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.

Sepsis är ett allvarligt tillstånd som uppstår när bakterier går från vävnad till blodbanan. Positiva blododlingar odlas på agarplattor och bakterier analyseras med Matrix Assisted Laser Desorption Ionisation Time-of-flight Mass spectrometry (MALDI-TOF MS) där prov blandas med en matrix och sedan bestrålas med laser. Proteinerna i provet joniseras och rör sig mot en detektor, vilket ger ett m/z-spektrum som jämförs med referensspektrum i en databas och ett score-värde erhålls över hur väl analyten liknar referensen. Arbetets syfte var att undersöka möjligheten att direktidentifiera bakterier från blod med en viss preparation innan analys med MALDI-TOF MS och på så vis möjliggöra snabbare preliminära svar samt undersöka möjligheten att särskilja Staphylocoocus aureus och koagulasnegativa stafylokocker. Innan analys med MALDI-TOF MS centrifugerades blod från positiva blododlingar blod i flera steg med 5 % natriumkloridlösning (NaCl-metoden). Dessutom testades ett kommersiellt kit (Sepsityper, Bruker Daltonics). Med NaCl-metoden sågs korrekt identifiering hos 66 % av inokulerade proverna. Av blododlingar innehållande med S. aureus respektive koagulasnegativa stafylokocker identifierades 60 % respektive 43 % av bakterierna korrekt. Med Sepsiptyper erhöll 58 % av proverna godkänt score-värde. Slutsatsen blev att det är möjligt att identifiera bakterier direkt från blod efter viss preparation, men metoden bör utvecklas mer då det fanns en signifikant skillnad i score mellan NaCl-metoden och nuvarande metod. Det är dock möjligt att skilja mellan Staphylococcus aureus och koagulasnegativa stafylokocker. Fler studier är nödvändiga för att avgöra möjligheten att föra in någon av metoderna i rutindiagnostiken.

Le répertoire des bactéries est mal connu en Afrique du fait des méthodes d’étude essentiellement basées sur des techniques de culture sur milieux simples associées à des tests biochimiques, ce qui ne permet pas son exploration. Il a néanmoins été récemment bouleversé par l’usage systématique de la spectrométrie de masse de type MALDI-TOF MS.Au cours de nos travaux de thèse de doctorat, nous avons utilisé deux types de spectromètres de masse: le MALDI-TOF Vitek MS, nouvellement installé à Dakar; le MALDI-TOF Microflex LT, installé à Marseille. Les résultats que nous avons obtenus ont montré, pour la première fois, que la technique du MALDI-TOF est efficace et tout à fait adaptée en Afrique pour le diagnostic spécifique de routine. Cette performance a conduit le laboratoire clinique de l’HPD à opter pour son utilisation à la place des traditionnelles techniques d’identification phénotypique telles que les galeries API. Nous avons également confirmé que le MALDI-TOF est un puissant outil d’identification des espèces bactériennes rarement impliquées dans les maladies infectieuses humaines. De plus, cet outil nous a permis de détecter sept nouvelles espèces de bactéries isolées pour la première fois chez l’homme. En Afrique, il faudrait donc multiplier l’installation de spectromètre de masse MALDI-TOF, ou mettre en place des réseaux autour de plateformes MALDI-TOF sous-régionales partagées entre plusieurs structures sanitaires et/ou de recherche. / The Africa bacteria repertory is unfamiliar because the available tools in this region are not allowed its best knowledge. In fact, bacteria are most often identified using culture techniques on simple media and biochemical tests which enable the identification of some common characters. These methods do not facilitate an exhaustive knowledge of the bacterial repertory; consequently they have recently been revolutionized by the systematic use of MALDI-TOF mass spectrometry (MS).In our thesis we used two mass spectrometers, respectively, MALDI-TOF Vitek MS currently installed at Dakar (Senegal) and MALDI-TOF Microflex LT installed in Marseille (France). In addition we have also confirmed that MALDI-TOF is a powerful tool for identifying bacterial species rarely involved in human infectious diseases. Thus in adopting the MALDI-TOF as a first-line tool in bacterial identification before Gram staining or other techniques of phenotypic identifications based on chemical characteristics, we discovered seven new species of bacteria isolated for first time in humans. Microbial identification using MALDI-TOF MS is currently feasible in Africa. Its performance and effectiveness in routine diagnosis of clinical microbiology laboratories have been proven. It is necessary either to increase the installation of MALDI-TOF, or establishing a network around a shared MALDI-TOF platform between several structures located in the same area, especially in the underdeveloped countries of Africa amortization of investment costs of the device, because it allowed reducing the time of reporting results and indirectly facilitating better care for patients.

