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.

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.

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.

Métodos microbiológicos tradicionais como isolamento, coloração de Gram e testes bioquímicos auxiliam na identificação do gênero Streptococcus, no entanto, as espécies apresentam ampla variação fenotípica, tornando difícil a identificação ou diferenciação das mesmas apenas por estes métodos. Uma das espécies mais importantes em suínos, Streptococcus suis, tem provocado grandes prejuízos em todo o mundo e tem sido descrito como uma importante zoonose em alguns países. S. suis está presente nas vias respiratórias superiores, colonizando principalmente tonsilas, cavidades oral e nasal facilitando a alta disseminação por contato direto, principalmente em leitões entre 4 e 12 semanas de vida. Os quadros clínicos mais frequentes em suínos infectados pelo S. suis são meningite, artrite e pneumonia. O objetivo do presente estudo foi identificar estirpes do gênero Streptococcus mediante as técnicas de reação em cadeia pela polimerase (PCR), sequenciamento parcial do gene 16S rRNA e espectrometria de massa MALDI-TOF (MALDI-TOF MS). As análises por PCR e por MALDI-TOF MS resultaram na identificação de 215 estirpes como S. suis e 35 como diferentes espécies pertencentes ao gênero Streptococcus. Os resultados da identificação das 35 estirpes pertencentes a outras espécies do gênero Streptococcus pelo MALDI-TOF MS foram confirmados pelo sequenciamento parcial do gene 16S rRNA, sendo que as duas técnicas apresentaram 100% de concordância. Os resultados obtidos indicam grande eficácia na utilização das técnicas avaliadas para a identificação de S suis e de outras espécies do gênero Streptococcus. A técnica de MALDI-TOF MS, apesar do custo elevado do equipamento, apresentou a vantagem de ser rápida, apresentar baixo custo por análise e reduzida utilização de material / Traditional microbiological methods such as isolation, Gram staining and biochemical tests help to identify the Streptococcus genus, however, the species present broad phenotypic variation, making it difficult for their identification or even differentiation just by these methods. One of the most important species in swine, Streptococcus suis, has led to great losses worldwide and has been described as an important zoonosis in some countries. S. suis is present in the upper airways, especially colonizing tonsils, oral and nasal cavities facilitating the high dissemination by direct contact, especially among piglets between 4 to 12 weeks of age. The most common clinical manifestations in pigs infected by S. suis are meningitis, arthritis and pneumonia. The aim of this study was to identify Streptococcus strains by polymerase chain reaction (PCR), 16S rRNA gene partial sequencing and MALDI-TOF mass spectrometry (MALDI-TOF MS). PCR and MALDI-TOF MS analysis resulted in the identification of 215 strains as S. suis and 35 as different species of the Streptococcus genus. The identification of the 35 strains belonging to other species of the genus by MALDI-TOF MS was confirmed by 16S rRNA gene partial sequencing, and both techniques presented 100% concordance. These results demonstrate the high efficiency in the use of the evaluated techniques for the identification of S. suis and the other species of the Streptococcus genus. The MALDI-TOF MS technique, despite the equipment high cost, presented the advantage of being fast, have low cost per analysis and reduced material usage

O objetivo deste estudo foi avaliar a técnica de espectrometria de massas por ionização/dessorção a laser assistida por matriz tempo-de-vôo (MALDI-TOF) para a identificação direta em amostras de leite (sem cultivo microbiológico) de bactérias causadoras de mastite. Para tanto foram realizados dois experimentos (1 e 2). No experimento 1, o objetivo foi determinar a sensibilidade diagnóstica da técnica MALDI-TOF MS para a identificação direta em amostras de leite de Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae e Escherichia coli. Foram realizadas contaminações experimentais de S. aureus, S. uberis, S. agalactiae, S. dysgalactiae e E. coli em amostras de leite, para a obtenção de contagens de 103 a 109 ufc/mL. As amostras de leite contaminadas foram processadas com o uso do kit Maldi Sepsityper® (Bruker Daltonics) e submetidas ao protocolo de lise bacteriana para posterior análise por MALDI-TOF MS. Espectros de massas foram coletados na faixa de massas de 2.000-20.000 m/z e foram analisados pelo programa MALDI Biotyper 3.0 (Bruker Daltonics) com as configurações padrão para obtenção da identificação bacteriana. A identificação direta de patógenos causadores de mastite a partir de amostras de leite foi possível em contagem ≥106 ufc/mL para S. aureus, ≥107 ufc/mL para E. coli, e ≥108 ufc/mL para S. agalactiae, S. dysgalactiae e