The prevalence of allergy is increasing and it is becoming a serious problem not on- ly in medicine, but also in social and economic terms. The most effective way to minimize the development of allergic diseases is preventive measures. In recent years, many studies have attempted to confirm or rebut the hypothesis that early administration of probiotic bacteria to newborns and pregnant women before birth could have preventive effects on the development of allergy. In the Czech Republic, the probiotic strain Escherichia coli O83:K24:H31 (EC O83), being registered with the State Health Institute for Drug Control under the name Colinfant Newborn, has long been used to prevent allergies and paediatri- cians have long been known and used it against various diarrhoea. The aim of this work was to elucidate the effect of EC O83 on CBMC (cord blood mononuclear cells) and to compare the ability of CBMC of healthy mothers (children with a relatively low risk of developing allergic disease) and allergic mothers (children at high risk of developing allergies) to form cytokines in response to EC O83 stimulation. Phytohemagglutinin was used as a positive control, Escherichia coli Nissle 1917 was used as a reference probiotic strain, which is much more known abroad than EC O83. Cytokine production was detected by...

L'objectif principal de ce travail etait d'explorer les bactéries pathogènes que recèle le tube digestif des gorilles. Pour ce faire, un total de 48 échantillons de selles, provenant du Cameroun, appartenant à 21 gorilles ont été analysés. D'abord la culturomique et le pyroséquençage ont été utilisés pour évaluer exhaustivement la diversité bactérienne. En appliquant la culturomique, 86 conditions de culture dont des milieux fabriqués à base de plantes tropicales, sur un échantillon de selles de gorille, 12 800 colonies microbiennes ont été isolées et testées, et 147 espèces bactériennes identifiées. De nombreux pathogènes opportunistes ont été observés, dont 8 qui sont fréquemment associés à des maladies chez l'homme: Mycobacterium bolletii, Proteus mirabilis, Acinetobacter baumannii, Klebsiella pneumoniae, Serratia marcescens, Escherichia coli, Staphylococcus aureus et Clostridium botulinum. En utilisant la PCR en temps réel pour cribler des pathogènes bactériens dans les 48 échantillons de selles de gorilles, des bactéries fastidieuses telles que Bartonella spp. Borrelia spp., Coxiella burnetii, Tropheryma whipplei ont été observées. Nous avons estimé la prévalence de ces agents pathogènes qui varie entre 4,76% et 85,7%. Ce travail a permis de savoir que l'homme et le gorille ont en commun plusieurs espèces bactériennes dont des pathogènes émergents. Par conséquent, les gorilles sauvages peuvent servir de réservoir et de source pour l'émergence et/ou la réémergence des bactéries pathogènes pour l'homme. / The main objective of this work is to explore the gorilla's potential role as a reservoir for pathogenic bacteria. We used both microbial culturomics and pyrosequencing to analyze the gorilla gut bacteria. By applying culturomics to one index gorilla, we tested 12,800 colonies and identified 147 different bacterial species, including 5 new species. Many opportunistic human pathogens were observed, including 8 frequently associated with human disease: Mycobacterium bolletii, Proteus mirabilis, Acinetobacter baumannii, Klebsiella pneumoniae, Serratia marcescens, Escherichia coli, Staphylococcus aureus and Clostridium botulinum. Using specific real-time PCR on 48 gorilla fecal samples, we also observed the fastidious pathogens Bartonella spp. Borrelia spp., Coxiella burnetii, Tropheryma whipplei. Using microsatellite analysis of the gorilla samples, we estimated that the prevalence of these pathogens was between 4.76% and 85.7%. Therefore, the gorilla shares many bacterial pathogens with humans, which suggests that wild gorillas might be a reservoir for the emergence and/or reemergence of these pathogens, especially in areas where human and gorilla habitats overlap and because of the increasing presence of humans in the African equatorial forests.

