Les canalopathies cardiaques congénitales constituent la principale hypothèse diagnostique dans les cas de mort subite inexpliquée chez les sujets de moins de 35 ans. Notre travail a eu pour objectif demettre au point une stratégie de détection post-mortem des mutations sur les gènes connus pour être impliqués dans les canalopathies cardiaques, applicable en routine, à partir de la principale source d’ADN post-mortem disponible en France à savoir les prélèvements fixés au formol et inclus en paraffine (FFIP). A partir d’une cohorte de 12 cas, deux techniques de détection de variants génétiques ont été évaluées, une technique de criblage par l’analyse des courbes de fusion haute résolution et une technique de génotypage par spectrométrie de masse MALDI-TOF, respectivement sur le gène KCNQ1 et le gène RyR2. Quelle que soit la technique utilisée, il n’est pas possible de s’affranchir du séquençage de type Sanger afin d’explorer les séquences d’intérêts qui n’ont pu être optimisées avec l’une ou l’autre des méthodes à la fois sur les prélèvements congelés et FFIP. L’arrivée des séquenceurs de nouvelles générations ouvrent ainsi de nouvelles perspectives dans ce domaine. / The congenital cardiac channelopathies constitute the principal diagnostic hypothesis in autopsynegative sudden unexplained death concerning people younger than 35 years old. The present study aimed to develop a strategy of mutations detection on known genes implicated in the cardiac channelopathies. This strategy of mutations detection had to be applicable to routine and has been studied on formalin-fixed and paraffin-embedded (FFPE) tissues which are the principal DNA source available in France. On a cohort of 12 cases, two technique of sequence variants detection wereevaluated: the screening method of High Resolution Melt and the genotyping method based on a MALDI-TOF mass spectrometry, respectively on KCNQ1 and RyR2 genes. Whatever the technique, there is a necessity of resorting to the Sanger sequencing to explore the sequence of interest none optimized with one or the other technology both on FFEP and frozen tissues. That’s why the next generation sequencing method should open new perspectives in the post-mortem diagnostic of cardiac channelopathies.

A espectrometria de massas baseada na tecnologia MALDI-TOF (do inglês, matrix-assisted laser desorption ionization-time of flight) (MALDI-TOF MS) tem sido cada vez mais incorporada à rotina de identificações microbiológicas nos laboratórios farmacêuticos de controle de qualidade, principalmente para as atividades do Programa de Monitoramento Ambiental de Salas Limpas. Isso porque o longo tempo necessário para a obtenção dos resultados por meio de métodos convencionais tem incentivado a procura por técnicas que permitam métodos rápidos. O objetivo deste trabalho foi avaliar a adequação da técnica MALDI-TOF MS para a identificação de bactérias isoladas do ambiente de salas limpas utilizadas em algumas etapas da produção de uma vacina viral. Treze espécies bacterianas conhecidas, normalmente isoladas das salas limpas estudadas, e cinco cepas ATCC foram identificadas pela técnica MALDI-TOF MS e por uma técnica bioquímica (BBL Crystal®). O desempenho da técnica MALDI-TOF MS foi superior ao da técnica bioquímica na identificação correta das espécies bacterianas (88,89% e 38,89%, respectivamente) e produziu menos identificações não confiáveis (5,55% e 22,22%, respectivamente). Os resultados evidenciaram que a técnica MALDI-TOF MS pode ser implementada para identificação rotineira de bactérias em um laboratório de controle de qualidade farmacêutico. Entretanto, a dependência de bases de dados exige estudos adicionais de isolados não identificados e, se apropriado, a adição destes a uma base de dados interna. O aperfeiçoamento de métodos de identificação microbiana é muito relevante no contexto de salas limpas, pois permitem ações corretivas e proativas essenciais para garantir a segurança microbiológica do processamento asséptico. / Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has been increasingly introduced in routine microbiological identifications of pharmaceutical quality control laboratories, mainly for the activities of the Environmental Monitoring Program of Clean Rooms. The long time needed to obtain the results through conventional methods has stimulated the search for techniques that allow rapid methods, as MALDI-TOF MS. Thus, the objective of this work was to evaluate the suitability of the MALDI-TOF MS technique for the identification of bacteria isolated from the environment of clean rooms used in some stages of the production of a viral vaccine. Thirteen bacterial species commonly isolated from clean rooms studied and five strains ATCC were identified by MALDI-TOF MS technique and by a biochemical technique (BBL Crystal® System). Performance of MALDI-TOF MS was better than biochemical technique for correct species identifications (88.89% and 38.89%, respectively) and produced fewer unreliable identifications (5.55% and 22.22%, respectively). MALDI-TOF MS can be implemented for routine identification of bacteria in a pharmaceutical quality control laboratory. However, as a database-dependent system, maybe some isolated not identified by this technique must be additionally studied and, if appropriate, added to an in-house database.

