Les maladies transmises par les arthropodes vecteurs sont responsables de centaines de milliers de cas d’infections humaines et de décès chaque année à travers le monde. Ces maladies, causées par des bactéries, virus ou parasites, parfois émergents ou réémergents, sont parfois peu connues ou sous-estimées. Les arthropodes peuvent être utilisés comme outil de suivi épidémiologique des micro-organismes qui leur sont associés et dont certains pourront être transmis à des hôtes vertébrés. L’identification des arthropodes reste cruciale dans les enquêtes entomologiques.Nous avons pu ainsi détecter de potentielles nouvelles bactéries dans les tiques de Tahiti et les triatomes de Guyane.Nous avons exploré le microbiote salivaire de près de mille moustiques de 3 pays différents par métagénomique 16S. Nous avons ainsi détecté un large nombre de bactéries pathogènes opportunistes mais aussi un très grand nombre de génotypes correspondant probablement à des espèces et genres bactériens nouveaux. Enfin notre axe majeur a été le développement de l’utilisation de la spectrométrie de masse MALDI-TOF en entomologie médicale. Pour pallier les limites des méthodes de référence d’identification des arthropodes existantes, nous avons validé l’utilisation de cet outil pour l’identification des moustiques (collectés sur terrain en Australie) et de puces (Espagne, Corse, Algérie). Nous avons également mis au point son utilisation pour l’identification de nouvelles familles d’arthropodes, comme les punaises de lits et les triatomes. Nous avons pu mettre en évidence, la capacité de la spectrométrie de masse MALDI-TOF pour différencier les anophèles infectés ou non par des plasmodies. / Vector-borne diseases are responsible for hundreds of thousands of cases of human infections and deaths each year worldwide. Generally, little is known about these diseases, caused by bacteria, viruses or parasites, sometimes emerging or re-emerging. Arthropods can be used as a tool for epidemiological monitoring of their associated microorganisms, some of which being able to be transmitted to vertebrate hosts. The identification of arthropods remains crucial in entomological investigations.We were able to detect potential new bacteria in ticks from Tahiti and triatomines in French Guiana.We explored the salivary microbiota of nearly a thousand mosquitoes from 3 different countries by 16S rRNA metagenomics. We have thus detected a large number of opportunistic pathogenic bacteria but also a very large number of genotypes probably corresponding to new bacterial species and genera. Finally, our major focus has been the development of the use of MALDI-TOF mass spectrometry in medical entomology. To overcome the limitations of existing arthropod identification reference methods, we validated the use of this tool for the identification of mosquitoes (collected in the field in Australia) and fleas (Spain, Corsica, Algeria). We have also developed its use for the identification of new families of arthropods, such as bed bugs (Cimicidae) and triatomines (Reduviidae). We were able to highlight the capacity of MALDI-TOF mass spectrometry to differentiate between anopheles infected or not by malaria parasites.

BACKGROUND: There is an increased need to replace materials derived from fossil sources by renewables. Sugar-cane derived carbohydrates are very abundant in Brazil and are the cheapest sugars available in the market, with more than 400 million tons of sugarcane processed in the year 2007. The objective of this work was to study the preparation of sugar acrylates from free sugars and free acrylic acid, thus avoiding the previous preparation of protected sugar derivatives, such as glycosides, or activated acrylates, such as vinyl acrylate. RESULTS: Lipase catalyzed esterification of three mono- and two disaccharides with acrylic acid, in the presence or absence of molecular sieves was investigated. The reactions were monitored by high-performance liquid chromatography (HPLC) and the products were analyzed by matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry. The main products are mono- and diacrylates, while higher esters are formed as minor products. The highest conversion to sugar acrylates was observed for the D-glucose and D-fructose, followed by D-xylose and D-maltose. Molecular sieves had no pronounced effect on the conversion CONCLUSIONS: A feasible method is described to produce and to characterize sugar acrylates, including those containing more than two acrylate groups. The process for production of these higher esters could potentially be optimized further to produce molecules for cross-linking in acrylate polymerization and other applications. The direct enzymatic esterification of free carbohydrates with acrylic acid is unprecedented.

No description available.

