Les entérovirus (EV) humains, membres de la famille des Picornaviridae, comprennent plus de 100 génotypes appartenant à 4 espèces : Enterovirus A, B, C et D. Ces virus sont à l’origine de pathologies très variées et occupent une place importante en santé publique. La méthode conventionnelle de typage des EV consiste en une réaction de séroneutralisation avec des antisérums spécifiques à partir de souches isolées en culture cellulaire ; cette technique est longue, coûteuse et limitée par sa capacité à identifier correctement les variants antigéniques et les nouveaux génotypes. De plus, elle est limitée aux génotypes cultivables. De nouvelles méthodologies de typage moléculaire par séquençage partiel du génome ont été récemment développées ; elles consistent à analyser une partie variable de la région codant une des protéines de capside (VP1 ou alternativement VP2 ou VP4). Cependant ces techniques sont le plus souvent réalisées à partir de souches isolées en culture cellulaire. Le but de ce travail a été de développer une technique de typage des EV directement sur des prélèvements cliniques en se basant sur le séquençage partiel de la région VP2 dont le laboratoire avait montré précédemment l’intérêt (Nasri et al., 2007). Pour le dessin des amorces, nous avons utilisé la stratégie CODEHOP (COnsensus DEgenerate Hybrid Oligonucleotide Primer) de manière à améliorer à la fois la spécificité et la sensibilité de la méthode d’amplification. Nous présentons ici un premier article décrivant la nouvelle technique de typage VP2 et rapportons son application au typage d’échantillons cliniques trouvés positifs par une PCR ciblant la région 5’ non codante du génome des EV sur une période de trois ans. Le deuxième article présente pour la première fois l’application d’une technique de typage direct à des échantillons environnementaux d’eaux usées. Le troisième article montre l’intérêt de coupler deux techniques de typage ciblant des régions différentes (VP1 et VP2) pour l’identification de souches d’EV isolées par culture cellulaire en Centre-Afrique. Malgré des problèmes de sensibilité, cette nouvelle technique de typage directement à partir d’échantillons peut rendre de grands services tant en clinique humaine que pour la surveillance environnementale / Human enteroviruses (EV), members of the Picornaviridae family, comprise more than 100 genotypes belonging to four species: Enterovirus A, B, C and D. These viruses are responsible for a wide range of pathologies and play an important role in Public Health. The classic method for typing EVs consists in a seroneutralisation assay with specific antisera using strains isolated by cell culture; this technique is cumbersome, expensive and unable to type currently antigenic variants and new serotypes. In addition, it is limitated to culturable serotypes. New methods of molecular typing by partial sequencing of the genome have been recently developed; they consist in analysing a variable part of the region coding for capsid protein (VP1 or alternatively VP2 or VP4). However, these techniques are usually performed on strains isolated by cell culture. The aim of this work was to develop a typing method able to work from clinical specimens by partial sequencing of the VP2 region, which had been shown to exhibit a good typing performance (Nasri et al., 2007). For the design of primers, we used the CODEHOP (COnsensus DEgenerate Hybrid Oligonucleotide Primer) strategy on order to improve the sensitivity and the specificity of the amplification assay. We present herein a first article that describes in details the new VP2 typing method and requests its use for typing clinical specimens found positive by a PCR assay targeting the 5’ non coding region of EVs over a period of three years. The second paper describes for the first time the direct use of a typing method on environtmental wastewater samples. The third article shows the interest of coupling 2 typing techniques targeting different regions (VP1 and VP2) of the EV genome for the identification of strains isolated by cell culture in Republic of Central Africa. Despite a loss of sensitivity, the new VP2 typing method used directly on specimens was found to be of great help both for human diagnosis and environmental surveillance