Fatty acids (FAs) represent an important class of metabolites, impacting on membrane building blocks and signaling compounds in cellular regulatory networks. In nature, prokaryotes are characterized with the most impressing FA structural diversity and the highest relative content of free fatty acids (FFAs). In this context, nitrogen-fixing bacteria (order Rhizobiales), the symbionts of legumes, are particularly interesting. Indeed, the FA profiles influence the structure of rhizobial nodulation factors, required for successful infection of plant root. Although FA patterns can be assessed by gas chromatography—(GC-) and liquid chromatography—mass spectrometry (LC-MS), sample preparation for these methods is time-consuming and quantification suffers from compromised sensitivity, low stability of derivatives and artifacts. In contrast, matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) represents an excellent platform for high-efficient metabolite fingerprinting, also applicable to FFAs. Therefore, here we propose a simple and straightforward protocol for high-throughput relative quantification of FFAs in rhizobia by combination of Langmuir technology and MALDI-TOF-MS featuring a high sensitivity, accuracy and precision of quantification. We describe a step-by-step procedure comprising rhizobia culturing, pre-cleaning, extraction, sample preparation, mass spectrometric analysis, data processing and post-processing. As a case study, a comparison of the FFA metabolomes of two rhizobia species—Rhizobium leguminosarum and Sinorhizobium meliloti, demonstrates the analytical potential of the protocol.

Penicillium nordicum è un importante contaminante di salumi, rappresentanando il 10 % e il 26 % della popolazione di Penicillium spp . isolati , rispettivamente dall'aria e dai prodotti carnei stagionati in un'indagine gestita in Italia ( Battilani et al. , 2007). Diverse colonie di P. nordicum isolate dai salumi hanno dimostrato di essere importanti produttori di ocratossina A , OTA ( Sansom e Frisvad , 2004 . Pietri et al, 2006 ; . Battilani et al , 2010). Attualmente, l'impostazione appropriata delle condizioni ambientali (temperatura, umidità relativa e circolazione dell'aria ), è l'unico strumento accettato per impedire la crescita incontrollata di P. nordicum all'interno degli impianti di stagionatura attraverso una accurata analisi dei punti critici di controllo e l’ideazione di un relativo piano HACCP (Hazard Analysis and Critical Control Points) ben struttutato ( Asefa et al , 2011; Virgili et al , 2012). Anche se il sistema HACCP è stato applicato con successo nel settore alimentare ci sono rischi per la sicurezza alimentare non attentamente considerati. Questo è particolarmente vero per quanto riguarda i rischi micotossigeni associati ai prodotti alimentari di origine animale. Il termine "rischi micotossigeni" è utilizzato da Asefa et al. ( 2011) per descrivere lieviti patogeni e metaboliti secondari tossici prodotti da specie fungine tossigene che contaminano i prodotti alimentari e incidono sulla sicurezza alimentare. La maggior parte dei piani HACCP nelle attività di trasformazione alimentare, come ad esempio la produzione di formaggi e di prodotti carnei stagionati, tiene in considerazione principalmente il rischio derivante da agenti batterici (Arvanitoyannis e Mavropoulos, 2000; Barbuti e Parolari, 2002) anche se tali prodotti alimentari vengono spesso contaminati da funghi micotossigeni e dai loro metaboliti (Spotti et al 1989; Spotti et al , 2001a; Battilani et al 2007). Pertanto, dovrebbe essere cruciale definire un piano HACCP specificamente incentrato sui rischi micotossigeni. L'identificazione, il controllo e la standardizzazione della micoflora superficie dei salumi è fondamentale per preservare la sicurezza delle produzioni e la salute dei consumatori . Questo è il contesto in cui deve essere valutata l’efficacia e l’affidabilità per l’identificazione delle popolazioni di Penicillium spp di interessante per la produzione alimentare. In questo contesto , il progetto di ricerca di questa tesi di dottorato ha cercato di approfondire le conoscenze su tali tematiche con l'intento di limitare il rischio micotossigeno nella catena di produzione dei prodotti carnei stagionati. Sono stati affrontati i seguenti argomenti: 1 . studio della composizione e dinamica della microflora fungina presente sulla superficie dei salumi (prodotto testato, salame) e l'aria di ambienti di stagionatura tenendo conto dell'influenza di alcuni parametri di processo (inoculo starter, temperatura, fase produttiva). 