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<p>This study reports on a proteomic analysis of sorghum proteomes in response to salt and hyperosmotic stresses. Two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify sorghum proteins using both sorghum cell suspension cultures and whole plants. The sorghum cell suspension culture system was used as a source of culture filtrate (CF) proteins. Of the 25 visualised CBB stained CF spots, 15 abundant and well-resolved spots were selected for identification using a combination of MALDI-TOF and MALDI-TOFTOF MS, and database searching. Of these spots, 14 were positively identified as peroxidases, germin proteins, oxalate oxidases and alpha-galactosidases with known functions in signalling processes, defense mechanisms and cell wall metabolism.</p>

<p>This study reports on a proteomic analysis of sorghum proteomes in response to salt and hyperosmotic stresses. Two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify sorghum proteins using both sorghum cell suspension cultures and whole plants. The sorghum cell suspension culture system was used as a source of culture filtrate (CF) proteins. Of the 25 visualised CBB stained CF spots, 15 abundant and well-resolved spots were selected for identification using a combination of MALDI-TOF and MALDI-TOFTOF MS, and database searching. Of these spots, 14 were positively identified as peroxidases, germin proteins, oxalate oxidases and alpha-galactosidases with known functions in signalling processes, defense mechanisms and cell wall metabolism.</p>

Dans ce travail, nous nous sommes intéressés à l’étude des dirofilarioses chez le réservoir canin « le chien » ainsi que chez les vecteurs « moustiques », en particulier, en ce qui concerne la détection, la surveillance et la prophylaxie. Le premier objectif était de développer une PCR duplex en temps réel ciblant le gène COI capable de détecter et de différencier simultanément D. immitis et D. repens. Ainsi, nous avons détecté par cet outil moléculaire, pour la première fois en France, D. immitis et D. repens chez des moustiques tigre "Aedes albopictus". Nous avons, de plus, confirmé la présence de l’infection à D. immitis chez les chiens du nord d'Algérie.Le deuxième objectif était d'évaluer l’intérêt de la spectrométrie de masse MALDI-TOF MS pour la détection de changements dans les profils protéiques d'Aedes aegypti infectés expérimentalement avec des nématodes filaires (D. immitis, Brugia malayi et B. pahangi). Les résultats obtenus montrent la capacité du MALDI-TOF MS à différencier des moustiques infectés et non infectés par les filaires. Ainsi, les meilleurs taux de classification correcte obtenus sont 94,1, 86,6, 71,4 et 68,7% pour les non infectés versus ceux infectés, respectivement, par D. immitis, B. malayi et B. pahangi.Le troisième objectif de ce travail était l’évaluation de l'efficacité anti-gorgement et insecticide d'un ectoparasiticide (Vectra® 3D) contenant trois principes actifs : le dinotéfurane, le pyriproxyfène et la perméthrine contre Ae. albopictus, l'une des principales espèces vectrices de Dirofilaria spp. Les résultats ont démontré que le DPP a une efficacité anti-gorgement et insecticide significative contre Ae. albopictus / In this work, we are interested in studying dirofilarial infections in dogs and vectors “Mosquitoes” especially detection, monitoring and prophylaxis. The first objective is to develop a real-time duplex PCR targeting the COI gene capable of simultaneously detecting and differentiating D. immitis and D. repens. Subsequently, we applied this tool to a canine dirofilariosis surveillance process in different endemic areas of Mediterranean basin (Corsica and Algeria). We have thus detected by this molecular tool for the first time in France, D. immitis and D. repens in Aedes albopictus mosquitoes. We reported, also, the presence of D. immitis in dogs from northern Algeria.The second aim was to assess whether the MALDI-TOF MS can detect changes in the protein profiles of Aedes aegypti infected experimentaly with filarial nematodes (D. immitis, Brugia malayi and B. pahangi). Obtained results showed the potential of MALDI-TOF MS as a reliable tool for differentiating non-infected and filariae-infected Ae. aegypti mosquitoes with a best correct classification rate obtained from the thorax-head part with 94.1 and 86.6, 71.4 and 68.7% for non-infected and D. immitis, B. malayi and B. pahangi infected mosquitoes respectively.The third aim of this work has focused on the evaluation of the anti-feeding and insecticidal efficacy of an ectoparasiticide (Vectra® 3D) containing three active ingredients: dinotefurane, pyriproxyfen and permethrin (DPP) against Ae. albopictus. Results demonstrated that the DPP combination has significant anti-feeding and insecticidal efficacy against Ae. albopictus for at least 4 weeks.

