La métabolomique propose une approche basée sur l’étude d’une réponse métabolique globale et dynamique d’un organisme à des stimuli biologiques ou à des mutations génétiques et ainsi constitue un outil de recherche translationnel à fort potentiel en oncologie. Nous avons appliqué l’approche métabonomique par RMN à haut champ à l’analyse d’échantillons sanguins issus d’études cliniques coordonnées par le Centre Léon Bérard et pour la cohorte prospective E3N (Etude Epidémiologique auprès de femmes de la MGEN) dans le but d’identifier de marqueurs systémiques du cancer à partir de fluides biologiques humains. Tout d’abord, l’analyse statistique des profils métaboliques de sérums de patientes atteintes d’un cancer du sein a permis de mettre en évidence une signature métabolique discriminant les patients ayant un cancer localisé des patientes métastatiques et de révéler des biomarqueurs associés à la sévérité de ce cancer. D’autre part, cette approche permet des investigations sur les effets de la chimiothérapie. Ainsi, lors de l’étude de sérums de patients atteints d’un cancer du rein métastatique traités par une chimiothérapie expérimentale, une signature métabolique caractéristique de la modification plus rapide du métabolisme de l’hôte a été mise en évidence pour ce traitement en comparaison à deux thérapies conventionnelles. Enfin, l’analyse de plasmas sanguins issus de la cohorte E3N s’intéresse à l’identification de biomarqueurs associés à la survenue du cancer du sein et de son étiologie. Après identification et quantification des sources de variations systématiques impactant les profils métaboliques obtenus, une analyse stratifiée de la cohorte « cas prospectifs »-contrôles est présentée dans cette thèse. / Metabolomics offers an approach based on the study of a comprehensive and dynamic metabolic response of an organism to biological stimuli or genetic mutations and thus constitutes a tool for translational research with a strong potential in oncology. We applied the metabonomic approach by high field NMR for the analysis of blood samples from clinical studies coordinated by the Centre Léon Bérard and for the prospective E3N cohort ( Etude Epidémiologique auprès de femmes de la MGEN ) in order to identify systemic cancer markers from human biological fluids . First, the statistical analysis of metabolic serum profiles from patients with breast cancer has highlighted a metabolic signature discriminating patients with localized cancer and metastatic patients and reveal biomarkers associated with severity of this cancer. Furthermore , this approach allows investigations on the effects of chemotherapy. Thus, during the study of sera from patients with metastatic renal cell carcinoma treated with experimental chemotherapy, a characteristic metabolic signature of faster modification of the host metabolism has been demonstrated for this treatment in comparison to two standard therapies. Finally, analysis of blood plasma from the E3N cohort interests the identification of biomarkers associated with the development of breast cancer and its etiology . After identification and quantification of sources of systematic variations affecting the metabolic profiles obtained, a stratified analysis of the "prospective case "- controls cohort is presented in this thesis.

Um total de 107 S. aureus isolados de casos de mastite, glândulas portadoras, pele do úbere, ordenhadores e insufladores, em rebanhos de São Paulo e Pernambuco, foram tipados por técnicas moleculares PCR-RFLP do gene coa e PCR de spa distinguiram seis perfis. Todas as amostras amplificaram genes coa, spa, icaA e 69% produziram biofilme glicose-induzido in vitro. PFGE identificou 31 perfis e 12 linhagens. Uma linhagem foi predominante (P < 0,0001) e amplamente disseminada em ambas as regiões. Um mesmo perfil foi isolado de mastite clínica, subclínica e portadoras. Houve heterogeneidade genética entre isolados de fazenda. Isolados de origem humana e animal constituíram populações distintas. Poucos isolados de leite, insufladores e pele do úbere tiveram igual perfil. Uma amostra extramamária, 77% dos isolados de leite e. 99% de S. aureus de portadoras produziram biofilme. Não foi detectada correlação entre produção de biofilme e CCS. O isolamento sucessivo do mesmo perfil de PFGE de glândulas assintomáticas por mais de 16 dias caracterizou o estágio de portador. / A total of 107 S. aureus isolated from bovine milk, udder skin, milkers and milking machine, from São Paulo and Pernambuco herds was typed by molecular techniques. PCR-RFLP coa gene and PCR spa gene distinguished six amplicons. All strains amplified coa, spa, icaA genes and, 69% produced in vitro glucose-induced biofilm. PFGE identified 31 pulsotypes, 12 lineages. One of the lineages was predominantly isolated (P<0.0001) and widely disseminated. A same pulsotype was isolated from clinical and, subclinical mastitis as well as from carriers. There was genetic heterogeneity among isolates from the herds. Strains from human and animal origin were genetically different. Few isolates from milk, milking machine and udder skin showed similar pulsotype. An extramammary strain, 77% of the milk isolates and, 99% of the S. aureus isolated from carriers produced biofilm. It was not detected any correlation between SCC and biofilm production. The successive isolation during more than 16 days of a same pulsotype from the asymptomatic glands characterized the carrier status.