2 . sviluppo di un metodo MALDI TOF MS per l'identificazione di Penicilium a livello di specie per le prospettive future di screening diretti della microflora presente sui salumi. 3 . confronto e integrazione di diverse tecniche, come l'analisi morfologica, l’analisi molecolare e l’analisi tramite spettrometria di massa, per l'identificazione delle specie di Penicillium presenti nei salumi. 4 . valutazione dei lieviti selezionati, isolati dalla superficie di prosciutto crudo, per competere con P. nordicum ed inibire l'accumulo di OTA nella prospettiva del loro uso come starter superficiali con funzione di agenti di biocontrollo. / Penicillium nordicum is an important contaminant of cured meat products, representing 10% and 26% of the Penicillium spp. isolated, respectively, from the air or the products in a survey managed in Italy (Battilani et al., 2007). Several P. nordicum cured meat isolates proved to be important producers of ochratoxin A, OTA (Sansom and Frisvad, 2004; Pietri et al., 2006; Battilani et al., 2010). Currently, the appropriate setting of environmental conditions (temperature, relative humidity and air circulation), is the only accepted tool to prevent the uncontrolled growth of P. nordicum inside dry-curing plants through a carefully structured Hazard Analysis Critical Control Point (HACCP) plan (Asefa et al., 2011; Virgili et al., 2012). Even if the HACCP system has been successfully applied in the food industry, there are food safety hazards not carefully considered. This is especially true with regard to mycotoxigenic hazards associated with animal food products. The term “mycotoxigenic hazards” is used by Asefa et al. (2011) to describe pathogenic yeasts and toxic secondary metabolites of toxigenic moulds that contaminate food products and affect food safety. Most HACCP plans in food processing activities, such as the production of cheese and dry-cured meat products, considered mainly bacterial agents (Arvanitoyannis and Mavropoulos, 2000; Barbuti and Parolari, 2002), even if such food products get often contaminated with mycotoxigenic fungi and their metabolites (Spotti et al 1989; Spotti et al., 2001a; Battilani et al 2007). Therefore, it should be crucial to define a HACCP plan specifically focused on the mycotoxigenic hazards. The identification, control and standardization of the surface mycoflora of cured meat products is mandatory to preserve the productions safety and the consumers health. This is the context of the effectiveness and reliability evaluation for the Penicillium spp. identification methods of interesting species for food production. In this context, the research project of this PHD thesis tried to fill some gaps of knowledge with the attempt to limit the mycotoxigenic risk in the cured meat products chain. The following topics were faced: 1. study of the composition and dynamic of fungal microflora present on the surface of cured meat products (salami) and the air of seasoning environments taking into account the influence of some process parameters (starter inoculum, curing temperature, stage of seasoning). 2. development of a MALDI TOF MS method for the identification of Penicilium at species level for future direct screening perspectives of the microflora present on cured meat products. 3. comparison and integration of different techniques, as morphological, molecular and mass spectral analysis, for the identification of Penicillium species in cured meat products. 4. evaluation of selected yeasts, isolated from dry-cured ham surface, to compete with P. nordicum and to inhibit OTA accumulation in the perspective of their use as surface starter biocontrol agents.