Les méthodes pour le diagnostic des infections à mycobactéries sont restées pratiquement inchangées pendant des décennies et n'auraient probablement pas progressé sans la réémergence inattendue de la tuberculose (TB) au cours des vingt dernières années du 20ème siècle. Le diagnostic de laboratoire de la tuberculose pulmonaire repose principalement sur la détection des organismes du complexe Mycobacterium tuberculosis (MTC) dans les prélèvements respiratoires par leur isolement et identification ou par les méthodes moléculaires. Pour les patients qui n’expectorent pas, des échantillons du tractus respiratoire peuvent être obtenus par des procédures invasives. En nous basant sur la survie connue de MTC dans le liquide gastrique, nous avons considéré que les MTC devraient également être détectables dans les selles. Nous avons recherché des MTC en parallèle dans des échantillons de tractus respiratoire et dans les selles récoltés des mêmes patients. Mycobacterium tuberculosis a été détecté après décontamination à la chlorhexidine dans des cultures sur des milieux à base d'œuf et par l'examen direct microscopique après coloration de Ziehl-Neelsen. Après la mise au point d’une technique originale d’extraction de l’ADN total à partir des selles, nous avons utilisé la détection de M. tuberculosis par PCR en temps réel en duplex utilisant le gène IS6110 comme cible moléculaire et un contrôle interne de présence d’inhibiteurs de la PCR. Ces travaux mettent en évidence l’intérêt et la sensibilité de l’analyse des selles pour le diagnostic de la tuberculose pulmonaire. Cette technique est aujourd’hui utilisée dans notre laboratoire en routine et est en cours d’évaluation par d’autres équipes.Actuellement, l'identification des mycobactéries basée sur l’analyse des caractéristiques biochimiques (test à l’uréase, test de la nitrate réductase, test de la résistance à la pyrazinamide (pza), test de la sensibilité, test de la résistance à la thiophen-2-carboxylic acid hydrazide (TCH) etc …) est le plus souvent remplacée par des méthodes de biologie moléculaire dont l’amplification par PCR d'un gène spécifique. Ces méthodes sont laborieuses et peuvent exiger un degré d'expertise élevé. Afin de simplifier l'identification précise des isolats de mycobactéries en laboratoire de routine, après avoir vérifié l’inactivation des MTC par la chaleur et l’éthanol pour pouvoir travailler hors du laboratoire P3, nous avons mis au point les conditions pour l'utilisation de la spectrométrie de masse « matrix-assisted laser desorption and ionisation-time-of-flight» (MALDI-TOF-MS) en association avec un logiciel bioinformatique spécifique, comme outil d’identification et de différenciation entre les membres du complexe MTC, du complexe Mycobacterium avium et les autres mycobactéries non-tuberculeuses. Nous avons ensuite appliqué cette technique afin d’identifier les mycobactéries à partir du milieu liquide Middlebrook 7H10 utilisé dans une étuve automatisée. L’identification des mycobactéries isolées dans notre laboratoire, est maintenant réalisée en routine par MALDI-TOF-MS. L’ensemble de ces travaux contribue à améliorer le diagnostic de routine de la tuberculose pulmonaire et les techniques mises au point sont utilisées en routine dans notre laboratoire, en particulier dans le cadre d’un « kit tuberculose ». / The diagnosis of mycobacterial infections remained practically unchanged during numerous decades and would not probably have progressed of the whole without the unexpected re-emergence of the tuberculosis (TB) during the last twenty years of the 20th century.The diagnosis of laboratory of pulmonary tuberculosis is mainly based on the detection of microorganisms of the Mycobacterium tuberculosis complex (MTC) in the respiratory specimens. For the patients who do not expectorate, samples of the respiratory tract can be only obtained according to invasive procedures. Based on the survival known for MTC organisms in the gastric liquid, we considered that MTC organisms would be detectable also in samples of stools. We compared the presence of bodies MTC in samples of respiratory tract and the specimens of stools collected from the same patients. MTC was detected in cultures on egg-based media after appropriate decontamination using the chlorhexidine and by the microscopic examination after Ziehl-Neelsen staining. After the set on of an original protocol of the total DNA extraction from stools, we were able to use by the detection with the real-time PCR of IS6110 using internal controls as PCR inhibitor control. This protocol illustrate the utility of stool analysis for the diagnosis of pulmonary tuberculosis, this technique is actually used in routine in our laboratory, it is also under investigation by other research teams.Phenotypic identification of mycobacteria based on the analysis of biochemical pattern (urase test, nitrate reductase test, resistance to pyrazinamide (pza) test, susceptibility to thiophen-2-carboxylic acid hydrazide (TCH) test, etc …) is more often replaced by molecular methods using DNA, including the development by PCR of a specific gene. These methods are generally laborious and can require a considerable degree of expertise. To simplify the identification specifies isolates of mycobacteria routinely in the laboratory, we estimated the use of the mass spectrometry "matrix-assisted laser desorption and ionization-time-of-flight" (MALDI-TOF MS) in association with a specific bioIT software, was capable of identifying and of distinguishing between the members of Mycobacterium tuberculosis complex, Mycobacterium avium complex and the non- tuberculosis mycobacteria. We verified the MTC inactivation by heating or by ethanol allowing the analysis of the inactivated MTC out of the P3 laboratory; we studied the cost-effectiveness of this method from the blood solid media by extrapolating this approach in the emergent countries. We then applied the same method to identify mycobacteria from Middlebrook 7H10 liquid media used in an automated oven. Identification of cultured Mycobacteria is now done in routine in our laboratory by MALDI-TOF-MS. These data contribute in improving the routine diagnosis of pulmonary tuberculosis and this protocol is then used routinely in our laboratory, particularly with the “kit de tuberculose” we set on.