La spectrométrie de masse de type MALDI-TOF vient d'être largement adoptée dans les laboratoires de microbiologie clinique, qu'ils soient de ville ou hospitalier. Le MALDI-TOF est maintenant utilisée en routine pour l'identification des micro-organismes. Les micro-organismes sont identifiés en quelques minutes à un coût minime et avec une meilleure précision qu'en utilisant les méthodes conventionnelles. Le MALDI-TOF permet une identification microbienne révolutionnaire mais il ne permet pas aisément la détection de résistances aux antimicrobiens ou de facteurs de virulence. Le travail de thèse a eu pour objectif d'adresser cette problématique : est-il possible d'identifier, de typer un micro-organisme et de détecter sa résistance ou sa virulence par spectrométrie de masse ? Pour cela, l'utilisation d'une chromatographie liquide conventionnelle couplée à un spectromètre de type triple-quadripôle travaillant en mode SRM a été proposée. Nous avons démontré que la caractérisation complète des micro-organismes était possible à l'aide d'une seule méthode. La preuve de concept a été apportée pour des bactéries à Gram positive, à Gram négative et pour des levures. L'approche a été démontrée pour la détection des mécanismes majeurs de résistance aux bêta-lactamines, aux glycopeptides et aux azolés. La détection de toxines a également été démontrée. Fort de ces démonstrations, une évaluation clinique a été réalisée avec des souches et des hémocultures cliniques. La caractérisation de staphylocoques dorés, une des espèces capitales dans les maladies infectieuses du fait de sa prévalence, de sa sévérité et de sa résistance aux antibiotiques, a été engagée pour illustrer l'intégration de l'approche ESI-MS. La méthode SRM développée a permis en moins de deux heures d'obtenir des résultats en parfait accord avec les méthodes conventionnelles. Ce concept ouvre de nouveaux horizons pour la spectrométrie de masse en microbiologie clinique / Whole cell mass spectrometry based on MALDI-TOF technology has been broadly embraced in clinical microbiology. MALDI-TOF is routinely used for microbial identification. WC-MS is faster, more accurate and cheaper for large laboratories than conventional biochemical tests. MALDI-TOF revolutionizes microbial identification but the selection of the right antibiotic treatment still requires time consuming antibiotic susceptibility testing. Thesis work is dedicated to answer to this problematic : it is possible to perform identification of a micro-organism to the species or sub-species level and to detect its resistance or its virulence by mass spectrometry ? To this purpose, a conventional bore chromatography coupled to a triple-quadripole mass spectrometer in SRM mode is proposed. We show that complete microorganism characterization was possible thanks to only one method. A proof of concept was given for Gram negative, for Gram positive and for yeasts. The approach was extended to major resistance mechanisms to beta-lactamin, to glycopeptides and to azoles. Toxins detection was also proved. Driven by this success, a clinical evaluation was carried out based on strains and blood culture. Staphylococcus aureus was chosen to illustrate ESI-MS approach integration. This common Gram positive commensal bacteria is responsible for both community- and hospital-acquired infections. The developed SRM method allows obtaining results in less than two hours in perfect agreement with conventional methods. This concept opens new horizons for mass spectrometry in clinical microbiology

Structure and functional study of glycans are highly challenging due to the difficulties in analyzing glycans and limited availability of samples for study. These limitations could be resolved by attaching DNA barcode to the glycan, which virtually represent glycan in further application, by increasing the sensitivity of detection by polymerase chain reaction (PCR), requiring minimal samples for analysis. Assuming bigger arena of DNA Encoded Glycan Libraries (DEGL) in future, we propose here a method for uniquely coding all glycans using computer program that can convert the structural information of glycans to DNA barcode. A unique and universal coding for glycans will benefit both synthesis and analysis of DEGLs. As a proof of principle study, a small DNA Encoded Glycan Library (DEGL) of blood and globo series glycan antigen and its application was demonstrated in detecting blood group and breast cancer from plasma.

