Abstract Rapid and reliable identification of microorganisms is of paramount importance for advancing homeland security. Mass spectrometry is emerging as an effective bioanalytical tool having unique capabilities in handing complex mixtures. Matrix-assisted layser desorption / ionization time-of-flight mass spectrometry ¡]MALDI/TOF MS¡^is commonly used in the analysis of bio-molecules owing to its merits of high sensitivity, wide mass range, and ease-of-operation. The objective of the first part of the thesis is to develop an analysis method to characterize the genus, strains, and subspecies of the infections gastroenteritis bacteria by using MALDI/TOF MS. MALDI/TOF MS can be applied to classify different microoganisms based on examination of ion patterns from different microoganisms and unique distinguishing protein ions as biomarkers for different strains. The second section, bacteriophages MS2 and T4 were used as materials for identification of their specific protein markers. Protocols including sample preparation, purification, combined in gel digestion and post source decay to which the mass spectrometer brought for analytical specificity were employed. According to the experimental results, classification of different microorganisms based on examination of ion patterns is feasible. Furthermore, the peak at m/z 5255 in the MALDI mass spectra of MS2 analysis and the peak at m/z 8600 of T4 can provide the unique distinguishing signals. The experimental mass spectral peaks were submitted to the database search, and one of these peaks was matched to a tail fiber assembly helper protein. Thus, the method developed and the mass spectra presented in this thesis can be potentially applied to the practical virus analysis.

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The aim of this study was to optimize the sample preparation and methods for analysis of oligosaccharides of hyaluronic acid with MALDI- TOF-MS. The analysis was carried out on an Autoflex speed MADLI-TOF- MS instrument with both linear and reflectron mode. Matrices used in this study were 2,5-DHB and Super-DHB and type of matrix was chosen depending on the size of the analyzed oligosaccharides. The application of sample and matrix on the target that gave the most homogenous crystallization was sandwich and the laser power in the MALDI was kept at 65 %. Since it is known that salts and buffers interfere with the analysis, sample clean-up such as solid phase extraction (SPE) in pipette tips and dialysis was performed. SPE worked best for low mass oligosaccharides and provided high intensity and little noise. With SPE a concentration of the analyte could be done which was the advantage over dialysis. Dialysis worked well for larger oligosaccharides and mixtures of different sized oligosaccharides. Another way of using MALDI for biomolecule analysis is with TLC-MALDI. A fast and accurate separation was achieved and analysis could be done directly from the plate. The optimized methods were evaluated according to linearity and precision, LOD, mass accuracy and matrix stability. The linearity and precision was good in a higher concentration range (50 μg/mL and higher), but the test for limit-of-detection (LOD) indicated that concentrations from 20-30 μg/mL could be analyzed with no interference from the background. The mass accuracy was within the acceptable limits according to Bruker Daltonics when a mass calibration was done for each analyzed sample. The stability of the matrix in solution was difficult to study because of the day-to-day variation in intensities given by the MALDI-TOF-MS technique.

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Geólogo / La nanogeociencia así como la mineralogía ambiental son áreas de investigación que gracias a nuevos avances tecnológicos se están expandiendo rápidamente en múltiples direcciones enfocadas en la detección, caracterización y comprensión de fases no cristalinas, pobremente cristalinas y fases mixtas amorfo-cristalinas, así como también materiales nanométricos. En este contexto, los procesos no clásicos de nucleación y crecimiento cristalino presentes en soluciones acuosas representan un campo emergente que surge como alternativa al enfoque clásico con el cual ha sido abordado este tema (diferentes etapas de unión de monómeros básicos -átomos, iones o moléculas- entre sí). Estos procesos utilizan polímeros y nanopartículas más pequeñas como unidades básicas precursoras del crecimiento cristalino. La formación de nanopartículas de aluminio (comunes tanto en entornos naturales como procesos industriales), y más concretamente la precipitación de hidrobasaluminita (Al4SO4(OH)10 · 12-36H2O), puede ser considerada como el proxy perfecto para el estudio de estos procesos no clásicos. Para caracterizar las especies poliméricas generadas durante la síntesis acuosa de nanopartículas de hidrobasaluminita la metodología utilizada en este trabajo es el análisis mediante ionización por electrospray acoplada a un espectrómetro de masas de tiempo de vuelo (ESI-TOF/MS). Esta técnica fue aplicada a cuatro soluciones de Al2(SO4)3 · 18H2O resultando en la identificación de 38 especies poliméricas catiónicas, ratificando la utilidad de esta técnica en el estudio de estos procesos no clásicos de cristalización permitiendo generar un primer volumen de datos para la elaboración de una base de datos con la masa y la fórmula de los polímeros de aluminio más comunes detectados en el sistema acuoso Al-S-O-H. Los resultados mostraron una especiación variable, donde las especies reconocidas abarcan polímeros basados en Al, Al2, Al3, Al4, Al5, Al6 y algunos más complejos como Al11 o Al13. Dentro de los parámetros estudiados el efecto del pH fue el más notable de todos. Observándose una clara dependencia de este factor en la formación de complejos de mayor tamaño y carga. Por otra parte, la concentración de Al en la solución también mostró injerencia sobre la especiación de las muestras. Finalmente, la metodología y resultados obtenidos, a pesar de las limitaciones asociadas, permiten acercarse a una caracterización más certera de los procesos que rigen la precipitación de hidrobasaluminita y la naturaleza de las especies que participan en éstos.