Background: Critically ill patients are at particular risk of developing bloodstream infection. Such infections are associated with the development of sepsis, leading to a marked increase in mortality rate. Early detection of the causative organism and appropriate antibiotic treatment are therefore critical for optimum outcome of patients with nosocomial infection. Current infection diagnosis is based on standard blood culture techniques. However, microbiological culture has a number of limitations, not least that it takes several days to confirm infection and is therefore not useful in directing the early treatment with antibiotics. New techniques based on the detection of pathogen DNA using real-time polymerase chain reaction (PCR) technology have the potential to address these limitations but their clinical utility is still to be proved. Objectives: Develop and evaluate novel PCR-based approaches to bloodstream infection diagnosis in critical illness based on detection and identification of bacterial and fungal DNA in blood. Methods: A range of commercial and 'in-house' PCR-based assays for detection of bacterial and fungal DNA were developed and/or optimised for use in clinical blood samples. These included LightCycler SeptiFast, a CE-marked multi-pathogen assay for common bloodstream pathogens, BactScreen and GramScreen, broad spectrum bacterial assays based on 16S rRNA gene and real-time PCR assays developed to detect a range of clinically important fungal pathogens. Novel approaches to speciation of pathogen DNA using melting temperature (Tm) profiling and high resolution melting analysis (HRMA) were developed. Clinical evaluation of assays was either on blinded clinical isolates or blood samples from critically ill patients with clinical suspicion of bloodstream infection against conventional microbiological culture. Several techniques aimed at improving extraction of pathogen DNA from blood were also investigated. Results: The CE-marked commercial assay SeptiFast showed analytical sensitivity and specificity of 79% and 83% respectively. Concordance with positive culture results was good but high levels of 'false positives' were detected possibly attributed to detection of free pathogen DNA not associated with viable pathogens. The predictive value of a negative SeptiFast test was 98% suggesting that absence of pathogen DNA is a strong indicator of absence of infection. Further studies were aimed at detailed optimisation and validation of 16S rRNA gene real-time PCR assays for bacterial DNA. BactScreen and GramScreen were able to detect a broad range of clinically important bacteria down to <50 CFU/ml blood. A preliminary comparative evaluation against SeptiFast showed BactScreen gave excellent concordance with blood culture results with minimal false positive results compared to SeptiFast. Efficient extraction of pathogen DNA was shown to be a key factor in determining analytical sensitivity and several protocols were evaluated. Low cost approaches to speciation of bacterial DNA were developed by combining broad range real-time PCR with HRMA. A novel HRMA method based on Tm profiling was shown to identify 89% and 96% of blinded clinical isolates at species or genus level respectively. Real-time PCR/HRMA approaches were also successfully developed for detection and identification of fungal pathogens including a range of Candida and Aspergillus species associated with bloodstream fungal infection. Conclusions: These studies have highlighted some of the key factors that need to be considered when developing and validating PCR based assays for pathogen DNA detection in blood. A set of novel tools have been developed for rapid detection and identification of bacterial and fungal pathogens that could address the challenges of infection diagnosis based on pathogen DNA detection. Further work is required, not least in development of more efficient pathogen DNA extraction and detailed clinical validation but the tools described here have the potential to provide cost effective solutions to aid infection diagnosis that would be complementary to current culture-based methods. The provision of time critical information could have a positive impact on clinical decision-making leading to more effective management and treatment of patients with suspected bloodstream infection.

Un prélèvement efficace de K+ à partir du sol est essentiel au développement des végétaux. Sur un sol riche en NaCl, le maintien d'un prélèvement sélectif et efficace de K+ à partir du sol et le contrôle de l'exportation de Na+ par la racine vers les feuilles constituent des fonctions essentielles pour la survie de la plante. Chez les plantes, les transporteurs HKT (High-affinity K+ Transporters) sont classés en deux sous-familles sur des bases phylogénétiques et de sélectivité ionique. Les membres de la sous-famille 1 transportent sélectivement Na+. Plusieurs d'entre eux ont été identifiés comme des acteurs majeurs de l'adaptation des plantes aux fortes salinités du sol en prévenant l'accumulation de Na+ dans les parties aériennes. Les membres de la sous-famille 2 co-transportent Na+ et K+. Leur rôle dans la plante, notamment dans le transport de K+, est encore mal compris. Je me suis intéressé à différents systèmes