La sécurité alimentaire est basée sur des systèmes de cultures durables. Les agents phytopathogènes présentent un risque sérieux pour la stabilité de l'agriculture mondiale. Dans ce contexte, la biosurveillance des agents phytopathogènes s'avère indispensable afin de connaitre et de comprendre la répartition, les routes et les facteurs de dispersion des populations phytopathogènes, et de prendre les mesures adaptées pour limiter leur propagation. Le genre bactérien Xanthomonas comprend un ensemble d'espèces phytopathogènes-spécifiques s'attaquant a une large gamme d'espèces végétales dont certaines sont importantes pour la production agricole. Les deux espèces d'étude, i.e les pathovars de Xanthomonas oryzae (Xo) et Xanthomonas axonopodis pathovar manihotis (Xam), pathogènes respectivement du riz et du manioc, font partie du « top 10 » des bactéries phytopathogènes d'importance majeure dans le monde. Des travaux portant sur l''épidémiosurveillance de ces bactéries phytopathogenes doivent pouvoir être mis en place en routine. L'objectif est de typer et de relier ces souches bactériennes à différentes échelles géographiques, ainsi que de détecter et caractériser les épidémies de manière précoce. Pour ce faire, plusieurs approches de typage moléculaire à haut niveau de résolution ont été explorées. Des marqueurs moléculaires basés sur les loci VNTR (Variable Number of Tandem repeats) ont été étudiés. Chez X. oryzae, un outil MLVA-25 (Multilocus VNTR Analysis) pour le pathovar Xanthomonas oryzae pv. oryzicola (Xoc) et un outil MLVA-16 pour les trois lignées génétiques de X. oryzae ont été développés. L'étude par le MLVA-16 de populations de X. oryzae a permis de caractériser des complexes clonaux généralement associés à de nouvelles épidémies. La description de nouvelles souches de Xoc en Afrique Centrale et Afrique de l'Est indique une provenance vraisemblablement d'origine asiatique. Chez Xam, la recherche de loci VNTR polymorphiques sur 65 génomes de Xam complets a abouti à la description de seize loci VNTR robustes donc cinq ont été ensuite utilisés pour l'étude de populations de Xam dans les plaines de l'est Colombien. Cette dernière étude met en avant une structuration des populations de Xam selon les régions. Enfin, une méthode de spoligotypage associée aux locus CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats) et des marqueurs minisatellites ont été développés chez les souches du pathovar Xanthomonas oryzae pv. oryzae (Xoo). Les analyses préliminaires ont permis de définir la composition de la cassette CRISPR et de proposer des outils de spoligotypage utiles pour les souches asiatiques de Xoo. D'autre part, 18 marqueurs minisatellites ont indiqué une corrélation significative avec les races des souches et peuvent servir à l'étude plus large de populations Xoo philippines ou asiatiques. En conclusion, des nouvelles approches de typage moléculaire ont été évaluées, mises au point et employées avec succès pour étudier les bactéries pathogènes du riz et du manioc appartenant au genre Xanthomonas. / Food and agriculture safety rely on durable cropping systems. Consequently, phytopathogens pose a serious risk for durable agriculture in the world. In this context, surveillance of phytopathogens is a mandatory prerequisite in order to understand and to predict pathogen repartition, dispersion routes and factors, and to trigger appropriate measures to reduce the pathogen's propagation. The genus Xanthomonas displays a large diversity of host-specific plant-pathogenic species that infect a wide range of plant species, including commercially grown crops. The two studied species, i.e. the rice-pathogenic Xanthomonas oryzae and the cassava-pathogenic Xanthomonas axonopodis pathovar manihotis (Xam), belong to the top-10 of phytopathogenic bacteria and are thus of major interest. Routine epidemiological surveillance of these bacteria has to be achieved in order to type and link strains at different geographic scales as well as to characterize outbreaks and epidemics. For this purpose, several high-resolution molecular typing approaches were explored. Firstly, VNTR (Variable Number of Tandem repeats)-based molecular markers were studied. For X. oryzae, multilocus VNTR analyses (MLVA) were developed: MLVA-25 for the pathovar oryzicola (Xoc) and MLVA- 16 for the three known lineages of X. oryzae. A large population study of X. oryzae by MLVA-16 allowed us to characterize genetic clonal complexes, which were likely associated with new epidemics. Also, the novel description of Xoc strains from central and east Africa indicated their probable Asian provenance. For Xam, the exploration of polymorphic VNTR loci in 65 available genome sequences allowed the description of sixteen robust VNTR loci. Among them, five highly polymorphic loci were further used in a population study of Xam in the eastern plain of Colombia. The results provided evidence of a geographical Xam population structuration. Secondly, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-associated spoligotyping and minisatellites markers were explored for a largely divergent set of Philippine strains of Xanthomonas oryzae pv. oryzae (Xoo). Both approaches were compared to genome-wide SNPs and races. Preliminary studies identified the composition of CRISPR arrays, which could be useful for a spoligotyping approach. On the other hand, 18 minisatellites markers revealed a significant correlation with races and could be used for a larger study of Philippine or Asian Xoo populations. In conclusion, novel molecular typing approaches were successfully evaluated, implemented and used to study rice- and cassava-pathogenic bacteria of the genus Xanthomonas.