O presente estudo teve por objetivos caracterizar os vírus Influenza circulantes na cidade de São Paulo e verificar a variabilidade genética do gene da neuraminidase (NA) dos Influenzavírus A. Um total de 3009 amostras de crianças internadas no Hospital Universitário da USP, no período de 1995 a 2006, foram submetidas à duplex RT-PCR para detecção dos Influenzavírus A e B (IA e IB). As amostras positivas para IA foram submetidas à subtipagem pela multiplex RT-PCR. Cento e trinta e três amostras (4,4%) foram positivas, sendo 88,0% (117/133) IA e 12,0% (16/133) IB. Entre as amostras IA, 94 eram H3N2, 7 H1N1 e 16 não foram subtipadas pela multiplex. Um total de 74 amostras (71 H3N2 e 3 H1N1) tiveram o gene da neuraminidase sequenciado (total ou parcialmente). As sequências obtidas foram submetidas à análise filogenética, sendo verificado o agrupamento das cepas circulantes em clusters por ano de isolamento, demonstrando sua evolução temporal. Quando comparadas com as cepas vacinais utilizadas no período, foi verificada uma boa similaridade. / The aim of this study was to characterize Influenza virus circulating in São Paulo city and the neuraminidase (NA) gene sequence of Influenzavirus A in 3009 samples from children hospitalized at USP University Hospital, from 1995 to 2006. Samples underwent duplex RT-PCR for detection and typing of Influenza virus A and B (IA and IB) and also were submitted to multiplex PCR for subtyping of IA. Among 3009 samples, 4.4% were Influenza virus, being 88.0% IA (117/133) and 12.0% IB (16/133). Among samples of IA, 94 were characterized as H3N2, 7 H1N1 and 16 were not subtyped. A total of 74 samples (71 H3N2 and 3 H1N1) had the NA gene sequenced (total or partially). Sequences obtained were compared to sequences of the world and sequences of vaccine strains. Brazilian samples were grouped in clusters with samples isolated in the same year. It was also found a good similarity between circulating strains and vaccine strains.

As espécies do Complexo Acinetobacter calcoaceticus-A. baumannii (ACB) são importantes causadoras de Infecções Relacionadas à Assistência à Saúde em todo o mundo. Detêm maior relevância os isolados com resistência aos antimicrobianos, os quais impactam negativamente no prognóstico, na mortalidade e custos associados ao cuidado com o paciente. O objetivo deste estudo foi avaliar a diversidade genética e o perfil de susceptibilidade antimicrobiana de 134 isolados multirresistentes de A. baumannii presentes no Hospital das Clínicas da Faculdade de Medicina de Botucatu, entre 2007 e 2014. A identificação de A. baumannii deu-se pela pesquisa dos genes blaOXA-51-like e gltA, detectados em 85% (n=114) dos isolados Os isolados de Acinetobacter não-baumannii foram identificados por sequenciamento gênico como A. nosocomialis (n=4; 3,1%), A. pittii, A. bereziniae (n=2; 1,7%, cada), A. ursingii, A. variabilis, A. gyllenbergii (n=1; 0,9% cada) e Acinetobacter spp (n=2; 1,7%). Os isolados de A. baumannii foram submetidos às técnicas de PCR multiplex para detecção de outras oxacilinases, pesquisa de ISAba1, teste de susceptibilidade antimicrobiana, tipagem molecular por eletroforese em campo pulsado (PFGE), por sequência trilocus (3LST) e por sequência multilocus (MLST). Detectou-se o gene blaOXA-23-like em 105 isolados (92,1%), estando 100% associados a ISAba1; blaOXA-72 em um isolado (0,9%) e blaOXA-231 em dois isolados (1,7%). A maior parte (n=66; 57,9%) dos isolados foi classificada como extensivamente resistentes (XDR). O PFGE agrupou os isolados em 11 clusters (A-K) e o MLST identificou os isolados pertencentes majoritariamente aos clones CC79 (42,4%), CC1 (16,6%), CC15 (12,1%) e ao ST317 (18,2%). Os resultados do MLST e 3LST concordaram em 95,6%. Foi verificada a ocorrência de diferentes perfis de PFGE em A. baumannii MDR e XDR, predominando cepas carreadoras de ISAba1/OXA-23-like e pertencentes aos CC1, CC15, CC79, ST317. Predominaram o ST317 nos anos iniciais e o CC79 (ST730) de 2011 a 2014. Estes resultados fornecem subsídios que ressaltam a necessidade de monitoramento e controle de patógenos multirresistentes / Species of Acinetobacter calcoaceticus-A. baumannii Complex (ACB) are important causes of Healthcare Associated Infections worldwide. More relevant are isolates with antimicrobial resistance, which have a negative impact on the outcome, mortality and costs associated with patient care. The aim of this study was to evaluate the genetic diversity and the antimicrobial susceptibility profile of 134 multiresistant ACB spcies strains present at Botucatu Medical School Teaching Hospital between 2007 and 2014. Identification of A. baumannii species was by detection of blaOXA-51-like and gltA genes, detected in 85% (n=114) of the isolates. Non-baumannii Acinetobacter species were identified by gene sequencing as A. nosocomialis (n=4, 3.1%), A. ursingii, A. variabilis, A. gyllenbergii (n=1, 0.9% each) and Acinetobacter spp (n=2; 1.7%). A. baumannii isolates were submitted to multiplex PCR for other oxacillinases and ISAba1 detection, antimicrobial susceptibility testing, molecular typing by pulsed field gel electrophoresis (PFGE), trilocus sequence typing (3LST) and multilocus sequence typing (MLST). blaOXA-23-like gene was detected in 105 isolates (92.1%), of which 100% were associated with ISAba1; blaOXA-72 was present in one isolate (0.9%) and blaOXA-231, in two isolates (1.7%). The majority (n=66; 57.9%) of isolates were classified as extensively resistant (XDR). The PFGE grouped the isolates into 11 clusters (A-K) and MLST identified the isolates belonging mainly to CC79 (42.4%), CC1 (16.6%), CC15 (12.1%) and ST317 (18.2%). MLST and 3LST results were 95.6% concordant. We verified the occurrence of different PFGE profiles in multidrug-resistant (MDR) and extensively drug-resistant (XDR) A. baumannii, predominantly presenting ISAba1/OXA-23-like genes and belonging to CC1, CC15, CC79 and ST317. There was a prevalence of ST317 in the early years and CC79 (ST730) from 2011 to 2014. Our results highlight the importance of surveillance and control of multiresistant pathogens