de transports de K+ et Na+, appartenant essentiellement à la famille HKT chez le riz. La caractérisation que j'ai effectuée a fait appel à plusieurs approches : électrophysiologie (voltage-clamp après expression en ovocyte de xénope), biologie cellulaire, génétique inverse et PCR en temps réel. L'analyse de l'expression par RT-PCR en temps réel de toute la famille HKT (4 membres dans chacune des deux sous-familles) a montré que ces transporteurs sont différemment exprimés au niveau des racines et des feuilles, et que leur niveau de transcrits est fortement et differentiellement régulé en conditions de stress salin ou osmotique et en présence d'hormones, ce qui suggère que ces différents systèmes jouent des rôles propres et diversifiés dans la plante. L'analyse plus détaillée d'OsHKT2;4, a montré par expression hétérologue dans l'ovocyte de xénope que ce système possède des propriétés fonctionnelles originales: il transporte sélectivement K+ à faibles concentrations de Na+, mais co-transporte Na+ et K+ à fortes concentrations de Na+ (>10 mM). L'analyse de l'expression d'OsHKT2;4 a révélé que ce transporteur est surexprimé en condition de carence en K+ et de stress salin, suggérant qu'OsHKT2;4 pourrait jouer un rôle important dans le transport de K+ dans ces deux conditions. Enfin, un patron d'expression nouveau pour un transporteur HKT a été révélé par l'analyse de plantes transgéniques exprimant le promoteur d'OsHKT2;4 fusionné aux gènes rapporteurs GUS ou GFP : en plus d'une localisation classique dans les tissus conducteurs, une forte expression est observée dans les stomates des gaines et des limbes foliaires, suggérant un rôle dans l'osmocontractilité de ces cellules.Mots clés: Oryza sativa, transport de potassium, transporteur HKT, Na+-K+ co-transporteur, électrophysiologie, ovocyte de xénope, localisation tissulaire, PCR quantitative, stress salin / Efficient uptake of K+ from the soil solution is essential for plant development. When plants are grown on a soil rich in NaCl, the maintenance of an efficient and selective uptake of K+ and the control of Na+ export from roots to shoots are crucial for plant survival. In plants, transporters belonging to the HKT (Highaffinity K+ Transport) family have been sorted in two subfamilies based on phylogenetic grounds and functional properties. Subfamily 1 members transport selectively Na+. Several of them have been shown to play major roles in plant adaptation to salt stress by preventing excessive accumulation of Na+ in shoots. Subfamily 2 members are thought to co-transport Na+ and K+, at least when expressed in heterologous systems. Their roles in planta, especially their potential role in K+ transport, are still largely unknown. I have been interested in different K+ and/or Na+ transport systems in rice, mostly belonging to the HKT family. For their characterization, different approaches have been used: electrophysiology (two-electrode voltage-clamp after expression in Xenopus oocytes), cell biology, reverse genetics and real-time PCR. Realtime RT-PCR analyses on the whole family of rice HKT transporters (4 members in both subfamilies) showed that the expression level in roots and leaves of these different systems is variable, and is differentially regulated by salt and osmotic stresses as well as by hormonal treatments, which suggests that these transporters have diverse and differentiated functions in the plant. A detailed analysis of OsHKT2;4 revealed original functional properties: this HKT transporter was indeed shown to be K+-selectively in the presence of low external Na+, but to switch to Na+ and K+ co-transport mode at high (>10 mM) Na+ concentrations. Expression analysis of OsHKT2;4 showed that this transporter is overexpressed upon salt stress and K+ shortage, which suggests that it could play an important role in K+ transport in these two conditions. At last, a new expression pattern for an HKT transporter was evidenced through the analysis of transgenic rice plants expressing OsHKT2;4 promoter fused to the GUS or GFP reporter genes: in addition to a classical localization in vascular tissues, expression of OsHKT2;4 was observed in stomata, suggesting a role for OsHKT2;4 in osmotic regulation in these cells

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The production of common quail is on increasing development and needs further genetics studies for productive direction. As the PCR technique in real time the most important in the analysis of animal gene expression, studies and experiments related components are needed. Among these components is the reference gene, normalizer data that has great impact on the results and has direct influence on the success of the analysis, which must have significant expression in tissue and invariably in all the experimental conditions. Among the experimental conditions, sex (male and female) can be considered, since there are research about responses in the animal gene expression in both sexes. Thus it is necessary to use a single set of reference genes for normalizing expression data from both samples (male and female), by eliminating the effect of sex. So, We aim with this study was to evaluate the stability and recommend reference genes for quantitative real-time PCR in