Alopecia areata (AA) is a putative autoimmune hair loss disorder. It mainly affects the scalp hair but can also involve body hair, and can also affect the nail and the eye. While there are may be several lines of evidence to support the autoimmune basis of AA, there is still very little information on the hair follicle autoantigen(s) involved in its pathogenesis. In this project, serum antibodies (AA=10, control=10) were used to immunoprecipitate AA-relevant target antigens from normal human scalp hair follicle extracts. These immunoprecipitates were analysed by LC-MALDI-TOF/TOF mass spectrometry for target protein identification. This part of the project involved substantial methods development. Trichohyalin was immunoprecipitated by all AA sera, but by only 5 normal sera. Importantly, the mean Mascot scores of the AA group was significantly higher than the normal group (p=0.005). Keratin 16 was also identified from immunoprecipitates as another potential AA-relevant target antigen. Functional studies by ex vivo whole hair follicle organ culture using commercial antibodies to trichohyalin and keratin 16 significantly inhibited hair fibre elongation compared to controls. Indirect immunofluorescence studies revealed that AA sera contained higher immunoreactivity against normal human scalp anagen hair follicles compared to normal sera. Immunoreactivities were mainly in the outer root sheath and inner root sheath, and less so to the medulla and hair bulb matrix. Double immunofluorescence studies of AA and normal serum with anti-trichohyalin antibody (AE15) revealed co-localisation of 9 of the AA sera antibodies with trichohyalin in the inner root sheath (mostly in Henle¿s, less in Huxley¿s/inner root sheath cuticle), but only weakly in 3 normal sera. This study supports the involvement of an antibody response to anagen-specific hair follicles antigens in AA. Moreover, there may be some evidence that these antibodies may have a pathogenic role.

No description available.

Alopecia areata (AA) is a putative autoimmune hair loss disorder. It mainly affects the scalp hair but can also involve body hair, and can also affect the nail and the eye. While there are may be several lines of evidence to support the autoimmune basis of AA, there is still very little information on the hair follicle autoantigen(s) involved in its pathogenesis. In this project, serum antibodies (AA=10, control=10) were used to immunoprecipitate AA-relevant target antigens from normal human scalp hair follicle extracts. These immunoprecipitates were analysed by LC-MALDI-TOF/TOF mass spectrometry for target protein identification. This part of the project involved substantial methods development. Trichohyalin was immunoprecipitated by all AA sera, but by only 5 normal sera. Importantly, the mean Mascot scores of the AA group was significantly higher than the normal group (p=0.005). Keratin 16 was also identified from immunoprecipitates as another potential AA-relevant target antigen. Functional studies by ex vivo whole hair follicle organ culture using commercial antibodies to trichohyalin and keratin 16 significantly inhibited hair fibre elongation compared to controls. Indirect immunofluorescence studies revealed that AA sera contained higher immunoreactivity against normal human scalp anagen hair follicles compared to normal sera. Immunoreactivities were mainly in the outer root sheath and inner root sheath, and less so to the medulla and hair bulb matrix. Double immunofluorescence studies of AA and normal serum with anti-trichohyalin antibody (AE15) revealed co-localisation of 9 of the AA sera antibodies with trichohyalin in the inner root sheath (mostly in Henle's, less in Huxley's/inner root sheath cuticle), but only weakly in 3 normal sera. This study supports the involvement of an antibody response to anagen-specific hair follicles antigens in AA. Moreover, there may be some evidence that these antibodies may have a pathogenic role.

