京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20793号 / 医博第4293号 / 新制||医||1025(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 妻木 範行, 教授 鈴木 茂彦, 教授 瀬原 淳子 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22499号 / 農博第2403号 / 新制||農||1077(附属図書館) / 学位論文||R2||N5279(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 佐藤 健司, 教授 菅原 達也, 准教授 豊原 治彦 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

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Syftet med detta examensarbete var att validera en metod som kvantitativt bestämmer PFAS ijord, slam och sediment. Valideringen omfattade en del olika tester för att utvärdera metodensrapporteringsgräns, precision, riktighet, mätosäkerhet och specificitet. Excel har använts för attberäkna och sammanställa resultaten från de olika testerna. Blankprover och spikade prover haranalyserats för att undersöka rapporteringsgräns. Kontrollprover har analyserats för attundersöka precision samt specificitet. För att undersöka riktighet har en rad olika tester utförts;ISTD-matchning för några av PFAS-analyter som saknade en direkt ISTD-match, jämförelsermot certifierat referensmaterial, analys av tidigare utförda tester som redan hade analyserats iALS Prag och spikade provmatriser. Mätosäkerheten uppskattade och utvärderades i samrådmed kvalitetsansvarig. Resultatet från metodvalideringen bekräftar att metoden har visat sig vara väl validerad.Samtliga tester som utfördes under valideringsprocessen har konsekvent uppfyllt och överträffatde ställda acceptanskriterium för valideringen. Kriterierna inkluderade bland annat relativstandardavvikelse (RSD), utbyte, bias och halt för olika testparametrar. Den omfattandemetodvalideringen gav starka bevis att metoden är tillförlitlig och kan användas för att genererapålitliga resultat inom PFAS-analys. / The purpose of this thesis was to validate a method for quantitatively determining PFAS in soil,sludge, and sediment. The validation involved a number of different tests to evaluate themethod's reporting limit, precision, accuracy, measurement uncertainty, and specificity. Excelwas used to calculate and summarize the results from the different tests. Blank and spikedsamples were analyzed to investigate the reporting limit. Control samples were analyzed toinvestigate precision and specificity. A variety of tests were performed to investigate accuracy;ISTD matching for some PFAS analytes that lacked a direct ISTD match, comparisons againstcertified reference material, analysis of previously performed tests that had already beenanalyzed at ALS Prague, and spiked sample matrices. The measurement uncertainty wasestimated and evaluated in consultation with the quality manager. The results of the method validation confirm that the method has been shown to be wellvalidated. All tests performed during the validation process have consistently met and exceededthe established acceptance criteria for the validation. Criteria included, among others, relativestandard deviation (RSD), recovery, bias, and concentration for various test parameters. Thecomprehensive method validation provided strong evidence that the method is reliable and canbe used to generate reliable results in PFAS analysis.

Les substances synthétisées par les bactéries du genre Bacillus présentent un grand intérêt en raison de leurs nombreuses activités antimicrobiennes. L’objectif de ce travail a consisté à mettre au point un protocole analytique fiable pour extraire ces substances, et les caractériser par spectrométrie de masse et par leurs activités biologiques. Une première approche par spectrométrie de masse MALDI-ToF ne nous a pas donné de résultats satisfaisants en raison d’un phénomène de suppression spectrale. Une seconde stratégie d’étude combinant deux techniques analytiques a alors été envisagée. Les composés et les familles lipopeptidiques ont d’abord été séparés par chromatographie liquide analytique et analysés en ligne en mode de couplage ESI-IT. Ensuite les fractions collectées ont été caractérisées par spectrométrie de masse MALDI-Q-ToF (et ESI-Q-ToF) pour déterminer les masses exactes et obtenir des spectres de fragmentation complémentaires des premiers. Ce procédé nous a permis d’établir des critères d’identification des composés et des familles de lipopeptides de structures connues. Cette caractérisation est basée sur des temps de rétention en chromatographie, des mesures de masses exactes et sur des schémas de fragmentations. Nous