Yeasts of genus Cryptococcus belong to the Basidiomycetes, a wide group with different geographical sharing in nature. Many of them rank among human pathogens that endanger immunocompromised patients. Thanks to the big diversity at the level of subspecies, various species of the genus Cryptococcus show different molecular characteristics. Their identification is difficult because of the presence of polysaccharide capsule that surrounds the cell of some species. This work deals with the identification of species occurring in meadows and gardens. The 79 yeasts samples are identified by MALDI-TOF MS. The influence of different culture media on the identification results is monitored simultaneously with the identification. Since a capsule is present in many species, another parameter to be monitored is the influence of the sample preparation method and the matrix on the identification. 51 samples of yeasts of the genus Cryptococcus are identified at the species level in the experimental part. Selected samples are further subjected to the determination of characteristics by conventional microbiological methods. The determined parameters are the presence of urease, radial growth constant, susceptibility to antimycotics, fermentation and assimilation of sugars, growth in the presence of alcohols and growth in the absence of vitamins. The yeast samples are classified into yeast species based on microbiological results. Biotyping resulted in identification of selected samples in the species Filobasidium magnum, Filobasidium oeirense and Filobasidium wieringae. Other samples showed ambiguous identification. As these species have the same properties, they could not be distinguished by the selected microbiological tests.

Cryptococcus laurentii has been classically considered a saprophytic species, although several cases of human and animal infection have been already reported. This species is reported to be heterogenous. The taxonomy of yeast Cryptococcus laurentii was always highly ambiguous. The application of molecular biology and bioinformatic methods led to dividing of searched strains to two distinct phylogenetic groups, some varieties were recognized as species and the locution „Cryptococcus laurentii group“ was introduced. The taxonomy of this group is likely not definitive and with advancing knowledge will change. Our aim was the identification of individual species within this group based on matrix-assisted laser desorption/ionization – time of flight mass spectrometry (MALDI – TOF MS), which has recently been described as a rapid, reliable, cost-effective and powerful tool for analyzing of microorganisms, even on variety level. Generally, the yeasts of genus Cryptococcus form highly resistant polysaccharide capsules and produce large amount of extracellular polysaccharides, therefore belong to so called „difficult“ cases for biotyping. The experimental protocol has been optimized for MS analysis of this genus on the selected strains of Cr. neoformans, Cr. laurentii and Cr. magnus from the Culture Collection of Yeasts (CCY).Thirty-three strains, originally classified as Cryptococcus laurentii has been identified by chosen method. These strains were distributed into six different groups according their spectra similarities. It was selected at least one strain of each group, which was classified based on the sequence analysis of the D1/D2 domains of the LSU rRNA gene. This strain (with known sequence) became representative for its group. Type strain of Cryptococcus laurentii (CCY 17-3-2) belongs to the group I. Its MS spectrum of ribosomal proteins differ from mass spectra of all other biotyped species, even with strains identified as Cryptococcus laurentii was the similarity of the spectra low, which could be caused by identification of two different varieties. The group II is represented by Cryptococcus laurentii CCY 17-3-17. Except this strain, thirteen more strains belong to the group II. The group III represents Cryptococcus flavescens CCY 17-3-29. This group included 12 additional strains with almost identical mass spectra. Group IV included only one strain (CCY 17-3-13), which was identified as Cryptococcus carnescens based on gene sequence analysis. Similarly, one representative (CCY 17-3-5) has the group V. Strain CCY 17-3-5 was identified as Cryptococcus flavus. The last group VI of three members represents strain 17-3-35 identified as Bulleromyces albus. While Cr. laurentii and Cr. flavescens belong to phylogenetic group I and Cr. carnescens to the phylogenetic group II, four strains giving two types of different MS spectra and identified as Cr. flavus (1 strain) and Bulleromyces albus (3 strains) were excluded from „Cr. laurentii group.“