different quails tissues of both sexes. The stability of 10 housekeeping genes (GAPDH, RPL5, MRPS27, MRPS30, TFRC, HMBS, EEF1, LDHA, B2M and UBC) was analyzed in four tissues (heart, thigh, brain and spleen) of males and females quails, from online available RefFinder tool. The RefFinder is based on the results of programs and methods commonly used for this purpose (Bestkeeper, NormFinder, GeNorm and ΔCq method) and the analysis was done separately for each tissue. The results confirm prior knowledge that different tissues express different genes, that it was noted from the different recommendations for each tissue. However, it is important to note that although the sex be able to influence the expression of genes, we observed stable constitutive genes for both sexes. The most stable housekeeping genes were: MRPS30, EEF1 and HMBS in thigh muscle; B2M, GAPDH and UBC in brain; MRPS30, TFRC and HMBS in heart; and EEF1, LDHA and HMBS in spleen, It is therefore recommended to be used as reference genes for gene expression studies of male and female quails. The recommendation of stable genes that can be used in both sexes quail facilitates the execution of subsequent genetic studies needed to assess the animals eliminating the effect of sex, influencing, consequently, in the development on the entire production. / A coturnicultura de corte está em crescente desenvolvimento e necessita de maiores estudos no campo genético para o direcionamento produtivo. Sendo a técnica da PCR em tempo real uma das mais importantes na análise da expressão gênica animal, estudos e experimentações relacionadas a seus componentes se fazem necessários. Dentre esses componentes encontra-se o gene de referência, normalizador de dados que possui grande impacto nos resultados e tem influência direta no sucesso da análise, devendo este possuir expressão significativa no tecido de forma invariável em todas as condições experimentais. Dentre as condições experimentais, o sexo (macho e fêmea) pode ser considerado, uma vez que existem pesquisas voltadas à busca de respostas do comportamento gênico animal de ambos os sexos. Dessa forma se faz necessária a utilização de um único conjunto de genes de referência para normalizar os dados de expressão de ambas as amostras (machos e fêmeas), eliminando o efeito de sexo. Assim, objetivou-se com esse trabalho avaliar a estabilidade e recomendar genes de referência para PCR quantitativo em tempo real em diferentes tecidos de codornas de corte de ambos os sexos. Foram analisadas as estabilidades de 10 genes constitutivos (GAPDH, RPL5, MRPS27, MRPS30, TFRC, HMBS, EEF1, LDHA, B2M e UBC) em quatro tecidos (coração, coxa, cérebro e baço), de codornas machos e fêmeas, a partir da ferramenta RefFinder disponível online. O RefFinder se baseia nos resultados dos programas e métodos comumente utilizados para esta finalidade (Bestkeeper, NormFinder, GeNorm e método ΔCq) e a análise foi feita separadamente para cada tecido. Os resultados confirmam o conhecimento prévio de que diferentes tecidos expressam genes diferentes, notável a partir das diferentes recomendações para cada tecido. Entretanto, é importante notar que, apesar de o sexo influenciar na expressão de genes, foi possível observar genes constitutivos estáveis para ambos os sexos. Os genes que se mostraram mais estáveis foram: MRPS30, EEF1 e HMBS no músculo da coxa; B2M, UBC e GAPDH no cérebro; MRPS30, TFRC e HMBS no coração; e EEF1, LDHA e HMBS no baço; sendo, portanto, recomendados para serem empregados como genes de referência em estudos de expressão gênica de codornas de corte machos e fêmeas. A recomendação de genes estáveis que podem ser utilizados em ambos os sexos de codornas facilita a execução de estudos genéticos posteriores que necessitem avaliar os animais eliminando o efeito de sexo, influenciando, consequentemente, no desenvolvimento de toda a cadeia produtiva.

O óxido nitroso (N2O) é um importante gás de efeito estufa (GEE) e, nos ecossistemas terrestres, é produzido principalmente pelo processo de desnitrificação. Esse ocorre em condições anaeróbias e, portanto, é fortemente estimulado pelo aumento do teor de umidade do solo. Entretanto, solos sob diferentes usos podem exibir taxas de emissão de N2O distintas, mesmo quando apresentam teores de umidade equivalentes. Ainda não está claro se isso se deve somente ao fato de os mesmos diferirem quanto a atributos físicos e químicos capazes de afetar a atividade dos organismos desnitrificantes ou se também se deve à diferenças com relação ao tamanho de suas populações. O presente trabalho foi desenvolvido com o objetivo de compreender as relações entre os fluxos de N2O, a umidade e a abundância de genes bacterianos envolvidos no processo de desnitrificação (nirK, norB e nosZ) em solos de floresta, pastagem e cultivo de cana-de-açúcar, utilizando um experimento de microcosmos. Amostras de solo foram coletadas na fazenda Capuava, situada no município de Piracicaba, SP. Os microcosmos estabelecidos a partir das mesmas foram mantidos com diferentes teores de umidade (original e ajustados para atingir 60% e 90% da capacidade de campo) e incubados a 30 °C por 30 dias. Ao longo do período de incubação, os fluxos de N2O a partir desses solos foram analisados por cromatografia