Antalet monoklonala antikroppar som används i läkemedel ökar kraftigt. Dessa läkemedel är dyra och risken för förfalskning är stor. Behovet att utveckla en metod för snabb och precis identifiering av monoklonala antikroppar är därför brådskande. För identifiering utfördes analyser med Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-ToF-MS), Capillary Gel Electrophoresis (CGE) and Capillary Zone Electrophoresis (CZE) på nio monoklonala antikroppar. Fokuset var att undersöka huruvida signifikanta fysiokemiska egenskaper och unika aminosyrasekvenser var närvarande och kunde urskiljas. Olika analyser med MALDI-ToF-MS användes till att både separera de monoklonala antikropparna baserat på dess fysiokemiska egenskaper, och annotera aminosyrasekvenser innehållande nyckelfragment. Med metoderna baserade på kapillärelektrofores uppnåddes också separation. CZE föredras framför CGE då mängden data som erhålls från CZE är större och provberedningen är enklare. Sammanfattningsvis utformades ett protokoll för identifieringsprocessen, vilket inleds med MALDI-ToF-MS-analyser av monoklonala antikroppar på reducerad form mot kända referenser. Därefter är en hypotes formulerad utifrån vilka antikroppar som ser mest lika ut. Slutligen analyseras dessa med CZE för fastställning av den monoklonala antikroppens identitet. / The number of monoclonal antibodies used in pharmaceuticals is increasing sharply. These medicines are expensive, and the risk of counterfeiting is high. The need to develop a method for rapid and precise identification of monoclonal antibodies is therefore urgent. For identification, analyses were performed with Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-ToF-MS), Capillary Gel Electrophoresis (CGE) and Capillary Zone Electrophoresis (CZE) on nine monoclonal antibodies. The focus was to investigate whether significant physiochemical features and unique amino acid sequences were present and could be distinguished. Various analyses with MALDI-ToF-MS were used to both separate the monoclonal antibodies based on their physicochemical properties and annotate amino acid sequences containing key fragments. With the methods based on capillary electrophoresis, separation was also achieved. CZE is preferred over CGE as the amount of data obtained from CZE is greater and sample preparation is simpler. In summary, an identification process protocol was designed and is initiated with MALDI-ToF-MS analyses of reduced-form monoclonal antibodies against known references. A hypothesis is then formulated based on which antibodies look the most similar. Finally, these are analysed by CZE to determine the identity of the monoclonal antibody.

Ein kompetitiver ELISA (Enzyme-linked Immunosorbent Assay) für den Nachweis von Carbamazepin (CBZ) mit einer Bestimmungsgrenze von ca. 30 ng/L wurde entwickelt und validiert. Dieser in Gewässern häufig auftretende anthropogene Marker wurde anschließend in einer Vielzahl an Proben aus Abwässern, Oberflächengewässern und Trinkwässern nachgewiesen. Der ELISA zeigte eine exzellente Präzision und erbrachte in allen Matrizes geringfügig höhere Analysenergebnisse als die Referenzmethode HPLC-MS/MS. Die beständige Überbestimmung der CBZ-Konzentration in Höhe von ca. 7 % konnte auf die Präsenz von Cetirizin und geringen Mengen des persistenten Metaboliten 10,11 Epoxy¬carbamazepin (EP-CBZ) zurückgeführt werden. Die Bindungseigenschaften des verwendeten Antikörpers wurden anhand der Kreuz¬reaktivi¬täten von 37 Substanzen eingehend untersucht. Nach Kopplung von Flüssig¬chromato¬graphie und ELISA konnte das strukturell nicht mit CBZ verwandte Anti¬histaminikum Cetirizin als Kreuzreaktand identifiziert werden. Der störende Einfluss dieses Kreuz¬reaktanden auf den CBZ-ELISA konnte nach einer Änderung des pH-Wertes im Proben¬puffer minimiert werden. Die pH-abhängige Selektivitätssteuerung ermöglichte überdies die Entwicklung eines Dual-Analyt-Immunoassays für die parallele Bestimmung von CBZ und Cetirizin. Darüber hinaus wurden die Metaboliten EP-CBZ, DiOH-CBZ, 2-OH-CBZ, 3-OH-CBZ und 10 OH-CBZ in Abwasser, Oberflächenwasser und Trinkwasser quantifiziert. DiOH-CBZ erwies sich als ähnlich persistent wie CBZ und