avons fait de même avec les composés non identifiés et ensuite évalué leur activité biologique en réalisant des tests de diffusion sur agar. Cette stratégie d’étude permet de faire un criblage de l’ensemble des biomolécules produites par des souches de Bacillus et de relier une structure à une activité biologique. Ce protocole, mis au point pour des cultures réalisées en milieu liquide, a ensuite été adapté aux cultures sur boîtes de Pétri pour pouvoir analyser les composés produits à l’échelle de la colonie bactérienne. / Compounds produced by Bacillus bacteria present a major interest because of their biological activities. The aim of this work was to develop a reliable analytical methodology to extract these compounds, and to characterize them by mass spectrometry and by their biological activities. A first approach by MALDI-ToF mass spectrometry doesn’t give satisfactory results because of strong spectral suppression effects. Therefore, we have designed a second strategy which combined two analytical technologies. First, compounds and lipopeptides families were separated by analytical liquid chromatography and analysed by ESI-IT. Then, collected fractions were characterized by MALDI-Q-ToF (and ESI-Q-ToF) in order to determine accurate mass measurements and obtain complementary product ion spectra. This process led us to establish identification criteria of compounds and lipopeptides families which have known structures. This characterization is based of retention times in chromatography, accrurate mass measurements and fragmentation schemes. We have achieved the same experiments with non identified compounds and then assessed their biological activity by agar well diffusion test.This strategy allows to obtain a screening of whole of the biomolecules produced by bacillus strains and establishes a link between structure and biological activity. This methodology, designed for cultures in liquid medium, was then adapted to cultures in Petri disches in order to analyse compounds producted at the bacterial colony scale.

A contaminação de corpos d\'água por fármacos é um tema de extrema relevância, tendo em vista problemas como a escassez de água, florações de cianobactérias tóxicas e lançamentos clandestinos de efluentes domésticos. Sendo assim, este trabalho teve como objetivo determinar a presença de cafeína (CAF), fluoxetina (FLX), levotiroxina (LVX) e bezafibrato (BZF) em mananciais do estado de São Paulo, bem como avaliar a toxicidade desses compostos à cianobactéria Microcystis aeruginosa LTPNA 08. Um método por LC-MS/MS foi desenvolvido e validado, de acordo com a RDC nº 166 da ANVISA, para a detecção de CAF, FLX, LVX e BZF em amostras ambientais. As represas Guarapiranga e Billings, bem como os rios Taiçupeba, Sorocaba, Baixo Cotia, Grande e Paraíba foram monitorados de abril a setembro de 2017. A toxicidade dos fármacos foi avaliada por meio do monitoramento do crescimento, produção de microcistinas e viabilidade celular da cianobactéria M. aeruginosa LTPNA 08. CAF foi detectada em todas as amostras analisadas, com concentrações que variaram de 6,6 ng.L-1 a 16,47 µg.L-1. No Rio Cotia foram verificadas as maiores concentrações de CAF, FLX e BZF (16,47 µg.L-1; 3,5 ng.L-1 e 322 ng.L-1, respectivamente). A LVX, cujos produtos de biotransformação não foram monitorados, não foi detectada em nenhuma amostra analisada. A concentração de 50 µg.L-1 de FLX inibiu o crescimento da cianobactéria em 82,3% (CE50: 31,4 µg.L-1). Em relação à produção de microcistinas totais, os fármacos inibiram a liberação da fração extracelular para a maior concentração testada ao longo do tempo de monitoramento, embora não tenham demonstrado efeito sobre a viabilidade celular. Sendo assim, considerando-se que fármacos estão presentes nos mananciais monitorados no estado de São Paulo e que a FLX pode causar efeito sobre a M. aeruginosa, os efeitos decorrentes da exposição a concentrações ambientais contínuas e cumulativas de fármacos em corpos d\'água devem ser estudados. Além disso, uma vez que a ocorrência destas substâncias e outros contaminantes antropogênicos no ambiente aquático natural é uma questão emergente devido aos efeitos adversos potenciais que estes compostos representam para a vida aquática e os seres humanos, os tipos e níveis destes compostos, que têm um impacto maior na qualidade da água, deve ser constantemente monitorada. Práticas de gestão que investem em saneamento e na