Identifier rapidement des microorganismes pathogènes dans des échantillons environnementaux est un enjeu majeur dans le domaine de la biodéfense. Dans ce contexte, la spectrométrie de masse MALDI-TOF peut offrir une réponse simple, rapide et peu coûteuse. L'enjeu de la thèse, dans le cadre du projet ANR franco-allemand GEFREASE, a été de développer des méthodes permettant l'identification des microorganismes pathogènes et notamment de mettre en place des approches ciblées pour la préparation d'échantillon à l'aide d'anticorps en amont de la spectrométrie de masse. Dans un premier temps, l'étude du protéome de la bactérie modèle, Francisella tularensis subsp. holarctica LVS, responsable de la tularémie, a permis d'identifier les protéines et les peptides les plus abondants donnant un signal intense par spectrométrie de masse. Ensuite l'étude protéogénomique de douze protéines cibles a permis de choisir trois biomarqueurs dont le profil des masses par spectrométrie de masse de type MALDI-TOF (approche top-down) est spécifique de l'espèce et de la sous-espèce des bactéries du genre Francisella. Par cette méthode la virulence d'une souche est donc rapidement déterminée puisqu'elle est dépendante de la sous-espèce à laquelle la bactérie appartient. Ce test mis au point présente l'avantage d'être simple et rapide. Dans un deuxième temps, la mise au point d'un protocole d'enrichissement de la bactérie modèle par immunocapture magnétique a permis de montrer qu'il est possible de concentrer des bactéries grâce à des billes magnétiques couplées à des anticorps dirigés contre la bactérie entière. Cette approche a été expérimentée dans le cas de mélanges de bactéries où la bactérie modèle était largement minoritaire et dans des échantillons de matrices alimentaires diverses telles que de l'eau minérale ou du lait. La méthodologie a été validée sur un agent de classe 3, Francisella tularensis subsp. tularensis. / The rapid identification of pathogenic microorganisms in environmental samples is a major issue in the biodefense field. MALDI-TOF mass spectrometry can offer a fast, straightforward and inexpensive answer. In the framework of the Franco-German ANR project GEFREASE, the purpose of the thesis was to develop methodologies allowing identification of pathogenic microorganisms and particularly to set up targeted approaches using antibodies for sample preparation beforehand mass spectrometry. First of all, the proteome study of Francisella tularensis subsp. holarctica LVS, responsible for tularemia, allowed us to identify the most abundant proteins and peptides, and for which the most intense signals are observed when using mass spectrometry. The proteogenomic study of twelve of these proteins enable us to choose three biomarkers for which the masses monitored by MALDI-TOF mass spectrometry (top down approach) allow deciphering the Francisella species and subspecies. The interest of this work is being able to conclude on a strain virulence based on the knowledge of the subspecies it belongs. The finalized test is easy and fast. Secondly, the development of a magnetic immunocapture of Francisella tularensis subsp. holarctica LVS allowed us to show that it is possible to concentrate bacteria using magnetic beads coupled to antibodies raised against the entire bacterium. This approach has been experimented in the case of bacterial mixtures where the model bacterium was largely in minority and for samples containing various food matrices such as mineral water or milk. The methodology has been validated on a class 3 agent, Francisella tularensis subsp. tularensis.

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.