La donnée de séquences nucléotidiques permet d'inférer des arbres phylogénétiques, ou phylogénies, qui décrivent leur lien de parenté au cours de l'évolution. Associer à ces séquences leur date de prélèvement ou leur pays de collecte, permet d'inférer la localisation temporelle ou spatiale de leurs ancêtres communs. Ces données et procédures sont très utilisées pour les séquences de virus et, notamment, celles du virus de l'immunodéficience humaine (VIH), afin d'en retracer l'histoire épidémique à la surface du globe et au cours du temps. L'utilisation de séquences échantillonnées à des moments différents (ou hétérochrones) sert aussi à estimer leur taux de substitution, qui caractérise la vitesse à laquelle elles évoluent.Les méthodes les plus couramment utilisées pour ces différentes tâches sont précises, mais lourdes en temps de calcul car basées sur des modèles complexes, et ne peuvent traiter que quelques centaines de séquences. Devant le nombre croissant de séquences disponibles dans les bases de données, souvent plusieurs milliers pour une étude donnée, le développement de méthodes rapides et efficaces devient indispensable. Nous présentons une méthode de distances, Ultrametric Least Squares, basée sur le principe des moindres carrés, souvent utilisé en phylogénie, qui permet d'estimer le taux de substitution d'un ensemble de séquences hétérochrones, dont on déduit ensuite facilement les dates des spéciations ancestrales. Nous montrons que le critère à optimiser est parabolique par morceaux et proposons un algorithme efficace pour trouver l'optimum global.L'utilisation de séquences échantillonnées en des lieux différents permet aussi de retracer les chaînes de transmission d'une épidémie. Dans ce cadre, nous utilisons la totalité des séquences disponibles (~3 500) du sous-type C du VIH-1 (VIH de type 1), responsable de près de 50% des infections mondiales au VIH-1, pour estimer ses principaux flux migratoires à l'échelle mondiale, ainsi que son origine géographique. Des outils novateurs, basés sur le principe de parcimonie combiné avec différents critères statistiques, sont utilisés afin de synthétiser et interpréter l'information contenue dans une grande phylogénie représentant l'ensemble des séquences étudiées. Enfin, l'origine géographique et temporelle de ce variant (VIH-1 C) au Sénégal est précisément explorée lors d'une seconde étude, portant notamment sur les hommes ayant des rapports sexuels avec des hommes. / Nucleotide sequences data enable the inference of phylogenetic trees, or phylogenies, describing their evolutionary re-lationships during evolution. Combining these sequences with their sampling date or country of origin, allows inferring the temporal or spatial localization of their common ancestors. These data and methods are widely used with viral sequences, and particularly with human immunodeficiency virus (HIV), to trace the viral epidemic history over time and throughout the globe. Using sequences sampled at different points in time (or heterochronous) is also a mean to estimate their substitution rate, which characterizes the speed of evolution. The most commonly used methods to achieve these tasks are accurate, but are computationally heavy since they are based on complex models, and can only handle few hundreds of sequences. With an increasing number of sequences avail-able in the databases, often several thousand for a given study, the development of fast and accurate methods becomes essential. Here, we present a new distance-based method, named Ultrametric Least Squares, which is based on the princi-ple of least squares (very popular in phylogenetics) to estimate the substitution rate of a set of heterochronous sequences and the dates of their most recent common ancestors. We demonstrate that the criterion to be optimized is piecewise parabolic, and provide an efficient algorithm to find the global minimum.Using sequences sampled at different locations also helps to trace transmission chains of an epidemic. In this respect, we used all available sequences (~3,500) of HIV-1 subtype C, responsible for nearly 50% of global HIV-1 infections, to estimate its major migratory flows on a worldwide scale and its geographic origin. Innovative tools, based on the principle of parsimony, combined with several statistical criteria were used to synthesize and interpret information in a large phylogeny representing all the studied sequences. Finally, the temporal and geographical origins of the HIV-1 subtype C in Senegal were further explored and more specifically for men who have sex with men.