Made available in DSpace on 2016-03-04T13:55:12Z (GMT). No. of bitstreams: 2 7.pdf: 2671790 bytes, checksum: 3ea789720d5aa831716c6bfe2ca4f9b6 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Instituto de Tecnologia em Fármacos/Farmanguinhos. Rio de Janeiro, RJ, Brasil. / O Instituto de Tecnologia em Fármacos (Farmanguinhos), unidade técnico-científica da Fundação Oswaldo Cruz, representa o maior laboratório farmacêutico oficial vinculado ao Ministério da Saúde brasileiro responsável pela produção de mais de um bilhão de medicamentos por ano. As legislações sanitárias vigentes definem que devem ser utilizadas instalações segregadas e dedicadas para a produção de medicamentos, como penicilinas, minimizando os riscos de contaminação cruzada, que consequentemente possam gerar danos à saúde. Por isso, Farmanguinhos possui, atualmente, uma área dedicada à produção de amoxicilina, assim como o sistema gerador de água purificada, que atende à referida fabricação, também é dedicado à mesma. A água é a matéria-prima de mais elevado volume empregada na produção farmacêutica, exercendo profundo impacto na qualidade do produto e na segurança do paciente. O controle microbiológico da qualidade da água utilizada em processos farmacêuticos torna-se fundamental, já que micro-organismos podem sobreviver e proliferar em sistemas de água, tornando-se fontes de contaminação microbiana e pirogênica. A identificação destes micro-organismos e as informações obtidas sobre os mesmos podem ser extremamente úteis na identificação da fonte de contaminação microbiana de um produto ou processo, alem de direcionar ações corretivas, se necessário. O objetivo deste trabalho foi avaliar a diversidade microbiana presente em sistema gerador de água purificada, dedicado à produção de penicilínicos, de Farmanguinhos. Para isto, as amostras de água coletadas a partir do ponto de entrada, ponto de água potável, e do ponto de saída do sistema, ponto de água purificada, foram analisadas microbiologicamente quanto à contagem de micro-organismos mesófilos e quanto à presença de patógenos: Escherichia coli; coliformes totais e Pseudomonas aeruginosa. As amostras foram analisadas entre janeiro e maio de 2013. Todas as colônias obtidas foram isoladas e submetidas à identificação, através de equipamento MALDI-TOF. Não foram observados resultados acima da especificação para ambos os pontos, no período analisado. Do ponto de água potável foram isoladas apenas três espécies bacterianas: Bacillus cereus, Delftia acidovorans e Acinetobacter sp. Uma diversidade microbiana maior foi isolada a partir do ponto de água purificada, como: Burkholderia sp, Delftia acidovorans, Stenotrophomonas maltophilia, Bacillus cereus, Pseudomonas oryzihabitans e Pseudomonas putida. A espécie Delftia acidovorans, associada a fenômenos de biocorrosão foi o micro-organismo mais encontrado, sendo identificado em cerca de 1/3 do total de isolados. O sistema de água de Farmanguinhos é sanitizado mensalmente com sanitizante à base de glutaraldeído. Pode-se concluir que a sanitização está sendo eficiente para manter a contagem total de micro-organismos mesófilos dentro dos limites especificados. No entanto, algumas ações podem ser tomadas para evitar a presença de biofilmes, o que aprimorará ainda mais a qualidade da água purificada gerada. / The Instituto de Tecnologia em Fármacos (Farmaguinhos), technical- scientific unit of Fundação Oswaldo Cruz, is the largest official pharmaceutical laboratory under the Brazilian Ministry of Health responsible for producing more than one billion drugs per year. The current sanitary laws define that segregated facilities must be dedicated and used for the production of drugs such as penicillin, thus the risk of serious damage to health, due to crosscontamination, can be minimized. Because of this, Farmanguinhos has a dedicated production of amoxicillin, as well as a purified water generator system that caters to such manufacturing is also dedicated to this area. Water is the raw material of the highest volume used in pharmaceutical production so that it has a profound impact on the product quality and patient safety. Therefore, the microbiological quality control of water used in pharmaceutical processes