S. uberis. No experimento 2, o objetivo foi avaliar o efeito da pré-incubação de amostras de leite de quartos mamários com mastite subclínica sobre a eficácia da identificação sem cultivo de patógenos causadores de mastite por MALDI-TOF MS. Foram selecionados 2 rebanhos leiteiros para coletas de leite de quartos mamários de todas as vacas em lactação. As amostras de leite foram submetidas às análises de: a) cultura microbiológica; b) pré-incubação seguida de identificação por espectrometria de massas diretamente do leite; c) contagem bacteriana total (CBT). Para a realização da CBT, as amostras de leite foram submetidas à citometria de fluxo; e para a identificação direta de patógenos causadores de mastite a partir do leite, as amostras foram submetidas ao desnate por centrifugação (10.000 Xg por 10 minutos), e pré-incubação a 37ºC por 12 horas. Posteriormente, as amostras foram processadas com o uso do kit Maldi Sepsityper® (Bruker Daltonics) e submetidas ao protocolo de lise bacteriana para posterior análise por MALDI-TOF MS. Do total de 810 amostras de leite analisadas por cultura microbiológica (método referência), 347 apresentaram crescimento bacteriano, sendo 305 identificadas como agentes de interesse na identificação direta pelo método MALDI-TOF MS: Staphylococcus coagulase negativa (n=191), S. aureus (n=31), S. agalactiae (n=42), S. uberis (n=37), S. dysgalactiae (n= 4). Sendo assim, 305 amostras foram analisadas pelo método de idenfificação direta MALDI-TOF MS, a qual apresentou baixa sensibilidade quando comparado com a cultura microbiológica (método referência): Staphylococcus coagulase negativa (14,08%), S. agalactiae (15,25%), S. uberis (1,69%) S. aureus (6,12%) e S. dysgalactiae (0%). A pré-incubação de amostras de leite não aumentou a sensibilidade de identificação direta de microrganismos causadores de mastite pelo método MALDI-TOF MS. / The purpose of the present study was to evaluate the technique of mass spectrometry by desorption / ionization assisted laser array time-of-flight (MALDI-TOF MS) for the direct identification in milk samples (no microbiological culture) of mastitis causing bacteria. Therefore, we carried out two experiments (1 and 2). In experiment 1, we determined the diagnostic sensitivity of MALDI-TOF MS technique for the direct identification in milk samples of Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactiae, Streptococcus dysgalactiae and Escherichia coli. Experimental contamination of S. aureus, S. uberis, S. agalactiae, S. dysgalactiae and E. coli in samples of milk to a concentration of 103-109 cfu/mL were performed. The contaminated milk samples were processed using kit Maldi Sepsityper® (Bruker Daltonics) and subjected to bacterial lysis protocol for analysis by MALDI-TOF MS. Mass spectra were collected in the mass range of 2000-20000 m/z and were analyzed by MALDI Biotyper 3.0 software (Bruker Daltonics) with default settings to obtain bacterial identification. Direct identification of mastitis causing pathogens from milk samples was possible at ≥106 cfu/mL for S. aureus, ≥107 cfu/mL for E. coli and ≥108 cfu/mL for S. agalactiae, S. dysgalactiae and S. uberis. In experiment 2, the objective was to evaluate the effect of pre-incubation of milk samples from mammary quarters with subclinical mastitis on the effectiveness of identification without cultivation, of mastitis-causing pathogens by MALDI-TOF MS. We selected mammary quarter milk samples from all lactating cows on two dairy herds. The milk samples were subjected to analyzes of: a) microbiological culture; b) pre-incubation followed by identification by mass spectrometry directly from milk; c) total bacterial count (TBC). Flow cytometry was used to determine TBC and; to directly identify the mastitis-causing pathogens from milk, fat was separated by centrifugation (10,000 Xg for 10 minutes) and; samples were pre-incubated at 37°C for 12 hours. Subsequently, the skim milk samples were submitted to kit Maldi Sepsityper® (Bruker Daltonics) and to the bacterial lysis protocol for analysis by MALDI-TOF MS. A total of 810 milk samples were analyzed by microbiological culture (reference method), of which 347 showed bacterial growth. Considering all culture positive samples 305 were identified as agents of interest in the direct identification by MALDI-TOF MS method: coagulase negative staphylococci (n = 191), S. aureus (n = 31), S. agalactiae (n = 42), S. uberis (n = 37) and S. dysgalactiae (n = 4). Therefore, 305 samples were directly identified by MALDI-TOF MS, which presented low sensitivity when compared to microbiological culture (Reference method): coagulase-negative staphylococci (14.08%), S. agalactiae (15.25 %), S. uberis (1.69%), S. aureus (6.12%) and S. dysgalactiae (0%). Pre-incubation of milk samples did not increase the sensitivity of the MALDI-TOF MS method directly identify mastitis-causing microorganisms.