Fungos endofíticos estão presentes em plantas de diversos ambientes e produzem compostos com amplas propriedades químicas e aplicações, tanto na área médico-farmacológica quanto na agronômica. Entretanto, ainda há muito a ser investigado sobre seu potencial biotecnológico, principalmente em locais pouco explorados. As ilhas apresentam um ambiente particular e altamente vulnerável, tornando-as locais promissores na busca de organismos pouco comuns ou ainda endêmicos. Sendo assim, neste trabalho foi realizado o isolamento e estudo químico e biológico de fungos endofíticos isolados de 2 espécies plantas do gênero Anthurium da Ilha de Alcatrazes-SP. Para isto, fragmentos foliares das plantas A. loefgrenii (HRCB 46467) e A. alctrazense (HRCB 46465 - endêmica da ilha) foram inoculados em 10 meios de cultivo com diferentes composições, resultando no isolamento de 106 fungos endofíticos. Por meio de análises químicas por MALDI-TOF-MS e ensaio biológico contra fitopatógenos, foram selecionados 3 fungos para estudo. Estes foram identificados por técnicas morfológicas e moleculares como sendo Penicillium citrinum (P2MSF2F3), Penicillium simplicissimum (P210-4F2) e Aureobasidium melanogenum (P7AF2F3). No estudo dos metabólitos secundários de P. citrinum foi isolado o composto citrinina, o qual apresentou atividade inibitória do crescimento micelial dos fitopatógenos Colletotrichum gloeosporioides (MIC= 125 μg mL-1), Colletotrichum lindemuthianum (MIC= 0,48 μg mL-1), Phomopsis sojae (MIC= 250 μg mL-1) e Fusarium oxysporum f. sp. phaseoli (MIC= 125 μg mL-1). Outras frações obtidas do meio metabólico de P. citrinum (fração F3a3 e citrinina) apresentaram atividade inibitória (100% de inibição) à formas promastigotas de Leishmania infantum. No estudo dos metabólitos secundários de P. simplicissimum foi obtida a fração F2b, ativa contra L. infantum (100% de inibição), da qual foram isolados os compostos andrastina A e penicisoquinolina, sendo o primeiro relato de sua produção por esta espécie, além de outros 5 compostos ainda não identificados. No estudo dos metabólitos secundários de A. melanogenum foi isolado o composto metil-orselinato, relatado pela primeira vez para este fungo. Do mesmo fungo foi obtida a fração F1d2l ativa contra L. infantum (100% inibição), da qual foram isolados 2 compostos ainda não identificados. Este é o primeiro relato de fungos isolados de antúrios da ilha de Alcatrazes, bem como do estudo de seus metabólitos secundários. Este trabalho apresenta contribuição no conhecimento sobre fungos endofíticos e seu potencial metabólico, com aplicações nas áreas agronômica e médico-farmacológica. / Endophytic fungi are present in plants in various environments and produce compounds with wide chemical properties and applications, both in the medical and in agronomic field. However, much remains to be investigated about their biotechnological potential, especially in unexplored places. Islands have a particular and highly vulnerable environment, making them promising sites in search of unusual or endemic organisms. Thus, this work represents the isolation and chemical and biological study of endophytic fungi isolated from 2 species of plants of the genus Anthurium of Alcatrazes island-SP. For this, leaf fragments from plants A. loefgrenii (HRCB 46467) and A. alcatrazense (HRCB 46465 - endemic plant from the Island) were inoculated onto 10 culture media with different composition, resulting in the isolation of 106 endophytic fungi. Three strains were selected to be studied through chemical analysis by MALDI-TOF-MS and bioassay against phytopathogens. These were identified by morphological and molecular techniques as Penicillium citrinum (P2MSF2F3), Penicillium simplicissimum (P210-4F2) and Aureobasidium melanogenum (P7AF2F3). In the study of secondary metabolites of P. citrinum it was isolated the compound citrinin, which showed inibitory activity against the plant pathogens Colletotrichum gloeosporioides (MIC= 125 μg mL-1), Colletotrichum lindemuthianum (MIC= 0.48 μg mL-1), Phomopsis sojae (MIC= 250 μg mL-1) and Fusarium oxysporum f. sp. phaseoli (MIC= 125 μg mL-1). Other fraction obtained from the metabolic extract of P. citrinum (F3a3 fraction and citrinin), showed inibitory activity (100% inibition) to Leishmania infantum promastigotes. In the study of secondary metabolites of P. simplicissimum it was obtained F2b fraction, active against L. infantum (100% inhibition), which were isolated andrastin A and penicisoquinoline compounds, the first report of its production for this species, as well 5 unidentified compounds. In the study of secondary metabolites from A. melanogenum was isolated the methyl-orsellinate compound, first reported for this fungus. From the same strain was obtained F1d2l fraction, active against L. infantum (100% inhibition), from which were isolated 2 unidentified compounds. This is the first report of fungi isolated from Alcatrazes Island anthuriums and the study of their secondary metabolites. This study presents contribution to knowledge of endophytic fungi and their metabolic potential with applications in the medical and agronomic fields.

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.