La chromoblastomycose (CBM) et la sporotrichose (SPT) sont des infections fongiques chroniques des tissus sous-cutanés. Elles touchent surtout les membres, après blessure végétale ou souillure tellurique. Les principaux agents fongiques responsables de CBM appartiennent aux genres Fonsecaea et Cladophialophora tandis que Sporothrix est à l’origine de la SPT. A Madagascar, les études menées entre 1955 et 1994 ont montré une prévalence de la CMB à 0,5/100 000 habitants faisant considérer ce pays comme le premier foyer mondial. Depuis, aucune donnée n’a permis d’actualiser ces observations. Malgré les nombreux cas de SPT observés par les médecins, son épidémiologie à Madagascar n’a jamais été décrite.L'objectif général était d'évaluer l’incidence actuelle de ces mycoses à Madagascar. Les objectifs spécifiques étaient de caractériser les espèces fongiques responsables et de mettre en place un réseau clinico-biologique pérenne permettant une gestion adaptée des patients et l’utilisation de méthodes moléculaires pour l’identification des souches.Une étude prospective conduite entre mars 2013 et juin 2017 a donné lieu à des consultations dans les régions ou dans le service de dermatologie de l’hôpital universitaire d’Antananarivo et a permis d’inclure des patients présentant des lésions sous-cutanées chroniques. Les méthodes conventionnelles de diagnostic mycologique ont été complétées par des méthodes moléculaires (PCR, séquençage, MALDI-TOF-MS). Les cas ont été classés au cours de réunions de concertation clinico-biologique.Au total, 148 patients d’âge moyen de 41 ans avec une prédominance d’hommes (75,0%) ont été inclus : 63 cas de SPT (42,5%) et 50 cas de CBM (33,8%) ont été confirmés. L’incidence annuelle de le CBM a été estimée à 0,38/100 000 habitants dans la région Sava au Nord où les cas prédominent avec au niveau du district Anosibe An’Ala (à l’Est) une incidence maximale de 1,12/100 000. Alors que la CBM était prédominante dans le Nord-Est, l'Est et le Sud de l'île, étonnamment, la SPT était presque exclusivement localisée sur les hautes terres centrales. L’incidence globale moyenne de SPT était de 0,17/100 000 habitants dans les hauts plateaux et de 0,07/100 000 pour l’ensemble de Madagascar. Pour la SPT, le risque est plus élevé chez les jeunes (< 18 ans) et les formes cutanéo-lymphatiques des membres supérieurs sont les plus fréquentes. Pour la CBM, le risque de contamination est élevé chez les agriculteurs et les professions de services et la majorité des lésions (82,9%) était localisée au niveau des membres inférieurs. Sur le plan mycologique, 63 Sporothrix schenckii, 7 Cladophialophora carrionii, 29 Fonsecaea sp dont 22 F. nubica ont été identifiés. Pour le genre Fonseceae, l’identification de l’espèce nubica remplace l‘espèce pedrosoi initialement proposée pour les isolats malgaches et souligne l’importance de l’identification moléculaire pour une classification précise des espèces.Cette première étude sur la SPT humaine à Madagascar a permis d’actualiser les données épidémiologiques mondiales et de classer Madagascar avec un niveau endémique modéré alors qu’il était considéré comme faible jusque-là. La CBM persiste à une incidence élevée et comparable à celle décrite jusqu’en 1994, illustrant l’absence de contrôle de cette mycose dans le pays. La constitution d’un réseau clinico-biologique local stable et les nouveaux outils diagnostics mis en place dans ce travail (PCR et le MALDI-TOF MS), vont faciliter la conduite de programmes de surveillance et de contrôle alors que l’OMS a récemment classé la CBM en maladie tropicale négligée. Une enquête environnementale est en cours pour détecter les sources de contamination dans l’environnement. Le réseau mis en place permettra dans un futur proche de nouvelles études thérapeutiques (nouveaux schémas