The goal of this thesis is to determine if Sinorhizobium meliloti can be useful as a sentinel soil microorganism for assessing the impacts of contaminant stressors on the metabolome of a microorganism. Not only is a good deal known about this organism, but it is an important organism in agriculture. Moreover, the currently available gene array and a large library of gene fusion can be used as facile pathways to explore genetic and genomic impacts in addition to metabolomic impacts of contaminants, should such studies be deemed worthwhile. In this study, the polar metabolome of the soil microorganism, Sinorhizobium meliloti, has been analyzed by LC-ESI-MS using a HILIC column coupled to a medium mass resolution time-of-flight mass spectrometer. This approach has resulted in the retention (k' > 0.7) of over 300 polar metabolites as detected in both positive ion and negative ion modes. These data do not include ions corresponding to adduct ions, isotopic features or ions resulting from in-source decay processes. The retained peaks showed excellent linear responses and did not suffer from ion suppression, a common problem in flow-injection ESI analysis. This methodology has been applied to the analysis of S. meliloti exposed to fluorene, a common PAH contaminant, and to a coal tar fraction containing low molecular mass PAHs. Multiple cultures of S. meliloti were grown on M9 glucose minimal medium in the absence and presence of fluorene (0.14 mg/L and 1.4 mg/L) and a PAH mixture (total PAH concentrations of 0.14 mg/L and 1.4 mg/L). Analyses of biological replicates were performed in pentuplicate. The retention times of the resulting chromatograms were aligned, peak areas determined and the resulting data processed using PCA and OPLS-DA methods. The retention time reproducibilities of peaks were within ± 10 seconds and the biological variabilities of over 700 components averaged 23% ± 15% (n=25) . The impacts of fluorene exposures and PAH mixture exposures on the S. meliloti metabolomes (polar) caused significant changes in the metabolome. The lower concentration exposures had less of an impact than the higher dosages. Low dosages of both fluorene and the PAH mixture produced a similar metabolic response in S. meliloti, while at higher dosages the responses were more specific to each toxin. The use of SUS plots coupled with S-plots of the OPLS-DA analysis were particularly advantageous for the identification of metabolites of interest. Changes were seen in the levels of adenine, adenosine, glutamate, and aspartate, among others. In the future, the profiles of the non-polar metabolites of each of sample will be analyzed using a previously developed 'shotgun lipidomics' method. / Thesis / Master of Science (MS)

Les moustiques sont les principaux vecteurs incriminés dans la transmission d’agents pathogènes à l’homme. L’identification précise des espèces de moustiques est importante pour distinguer les espèces vectrices des non vectrices. La détermination de l’origine du repas sanguin des moustiques vecteurs est indispensable dans la compréhension du comportement des espèces vectrices. Nous avons mise à jour la littérature actuelle sur la faune Culicidienne du Mali. Ainsi, nous avons listé 106 espèces de moustiques actuellement enregistrée au Mali dont 28 Anophelinae et 78 Culicinae. Nous avons ensuite évalué l’efficacité du MALDI-TOF MS à identifier des moustiques collectés au Mali et déterminer leur source de repas sanguin. Nous avons confirmé la robustesse du MALDI-TOF MS à identifier un grand nombre de sang d’animaux. Nous avons artificiellement gorgé des femelles de An. gambiae et An. coluzzii sur différents types de sang d’animaux. Nous avons obtenu 100% d'identification correcte du repas de sang pour les spécimens collectés 1h à 24h après le gorgement. Ensuite nous avons expérimentalement gorgés An. gambiae, An. coluzzii et Ae. albopictus sur des repas de sang successif et mixte par MALDI-TOF MS. Nos résultats révèlent que le MALDI-TOF MS est tout à fait capable d’identifier le repas mixte. Mais en ce qui concerne le repas successif seul le dernier repas de sang est identifié. Enfin nous avons utilisé la culturomique et le MALDI-TOF pour l’étude du microbiote digestif de moustiques collectés sur le terrain au Mali et à Marseille. Cette approche a révélé une grande diversité du microbiote digestif des moustiques An. gambiae, Ae. albopictus et Cx. quinquefasciatus. / Mosquitoes are the main vectors involved in the transmission of pathogens to humans. Accurate identification of mosquito species is crucial to distinguish between vector and non-vector species. The mosquito blood meal determination is fundamental in understanding the behavior of vector species. Thus, we have listed 106 mosquito species currently recorded in Mali, including 28 Anophelinae and 78 Culicinae. Then, we evaluated the effectiveness of MALDI-TOF MS for identified mosquitoes collected in Mali and to determine their blood meal source. The results obtained show the ability of MALDI-TOF MS to identify mosquitoes collected in Mali and their source of blood meal. Subsequently, we were able to confirm the robustness of MALDI-TOF MS to identify other animal blood samples. We artificially engorged Anopheles gambiae and Anopheles coluzzii on eight animal bloods samples. We obtained 100% correct identification of the blood source for samples taken 1 to 24 hours after feeding. Then, we experimentally engorged An. gambiae, An. coluzzii and Ae. albopictus on successive and mixed blood meals using MALDI-TOF MS. The results revealed that MALDI-TOF MS is able to identify mixed blood meals. In addition we used MALDI-TOF and culturomics for the microbiota study of the mosquito collected in the field, notably in Marseille and Mali. The culturomics approach revealed a great diversity of the digestive microbiota of the An. gambiae, Ae. albopictus and Cx. quinquefasciatus mosquitoes.