redução da descarga de efluentes não tratados, e um plano de proteção de recursos hídricos com o objetivo de garantir a segurança da água seriam medidas essenciais para reduzir o aporte de contaminantes nos corpos d\'água do estado de São Paulo. / Contamination of water bodies by drugs is a subject of extreme relevance considering related problems such as water scarcity, harmful cyanobacterial blooms and discharge of untreated domestic effluents. Therefore, the aim of this work was to determine the presence of caffeine (CAF), fluoxetine (FLX), levothyroxine (LVX) and bezafibrate (BZF) in springs in the State of São Paulo, and to evaluate the toxicity of these compounds in cyanobacteria Microcystis aeruginosa LTPNA 08. A LC-MS/MS method was developed and validated according to RDC nº 166 of ANVISA to assess the concentration of CAF, FLX, LVX and BZF in environmental samples. Guarapiranga and Billings reservoirs, as well as the Taiçupeba, Sorocaba, Baixo Cotia, Grande and Paraíba rivers were monitored from April to September 2017.The drugs toxicity in M. aeruginosa LTPNA 08 was assessed by monitoring their effects on cyanobacterial growth, microcystins production and cell viabilityby flow cytometry. CAF was detected in all analyzed samples at concentrations ranging from 6.6 ng to 16.47 µg.L-1.Among studied sites, Cotia river showed the highest concentrations of CAF, FLX and BZF (16.47 µg.L-1, 3.5 ng.L-1 and 322 ng.L-1, respectively). LVX, which biotransformation products were not monitored, was not detected in any of the analyzed samples. Regarding the drugs toxicity, 50 µg.L-1 of FLX inhibited the cyanobacterial grow thin 82.3% (EC50 of 31.4 µg.L-1). Although no effect on cell viability was seen by flow cytometry, the highest concentrations of all compounds tested were able to inhibit the release of microcystins. Therefore, considering that some of the drugs monitored showed to be present in water sources in São Paulo State and that FLX affects cyanobacteria M. aeruginosa growth, the effects of continuous and cumulative exposure at environmental drug concentrations of in water bodies should be evaluated. Also, since the occurrence of these substances and other anthropogenic contaminants in the natural aquatic environment is an emerging issue due to the potential adverse effects these compounds pose to aquatic life and humans, thet ypes and levels of these compounds, which have a greater impact on water quality, should be constantly monitored. Management practices investing in sanitation and in reducing discharge of untreated effluents, as well as a plan for water resources protection with the goal of ensuring water security would be essential measures in reducing drugs loading into water bodies situated in São Paulo State.

A película adquirida (PA) é um filme formado pela adsorção seletiva de proteínas, glicoproteínas e lipídeos à superfície dentária. A presença de proteínas na PA forma uma interface protetora sobre a superfície do dente, participando em todos os eventos interfaciais que ocorrem na cavidade bucal, tais como des- e remineralização, lubrificação das superfícies dos dentes, e aderência bacteriana. Com o advento da proteômica, tem havido um aumento considerável no conhecimento acerca do perfil proteico de PAs adquiridas formadas sobre o esmalte dentário, em diferentes situações, mas nenhum trabalho até o momento descreveu o perfil proteômico de PAs formadas sobre a dentina. Este estudo foi pioneiro em comparar o perfil proteico de PAs formadas in situ sobre o esmalte e a dentina, nos tempos de 10 minutos e 2 horas, utilizando análise proteômica quantitativa livre de marcadores. Os experimentos foram realizados por três dias consecutivos. Em cada dia, os 9 voluntários receberam profilaxia dentária e em seguida utilizaram um aparelho vestibular com 6 blocos de esmalte e 6 de dentina humanos por 10 minutos ou 2 horas. Após esses períodos, a PA formada era coletada com auxílio de um papel filtro de eletrodos embebido em ácido cítrico 3%. Para as análises foi realizado um pool com os papéis dos 9 voluntários de todos os dias, para cada substrato e tempo de formação. Após a extração e digestão das proteínas, a separação dos peptídeos foi realizada por nano-HPLC (nano-Cromatografia Líquida de Alta Performace), interligada a um espectrômetro de massa (nLC-ESI-MS/MS). Os dados MS/MS obtidos foram processados e pesquisados em bancos de dados de proteínas humanas (UniProt e