and biological characterization of the glycosylated hPRL from HEK293 cells, including analysis of the carbohydrate structure. For this purpose, a transfection was performed on HEK293T (adhered) cells with the 3.4-TOPO pcDNA vector. Expression of 21.26 ± 8.3 μg/mL hPRL in the serum free conditioned medium was obtained. The hPRL was purified by immobilized metal affinity chromatography (IMAC), eluting 92% of the hPRL in a single fraction which analyzed by HPSEC, showed 97% purity. The N-glycans profile of the sample showed six species, all with sialic acid termination, complex type, being bi, tri and tetra antennary, with a relative predominance of N2G2S1 (29.4%). In vitro bioactivity of G-hPRL HEK293 demonstrated to be ≅ 16-fold lower than G-hPRL produced in CHO cells.

Der zeitnahe und möglichst sichere Nachweis bakterieller Krankheitserreger und deren Empfindlichkeit gegenüber verfügbaren antibakteriell wirksamen Chemotherapeutika (Antibiotika) stellt einen Hauptaufgabenbereich der medizinischen mikrobiologischen Routinediagnostik dar. Hierzu wurden im Laufe der Jahre unterschiedliche Methoden entwickelt, womit von der genauen Beschreibung der Kolonie- und mikroskopischen Morphologie, Anfärbbarkeit und Formation über die Charakterisierung der biochemischen Leistungsfähigkeit bis hin zur genauen Sequenzierung des gesamten Genoms ein enormer Fortschritt zu verzeichnen war. Seit Mitte der 1990er Jahre etablierte sich die Massenspektrometrie als phänotypisches Nachweisverfahren und gewann zunehmend an Bedeutung. Ebenso konnten Erfolge beim Nachweis Antibiotika resistenter Bakterien verzeichnet werden. Um das Potential dieser noch jungen Nachweismethode weiter zu erforschen, wurden in dieser Arbeit Spezies der Familie Enterobacteriaceae und der Nonfermenter in eine eigene massenspektrometrische Datenbank aufgenommen, um diese als Grundlage zur Validierung des Identifizierungspotentials der Methode mittels Blindstudie zu nutzen. Im selben Arbeitsschritt wurde der Versuch unternommen, Antibiotika resistente Stämme im Zuge der Speziesidentifizierung zu detektieren, um so Aussagen über eine mögliche Einschränkung der therapeutischen Möglichkeiten und gegebenenfalls notwendigen Hygienemaßnahmen treffen zu können.

Sepsis är ett allvarligt tillstånd som uppstår när bakterier går från vävnad till blodbanan. Positiva blododlingar odlas på agarplattor och bakterier analyseras med Matrix Assisted Laser Desorption Ionisation Time-of-flight Mass spectrometry (MALDI-TOF MS) där prov blandas med en matrix och sedan bestrålas med laser. Proteinerna i provet joniseras och rör sig mot en detektor, vilket ger ett m/z-spektrum som jämförs med referensspektrum i en databas och ett score-värde erhålls över hur väl analyten liknar referensen. Arbetets syfte var att undersöka möjligheten att direktidentifiera bakterier från blod med en viss preparation innan analys med MALDI-TOF MS och på så vis möjliggöra snabbare preliminära svar samt undersöka möjligheten att särskilja Staphylocoocus aureus och koagulasnegativa stafylokocker. Innan analys med MALDI-TOF MS centrifugerades blod från positiva blododlingar blod i flera steg med 5 % natriumkloridlösning (NaCl-metoden). Dessutom testades ett kommersiellt kit (Sepsityper, Bruker Daltonics). Med NaCl-metoden sågs korrekt identifiering hos 66 % av inokulerade proverna. Av blododlingar innehållande med S. aureus respektive koagulasnegativa stafylokocker identifierades 60 % respektive 43 % av bakterierna korrekt. Med Sepsiptyper erhöll 58 % av proverna godkänt score-värde. Slutsatsen blev att det är möjligt att identifiera bakterier direkt från blod efter viss preparation, men metoden bör utvecklas mer då det fanns en signifikant skillnad i score mellan NaCl-metoden och nuvarande metod. Det är dock möjligt att skilja mellan Staphylococcus aureus och koagulasnegativa stafylokocker. Fler studier är nödvändiga för att avgöra möjligheten att föra in någon av metoderna i rutindiagnostiken.