Objetivo: i) Estudar os casos de tuberculose (TB) por transmissão recente em brasileiros e migrantes sul-americanos, quantificar a transmissão cruzada recente entre ambas as populações e estudar seus fatores associados; ii) Avaliar, isoladamente, os fatores associados à transmissão recente da TB e o impacto da migração e de outras vulnerabilidades nos casos da doença no município. Métodos: Estudo transversal em indivíduos brasileiros e migrantes de origem sul-americana, residentes de distritos centrais do município de São Paulo (MSP) com grande proporção de populações vulneráveis e elevada presença recente de migrantes. Estudaram-se os casos de TB pulmonar por M. tuberculosis (Mtb) notificados entre 2013 e 2014, confirmados por meio de cultura de escarro. Foram utilizados os dados do Sistema Estadual de Notificação (SINAN-TB), de unidades de saúde e do laboratório central do Instituto Adolfo Lutz. Analizaram-se as variáveis sociodemográficas, clínicas e a presença de clusters simples e mistos, obtidos pelo agrupamento dos isolados de Mtb por RFLP-IS6110. i) Descreveu-se a distribuição dos clusters mistos e outras variáveis em brasileiros e migrantes sul-americanos e, investigou-se os fatores independentemente associados à presença de clusters mistos na amostra por meio de regressão logística simples e múltipla; ii) Investigaram-se os fatores independentemente associados à ocorrência de clusters mediante regressão logística simples e múltipla e estimou-se o impacto da transmissão recente da TB por meio da Fração Atribuível Populacional (PAF). Resultados: Foram amostrados 347 indivíduos, 19 por cento dos casos na área estudada. Desses, 76 por cento eram brasileiros e 24 por cento migrantes sul-americanos. O primeiro estudo mostrou que a proporção de clusters foi de 40,5 por cento , sendo 28 por cento desses compartilhados por brasileiros e migrantes sul-americanos (clusters mistos). Clusters mistos foram mais frequentemente encontrados em migrantes sul-americanos do que em brasileiros (OR=6,05), porém sugeriu-se transmissão cruzada em ambas as direções. A análise de sensibilidade, removendo usuários de drogas, HIV positivos e alcóolicos, teve pouco efeito no número de clusters mistos porém reduziu o de clusters simples em vi brasileiros, sugerindo maior transmissão recente do Mtb entre indivíduos brasileiros e migrantes não pertencentes a esses grupos de risco. O segundo estudo sugeriu que o uso de drogas está independentemente e diretamente associados a clusters sugestivos de transmissão recente (ORaj=3,18) e o alcoolismo (ORaj=0,30) está inversamente associado à transmissão recente. Foi encontrada baixa prevalência de comorbidades e outros fatores de risco entre migrantes sul-americanos, tanto em clusters quanto em perfis únicos. A transmissão recente entre usuários de drogas foi atribuída a cerca de 15 por cento dos casos de TB em brasileiros. Conclusões: Verificou-se que a transmissão recente, tanto entre brasileiros quanto entre migrantes sul-americanos, desempenha um papel importante nas regiões centrais do MSP. No entanto, sugere-se que a migração possa desempenhar um papel menos importante na transmissão/reativação da TB do que o uso de drogas e outras condições de vulnerabilidade. Dessa forma, no contexto dos crescentes movimentos migratórios para grandes cidades de países de média e baixa renda, a migração se soma à vulnerabilidade social, reforçando a necessidade de políticas intersetoriais para controle da TB. / Objective: i) to study recent tuberculosis (TB) transmission in Brazilians and South-American migrants, to estimate the proportion of recent cross-transmission occurring between both populations and to study potential associated factors to cross-transmission; ii) to study associated factors to cross-transmission and to estimate the impact of South-American migration and other vulnerabilities on TB cases. Methods: we conducted a cross-sectional study with TB patients in a central area of São Paulo (MSP) with a strong presence of South-American migrants and vulnerable populations. We selected notified pulmonary TB (PTB) cases confirmed by sputum culture between 2013 and 2014 among Brazilians and South-American migrants. We used the Tuberculosis State Notification System (SINAN-TBWEB), health facilities and the State Reference Laboratory (Instituto Adolfo Lutz) as data sources. We typed M. tuberculosis isolates by IS6110-RFLP, which were clustered by similarity. Clusters were considered mixed when isolates from at least one Brazilian and one South-American migrant were similar; and were considered simple when the cluster contained isolates from patients of only one nationality. We study demographic and clinical variables from TB patients and the presence of clusters within the sample. i) We described the distribution of