Escherichia fergusonii é um patógeno emergente na medicina humana e veterinária, nos relatos de casos de infecção em aves são escassos. O objetivo desse trabalho foi identificar e caracterizar estirpes de E. fergusonii isoladas de anatídeos de vida livre, aves cativas silvestres e aves comerciais criadas em sistemas convencionais e orgânico. Dentre as 431 amostras, foram isoladas 29 estirpes de Escherichia fergusonii, sendo a maior prevalência em galinhas no sistema de criação convencional. O perfil de sensibilidade das amostras foi avaliado, revelando diferenças entre os grupos, com a presença de estirpes de frangos no sistema de criação convencional apresentando maiores índices de resistência aos antimicrobianos testados, enquanto estirpes de irerês apresentaram sensibilidade a todos os antibióticos. Foram identificadas duas estirpes multirresistentes com resistência a mais de três classes de antimicrobianos. A caracterização molecular dessas amostras revelou estirpes de irerês (Dendrocygna viduata) com maior presença de genes de virulência (iroN, cvi, cva, astA e ompT). O ensaio de produção de hemolisinas em ágar sangue revelou amostras negativas, sem formação de hemólise ao redor da colônia. A técnica de AFLP classificou as estirpes em clados distintos sugerindo padrões de heterogeneidade entre as amostras. Esse é o primeiro relato de achados de Escherichia fergusonii em irerês de vida livre e gavião-pombo pequeno. / Escherichia fergusonii is an emerging pathogen in human and veterinary medicine, in cases reports associated with birds are scarce. The aims of this work were to identify and to characterize strains of E. fergusonii isolated from free-living ants, wild captive birds and poultry from conventional and organic farms. Among the 431 samples, 29 Escherichia fergusonii were isolated, being the highest prevalence in chickens from conventional breeding systems. The sensitivity profile of the samples was evaluated, revealing differences between the groups, with the higher antimicrobial resistance indexes in isolates from chicken in the conventional farms, while strains of white-faced whistling-duck showed sensitivity to all antibiotics. Two multiresistant strains with resistance to more than three classes of antimicrobial agents were identified. The molecular characterization of these samples revealed strains of white-faced whistlingduck (Dendrocygna viduata) with higher presence of virulence genes (iroN, cvi, cva, astA and ompT). The assay of hemolysin production on blood agar revealed negative strains, with no hemolysis forming around the colony. The AFLP technique classified the strains into distinct clades suggesting patterns of heterogeneity between the strains. This is the first report of Escherichia fergusonii in free-living white-faced whistling duck and white-necked hawk.

Este trabalho descreve a montagem de um espectrômetro de massa por tempo de voo para análise de macromoléculas, projetado e desenvolvido no Laboratório de Instrumentação e Partículas (LIP) da USP, e apresenta os primeiros resultados obtidos com o equipamento. O espectrômetro é constituído pelo acoplamento de uma fonte de íons Matrix Assisted Laser Desorption Ionization (MALDI) com um acelerador de partículas do tipo tandem. O objetivo é aumentar a energia dos íons moleculares produzidos na fonte visando a uma maior eficiência de detecção de moléculas de massas elevadas. Amostras padrão de iodeto de césio (CsI) foram utilizadas para caracterização e verificação das condições de funcionamento do aparelho. O início dos estudos de moléculas de massas grandes se deu com medidas de amostras de insulina. Um método de simulação foi desenvolvido para auxiliar a análise dos espectros de massa e seus resultados foram comparados com os dados experimentais obtidos com o espectrômetro. O trabalho aponta novas possibilidades para a aplicação de aceleradores de partículas em análise de massa de macromoléculas. / The present work describes the assembly of a time-of-flight mass spectrometer for analysis of macromolecules, designed and developed at the Instrumentation and Particle Laboratory (LIP), and presents the first results obtained with the equipment. The spectrometer is based on the coupling of a Matrix Assisted Laser Desorption Ionization (MALDI) ion source with a tandem type particle accelerator. The objective is to increase the molecular ions energy produced by the source, and thus improve the high mass detection efficiency. Standard samples of cesium iodide (CsI) were used for characterization and verification of the equipment operation setup. Measures of insulin samples marked the initial studies of large masses. A simulation method was developed to assist in mass spectra identification and the results were compared with experimental data obtained with the spectrometer. The results open new possibilities for the application of particle accelerators in macromolecules mass analysis.