thérapeutiques) ou génétique (facteurs d’hôtes) sur la CBM et SPT mais également sur d’autres mycoses endémiques et encore négligées à Madagascar / Chromoblastomycosis (CBM) and sporotrichosis (SPT) are chronic subcutaneous or cutanéo-lymphatic infections found mostly in tropical and subtropical regions. Studies carried out by the Institut Pasteur of Madagascar between 1955 and 1994 provided an inventory of the number of cases of CBM and identified this country as the leading focus of this mycosis worldwide. Mean incidence was estimated at about 0.5/100,000 inhabitants at the time. No new data have been obtained to update the epidemiological situation. About SPT, only sporadic cases have been reported in Madagascar, due to the absence of specific surveillance. CBM is usually caused by dematiaceous fungi, principally Fonsecaea spp. and Cladophialophora spp. The causal agent of SPT is Sporothrix schenckii, a dimorphic hyphomycete.The objectives of this study was to update the data on epidemiology and to evaluate the current burden of these two fungal infections. In addition, we aimed to set up a durable local bio-clinical network and to implement molecular tools to ensure reliable species identification (PCR, sequencing, mass spectrometry).A prospective study, involving the recruitment of patients with suspect lesions was undertaken from March 2013 to June 2017. Patients were included in the dermatology department of the univerity hospital of Antananarivo and through field campaigns in rural areas. Clinical samples were collected and analyzed with conventional mycological methods and molecular tools. Classification of the cases was achieved by the confrontation of mycological and clinical features.Among the 148 patients (mean age 41; male 75.0%): 63 SPT cases (42.5%) and 50 CBM cases (33.8%) were diagnosed. The highest annual incidence of CBM was estimated at 0.38/100,000 inhabitants in the Sava region located to the north. At the district level, the peak incidence was 1.12/100,000 at Anosibe An’Ala, eastern part of the country. Whereas CBM predominated at the periphery of the island, SPT was surprinsgly concentrated in the highlands where the mean incidence was 0.17/100,000 inhabitants. The incidence in the whole country was 0.07/100,000. SPT likelihood of infection was higher in young (<18 years) and the cutaneo-lymphatic forms of the upper limbs were the most frequent. For CBM, farmers and service workers were at high risk and the lesions were mostly (82.9%) located to the lower limbs. The mycological analyses revealed 63 strains of Sporothrix schenckii, 7 of Cladophialophora carrionii, 29 of Fonsecaea sp including 22 F. nubica. F. nubica identification corrected the one of F. pedrosoi previously found in Madagascar, highlighting the need for a molecular analysis of the strains.This is the first study describing human SPT in Madagascar. The SPT burden can now be considered as moderate instead of low as it was described before. It reveals an unexpected concentration of the patients in the central highlands. CBM burden was found at a high level, regrettably similar to the one described 20 years ago, showing the lack of control of this infection. The implementation of a durable bio-clinical network and of the new molecular tools (PCR et le MALDI-TOF MS) developed in this work will easy the development of surveillance programs, especially as the WHO recently added CBM in the neglected tropical diseases list. The network that was built for this work will be used for further therapeutic trials on new schedules of treatment, new drugs or new formulations as well as genetic studies about predisposing factors of CBM and SPT and others deep fungal infections that are still neglected in Madagascar. Yet, an environmental survey is ongoing to describe the sources of contamination.