simple and mixed clusters and other variables amongst Brazilians and South-American migrants and study associated factors to cross-transmission using logistic regression; ii) we estimated the proportion of recent transmission in the sample and investigated the factors associated with the presence of clusters by multiple logistic regression. Results: We sampled 347 individuals, 19 per cent of cases that occurred in the study area. Nearly 76 per cent were Brazilians and 24 per cent South-Americans. The first study demonstrated that 40.5 per cent of cases were clustered, of which approximately 30 per cent were mixed. South-American migrants were more likely to have mixed clusters than Brazilians (OR=6.05) and the results suggest that TB transmission occurred in both directions. We conducted a sensitivity analysis removing drug users, HIV positive and alcoholics, which suggested higher transmission amongst Brazilian and migrants outside these groups. In the second study we demonstrated that drug users were more likely to viii belong to clusters (ORadj=3.18) while individuals who abuse alcohol were more likely to belong to unique profiles (ORadj=0.30). South American migrants had lower prevalence of comorbidities and other risk factors. Nearly 15 per cent of PTB cases among Brazilians were attributed to recent transmission among drug users. Conclusions: We conclude that general recent transmission of TB and cross-transmission between Brazilian and South American migrants play an important role in the central regions of MSP. Nevertheless, we suggest that social vulnerability and drug use in TB transmission may be more important than migration status itself. In the context of increasing migration to large cities of low- and middle-income countries, our findings add migration to other social vulnerabilities, reaffirming the need for inter-sectoral policies on TB control.

O Vírus Sincicial Respiratório (RSV) é reconhecido como agente causador de infecção nosocomial entre receptores de pacientes de células-tronco hematopoéticas causando morbidade e mortalidade consideráveis nesses pacientes. O objetivo desse estudo foi caracterizar as cepas do RSV isoladas de receptores de transplante de células-tronco hematopoéticas (TCTH) do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo durante sua estação. As cepas do RSV foram tipadas (em grupo A ou B) e genotipadas. Das sete cepas analisadas dos receptores de TCTH durante o ano de 2001, somente duas pertenciam ao grupo B, as outras cinco eram pertencentes ao grupo A. Dessas sete cepas, três eram altamente relacionadas e haviam infectados pacientes que freqüentavam o ambulatório. Em 2002, das doze cepas analisadas, três pertenciam ao grupo A e as outras nove pertenciam ao grupo B. Sete cepas eram altamente relacionadas entre elas e eram também de pacientes de ambulatório sugerindo que a transmissão em hospital-dia era mais provável. Enfim, múltiplços genótipos do RSV co-circularam nas unidades de TCTH (ambulatório e enfermaria) do Hospital das Clínicas entre 2001 e 2002. A transmissão nosocomial foi mais provável ocorrer no ambulatório da unidade de TCTH quando comparada à enfermaria. Políticas de controle de infecção devem ser também implementadas em ambulatórios para evitar transmissão nosocomial do RSV e outros vírus respiratórios em pacientes de ambulatório. / Respiratory Syncytial Virus is recognized as the leading cause of nosocomial respiratory infection among recipients of hematopoietic stem cell transplant (HSCT) causing considerable morbity and mortality among theses patients. The aim this study was characterize the strains of Respiratory Syncytial Virus in recipients Hematopoietic Stem Cell Transplant Unit (HSCT) at Hospital das Clínicas, University of São Paulo Medical School during RSV season in symptomatic HSCT recipients at Hospital das Clínicas. The strains of RSV was typed (in group A or B) and genotyped. Of the seven strains analyzed from HSCT recipients during 2001, only two belonged to group B, the other five belonged to group A. Of these seven strains, three were closely related and were from outpatients. In 2002 , of the twelve strains analyzed, three belonged to group A and the other nine belonged to group B. Seven strains were closely related and were also from outpatients suggesting that nosocomial transmission in hospital-day was more likely. In conclusion, multiples genotypes of RSV co-circulated in the Hematopoietic Stem Cell Transplant units (ward and dayhospital) of Hospital das Clínicas between 2001 and 2002. Nosocomial transmission was more likely to occur at the HSCT Day-hospital as compared to the HSCT ward. Infection control practices should be also implemented at Day-hospital Units to avoid nosocomial transmission of RSV and other respiratory viruses in outpatient units.