becomes crucial, since microorganisms can survive and proliferate in water systems, becoming sources of microbial and pyrogenic contamination. The identification of these microorganisms and the information obtained about them can be extremely useful in identifying the source of microbial contamination of a product or process, in addition to direct corrective actions, if necessary. The objective of this study was to evaluate the microbial diversity present in the purified water generator system, dedicated to the area of manufacturing penicillin-based drugs of Farmanguinhos. To aim this objective, water samples collected from the entry point of the system, point of drinking water, and the point of departure, point of purified water, were analyzed microbiologically for the count of mesophilic and for the presence of pathogens: Escherichia coli; coliforms and Pseudomonas aeruginosa, respectively. The samples were analyzed between January and May of 2013. All colonies obtained were isolated and subjected to identification by MALDI - TOF equipment. No results above specification for both points were observed in the analyzed period. From the point of drinking water only three bacterial species were isolated: Bacillus cereus, Acinetobacter sp and Delftia acidorovans. A higher microbial diversity was observed from the purified water source, such as Burkholderia sp, Delftia acidovorans, Stenotrophomonas maltophilia, Bacillus cereus, Pseudomonas putida and Pseudomonas oryzihabitans. The species Delftia acidovorans associated with biocorrosion phenomena was the microorganism most frequently found and was identified in about 1/3 of the total isolates. The water system of Farmaguinhos water is sanitized monthly with sanitizing agent comprising glutaraldehyde. It can be concluded that sanitization is being effective to maintain the total count of mesophilic within the specified limits. However, some actions can be taken to avoid the presence of biofilms, which will improve even more the quality of purified water generated.

Les résines végétales sont des sécrétions de végétaux qui ont été utilisées par l’homme de l’Antiquité à nos jours dans de nombreuses applications (pharmaceutique, cosmétique et artistique). Ces exsudats sont composés majoritairement de terpènes. L'identification et la quantification de l'ensemble de ces composés dans les extraits végétaux reste un défi du fait de leur très grande diversité structurale. L’objectif de ce travail a été de développer de nouvelles approches analytiques pour identifier et quantifier les composés terpéniques présents dans ce matériel végétal afin d’en assurer le contrôle qualité et la certification. Deux méthodes séparatives ont été sélectionnées: la TLC et la GC. Pour ces deux techniques on s’est intéressé à toutes les potentialités de leur couplage avec la spectrométrie de masse. Le développement en TLC-1D et TLC-2D a permis le « screening » rapide des résines végétales et la faisabilité du couplage avec le MALDI-TOF-MS a été mise en évidence pour l’identification des marqueurs majoritaires (acides triterpéniques). La GC a permis une caractérisation plus aboutie des résines en mettant en place une méthode d’analyse exhaustive des terpènes des plus volatils au non-volatils. L’optimisation des différentes étapes de la méthodologie GC-MS s’est effectuée en se basant sur la méthode des plans d’expérience ainsi que sur des analyses statistiques tels que l’ACP et la CAH. Dans un souci d’apporter des éléments plus précis pour distinguer les résines les plus proches, la quantification de leurs marqueurs majoritaires a été établie après une validation complète de la méthode GC. L’ensemble de ce travail a permis de développer des outils pour une caractérisation rapide des extraits de résines permettant de différencier les espèces même les plus proches. / Resins are hydrocarbon secretions of many plants and well known for their protective benefits. They have been used as raw materials for a wide range of applications (pharmaceutic, cosmetic and artistic). Plant resins are complex mixtures of organic substances mainly terpenoid compounds which constitute the most abundant and structurally diverse group of plant secondary metabolites. The chemical