Neste trabalho é descrito um método prático e eficiente para dissociar, em subunidades &alpha; e &beta;, quantidades pequenas (da ordem de microgramas) dos hormônios foliculotrofina (hFSH), luteotrofina (hLH) e tireotrofina (hTSH) humana, nativos e recombinantes. A dissociação destes hormônios foi conseguida incubando-os, durante 16 horas, a 37ºC, com diferentes concentrações de ácido acético: 3M, 5M e 0,4M respectivamente para o hFSH, hLH e hTSH. Nestas condições, uma eficiência de dissociação acima de 98% foi obtida. Esta eficiência foi calculada com base nas determinações de massa dos heterodímeros e das subunidades, realizadas por MALDI-TOF-MS. Uma separação rápida e quantitativa das subunidades, com rendimentos da ordem de 80-90%, foi conseguida por cromatografia líquida de alta eficiência em fase reversa (RP-HPLC) em uma coluna C4. As subunidades foram caracterizadas quanto à pureza, hidrofobicidade, massa molecular e distribuição de carga por HPLC de exclusão molecular e fase reversa, SDS-PAGE e focalização isoelétrica. Quando analisadas quanto à hidrofobicidade, as subunidades mostraram-se aproximadamente iguais, enquanto as subunidades &beta; dos três heterodímeros apresentaram a seguinte escala de hidrofobicidade: &beta;-hFSH < &beta;-hTSH < &beta;-hLH. Com relação à massa molecular relativa (Mr), as subunidades &alpha; e &beta; do hFSH apresentaram as maiores Mr enquanto as subunidades do hLH as menores. A distribuição dos isômeros de carga das subunidades dos três hormônios ocorreu em uma região ácida, para o hFSH, em uma região básica, para o hLH e em uma região intermediária, para o hTSH. As subunidades &alpha; dos três hormônios, quando analisadas via SDS-PAGE, apresentaram praticamente a mesma mobilidade eletroforética, enquanto as subunidades &beta; apresentaram diferentes taxas de migração (mR), sendo mR &beta;-hFSH < mR &beta;-hTSH < mR &beta;-hLH. Diferenças relativas à massa molecular, hidrofobicidade, migração eletroforética e distribuição de carga foram encontradas entre as preparações recombinantes e hipofisárias dos três hormônios. O método descrito é suave, prático e flexível e pode ser adaptado à dissociação de outras glicoproteínas heterodiméricas recombinantes ou nativas. Permite não só estudos e caracterização direta de cada subunidade, como também detectar a presença de subunidades livres em preparações farmacêuticas, que são contaminantes indesejáveis, sendo, portanto, uma ferramenta extremamente útil para o controle de qualidade de produtos farmacêuticos. / In this work a practical and efficient method for the dissociation into &alpha;-and &beta;-subunits of small amounts (microgram range) of pituitaryderived and recombinant human follicle-stimulating hormone (hFSH), human luteotropin (hLH) and human thyrotropin (hTSH) is described. Dissociation was achieved by overnight treatment of the glycoproteins, at 37ºC, with acetic acid in different concentrations: 3M, 5M and 0,4M for hFSH, hLH and hTSH respectively. In these conditions, a dissociation efficiency of > 98% was attained. This efficiency was calculated on the basis of relative mass determinations of the heterodimers and subunits carried out via mass spectrometry (MALDI-TOF-MS). The &alpha;-and &beta;-subunits were rapidly and quantitatively separated by reversed-phase high-performance liquid chromatography (RP-HPLC) on a C4 column with yields of the order of 80-90%. The isolated subunits were characterized concerning their purity, hidrophobicity, molecular mass and charge distribution, via size exclusion and RP-HPLC, SDS-PAGE and isoelectric focusing. When analyzed with relation to the hydrophobicity, the &alpha;-subunits presented approximately the same hydrophobicity, while &beta;-subunits showed the following scale: &beta-hFSH < &beta;-hTSH < &beta;-hLH. Concerning molecular mass, &alpha;- and &beta;-subunits of hFSH were shown to have the highest while hLH subunits the lowest. Charge isomers of the subunits of the three glycohormones were predominantly distributed in an acidic region for hFSH, in a basic region for hLH, and in a wider pH range (acidic and basic) for hTSH. Similar migration rates (mR), analyzed via SDS-PAGE, were observed for the &alpha;-subunits of the three hormones. A greater variation was found for the &beta;-subunits: mR &beta;-hFSH < mR &beta;-hTSH < mR &beta;-hLH. Differences between recombinant and pituitary preparations of three hormones were observed with relation to molecular mass, hydrophobicity, electrophoretic migration and charge distribution. The described method is mild, practical and flexible and can be adapted to dissociate any recombinant or native heterodimeric glycoprotein, allowing studies and direct characterization of each subunit as well as the detection of free subunits that are undesired contaminants in pharmaceutical preparations, being also an extremely useful tool for the quality control of pharmaceutical products.

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.