wurde in besonders hohen Konzentrationen gefunden. Außerdem wurden mehrere weitere bislang nicht identifizierte Abbauprodukte von CBZ gefunden. Da weder Probenvorbereitung noch Probenanreicherung erforderlich sind, ist der Test schnell und kostengünstig durchführbar. Die für den Test nötigen Probenvolumen sind mit weniger als 1 mL sehr gering. Diese Eigenschaften erlauben ein Hochdurchsatzscreening und machen die Methode interessant für den Einsatz im Gewässermonitoring. / A competitive ELISA (enzyme-linked immunosorbent assay) for the quantitation of carbamazepine (CBZ) was developed and validated. A limit of quantitation (LOQ) of ca. 30 ng/L allowed for the quantitation of CBZ in many samples from wastewater, surface water and drinking water. The method was found to be excellently precise, but it displayed slightly higher results than obtained by the reference method liquid chromatography-tandem mass spectrometry (LC-MS/MS). The nearly constant overestimation of 7 % could be attributed to the presence of small amounts of cetirizine and the persistent metabolite 10,11 epoxy¬carbamazepine (EP-CBZ). The binding properties of the antibody were studied by determining the cross-reactivities of 37 compounds. Hyphenating liquid chromatography to ELISA led to the discovery of the cross-reactive antihistamine cetirizine that shares no obvious structural similarity with CBZ. The bias caused by cetirizine was eliminated by changing the pH value of the sample buffer. Moreover, the antibody’s pH-dependent selectivity enabled a dual-analyte immunoassay for the parallel determination of CBZ and cetirizine. Furthermore, the metabolites EP-CBZ, DiOH-CBZ, 2-OH-CBZ, 3-OH-CBZ and 10-OH-CBZ were quantified in wastewater, surface water and drinking water. DiOH-CBZ showed the highest concentrations of all analaytes investigated and was found to be equally persistent as CBZ. In addition, several further degradation products of CBZ were found that could not be identified. The ELISA allowed the detection of diurnal and seasonal fluctuations of analyte concentrations in wastewater and surface water. The anthropogenic marker CBZ enabled to trace wastewater from the source to the receiving waters. Since neither sample pretreatment nor enrichment is necessary, the method is very fast and cost-effective. Only a small sample volume (less than 1 mL) is needed making this ELISA an appropriate high-throughput screening tool for environmental monitoring.

(Doctoral Thesis, 2016, Mgr. Tomáš Faukner, IONIC POLYACETYLENE TYPE POLYMERS AND POLYMER NETWORKS BY CATALYST FREE QUATERNIZATION POLYMERIZATION) The composition and structure of a series of ionic π-conjugated poly(monosubstituted acetylene)s prepared via catalyst-free quaternization polymerization (QP) of 2-ethynylpyridine (2EP) activated with equimolar amount of alkyl halide [RX = ethyl bromide, ethyl iodide, nonyl bromide and haxadecyl (cetyl) bromide] as a quaternizing agent (QA) have been studied in detail. The performed QPs gave ionic polymers well soluble in polar solvents, with approximately half of pyridine rings quaternized, which implies that also non-quaternized monomers were involved in the process of QP. The configurational structure of polyacetylene main chains was suggested based on 1 H NMR, IR as well as Raman (SERS) spectral methods. The QPs in bulk gave more expected irregular cis/trans polymers while the QPs in acetonitrile solution gave high-cis polymers. A series of prepared symmetrical bi-pyridylacetylene based monomers has been polymerized via QP approach resulting into a series of new ionic π-conjugated poly(disubstituted acetylene) type materials. It is therefore obvious that the mechanism of quaternization activation frequently applied on monosubstituted...

The theoretical part of work is focused on the issue of biomass which can be used for energy purposes, inparticular agricultural waste, as well as can serve as a substrate for biogas station. It also deals with proteomics, its goals and approaches, separation methods. The aim of this work was to measure each sample of enzyme activity of biomass, which are used as a raw materials for biogas plants and their proteomic identification.