Lychnophora (Vernonieae: Asteraceae) é um gênero micro-endêmico dos \"campus rupestris\" do bioma Cerrado. Os extratos foliares de Lychnophora ericoides (\"Arnica-da-serra\") são usados na terapêutica popular principalmente como analgésico. Seus óleos essenciais são quimicamente ricos em sesquiterpenos. Tais compostos, principalmente os de esqueletos bisabolano e cadinano são produtos biossintéticos do precursor cátion nerolidila. Óleos essenciais de folhas de L. ericoides são bioativos frente a invertebrados do Táxon Acari. Um de seus componentes majoritários, o orto-acetoxi bisabolol, é antinociceptivo em ensaios in vitro. Terpenos são valorizados pela indústria de química fina, haja vista sucessos como Taxol® e Acheflan®. Nossos objetivos em Fitoquímica e Ciências Farmacêuticas se alinham a esse interesse. As terpeno-sintases (TerpS) envolvidas na biossínteses dos compostos são ainda alvo de pesquisas com em Bioquímica. Sua identificação é frequentemente conclusiva em diversos aspectos. Como exemplo, citamos a compreensão dos mecanismos da variabilidade metabólica temporal em espécies nativas e selvagens e os avanços na bioengenharia de Produtos Naturais (PN). Frente a este cenário de fronteira este manuscrito traz um resumo da investigação que objetivou determinar se há diversas isoformas de TerpS operando na produção temporalmente variável de sesquiterpenos bioativos em tecidos foliares de Lychnophora ericoides ou se não as há. Acessamos diferentes amostras selvagens de L. ericoides in situ. Nossos esforços em responder a questão supracitada foram divididos em tarefas, a seguir: (1) Determinação da fração rica em sesquiterpenos inspirada na \"Química Analítica Verde\"; (2) MALDI imaging das proteínas das folhas; (3) Estratégia \"omics\" combinada na identificação e clonagem das TerpS. Visitamos campos de ocorrência (CampO) georreferenciados bem como desconhecidos. O desenvolvimento de método de quantificação por Headspace-SPME permitiu uma comparação rápida e livre de solventes de amostras vegetais mínimas (10 mg). A separação e identificação foi conduzida por Gas Chromatography-Mass Spectrometry (GC-MS). As amostras de Diamantina, CampO mais Boreal de todos, apresentam a maior quantidade de derivados bisabolano. A hidrodestilação do material vegetal excedente ao HS-SPME forneceu óleos essenciais para o isolamento de moléculas. Dados espectrais diversos fundamentam a descrição de um derivado cadinano, o 11-dehidro cadinol, entre outros. O MALDI imaging determinou como exequível a geração de imagens moleculares em folhas de L. ericoides. Foram geradas imagens subepidermais dos íons com m/z que se assemelham a cadinano e bisabolano synthases (CadS and BS). A limitação do emprego da técnica é a determinação previa da massa nominal da proteína nativa. Polyacrylamide Gel Electrophoresis (1D-PAGE) gerou mapas de proteínas separadas por tamanho (A, B, C, e D) (30KDa> m/z <80KDa). A digestão com tripsina das bandas geraram peptídeos para análise em MS. Íons resultantes alimentaram o algorítimo MASCOT. Uma isoforma de germacrano-sintase foi identificada. Prospecção de genes codificadores com o cDNA (Ubiquitina +) determinou uma BS, de comprimento 1600pb, amplificada com primers de design para genes de Helianthus spp. A BS de L. ericoides foi clonada. Concluindo, destacamos dois pontos. (1) O controle enzimático na produção dos sesquiterpenos poderá enfim ser averiguada a nível do transcriptoma. (2) A busca pela produção biotecnológica de PN sofreu um pequeno incremento. Esperamos ter contribuído humildemente na produção sustentável de PN e por conseguinte na preservação da biodiversidade. / Lychnophora (Vernonieae: Asteraceae) is micro endemic to \"campus rupestris\" from Brazilian \"Cerrado\". Leaves extracts of Lychnophora ericoides (\"Arnica-da-serra\") are used as folk medicine and mainly as wound healer. Its essential oils were chemically profiled as sesquiterpene-rich. Such sesquiterpenes, both bisabolene-like and cadinane-like carbon skeletons, are derivatives of the nerolidyl cation. L. ericoides leaves essential oils are bioactive against invertebrate Acari. An anti-hypernociceptive ability of its component orto-acetoxy bisabolol was also displayed in vitro. Terpenes are valuable in fine chemistry industry, e.g. Taxol® and Acheflan®. Our phytochemical and pharmaceutical goals are aligned to such an interest. Terpene synthases (TerpS) behind their biosynthesis are target of researches in plant sciences and biochemistry. Identification of TerpS often led to conclusions with diverse impacts. Fundamental concepts on time-dependent shifts of terpene productions in wild type species and advances towards plant metabolites bioengineering are examples. Facing such a frontier field we share here an abstract for the investigation aimed to determine whether there were many isoforms of sesquiterpene synthases operating at leaves tissues in Lychnophora ericoides shift-able production of bioactive sesquiterpenes or whether there were not. We have accessed different in situ samples of L. ericoides. Efforts to answer the above question were sectioned as follows: (1) \"Green-Analytical-Chemistry\" oriented profiling of sesquiterpene-rich fraction; (2) MALDI imaging of proteins in leaves; (3) Combined \"omics\" approaches towards identifying TerpS and gene cloning. We visited both known and novel L. ericoides sites of occurrence (SoO). The development of a quantitative Headspace-Solid Phase Micro Extraction (HS-SPME) method enabled a rapid and solvent-free comparison of minimized samples (10mg). Separation and identification were carried out using Gas Chromatography-Mass Spectrometry (GC-MS). Samples harvested in Diamantina, Northern Most SoO, accumulate the highest amount of bisabolene-like derivatives. Hydrodistillation of leftovers material from HS-SPME yield essential oils used to purify unknown compounds. Based on a diverse spectra collection we report a novel cadinane-like derivative, one 11dehydro Cadinol. MALDI imaging has been determined as suitable for imaging proteins in L. ericoides. In a prospective fashion we generate sub-epidermal images of ions within the m/z frame comprising reported isoforms of both cadinane and bisabolane synthases (CadS and BS). The limitation to its use is the awareness of molecular weight of targeted native proteins. Polyacrylamide Gel Electrophoresis (1D-PAGE) protein mapping determined broad bands of protein distribution in different mass ranges (A, B, C, and D) (30KDa> m/z <80KDa). Bands tryptic digestion, followed by sample clean up, generated peptide pools feasible for MS. The output ions data feed MASCOT algorithm. A germacrane synthase could have been identified. When prospecting encoding genes, viable cDNA (Ubiquitin +) was used. A BS, lengthened 1600bp, was amplified with BS primers designed for Helianthus spp. genes. BS presented in L. ericoides was successfully cloned. In conclusion we headline two topics. (1) Hypothetical enzymatic control of the sesquiterpenes production can now be further investigated at transcriptome level. (2) The seek of a platform that guarantee natural products production in a controlled system has been moved forward. Future production of valued compounds can slightly rely in our humble contribution to support biodiversity conservation.