Neste trabalho é descrito um método prático e eficiente para dissociar, em subunidades &alpha; e &beta;, quantidades pequenas (da ordem de microgramas) dos hormônios foliculotrofina (hFSH), luteotrofina (hLH) e tireotrofina (hTSH) humana, nativos e recombinantes. A dissociação destes hormônios foi conseguida incubando-os, durante 16 horas, a 37ºC, com diferentes concentrações de ácido acético: 3M, 5M e 0,4M respectivamente para o hFSH, hLH e hTSH. Nestas condições, uma eficiência de dissociação acima de 98% foi obtida. Esta eficiência foi calculada com base nas determinações de massa dos heterodímeros e das subunidades, realizadas por MALDI-TOF-MS. Uma separação rápida e quantitativa das subunidades, com rendimentos da ordem de 80-90%, foi conseguida por cromatografia líquida de alta eficiência em fase reversa (RP-HPLC) em uma coluna C4. As subunidades foram caracterizadas quanto à pureza, hidrofobicidade, massa molecular e distribuição de carga por HPLC de exclusão molecular e fase reversa, SDS-PAGE e focalização isoelétrica. Quando analisadas quanto à hidrofobicidade, as subunidades mostraram-se aproximadamente iguais, enquanto as subunidades &beta; dos três heterodímeros apresentaram a seguinte escala de hidrofobicidade: &beta;-hFSH < &beta;-hTSH < &beta;-hLH. Com relação à massa molecular relativa (Mr), as subunidades &alpha; e &beta; do hFSH apresentaram as maiores Mr enquanto as subunidades do hLH as menores. A distribuição dos isômeros de carga das subunidades dos três hormônios ocorreu em uma região ácida, para o hFSH, em uma região básica, para o hLH e em uma região intermediária, para o hTSH. As subunidades &alpha; dos três hormônios, quando analisadas via SDS-PAGE, apresentaram praticamente a mesma mobilidade eletroforética, enquanto as subunidades &beta; apresentaram diferentes taxas de migração (mR), sendo mR &beta;-hFSH < mR &beta;-hTSH < mR &beta;-hLH. Diferenças relativas à massa molecular, hidrofobicidade, migração eletroforética e distribuição de carga foram encontradas entre as preparações recombinantes e hipofisárias dos três hormônios. O método descrito é suave, prático e flexível e pode ser adaptado à dissociação de outras glicoproteínas heterodiméricas recombinantes ou nativas. Permite não só estudos e caracterização direta de cada subunidade, como também detectar a presença de subunidades livres em preparações farmacêuticas, que são contaminantes indesejáveis, sendo, portanto, uma ferramenta extremamente útil para o controle de qualidade de produtos farmacêuticos. / In this work a practical and efficient method for the dissociation into &alpha;-and &beta;-subunits of small amounts (microgram range) of pituitaryderived and recombinant human follicle-stimulating hormone (hFSH), human luteotropin (hLH) and human thyrotropin (hTSH) is described. Dissociation was achieved by overnight treatment of the glycoproteins, at 37ºC, with acetic acid in different concentrations: 3M, 5M and 0,4M for hFSH, hLH and hTSH respectively. In these conditions, a dissociation efficiency of > 98% was attained. This efficiency was calculated on the basis of relative mass determinations of the heterodimers and subunits carried out via mass spectrometry (MALDI-TOF-MS). The &alpha;-and &beta;-subunits were rapidly and quantitatively separated by reversed-phase high-performance liquid chromatography (RP-HPLC) on a C4 column with yields of the order of 80-90%. The isolated subunits were characterized concerning their purity, hidrophobicity, molecular mass and charge distribution, via size exclusion and RP-HPLC, SDS-PAGE and isoelectric focusing. When analyzed with relation to the hydrophobicity, the &alpha;-subunits presented approximately the same hydrophobicity, while &beta;-subunits showed the following scale: &beta-hFSH < &beta;-hTSH < &beta;-hLH. Concerning molecular mass, &alpha;- and &beta;-subunits of hFSH were shown to have the highest while hLH subunits the lowest. Charge isomers of the subunits of the three glycohormones were predominantly distributed in an acidic region for hFSH, in a basic region for hLH, and in a wider pH range (acidic and basic) for hTSH. Similar migration rates (mR), analyzed via SDS-PAGE, were observed for the &alpha;-subunits of the three hormones. A greater variation was found for the &beta;-subunits: mR &beta;-hFSH < mR &beta;-hTSH < mR &beta;-hLH. Differences between recombinant and pituitary preparations of three hormones were observed with relation to molecular mass, hydrophobicity, electrophoretic migration and charge distribution. The described method is mild, practical and flexible and can be adapted to dissociate any recombinant or native heterodimeric glycoprotein, allowing studies and direct characterization of each subunit as well as the detection of free subunits that are undesired contaminants in pharmaceutical preparations, being also an extremely useful tool for the quality control of pharmaceutical products.

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.