A literature review was done to find that there are still issues with writing passwords. From the information gathered, it is stated that using keystroke characteristics could have the potential to add another layer of security to compromised user accounts. The world has become more and more connected and the amount of people who store personal information online or on their phones has steadily increased. In this thesis, a solution is proposed and evaluated to make authentication safer and less intrusive. Less intrusive in this case means that it does not require cooperation from the user, it just needs to capture data from the user in the background. As authentication methods such as fingerprint scanning and facial recognition are becoming more popular this work is investigating if there are any other biometric features for user authentication.Employing Artificial Intelligence, extra sensor metrics and Machine Learning models with the user's typing characteristics could be used to uniquely identify users. In this context the Neural Network and Support Vector Machine algorithms have been examined, alongside the gyroscope and the touchscreen sensors. To test the proposed method, an application has been built to capture typing characteristics for the models to train on. In this thesis, 10 test subjects were chosen to type a password multiple times so that they would generate the data. After the data was gathered and pre-processed an analysis was conducted and sent to train the Machine Learning models. This work's proposed solution and presented data serve as a proof of concept that there are additional sensors that could be used to authenticate users, namely the gyroscope. Capturing typing characteristics of users, our solution managed to achieve a 97.7% accuracy using Support Vector Machines in authenticating users.

Aerosols show type-specific characteristics, which depend on intensive aerosol optical and microphysical properties that influence the radiation processes in the atmosphere in several ways. There are still large uncertainties in the calculation of the aerosol direct radiative effect. The classification of aerosols and the characterization of the vertical aerosol distribution is needed in order to provide more accurate information for radiative-transfer simulations. In the framework of the present thesis, the vertical and spatial distribution as well as optical properties of atmospheric aerosols over the European continent were investigated based on lidar measurements. Possibilities for an aerosol classification or so-called aerosol typing were presented and major aerosol types were specified. Former studies about the classification of aerosols were summarized and representative values for aerosol-type-dependent parameters were given. Case studies were used to demonstrate how observations of the European lidar network EARLINET from 2008 until 2010 were analyzed for aerosol layers and how model simulations and auxiliary data including the assessment of meteorological conditions were applied to determine the origin of each single aerosol layer. Thus, aerosol-type dependent parameters were evaluated and a novel method for the typing of aerosols was developed, which can be used, e.g., within algorithms of satellite data retrievals. Additionally, conversion factors were determined, which are needed for the harmonization of satellite data of present and upcoming missions. Furthermore, findings of the aerosol typing based on EARLINET data were compared to results of the aerosol classification scheme for satellite-borne lidar measurements onboard CALIPSO. It could be shown that deficient classifications of the aerosol type emerged systematically within the automated CALIPSO algorithm. Those wrong classification leads to an underestimation of the single-scattering albedo and hence to an overestimation of the warming effect of the respective aerosol layer. This overestimated warming effect has to be kept in mind for simulations of the global aerosol radiative effect based on CALIPSO data. / Die Bestimmung des direkten Strahlungsantriebs von Aerosolen ist mit großen Unsicherheiten behaftet. Inwiefern Aerosole die Strahlungsprozesse in der Atmosphäre beeinflussen ist abhängig von ihren optischen und mikrophysikalischen Eigenschaften. Zur Optimierung von Strahlungstransfersimulationen werden daher ergänzende Informationen über typspezifische Aerosoleigenschaften sowie die vertikale Aerosolverteilung benötigt. Im Rahmen der vorliegenden Arbeit wurden anhand von Lidarmessungen die vertikale und räumliche Verteilung atmosphärischer Aerosole über Europa analysiert sowie deren optische Eigenschaften ermittelt. Einleitend werden Möglichkeiten der Aerosolklassifizierung erläutert und Aerosoltypen spezifiziert, die über Europa beobachtet werden können. Vorherige Studien zur Aerosolklassifizierung sind in einer Literaturübersicht zusammengefasst. Anhand von Fallstudien wurde zunächst die Analyse von Beobachtungen des europäischen