Le genre Bordetella comporte à l’heure actuelle neuf espèces, majoritairement responsables d’infections respiratoires. B. pertussis, agent de la coqueluche, est le modèle de travail de cette thèse avec d’autres espèces telles que B. holmesii et B. avium. Les endotoxines bactériennes sont les composants essentiels de la membrane externe des bactéries à Gram-négatif. Du point de vue chimique, ce sont des lipopolysaccharides (LPS) qui provoquent un grand nombre de désordres physiopathologiques allant de la simple fièvre, à faible dose, jusqu’au choc endotoxinique mortel, à forte dose. L’analyse structurale des LPS de Bordetella est la spécialité majeure du laboratoire et la structure des endotoxines de la plupart des espèces de ce genre y a été décrite. Il est admis que le lipide A, qui constitue la région hydrophobe des LPS, est responsable de la majorité des activités biologiques de ces molécules. Ainsi, la moindre variation structurale de ces molécules a une répercussion importante sur la reconnaissance hôte-pathogène, les activités biologiques et la virulence bactérienne. A titre d’exemple, il a été mis en évidence que la modification spécifique, par substitution des groupes phosphate du lipide A, avec de la glucosamine, jouait un rôle majeur dans la modulation de la réponse immunitaire. Cette originalité structurale qui a été mise en évidence dans notre équipe chez B. avium, B. bronchiseptica puis chez B. pertussis; elle semble être un trait spécifique des Bordetelles. Il faut savoir que la coqueluche fait des ravages dans les pays sous-développés et touche les nouveau-nés dans de nombreux pays, comme la France, d’une maladie infectieuse mortelle dans les cas graves. Le vaccin qui ne peut être injecté qu’à l’âge de 2-3 mois et dont les rappels ne sont pas régulièrement suivis est imparfait. Les spécialistes du domaine ont reconnu qu’un complément antigénique serait nécessaire pour le rendre plus efficace. Au cours de cette thèse, nous avons analysé la structure des LPS d’isolats cliniques de B. pertussis afin d’étudier leur évolution et leur adaptation avec le temps ainsi que leur application vaccinale potentielle. De plus, concernant deux souches de B. pertussis, BP338 et BP18-323 nous avons contribué à la mise en évidence de nouveaux gènes impliqués dans la biosynthèse de la GlcN substituant les groupes phosphate de la région lipidique et à expliquer la différence de longueur du seul acide gras distinct entre les deux souches. L’étude de l’influence de ces éléments structuraux sur l’activation du complexe, TLR4/MD-2 apporte de nouveaux éclairages sur les interactions entre les lipides A et ce récepteur. Nos études sur les isolats cliniques de B. holmesii, pathogène opportuniste responsable d’affections de type coqueluche, montrent une grande hétérogénéité structurale du lipide A au sein d’un même isolat. Nous avons montré dans cette souche la présence d’un marqueur spécifique des souches de Bordetella, il s’agit d’un acide gras présent uniquement chez les lipides A des isolats humains. Nos travaux effectués sur des isolats cliniques de B. pertussis appartenant aux ères pré- et post-vaccinales et provenant de différents pays, montrent une perte du matériel génétique avec une déficience de certains antigènes majeurs. Nous avons démontré, via des méthodes physico-chimiques, que ces modifications ne concernaient pas les LPS de ces isolats. La stabilité de ces antigènes ainsi que nos méthodes de purification, nous permettent de proposer que ces LPS détoxifiés soient de bons candidats pour améliorer l’efficacité des vaccins coquelucheux acellulaires. Enfin, toutes les études structurales présentées dans cette thèse ont permis de mieux comprendre la régulation de certains gènes en réponse à un stress extérieur. Elles participent, sur l’exemple d’un pathogène majeur, au déchiffrage des mécanismes moléculaires qui mènent à la virulence et à l’adaptation bactérienne. / The Bordetella genus is actually composed of nine species responsible for respiratory infections. B. pertussis, the agent of whooping cough, is the main model of this thesis along with other species such as B. holmesii and B. avium. Bacterial endotoxins are the major components of Gram-negative bacteria external membrane. From a chemical point of view, they are lipopolysaccharides (LPS) causing a high number of pathophysiological disorders ranging from low fever at weak doses, to lethal endotoxic choc at high ones. Structural analysis of the Bordetellae LPS is the major specialty of our group where the endotoxin structures of most species of the genus were described. It is well-known that lipid A, which constitutes the hydrophobic moiety of LPS, is responsible for the majority of biological activities of these molecules. Thus, any structural change of these molecules has an important impact on host-pathogen recognition, biological activities and bacterial virulence. For example, it has been demonstrated that the specific modification by grafting glucosamine on lipid A phosphate groups plays a major role in modulating the immune response. This structural peculiarity was highlighted by our team first in B. avium, B. bronchiseptica then in B. pertussis; it seems to be a unique trait of Bordetella. It should be noted that pertussis wreaks havoc in developing countries and affects newborns in several others, including France, where this infectious disease causes a significant death toll. The vaccine, which cannot be injected before the age of 2-3 months, could be improved and boosters are not regularly monitored. Experts in the domain have recognized the lack of an antigenic complement to make it more effective. In this thesis, we analyzed the structure of LPS from B. pertussis clinical isolates to study their evolution and adaptation over time along with their potential use in the design of new vaccines. In addition, regarding two strains of B. pertussis, BP338 and BP18-323, we have contributed to the identification of new genes involved in the biosynthesis of GlcN substituting the phosphate groups of the lipid moiety, which helped explaining the difference in the length of the single fatty acid differing between the two strains. The analysis of the influence of these structural elements on the activation of the receptor complex, TLR4/MD-2 sheds new light on the interactions between lipids A and this receptor. Our studies on clinical isolates of B. holmesii, an opportunistic pathogen responsible for pertussis-like illness, show great structural heterogeneity in the lipid A of these isolates. We showed the presence of a specific marker of Bordetella species, namely a fatty acid present only in the lipid A of human isolates. Our works on B. pertussis clinical isolates belonging to pre- and post-vaccine eras and coming from different countries show a loss of genetic material with a deficiency in certain major antigens. We have demonstrated, via physico-chemical methods, that these modifications did not affect the LPS of these isolates. The stability of these antigens and our ability to purify them, allow us to propose that detoxified LPS could be good candidates for improving the effectiveness of acellular pertussis vaccines. Finally, all structural studies presented in this thesis have provided insight into the regulation of certain genes in response to external stress. Our compiled work on a major pathogen is an important step in deciphering the molecular mechanisms leading to bacterial virulence and adaptation.