Les peptides vecteurs ou CPP sont de petits peptides, en général de taille inférieure à 30 acides aminés. Parmi les nombreux CPP décrits dans la littérature, les peptides riches en arginine ont fait l'objet d'une attention particulière. Plusieurs modifications chimiques du squelette peptidique conduisant à une distribution spatiale différente des groupes fonctionnels, ou encore l'introduction de chaînes aliphatiques ont été effectuées pour accroitre la capacité du peptide à traverser la membrane de la cellule. L'objectif de ce travail a été le développement de nouveaux peptides vecteurs cycliques contenant un domaine cationique minimal et pouvant être acylés par une chaîne aliphatique. Quinze nouveaux transporteurs cycliques, dont les peptides vecteurs classiques Pénétratine et R6W3ont été synthétisés. La cyclisation tête-queue par ligation chimique native a été rendue possible par l'introduction d'un résidu cystéine et d'une fonction thioester (ou précurseur) respectivement aux extrémités N et C-terminales des différentes séquences de CPP. Leur aptitude à transporter le peptide bioactif PKCi dans des cellules CHO a été évaluée par quantification de la cargaison internalisée en utilisant la spectrométrie de masse MALDI-TOF. Les résultats indiquent une meilleure internalisation essentiellement par voie d'endocytose dépendante des glycosaminoglycanes, suite à la cyclisation des CPP comparés à leur version linéaire. De toute la série des lipopeptides testés dans ce projet, deux séquences se distinguent par leur capacité remarquable à franchir les membranes cellulaire : les CPP [C12-R4] et [C12-R7]. / Cell-penetrating peptides (CPPs) are short, cationic or amphipatic peptides, usually containing less than 30 amino acids, which are able to deliver various bioactive cargoes inside cells. Among the many CPPs described in the literature, the arginine-rich peptides have attracted particular attention. Several chemical modifications of the peptide backbone leading to different spatial distributions of the CPP functional groups, or the introduction of aliphatic chains have been made to enhance their internalization efficiency. The aim of this work was the synthesis of new cyclic CPPs containing a minimal cationic domain and their functionalisation with an aliphatic chain. We have synthesised a small library of fifteen new cyclic carriers including the classical CPPs Penetratin and R6W3 using native chemical ligation (NCL) in solution. The introduction of an N-terminal Cys residue and of a C-terminal thioester (or precursor) in the initial linear peptide sequence allowed the head-to-tail cyclisation. The efficiency of cargo delivery in CHO cells was measured by MALDI-TOF mass spectrometry. We found that cyclisation of CPPs improved their internalisation efficiency mostly by glycosaminoglycan-dependent endocytosis. Among the whole series of lipopeptides tested in this project, two sequences are distinguished by their remarkable ability to cross cellular membranes: the peptides [C12-R4] and [C12-R7].

Herein, we report on the mechanochemical Scholl reaction of dendritic oligophenylene precursors to produce benchmark nanographenes such as hexa-peri-hexabenzocoronene (HBC), triangular shaped C60 and expanded C222 under solvent-free conditions. The solvent-free approach overcomes the bottleneck of solubility limitation in this well-known and powerful reaction. The mechanochemical approach allows tracking the reaction process by in situ pressure measurements. The quality of produced nanographenes has been confirmed by MALDI-TOF mass spectrometry and UV-Vis absorption spectroscopy. This approach paves the way towards gram scale and environmentally benign synthesis of extended nanographenes and possibly graphene nanoribbons suitable for application in carbon based electronics or energy applications.

The aim of the diploma thesis was the diagnostics of plasma generated in the atmosphere simulating conditions on Mars. This diploma thesis is focused on the simulation of Mars’ atmosphere at atmospheric pressure and normal laboratory temperature. Due to the similar conditions of Mars' atmosphere with Earth, this planet has been explored in the past as well as up to now. Mars' atmosphere is composed mostly of carbon dioxide, which makes up more than 90 % of Mars' atmosphere. A glow discharge generated in a special reactor at atmospheric pressure at a flow of pure CO2 was used to simulate the atmosphere of Mars. Part of the measurement was performed only in pure CO2 with changing current of 20, 25, 30, 35 and 40 mA. Part of the measurements was focused on the study of the effect of the addition of various gases, such as nitrogen, hydrogen and methane, at changes in their flow rates of 1, 2, 3, 4 and 5 sccm. The products formed in the special reactor were analysed using a mass spectrometer with proton ionization and with a flight time analyser. Optical emission spectrometry was used for plasma diagnostics and composition. Mainly simple aliphatic hydrocarbons, alcohols, aldehydes, and ketones were detected. With increasing flow rates of the individual gases, more complex aromatic compounds with higher molecular weights were formed. Corresponding mass and optical emission spectra were measured simultaneously.