Made available in DSpace on 2016-01-26T12:51:28Z (GMT). No. of bitstreams: 1 adrianomondini_tese.pdf: 12162182 bytes, checksum: fe0a4d238020f614624084ebb47e1011 (MD5) Previous issue date: 2010-03-05 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Dengue belongs to the Flavivirus genus and is the most common arboviral infection worldwide. It can be caused by four antigenically different serotypes (DENV 1-4). These serotypes are transmitted mainly by the bite of Aedes aegypti mosquitoes. The vector is widely associated with human activity and the influence of organized social space favors the interaction among vector, virus and man, making populated areas sources of dengue dispersion. In this study, we performed a molecular, spatial and temporal study of DENV transmission through positive samples of blood and infected mosquitoes captured in São José do Rio Preto/SP in a period of four years. Material and Methods: Serum samples of patients presenting dengue like symptoms and pools of mosquitoes had their viral RNA extracted and were tested by Multiplex- RT-PCR with Flavivirus generic primers based on non-structural protein (NS5) in the first round, followed by Nested assays with species-specific primers for the identification of DENV 1-3, yellow fever virus, Saint Louis encephalitis virus (SLEV) among others. Positive samples were analyzed spatially and phylogenetically. Results and Discussion: We analyzed 613 blood samples for four years: 199 in 2006, 94 in 2007, 313 in 2008 and 10 in 2009. The positivity was high in 2006 and 2007, with 106 and 51 infected patients, respectively. The major dengue serotype circulating during the 2006 and 2007 epidemics was DENV-3 and few cases of DENV-2, which is an indication of its recent introduction in the municipality. We also reported the first outbreak of SLEV in Brazil in 2006. Among DENV patients in 2008, only seven were infected by DENV-3 and 90 were infected by DENV-2, suggesting the reemergence of this serotype. We detected the circulation of DENV-1 in two Abstract xxv patients in 2008 and in four patients in 2009. Nearly 1200 mosquitoes were captured from December 2007 to March 2008. We have captured 814 Aedes aegypti mosquitoes, which were divided in 463 pools. Only 3.67% of them were positive for DENV-3 and DENV-2. Pools containing only male mosquitoes were positive for DENV, indicating the presence of transovarial transmission. We obtained sequences from 82 patients among 174 blood samples. We were able to geo-code 46 sequences. The alignment generated a 399-nucleotide long dataset with 134 taxa. The phylogenetic analysis indicated that all samples were of DENV-3 and related to strains circulating on the isle of Martinique in 2000 2001. Sixty DENV-3 from São José do Rio Preto formed a monophyletic group (lineage 1), closely related to the remaining 22 isolates (lineage 2). We assumed that these lineages appeared before 2006 in different occasions. The possibility of inferring the spatio-temporal dynamics from genetic data has been generally little explored, and it may shed light on DENV circulation. The use of both geographic and temporally structured phylogenetic data provided a detailed view on the spread of at least two dengue viral strains in a populated urban area. / Dengue pertence ao gênero Flavivirus e é a infecção por arbovírus mais comum no mundo todo. Pode ser causada por quatro sorotipos antigenicamente distintos (DENV 1-4). Estes sorotipos são transmitidos pela picada do mosquito Aedes aegypti. O vetor está amplamente associado a atividade humana e a influencia do espaço urbano favorece a interação entre o vetor, o vírus e o homem, tornando áreas populosas, grandes centros de dispersão do dengue. Neste estudo, foi realizada um estudo molecular, espacial e temporal da transmissão de DENV através de amostras positivas de sangue e de mosquitos infectados capturados em São José do Rio Preto/SP, num período de quatro anos. Materiais e métodos: Soro de pacientes apresentando sintomas de dengue e pools de mosquitos tiveram seu RNA viral extraído e foram testados por Multiplex-RT-PCR, com primers genéricos de Flavivirus baseados na proteína não estrutural 5 (NS5) numa primeiro ciclo,seguida por ensaios Nested com primers específicos para DENV, para o vírus da febre amarela, para o vírus da encefalite de Saint Louis, entre outros. As amostras positivas foram analisadas espacial e filogeneticamente. Resultados e discussão: Analisamos 613 amostras de soro durante 4 anos: 199 em 2006; 94 em 2007; 313 em 2008 e 10 em 2009. A positividade foi alta em 2006 e 2007, com 106 e 51 pacientes infectados, respectivamente. O principal sorotipo circulante durante as epidemias de 2006-2007 foi DENV-3 e poucos casos de DENV-2, o que pode ser a indicação de sua recente introdução no município. Nós também descrevemos a primeira epidemia de SLEV no Brasil em 2006. Dentre os pacientes com DENV em 2008, apenas sete estavam infectados com DENV-3 e 90 com DENV-2, sugerindo a reemergência do sorotipo. Nós Resumo xxiii detectamos a circulação de DENV-1 em dois pacientes em 2009 e em quatro pacientes em 2009. Aproximadamente 1200 mosquitos foram capturados entre Dezembro 2007 e Março de 2008. Capturamos 814 mosquitos Aedes aegypti, que foram divididos em 463 pools. Apenas 3,67% deles foram positivos para DENV-2 e DENV-3. Pools contendo apenas machos foram positivos para DENV, indicando a presença de transmissão transovariana. Nós obtivemos sequências de 82 pacientes dentre 174 amostras de sangue. Nós fomos capazes de geocodificar 46 sequências. O alinhamento gerou gerou nucleotídeos com 399 bp com 134 taxa. A análise filogenética indicou que todas as amostras foram de DENV-3 e estavam relacionadas às cepas circulantes na ilha da Martinica em 2000-2001. Sessenta pacientes com DENV- 3 de São José do Rio Preto formaram um grupo monofilético (linhagem 1), intimamente relacionado com os outros 22 isolados (linhagem 2). Nós assumimos que estas linhagens apareceram antes de 2006 em ocasiões diferentes. A possibilidade de inferir a dinâmica espaço-temporal através de dados genéticos é relativamente pouco explorada e pode esclarecer acirculação de DENV. O uso de dados filogenéticos estruturadosgeograficamente e temporalmente forneceu uma visão detalhada na dispersão de, pelo menos, duas cepas virais distintas numa área urbana.