Fungos endofíticos estão presentes em plantas de diversos ambientes e produzem compostos com amplas propriedades químicas e aplicações, tanto na área médico-farmacológica quanto na agronômica. Entretanto, ainda há muito a ser investigado sobre seu potencial biotecnológico, principalmente em locais pouco explorados. As ilhas apresentam um ambiente particular e altamente vulnerável, tornando-as locais promissores na busca de organismos pouco comuns ou ainda endêmicos. Sendo assim, neste trabalho foi realizado o isolamento e estudo químico e biológico de fungos endofíticos isolados de 2 espécies plantas do gênero Anthurium da Ilha de Alcatrazes-SP. Para isto, fragmentos foliares das plantas A. loefgrenii (HRCB 46467) e A. alctrazense (HRCB 46465 - endêmica da ilha) foram inoculados em 10 meios de cultivo com diferentes composições, resultando no isolamento de 106 fungos endofíticos. Por meio de análises químicas por MALDI-TOF-MS e ensaio biológico contra fitopatógenos, foram selecionados 3 fungos para estudo. Estes foram identificados por técnicas morfológicas e moleculares como sendo Penicillium citrinum (P2MSF2F3), Penicillium simplicissimum (P210-4F2) e Aureobasidium melanogenum (P7AF2F3). No estudo dos metabólitos secundários de P. citrinum foi isolado o composto citrinina, o qual apresentou atividade inibitória do crescimento micelial dos fitopatógenos Colletotrichum gloeosporioides (MIC= 125 μg mL-1), Colletotrichum lindemuthianum (MIC= 0,48 μg mL-1), Phomopsis sojae (MIC= 250 μg mL-1) e Fusarium oxysporum f. sp. phaseoli (MIC= 125 μg mL-1). Outras frações obtidas do meio metabólico de P. citrinum (fração F3a3 e citrinina) apresentaram atividade inibitória (100% de inibição) à formas promastigotas de Leishmania infantum. No estudo dos metabólitos secundários de P. simplicissimum foi obtida a fração F2b, ativa contra L. infantum (100% de inibição), da qual foram isolados os compostos andrastina A e penicisoquinolina, sendo o primeiro relato de sua produção por esta espécie, além de outros 5 compostos ainda não identificados. No estudo dos metabólitos secundários de A. melanogenum foi isolado o composto metil-orselinato, relatado pela primeira vez para este fungo. Do mesmo fungo foi obtida a fração F1d2l ativa contra L. infantum (100% inibição), da qual foram isolados 2 compostos ainda não identificados. Este é o primeiro relato de fungos isolados de antúrios da ilha de Alcatrazes, bem como do estudo de seus metabólitos secundários. Este trabalho apresenta contribuição no conhecimento sobre fungos endofíticos e seu potencial metabólico, com aplicações nas áreas agronômica e médico-farmacológica. / Endophytic fungi are present in plants in various environments and produce compounds with wide chemical properties and applications, both in the medical and in agronomic field. However, much remains to be investigated about their biotechnological potential, especially in unexplored places. Islands have a particular and highly vulnerable environment, making them promising sites in search of unusual or endemic organisms. Thus, this work represents the isolation and chemical and biological study of endophytic fungi isolated from 2 species of plants of the genus Anthurium of Alcatrazes island-SP. For this, leaf fragments from plants A. loefgrenii (HRCB 46467) and A. alcatrazense (HRCB 46465 - endemic plant from the Island) were inoculated onto 10 culture media with different composition, resulting in the isolation of 106 endophytic fungi. Three strains were selected to be studied through chemical analysis by MALDI-TOF-MS and bioassay against phytopathogens. These were identified by morphological and molecular techniques as Penicillium citrinum (P2MSF2F3), Penicillium simplicissimum (P210-4F2) and Aureobasidium melanogenum (P7AF2F3). In the study of secondary metabolites of P. citrinum it was isolated the compound citrinin, which showed inibitory activity against the plant pathogens Colletotrichum gloeosporioides (MIC= 125 μg mL-1), Colletotrichum lindemuthianum (MIC= 0.48 μg mL-1), Phomopsis sojae (MIC= 250 μg mL-1) and Fusarium oxysporum f. sp. phaseoli (MIC= 125 μg mL-1). Other fraction obtained from the metabolic extract of P. citrinum (F3a3 fraction and citrinin), showed inibitory activity (100% inibition) to Leishmania infantum promastigotes. In the study of secondary metabolites of P. simplicissimum it was obtained F2b fraction, active against L. infantum (100% inhibition), which were isolated andrastin A and penicisoquinoline compounds, the first report of its production for this species, as well 5 unidentified compounds. In the study of secondary metabolites from A. melanogenum was isolated the methyl-orsellinate compound, first reported for this fungus. From the same strain was obtained F1d2l fraction, active against L. infantum (100% inhibition), from which were isolated 2 unidentified compounds. This is the first report of fungi isolated from Alcatrazes Island anthuriums and the study of their secondary metabolites. This study presents contribution to knowledge of endophytic fungi and their metabolic potential with applications in the medical and agronomic fields.