Ce travail est composé de 3 parties. La première est une étude épidémiologique avec la détection moléculaire des spécimens appartenant à six espèces d’Argasidés collectées en Algérie et identifiées morphologiquement et par biologie moléculaire. Nous avons pu détecter Borrelia hispanica dans des Ornithodoros occidentalis et Borrelia cf turicatae dans des Carios Carpensis. Dans des Argas persicus nous avons pu identifier un nouveau génotype de Bartonella spp ainsi qu’un génotype appartenant à une nouvelle espèce dans la famille des Anaplasmataceae. Dans la 2e partie, nous avons évalué la capacité vectorielle des punaises de lit à transmettre Borrelia recurrentis, l’agent de la fièvre récurrente. Pour ce fait, nous avons utilisé un modèle expérimental d’infection artificielle de Cimex lectularius par B. recurrentis pour ensuite détecter la présence de la bactérie dans les fèces. Nous avons utilisé quatre approches : la détection par qPCR, la culture à partir des fèces, la FDA (Fluorescein Diacetate) et l’inoculation des fèces aux souris. Nous avons également utilisé l’Immunofluorescence pour localiser la bactérie dans le corps de la punaise. Nous avons constaté que les punaises de lit acquièrent la bactérie et excrètent des microorganismes vivants dans les fèces. Elles peuvent être considérées comme vecteur potentiel de Borrelia recurrentis. La troisième partie s’intéresse à l’évaluation de la capacité du MALDI-TOF MS à identifier les puces, les punaises et les pathogènes associés. / This work focuses on three main parts, a first part presents an epidemiological study of bacteria associated with soft ticks in Algeria, or we identified morphologically and confirmed by molecular biology six species of Argasidae. In addition, looking further we could detect Borrelia hispanica in Ornithodoros occidentalis and Borrelia cf turicatae in Carios Carpensis. On the other hand, in Argas persicus a new genotype of Bartonella spp has been identified as well as a new species of Anaplasmatacea bacteria.A second part evaluates the vectorial capacity of bed bugs to transmit Borrelia recurrentis, the agent of the relapsing fever. For this reason an experimental model of artificial infection of Cimex lectularius by Borrelia recurrentis has been developed, to study the presence of bacteria in feces. In this model, four approaches were used: qPCR, fece’s culture, FDA (Fluorescein Diacetate) and fece’s inoculation to mice. Immunofluorescence was also used to detect the location of the bacteria in the body of the bed bug. We confirmed that bed bugs acquire the bacteria and excrete live microorganisms in the feces. They can be considered as potential vector of Borrelia recurrentis.The third part is an assessment of the capacity of MALDI-TOF MS to identified fleas, bed bugs and associated pathogens. This innovative tool, which has revolutionized medical entomology and has shown its efficiency to identify several species of arthropods, has also been able to distinguish between infected and uninfected fleas and bugs, and even distinguish between fleas and bugs infected by the same species of bacteria.