Lidarnetzwerkes EARLINET von 2008 bis 2010 auf das Vorhandensein von Aerosolschichten verdeutlicht. Die Herkunft jeder einzelnen Aerosolschicht wurde anschließend unter Verwendung von Modellrechnungen sowie weiteren Informationen bestimmt und aerosoltypspezifische Kenngrößen berechnet. Mit Hilfe dieser Kenngrößen ist es möglich, den Typ des Aerosols abzuleiten. Daraus wurde eine neuartige Methode zur Typisierung von Aerosolen entwickelt, die z.B. in Algorithmen zur Verarbeitung von Satellitendaten verwendet werden kann. Zusätzlich wurden Umrechnungsfaktoren bestimmt, die zur Zusammenführung und zum Vergleich von Daten aktueller und zukünftiger Satellitenmissionen benötigt werden. Die Ergebnisse der Aerosoltypisierung auf Basis von EARLINET-Daten wurden anschließend mit Ergebnissen der automatischen Typisierung weltraumbasierter Lidarmessungen des CALIPSO-Satelliten verglichen. Es konnte gezeigt werden, dass innerhalb des CALIPSO-Algorithmus systematisch fehlerhafte Klassifizierungen des Aerosoltyps auftreten. Diese falsche Klassifizierung führt zu einer Unterschätzung der Einfachstreualbedo und zu einer Überschätzung der erwärmenden Wirkung der betreffenden Aerosolschicht. Die überschätzte Wärmewirkung hat wiederum fehlerhafte Ergebnisse bei Strahlungstransferrechnungen, die auf CALIPSO-Daten basieren, zur Folge.

Cystic fibrosis (CF) is the most common autosomal recessive genetic disorder in Caucasians. Lower respiratory tract of CF patients is colonized by specific bacteria, often leading to chronic infection and lung tissue damage. In this thesis we characterized 338 isolates of S. aureus from 92 Czech CF patients isolated in 2011-2013. Using spa typing and PFGE we detected high clonal heterogenity of this collection with the exception of MRSA strains (resistant to oxacillin; 5% prevalence) which were clonally related. The prevalence of S. aureus MLSB resistance in our collection was high (69 %), which is a serious problem due to common usage of these antimicrobials in clinical practice. A half of the MLSB resistant strains lacked any known determinant of this resistance (ermA, ermC, ermT, msrA). Sequencing of the ribosomal genes revealed a high number of S. aureus strains carrying target site mutations resulting in MLSB resistance (37 %). This is new important information about the staphylococcal strains associated with chronic infections in Czech Republic. Focusing on mutability of analysed strains, we also detected several strains with point mutations or deletions in their mutator genes mutS a mutL. Hypermutability could be responsible for the high rate of ribosomal mutations and for the presence of...

To understand transmissible human diseases, disciplines such as epidemiology and the surveillance of affected cases are as essential as the knowledge about the pathogenesis and the course of a disease. Epidemiologists categorize and estimate factors for public health risks by taking metadata into account including geographic aspects, health and social states to study a disease transmission and prevent further cases. In addition, a focus on the causative agents itself is necessary in order to understand their ecology and hence their virulence traits. The causative agents for a severe pneumonia named Legionnaires’ disease (LD) are bacteria of the genus Legionella. The putative sources of LD infection are any aerosol-generating natural or man-made fresh water systems. Due to this ubiquitous distribution of legionellae, it is difficult to find the source of infection. Therefore, it is necessary to isolate the bacterium from the suffering patients to further characterize it in the laboratory and to compare the clinical isolates with isolates obtained from probable environmental sources. The predominant species isolated from LD patients is Legionella pneumophila serogroup (Sg) 1. Intensive genotyping of L. pneumophila Sg1 isolates by using the current gold standard method, the sequence-based typing scheme (SBT), revealed limitations in the discrimination of several sequence types (ST) which could not be compensated for by additional phenotypic typing scheme. In practical terms, this means that several clones or STs are disproportional frequently found in both, patients and water systems, and cannot be distinguished by current methods. Therefore, a distorted picture of endemic and globally-spread clones is generated and current typing methods cannot add substantial information during the identification of the infectious source. The aim of this thesis is to develop and implement new typing methods for L. pneumophila isolates with a higher resolution than the gold standard methods. A DNA-DNA hybridization based microarray was designed and equipped with probes that target specifically L. pneumophila virulence factors and genes that are involved in the biosynthesis of lipopolysaccharide structures. Legionellae can be subgrouped on the basis of their lipopolysaccharide structures. Here, the usually phenotypic characterization of