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.

Ziel der vorliegenden Arbeit war die Identifizierung genetischer Faktoren, die das Plasmaproteom regulieren. Die Untersuchungen wurden im Modellsystem einer F2-Kreuzung zweier Inzucht-Mausstämme (FVB.LDLR-/-, C57BL/6.LDLR-/-) durchgeführt, die sich in ihrer Atheroskleroseausprägung unterscheiden. Von jedem der 453 Tiere der F2-Generation wurden Plasmaproteomprofile mittels Massenspektrometrie (MALDI-TOF) generiert. Diese Spektren wurden in zwei unabhängigen Datenanalysen ausgewertet und eine Kopplungsanalyse (QTL-Analyse, quantitative trait loci) der Phänotypen mit jeweils 192 genetischen Markern in jedem der F2-Tiere durchgeführt. So wurden die Datensätze von Proteom und Genom miteinander kombiniert, um Genorte, die mit unterschiedlich regulierten Proteinen in Verbindung stehen, zu identifizieren. Dieser Ansatz ist bisher in der Literatur nicht beschrieben worden. In der vorliegenden Arbeit wird sowohl die Methodik der statistischen Auswertung als auch die weitere Analyse der generierten Daten beschrieben. Es wurden zahlreiche hochsignifikante Kopplungssignale gefunden, von denen zwei durch die Identifizierung von Proteinen verifiziert werden konnten. Es handelt sich hierbei um das Apo-A2 des HDL auf Chromosom 1 und Hämoglobin subunit alpha auf Chromosom 11. Eine Kolokalisation der gefundenen Proteine mit Loci der Atherosklerosedisposition konnte nicht identifiziert werden. Dieser Ansatz zeigt erstmals, dass eine hypothesenfreie Verbindung proteomischer und genomischer Daten möglich ist und zur Identifizierung genetisch regulierter Plasmaproteine beitragen kann.

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.

Une alternative aux produits industriels de type phénol ou résorcinol peut être des tanins ou de la lignine. Les deux sont des polyphénols naturels, le tanin est extrait de différentes parties de plantes, tandis que la lignine est habituellement obtenue comme sous-produit dans les industries papetières. Ces deux produits sont la base principale sur laquelle j’ai travaillé pendant le développement de cette thèse. Dans une première partie, une étude de caractérisation et de différenciation entre différents tannins ayant la même origine mais que présentent un comportement différent lorsqu'ils sont utilisés dans la même application dans les mêmes conditions a été effectuée. Cette étude met en évidence la GPC comme technique fondamentale pour la différenciation des tanins de quebracho sulfités. D'autre part, les interactions entre différentes substances avec du tanin et de la lignine ont été étudiées. Comme l’étude de la réaction entre les diamines (telles que l'hexaméthylènediamine) avec du tanin et de la lignine pour obtenir des résines polycondensées. En outre, dans cette section ont été obtenus des polyuréthanes avec au moins 70% de substances naturelles dans leur préparation sans utiliser d'isocyanate dans le procédé. De plus, des aldéhydes dérivés de la lignine, comme la vanilline, ont été utilisés avec le tanin de pin pour la fabrication d'adhésifs dans la préparation de panneaux de particulaires, obtenant des résultats satisfaisants selon les normes européennes et des substances complètement naturelles. Enfin, dans le cadre d'un projet industriel les étapes initiales pour le développement d'une mousse de tanin rigide applicable par projection pour l'isolation thermique des bâtiments ont été réalisées. Lorsqu'un nouveau système de moussage mécanique a été développé pour des mousses de tanin basées sur des mousses de lutte contre incendie à base de tanin ou dans les mousses des opérations d’ouverture du tunnel, ce nouveau système de moussage évite les problèmes de retrait lors de la formation de la mousse / An alternative to industrial phenol or resorcinol industrial products may be tannins or lignin. Both are natural polyphenols, the tannin is extracted from different parts of plants, while lignin is usually obtained as a secondary product in the pulp and paper mill. These two products are the main basis on which I have worked during the development of this thesis. In a first part, a study of characterization and differentiation between different tannins with the same origin and that present a different behavior when used in the same application under the same conditions has been done. Highlighting the GPC as a fundamental technique for the differentiation between sulphited quebracho tannins. On the other hand, the interactions between different substances with tannin and with lignin have been studied. As the study of the reaction between diamines (such as hexamethylenediamine) with tannin and lignin to obtain a polycondensed resins. Also, in this section have been obtained polyurethanes with at least 70% of natural substances in their preparation without using any isocyanate in the process. In addition, aldehydes derived from lignin, such as vanillin, have been used next to pine tannin for the manufacture of adhesives in the preparation of particleboards, obtaining satisfactory results according to European standards and from completely natural substances. Finally, within an industrial project the initial steps have been carried out for the development of a rigid tannin foam applicable by projection for the thermal insulation of buildings. Where a new mechanical foaming system has been developed for tannin foams based in fire-fighting foams or in the foams of the tunneling operations, this new system of foaming avoids the problems of shrinkage during the formation of the foam