characterization of this material results in long and difficult separation due to the wide range of polarity and volatility of its constituents. The aim of this work was to develop new analytical approaches to improve the identification of resins certifying their origin and ensuring the quality control. For that purpose two analytical methods were selected: TLC and GC approaches hyphenated to mass spectrometry. TLC-1D and TLC-2D allow a rapid screening and first visual differences of resins. The innovating TLC coupling to MALDI-TOF-MS gives a clear identification of major markers (triterpenic acids). In order to have complementary information about the composition of resins, a gas chromatography-mass spectrometry (GC-MS) method was developed to analyze volatile to non-volatile compounds. The various stages of optimization were based on experimental design and statistical (PCA and HAC) approaches. For closely related resins, a quantitative approach was investigated based on a complete validation for major markers. This work allows the development of two complementary techniques that give a powerful approach for fast and reliable differentiation of various resins even the closest ones.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Alguns Bastonetes Gram-negativos não fermentadores (BGNNF) costumam ser considerados clinicamente pouco significantes e a sua implicação em infecções é subestimada. Devido à similaridade fenotípica, mudanças taxonômicas, baixa reatividade bioquímica e limitações nos bancos de dados em sistemas comerciais, a identificação de BGNNF é frequentemente equivocada, culminando com a denominação de diferentes micro-organismos apenas como BGNNF, por falta de melhor diferenciação. O objetivo desse estudo foi avaliar, por métodos fenotípico convencional, proteômico e molecular, a identificação de BGNNF incomuns isolados em hemoculturas de pacientes atendidos em um hospital universitário no Rio de Janeiro. Foram selecionadas 78 amostras isoladas de hemoculturas caracterizadas no laboratório clinico como BGNNF para a identificação por sequenciamento dos genes 16S RNA e recA, por um conjunto amplo de testes fenotípicos manuais e por MALDI-TOF MS. Os micro-organismos predominantes na amostragem foram genotipados pela técnica de eletroforese em gel de campo pulsado (PFGE). Pelo sequenciamento do gene 16S rRNA, a maioria das amostras (n=31; 40%) foi incluída no gênero Burkholderia, seguido de Pseudomonas stutzeri (10%) e Delftia acidovorans (4%). Os demais isolados foram agrupados em 27 diferentes espécies. O sequencimento do gene recA identificou a maioria das espécies de Burkholderia como Burkholderia contaminans (n=19; 24%). Os testes fenotípicos incluíram as 31 amostras apenas no CBc e para as outras 47 amostras, a concordância com o sequenciamento do gene 16S rRNA em nível de espécie foi de 64% (n=30) e apenas em gênero a concordância foi de 17% (n=8). A análise comparativa geral da identificação por MALDI-TOF MS com o sequenciamento do gene16S rRNA mostrou que 42% (n=33) das 78 amostras foram concordantes em nível de espécie e 45% (n=35) apenas em gênero. Excluindo as amostras do CBc, houve um aumento da concordância em nível de espécie para 60%. As discordâncias parecem ser devido às diferenças nos perfis proteicos das amostras em relação às amostras-referência do banco de dados do equipamento e podem ser aprimorados com a atualização de perfis no sistema. A análise do polimorfismo genético de B. contaminans mostrou a ausência de um clone disseminado causando surto, além da provável origem ambiental das infecções. Os setores de nefrologia e hemodiálise contribuíram com maior número de pacientes com amostras positivas (5 pacientes e 9 amostras). Os grupos clonais BcoD e BcoE foram encontrados em pacientes assistidos no mesmo setor com diferença de quatro meses (BcoD, nefrologia) e 1,5 ano (BcoE, hemodilálise), entre as culturas, respectivamente. As discordâncias entre as técnicas ocorreram principalmente devido a dificuldade de identificação das espécies do CBc. Os BGNNF incomuns são de difícil caracterização independente da metodologia usada e nenhum método por si só foi capaz de identificar todas as amostras. / Some nonfermenting Gram-negative Bacilli (NFGNB) are considered of low clinical significance, and their implication in infections is usually underestimated. Due to their phenotypic similarities, frequent taxonomic changes and low biochemical reactivity, as well as to limitations of bacterial identification commercial system databases, these NFGNB are frequently misidentified and are collectively referred to as NFGNB group, in the lack of a better differentiation. The aim of the present study was to evaluate the performance of the conventional phenotypic method, the proteomic matrix-assisted laser desorption ionization time of flight mass spectometry method (MALDI-TOF MS) and of molecular methods (16S RNA and recA gene sequencing) in the identification of 78 unusual NFGNB isolated from blood cultures of pacients treated at an university hospital in Rio de Janeiro. Clonality of the predominant species identified within these isolates was determined by pulsed-field gel electrophoresis (PFGE). By the 16S rRNA gene sequence analysis, most strains (n = 31; 40%) were included in the Burkholderia spp. followed by Pseudomonas stutzeri (n = 8; 10%), Delftia acidovorans (n = 3; 4%) and Stenotrophomonas maltophilia (n = 3; 4%). The remaining bacterial isolates were included in 27 different species. By the recA gene sequencing technique, most bacteria from the Burkholderia cepacia complex (BCC), samples were classified as Burkholderia contaminans (n=19; 24%). Phenotypic tests provided accurate identification of all 31 isolates included in the BCC by the 16S rRNA gene sequence analysis. For the other 47 samples, agreement of the results obtained with these two techniques in species and genus level identifications occurred in 30 (63,8%) and 17 samples (36,2%), respectively. The results obtained by the MALDI-TOF MS and 16S rRNA gene sequencing methods agreed at species and genus levels in 33 (42%) and 35 isolates (45%), respectively. When bacteria from the BCC were excluded from the analysis, the agreement between the two techniques at species level increased to 60%. Misidentification by the MALDI-TOF MS method may be due to differences in protein spectra between the samples and the reference strains in the equipment database. PFGE analysis of B. contaminans isolates revealed the absence of a disseminate clone causing an outbreak, and the probable environmental source of infections. The nefrology ang dialisis sectors contributed to the greatest number of patients with positive cultures (5 pacients and 9 isolates). Clones BcoD and BcoE were found in blood cultures of pacientes treated in a same sector with differences of 4 months (BcoD, nefrology) and 1.5 year (BcoE, dialisis). The misidentifications occurred mainly due to the hard differentiation of BCC species. Unusual NFGNB are of difficult characterization whatever the methodology used and no method alone was able to identify all the isolates.

En Afrique, les fièvres récurrentes causées par différentes espèces bactériennes du genre Borrelia sont des infections négligées transmises par les arthropodes et sont responsables de manifestations cliniques variant d’une septicémie mortelle à des formes plus bénignes et d'autres manifestations cliniques, en particulier d'avortement chez les femmes enceintes. Quatre espèces différentes de Borrelia, initialement séparées les unes des autres sur la base de leur répartition géographique et de leur vecteur, sont actuellement cultivées de prélèvements cliniques et de vecteurs : Borrelia crocidurae, Borrelia duttonii, Borrelia recurrentis et Borrelia hispanica. Ces différentes espèces circulent sur le continent africain en parallèle avec au moins six espèces non encore cultivées et détectées dans des vecteurs. Notre travail est une contribution à l’investigation des fièvres récurrentes à Borrelia en Afrique. Dans cette perspective, nous avons mis au point la détection rapide en spectrométrie de masse MALDI-TOF des Borrelia dans les tiques en créant au préalable une base de données Borrelia MALDI-TOF-MS. La base de données de Borrelia et un logiciel de soustraction IHU ont été utilisés pour détecter B. crocidurae dans 20 tiques Ornithodoros sonrai, y compris huit tiques qui ont été testées positives pour B. crocidurae par PCR-séquençage, ce