Mycoplasma agalactiae est responsable de l'agalactie contagieuse, maladie des petits ruminants difficilement contrôlée et figurant sur la liste de l’OIE. Afin d’évaluer la diversité génétique de ce pathogène, 101 isolats ont été comparés par trois techniques (VNTR, RFLP, répertoire vpma). Les résultats révèlent une grande homogénéité génétique dont la souche type PG2 est représentative. Quelques isolats font exception telle la souche 5632 que nous avons séquencée et analysée ici. La comparaison des génomes et des protéomes entre 5632 et PG2 indiquent que la plasticité de ces génomes est liée à d’importants échanges d'ADN et à la présence de nombreux éléments génétiques mobiles (10% du génome). Ces analyses révèlent également une forte dynamique au sein de répertoires de gènes codant des protéines de surfaces. Pour les mycoplasmes, bactéries minimales dépourvues de paroi, ces évènements ont certainement joués un rôle dans leur survie et leur adaptation à des hôtes complexes. / Mycoplasma agalactiae is responsible of contagious agalactia, a disease of small ruminants that is still difficult to control and is listed by the OIE. In order to evaluate the genetic diversity of this pathogen, 101 isolates were compared using three techniques (VNTR, RFLP, vpma repertoire). Results revealed a high genetic homogeneity with the PG2 type strain as representative. Some isolates however diverged such as the 5632 which was sequenced and analysed here. Whole comparative genomic and proteomic analyses of the 5632 and PG2 strains indicate that their genomic plasticity resides in important genes flux and in the presence of several mobile genetic elements (10% of the genome). These analyses also revealed that specific loci encoding repertoire of surface proteins are highly dynamic. For these minimal bacteria that lack a cell-wall, these events have most likely played a major role in their survival and adaptation to complex hosts.

Made available in DSpace on 2014-07-29T15:26:24Z (GMT). No. of bitstreams: 1 tese valeria.pdf: 2053227 bytes, checksum: 9897e5628e288900b234c5acbe0fb42b (MD5) Previous issue date: 2008-08-06 / Nowadays, dengue constitute the major public health problem, because is relevant cause of illness and death between thousands people that resident in the tropical and subtropical regions in world. The dengue virus is classified as four serotypes (DENV-1, 2, 3 and 4) according to antigenic differences and characterized intra-typical groups called genotypes. The laboratorial surveillance enables the diagnostic confirmation of dengue infection and monitoring serotypes circulating through the routine diagnostic techniques. Recently, the use molecular techniques has contributed to characterize and monitoring of the genotypes potentially virulent during epidemic and knowledge of biology of dengue virus. This thesis was organized in an introduction section, that include a literature review on dengue, and two manuscripts that describing the research conducted with focus on laboratory diagnostic and molecular epidemiology. The first manuscript entitled Laboratorial Surveillance of Dengue Virus in Central-Brazil, 1994-2003, was published at Journal of Clinical Virology, 2006 37 (3): 179-83. In this study we present the results of the virological surveillance for dengue cases conducted in the city of Goiânia (~1,200,000 population) from 1994 to 2003. Suspected cases were from the main public infectious disease reference hospital and outpatient clinics covering the metropolitan area. Serological and virus isolation tests were conduced at the regional reference laboratory. Our objective was to report dengue circulating serotypes from 1994 to 2003 and the role of distinct serotypes on dengue clinical outcomes in Central Brazil and to characterize serotypes and genotypes by reverse transcriptase PCR (RT-PCR) and by restricted site-specific PCR (RSS-PCR) patterns in selected samples. Laboratory surveillance identified mainly DEN-1 serotype from 1994 to 2002 shifting to a high circulation of DEN-3 in 2003. The adults (87,4%) were the most affected group and dengue fever accounted for the majority of the cases. Diagnosis of dengue was confirmed in ~50% of the suspected and enhanced by RT-PCR. RSS-PCR patterns for DEN-1 and DEN-3 corresponded to the circulating subtypes in the country. The infection DENV-3 did not suggest a major role of infecting DEN-3 in increasing disease severity during its first-year spread in Central Brazil. The second manuscript, to be submitted for publication is entitled: Epidemiologia Molecular do Vírus Dengue tipo 3 em Goiânia GO, 2005-2006. The objective of this manuscript was to characterize the DENV-3 genotype circulating isolated from well-characterized clinical and laboratory