Métodos microbiológicos tradicionais como isolamento, coloração de Gram e testes bioquímicos auxiliam na identificação do gênero Streptococcus, no entanto, as espécies apresentam ampla variação fenotípica, tornando difícil a identificação ou diferenciação das mesmas apenas por estes métodos. Uma das espécies mais importantes em suínos, Streptococcus suis, tem provocado grandes prejuízos em todo o mundo e tem sido descrito como uma importante zoonose em alguns países. S. suis está presente nas vias respiratórias superiores, colonizando principalmente tonsilas, cavidades oral e nasal facilitando a alta disseminação por contato direto, principalmente em leitões entre 4 e 12 semanas de vida. Os quadros clínicos mais frequentes em suínos infectados pelo S. suis são meningite, artrite e pneumonia. O objetivo do presente estudo foi identificar estirpes do gênero Streptococcus mediante as técnicas de reação em cadeia pela polimerase (PCR), sequenciamento parcial do gene 16S rRNA e espectrometria de massa MALDI-TOF (MALDI-TOF MS). As análises por PCR e por MALDI-TOF MS resultaram na identificação de 215 estirpes como S. suis e 35 como diferentes espécies pertencentes ao gênero Streptococcus. Os resultados da identificação das 35 estirpes pertencentes a outras espécies do gênero Streptococcus pelo MALDI-TOF MS foram confirmados pelo sequenciamento parcial do gene 16S rRNA, sendo que as duas técnicas apresentaram 100% de concordância. Os resultados obtidos indicam grande eficácia na utilização das técnicas avaliadas para a identificação de S suis e de outras espécies do gênero Streptococcus. A técnica de MALDI-TOF MS, apesar do custo elevado do equipamento, apresentou a vantagem de ser rápida, apresentar baixo custo por análise e reduzida utilização de material / Traditional microbiological methods such as isolation, Gram staining and biochemical tests help to identify the Streptococcus genus, however, the species present broad phenotypic variation, making it difficult for their identification or even differentiation just by these methods. One of the most important species in swine, Streptococcus suis, has led to great losses worldwide and has been described as an important zoonosis in some countries. S. suis is present in the upper airways, especially colonizing tonsils, oral and nasal cavities facilitating the high dissemination by direct contact, especially among piglets between 4 to 12 weeks of age. The most common clinical manifestations in pigs infected by S. suis are meningitis, arthritis and pneumonia. The aim of this study was to identify Streptococcus strains by polymerase chain reaction (PCR), 16S rRNA gene partial sequencing and MALDI-TOF mass spectrometry (MALDI-TOF MS). PCR and MALDI-TOF MS analysis resulted in the identification of 215 strains as S. suis and 35 as different species of the Streptococcus genus. The identification of the 35 strains belonging to other species of the genus by MALDI-TOF MS was confirmed by 16S rRNA gene partial sequencing, and both techniques presented 100% concordance. These results demonstrate the high efficiency in the use of the evaluated techniques for the identification of S. suis and the other species of the Streptococcus genus. The MALDI-TOF MS technique, despite the equipment high cost, presented the advantage of being fast, have low cost per analysis and reduced material usage