qui ouvre la voie à l'utilisation du MALDI-TOF-MS pour la double identification des vecteurs et des agents pathogènes vectorisés, dont il s’agissait du premier exemple maintenant étendu à d’autres modèles dans notre laboratoire. / In Africa, relapsing fever borreliae are neglected arthropod-borne pathogens causing mild to deadly septicemia and other clinical manifestations, particularly abortion in pregnant women. Four different species of Borrelia, initially distinguished one from another on the basis of geography and vector, are currently cultured causative agents in Africa: Borrelia crocidurae, Borrelia duttonii, Borrelia recurrentis et Borrelia hispanica. These different species are circulating in parallel to at least six not-yet cultured species in vectors. Our work consisted in the investigation of recurrent fevers borreliosis in Africa. We have developed rapid detection in MALDI-TOF mass spectrometry of Borrelia in ticks by creating a prior a Borrelia MALDI-TOF-MS database. The Borrelia database and a custom software program that subtracts the uninfected O. sonrai profile were used to detect B. crocidurae in 20 O. sonrai ticks, including eight ticks that tested positive for B. crocidurae by PCR-sequencing; which paves the way for the use of MALDI-TOF-MS for the dual identification of vectors and vectorized pathogens. We have also illustrates a non-specialized circulation of B. crocidurae borreliae within a collection of 35 O. sonrai ticks in West Africa. These ticks were genotyped by 16S rRNA mitochondrial gene sequencing while B. crocidurae was genotyped by Multispacer Sequence Typing (MST). The 35 ticks were grouped into 12 genotypes strong geographic structuring and 35 B. crocidurae into 29 genotypes without strict geographic structure. One O. sonrai genotype carried several B. crocidurae genotypes and one B. crocidurae genotype was found in different O. sonrai genotypes.

Philosophiae Doctor - PhD / This study reports on a proteomic analysis of sorghum proteomes in response to salt and hyperosmotic stresses. Two-dimensional gel electrophoresis (2DE) in combination with mass spectrometry (MS) was used to separate, visualise and identify sorghum proteins using both sorghum cell suspension cultures and whole plants. The sorghum cell suspension culture system was used as a source of culture filtrate (CF) proteins. Of the 25 visualised CBB stained CF spots, 15 abundant and well-resolved spots were selected for identification using a combination of MALDI-TOF and MALDI-TOFTOF MS, and database searching. Of these spots, 14 were positively identified as peroxidases, germin proteins, oxalate oxidases and alpha-galactosidases with known functions in signalling processes, defense mechanisms and cell wall metabolism. / South Africa

Der zeitnahe und möglichst sichere Nachweis bakterieller Krankheitserreger und deren Empfindlichkeit gegenüber verfügbaren antibakteriell wirksamen Chemotherapeutika (Antibiotika) stellt einen Hauptaufgabenbereich der medizinischen mikrobiologischen Routinediagnostik dar. Hierzu wurden im Laufe der Jahre unterschiedliche Methoden entwickelt, womit von der genauen Beschreibung der Kolonie- und mikroskopischen Morphologie, Anfärbbarkeit und Formation über die Charakterisierung der biochemischen Leistungsfähigkeit bis hin zur genauen Sequenzierung des gesamten Genoms ein enormer Fortschritt zu verzeichnen war. Seit Mitte der 1990er Jahre etablierte sich die Massenspektrometrie als phänotypisches Nachweisverfahren und gewann zunehmend an Bedeutung. Ebenso konnten Erfolge beim Nachweis Antibiotika resistenter Bakterien verzeichnet werden. Um das Potential dieser noch jungen Nachweismethode weiter zu erforschen, wurden in dieser Arbeit Spezies der Familie Enterobacteriaceae und der Nonfermenter in eine eigene massenspektrometrische Datenbank aufgenommen, um diese als Grundlage zur Validierung des Identifizierungspotentials der Methode mittels Blindstudie zu nutzen. Im selben Arbeitsschritt wurde der Versuch unternommen, Antibiotika resistente Stämme im Zuge der Speziesidentifizierung zu detektieren, um so Aussagen über eine mögliche Einschränkung der therapeutischen Möglichkeiten und gegebenenfalls notwendigen Hygienemaßnahmen treffen zu können.