samples in Goiânia-GO/Brasil. Seven samples had sequences of the prM/M/E region obtained and comparative analysis was performed with the reference strains. The results showed the homology of the genomics sequences with genotype III strains. The nucleotide identity of the all the samples varied from 97.0% to 99.6% and the amino acid sequences from 97.5% to 99.5%. The analysis of the nucleotide sequence revealed silent mutation and 14 amino acid changes in the protein deduced from gene prM/M/E. In conclusion, the study confirms that the strains of DENV-3 in Central Brazil relate to genotype III. The genomic changes along Domain III of the protein E were observed, which could affect the pathogenicity, but were not consistent between samples of DCC and DHF. Samples of patients with dengue fever had mutations related to viral attenuation. More investigation is necessary to evidence of genomic changes found in relationship with clinical forms. / Atualmente, a dengue representa um dos maiores problemas em saúde pública, pois é causa de doença e morte entre milhares de pessoas que residem nas regiões tropicais e subtropicais do mundo. Os vírus dengue existem como quatro sorotipos virais (DENV-1 a DENV-4) de acordo com diferenças antigênicas, sendo caracterizados grupos intra-típicos denominados genótipos. A vigilância laboratorial possibilita a confirmação diagnóstica de infecção por dengue e o monitoramento de sorotipos circulantes, através de técnicas diagnósticas de rotina. Recentemente, o uso de técnicas moleculares tem contribuído para a caracterização e monitoramento de genótipos potencialmente virulentos na vigência de epidemias e melhor entendimento da biologia do vírus. Esta tese de doutorado compreende uma introdução que contempla uma revisão da literatura sobre dengue e dois manuscritos que descrevem as pesquisas realizadas com enfoque no diagnóstico laboratorial e epidemiologia molecular. O primeiro manuscrito intitulado Laboratorial Surveillance of Dengue Virus in Central-Brazil, 1994-2003, foi publicado no Journal of Clinical Virology, 2006 37 (3): 179-83. Neste estudo, apresentamos os resultados de uma vigilância laboratorial conduzida na cidade de Goiânia na região Centro-Oeste do Brasil, desde a introdução dos vírus dengue em 1994 até 2003. Casos com suspeita clínica de dengue foram atendidos em unidades de saúde e hospitais de referência da região metropolitana e os testes sorológicos e/ou isolamento viral realizados no laboratório de referência estadual LACEN-GO. Nosso objetivo foi descrever sobre os sorotipos virais circulantes de dengue nos anos de 1994 a 2003 e o seu papel nos desfechos clínicos de dengue na região Centro-Oeste do Brasil e caracterizar sorotipos e genótipos ou subtipos genômicos pela RT-PCR e RSS-PCR em amostras selecionadas. O sorotipo DENV-1 foi identificado como prevalente nos anos 1994 a 2002, sendo substituído por DENV-3 em 2003. Os adultos (87,4%) foram mais atingidos e a dengue clássica a mais diagnosticada. A dengue foi confirmada em 50% dos casos suspeitos de dengue incrementado pela RT-PCR. Os genótipos ou subtipos genômicos do DENV-1 e DENV-3 corresponderam circulantes no país. A infecção pelo DENV-3 não sugere um papel relevante no aumento da gravidade da doença durante seu primeiro ano de dispersão no Centro-Oeste do Brasil. O segundo manuscrito, a ser submetido à publicação, foi intitulado Epidemiologia Molecular do Vírus Dengue tipo 3 em Goiânia GO, 2005-2006. O objetivo desse trabalho foi caracterizar o genótipo circulante do DENV-3 isolado de amostras bem caracterizadas clínica e laboratorialmente em Goiânia-GO/Brasil e analisar as alterações genômicas encontradas. Sete seqüências regiões prM/M/E, foram obtidas e analisadas em comparação com cepas protótipo do DENV-3. As seqüências do estudo apresentaram maior homologia com a cepas do genótipo III. O percentual de identidade nucleotídica das amostras do DENV-3 seqüenciadas variou de 97,0% a 99,6% e de aminoácidos 97,5% a 99,5%, mostrando-se conservadas. A análise da seqüência nucleotídica das amostras do estudo revelou mutações silenciosas e 14 substituições de aminoácidos na seqüência da proteína deduzida do gene prM/M/E. Em conclusão, o estudo confirma que as cepas de DENV-3 da região centrooeste pertencem ao genótipo III. Alterações genômicas significativas foram observadas ao longo do domínio III da proteína E, as quais poderiam afetar a patogenicidade, entretanto não foram consistentes entre as amostras de DCC e FHD. As amostras de pacientes com dengue clássica apresentaram alterações em resíduos de aminoácidos no domínio III relatados como pontos de atenuação viral. Maiores investigações serão necessárias para confirmar as alterações encontradas e sua relação com as formas clínicas.