TrEMBL), utilizando o algoritmo SEQUEST no software Proteome Discoverer 1.3. Para a PA formada sobre o esmalte, foram identificadas 160 e 64 proteínas, nos tempos de formação de 10 minutos e 2 horas, respectivamente. Os respectivos números de proteínas identificadas para a dentina foram 86 e 52, respectivamente. Nos tempos de 10 minutos e 2 horas, respectivamente, 25 e 11 proteínas foram comuns a ambos os substratos e foram submetidas à quantificação livre de marcadores (SIEVE), revelando que a maioria das proteínas com diferença de expressão entre os dois substratos teve sua expressão aumentada na dentina. Foram identificadas ainda, no tempo de 10 minutos de formação da PA, 135 e 61 proteínas exclusivas ao esmalte ou à dentina, respectivamente. O número correspondente de proteínas exclusivas para o tempo de 2 horas foi de 53 e 41 proteínas, para o esmalte e dentina, respectivamente. Dentre as proteínas exclusivas da dentina, foram identificadas várias proteínas relacionadas ao complexo cálcio/calmodulina, assim como proteínas associadas à tumorigênese e à fosforilação/desfosforilação de proteínas. Em adição, muitas das proteínas identificadas no presente estudo, tanto para o esmalte quanto para a dentina, ainda não foram caracterizadas e, portanto, não têm função conhecida na PA. Sua caracterização e estudos funcionais futuros poderão trazer novos horizontes no entendimento da importância da PA para a proteção da estrutura dentária, bem como do papel da PA como sítio de biomarcadores para doenças bucais e sistêmicas. / The acquired pellicle (AP) is a film that results from selective adsorption of proteins, glicoproteins and lipids on the tooth surface. The presence of proteins in the AP forms a protective interface on the tooth surface that participates in all the surface events occurring in the oral cavity, such as de- and remineralization, lubrification of the tooth surfaces and bacterial adherence. With the advent of Proteomics, considerable increase in the knowledge of the protein profile of the AP formed on tooth enamel, under different circunstances, has been observed. However, so far the proteomic profile of the AP formed on dentin has not been described. This is the first study to compare the proteomic profile of APs formed in situ for 10 minutes and 2 hours, on enamel and dentin, using quantitative label-free proteomics. The experiments were conducted for 3 consecutive days. Each day, 9 volunteers were submitted to dental prophylaxis and in sequence wore a vestibular device containing 6 human enamel and 6 human dentin blocks for 10 minutes or 2 hours. After these periods, the PA formed was collected with an electrode filter paper soaked in 3% citric acid. The papers from the 9 volunteers, for each substrate and time of pellicle formation were pooled and used for analysis. After protein extraction and digestion, peptides were separated by nano-HPLC (High-performance liquid chromatography) coupled to a mass spectrometer (nLC-ESI- MS/MS). The obtained MS/MS spectra were searched against human protein databases (UniProt and TrEMBL) using SEQUEST algorithm in Proteome Discoverer 1.3 software. For the AP formed on enamel, 160 and 64 proteins were identified for the times of pellicle formation of 10 minutes and 2 hours, respectively. The respective numbers of identified proteins for dentin were 86 and 52, respectively. For the times of 10 minutes and 2 hours, respectively, 25 and 11 proteins were common to both substrates. They were submitted to label-free quantification, which revealed that most of the proteins with differential expression were overexpressed in the dentin. For APs formed for 10 minutes, 135 and 61 proteins were identified exclusively for enamel or dentin, respectively. The corresponding number for the 2-hour APs was 53 and 41 proteins, respectively. Among the proteins identified exclusively in dentin, many proteins related with calcium/calmodulin complex, as well as proteins associated with tumorigenesis and protein phosphorylation/dephosphorylation were found. In addition, many of the identified proteins, both for enamel and dentin, remain uncharacterized and, therefore have no described function in the AP. In the future, their characterization and functional studies might open new avenues for the understanding of the importance of the AP for the protection of the dental structure, as well as for the use of the AP as a site for biomarkers of oral and systemic diseases.