L. pneumophila Sg1 is successfully transmitted to a DNA-based genotypic method. Furthermore, the detailed validation of the DNA-microarray revealed a higher discriminatory power in comparison to the gold standard methods. It enables previously indistinguishable clones to be subdivided, providing valuable information about probable sources of infection. The second new tool for typing of L. pneumophila is based on the core genome of the bacteria. An extended SBT-scheme was extracted from the core genome and accordingly named core genome multilocus sequence typing (cgMLST). This genome wide gene-by-gene typing approach allows a high genomic resolution of L. pneumophila isolates by retaining epidemiological concordance. A major advantage of this genome-based method is the detection of large recombination events within the analysed genomes, which is, so far, reserved for whole genome sequencing. The population structure of legionellae is largely driven by recombination and horizontal gene transfer rather than by spontaneous mutations. Therefore, the detection of recombination events is essential for typing of L. pneumophila isolates. In addition, the cgMLST-scheme assigns a core genome sequence type to the analysed isolate and allows backwards compatibility with the current SBT-scheme. Both methods proved to be fast, reliable and robust typing methods through their application during outbreak investigations. Furthermore, both systems are particularly suited as routine molecular typing tools for the surveillance of single cases. The raw data are verified and translated into uniform portable codes, which enables the easy transfer and comparison of results. The standardized and portable quality of the results of both methods enables the establishment of a curated global database. This qualifies both methods as potential new gold standard methods for the genotyping of L. pneumophila isolates.

Both dependent types and linear types have their desirable properties. Department types can express functional dependencies of inputs and outputs, while linear types offer control over the use of computational resources. Combining these two systems have been difficult because of their different interpretations of context presence of variables. Quantitative Type Theory (QTT) combines dependent types and linear types by using a semiring to track the kind of use of every resource. We extend QTT with the additive pair and additive unit types, express the complete QTT rules in bidirectional form, and then present our interpreter of a simple language based on QTT. 1

Abstrakt Syftet med denna studie är att bidra med kunskap om lärares uppfattning och förståelse om och för handskriftens betydelse för elevers finmotoriska utveckling i svenskundervisningen i årskurs 1 – 4.  Detta för att belysa vikten av att fortsätta använda handskriften i skrivinlärningen. I denna studie har en kvalitativ metod använts vid intervjuer av respondenterna. Studiens teori baseras på det sociokulturella perspektivet och pragmatismen. En fenomenografisk forskningsansats har använts i studiens bearbetning, tolkning och analys av den insamlade datan. Resultatet har diskuterats mot bakgrund och tidigare forskning samt utifrån det sociokulturella perspektivet och pragmatismens syn på lärande. Resultatet visar att oavsett vilken metod lärare väljer att använda, handskrift eller tangentsinlärning, behöver eleverna olika övningar för att träna handen och finmotoriken, eftersom eleverna behöver kunna forma bokstäverna för att kunna skriva enkla texter med en läslig handstil. I resultatet framkommer det även att några lärare använder en form av ASL-metod i sin undervisning och är eniga om att handskriften behöver användas parallellt med tangentbordet. / Abstract The purpose of this study is to contribute to the knowledge about teachers´perception and understanding about the significance of the use of handwriting for the development of students' fine motor skills in the Swedish language classroom, in primary school during years 1 – 4. This is to highlight the importance of the continued use of handwriting when learning to write. In this study, a qualitative method was used in the interviews with the respondents. The study's theory is based on the sociocultural perspective and pragmatism. A phenomenographical research approach has been used in the processing, interpretation and analysis of the collected data. The results have been discussed in the context of previous research and based on socio-cultural and pragmatism's views on learning. The results show that regardless of which method teachers choose to use, handwriting or typing, students need complementary exercises to train the hand and the fine motor skills, as students need to be able to shape the letters to be able to write simple texts in a readable handwriting. The results also show that some teachers use some form of iWTR method in their teaching and they agree that handwriting needs to be used in parallel with a keyboard.