Le MALDI-TOF MS (Matrix Assisted, Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) est une technique protéomique qui est utilisée en routine pour l’identification des microorganismes dans les laboratoires de microbiologie. Ainsi, dans ce travail nous avons évalué le MALDI-TOF MS pour l’identification du repas sanguin des moustiques. Dans la première partie de notre travail, une revue bibliographique a été effectuée sur les différentes méthodes (sérologiques, biologie moléculaire) connues dans les études de préférence trophiques des arthropodes. La deuxième partie fut l’optimisation du MALDI-TOF MS pour l’identification de l’origine du repas sanguin des moustiques. Pour l’optimisation, Anopheles gambiae Giles et Aedes albopictus ont été artificiellement nourris sur le sang de plusieurs hôtes vertébrés en utilisant l’appareil Hemotek durant deux heures sous les conditions standard. Nos résultats ont montré que la comparaison des spectres provenant des moustiques nourris sur le même type de sang révèle une grande reproductibilité des profils protéiques. L’interrogation des MS spectres contre la base de données a révélé une identification correcte de l'origine du repas sanguin pour les spécimens collectés moins de 24 heures après la prise du repas sanguin. Pour les échantillons collectés sur le terrain, le MALDI-TOF MS a permis de détecter dans le repas de sang des moustiques une grande diversité d’hôtes domestiques. En conséquence la technique MALDI-TOF MS serait un outil efficace pour les études de surveillance épidémiologique des maladies vectorielles et l'identification de la préférence trophique de spécimens fraichement gorgés. / MALDI-TOF MS (Matrix Assisted, Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry) is a proteomic technique that routinely used for microorganisms identification in clinical microbiology laboratory. Recently, the MALDI-TOF MS was successfully used as a innovative tool for arthropod identification. Thus, in this work we evaluated the MALDI-TOF MS to identify the blood meal sources from engorged mosquitoes. In the first part of our work, a bibliographical review was carried out on the different methods (serological, molecular biology) known in the trophic preference determination of hematophagous arthropods. The second part was optimization of the MALDI-TOF MS for identifying the origin of the blood meal of mosquitoes. For optimization, the Anopheles gambiae Giles and Aedes albopictus were artificially fed on several vertebrate hosts blood using the Hemotek device for two hours under standard conditions. Our results showed intra-species reproducibility and inter-species specificity of MS spectra from mosquitoes engorged on the same or different vertebrate hosts. The MS spectra querying against the database reveal a correct identification of the the blood meal origin from the specimens collected less than 24 hours post-feeding. For field samples, MALDI-TOF MS allowed to detect the mosquitoes blood meal fed on wide variety of domestic hosts. Consequently the MALDI-TOF MS technique would be an effective tool for epidemiological surveys of vector-borne diseases and the identification of the trophic preference of mosquito freshly engorged.