gasosa. Amostras coletadas do interior dos microcosmos, antes e depois da aplicação dos tratamentos, foram comparadas quanto à estrutura de suas comunidades bacterianas, utilizando a técnica de T-RFLP, e quanto à abundância dos genes 16S rRNA, nirK, norB e nosZ, através da técnica de qPCR. Somente os solos que tiveram sua umidade ajustada para 90% da capacidade de campo exibiram incrementos significativos na produção de N2O. Em tais amostras, também foi verificada a alteração da estrutura das comunidades bacterianas e do número de cópias dos genes norB e nosZ. Apenas este último, no entanto, apresentou uma correlação positiva com a umidade do solo. A abundância dos genes avaliados não apresentou correlações significativas com as taxas de emissão do GEE. Por outro lado, as emissões cumulativas de N2O se correlacionaram positivamente com as quantidades de genes desnitrificantes presentes inicialmente nas amostras de solo. Estes genes se mostraram mais abundantes nas amostras de pastagem e floresta, as quais apresentavam maiores teores de matéria orgânica, carbono, nitrogênio, nitrato e argila do que aquelas provenientes da área cultivada com cana-de-açúcar. Tais resultados demonstram que o conteúdo de água do solo afeta a taxa de emissão de N2O, mas que isso não se deve a alterações na abundância das bactérias envolvidas no processo, como as que carregam os genes nirK, norB e nosZ. Aparentemente, no entanto, quantidade de GEE que o solo é capaz de produzir está relacionada ao tamanho das populações desses organismos desnitrificantes. / Nitrous oxide (N2O) is an important greenhouse gas (GHG) and, in terrestrial ecosystems, it is mainly produced by denitrification. This process occurs under anaerobic conditions and, therefore, is strongly stimulated by the increase of the soil moisture content. However, soils under different uses may exhibit distinct N2O emission rates, even when they have the same moisture content. It is still not clear whether this is due solely to the fact that they differ in relation to physical and chemical properties that affect the activity of denitrifying organisms or whether this is also due to differences in the size of their populations. The aim of this work was to evaluate the relations between N2O fluxes, moisture and abundance of bacterial genes involved in denitrification process (nirK, norB e nosZ) in soil samples from forest, pasture and sugarcane field, through a microcosm experiment. These samples were collected at Fazenda Capuava, located in Piracicaba, SP. Microcosms established from them were maintained with different moisture contents (original and adjusted to achieve 60% and 90% of field capacity) and incubated at 30 °C for 30 days. During the incubation period, the N2O fluxes from soils were analyzed by gas chromatography. Soil samples from microcosms, collected before and after application of the treatments, were compared regarding the structure of their bacterial communities, by using T-RFLP technique, and the abundance of 16S rRNA, nirK, norB and nosZ genes, through qPCR technique. Only samples that had their moisture content adjusted to 90% of field capacity exhibited significant increases in N2O production. In these samples, changes in the structure of bacterial communities and in the copy numbers of norB and nosZ genes were also detected. Only the latter gene, however, showed a positive correlation with soil moisture. The abundance of the quantified genes showed no significant correlations with the gas emission rates. On the other hand, the cumulative N2O emissions were positively correlated with the amounts of denitrifying genes initially present in the samples. These genes were more abundant in pasture and forest soils, which had higher levels of organic matter, carbon, nitrogen, nitrate and clay than those from sugarcane cropping area. These results indicate that soil water content affects the N2O emission rates. However it is not due to changes in the abundance of bacteria involved in the process, such as those that bear the nirK, norB and nosZ genes. Apparently, it is the size of these organisms\' populations that determines the amount of GHG that the soil is able to produce.

A vida moderna tem gerado mudanças importantes nos hábitos alimentares em todo o mundo e observa-se um aumento da demanda por alimentos prontos para o consumo, tais como frutas e hortaliças minimamente processadas. As condições de embalagem e armazenamento destes produtos podem favorecer a multiplicação de bactérias psicrotróficas, destacando-se o patógeno Listeria monocytogenes. Para estudos de avaliação de riscos microbiológicos relativos a L. monocytogenes, dados quantitativos são fundamentais, mas, os métodos para enumeração disponíveis atualmente são trabalhosos e morosos. Há grande interesse no desenvolvimento de métodos rápidos para a determinação das populações de L. monocytogenes, o que pode reduzir significativamente o tempo de análise. Neste trabalho, foram analisadas 162 amostras de hortaliças minimamente processadas adquiridas no comércio varejista de Ribeirão Preto/SP, no período de setembro de 2007 a agosto de 2008. Foram realizados ensaios convencionais para enumeração de coliformes totais e termotolerantes, Escherichia coli e bactérias aeróbias psicrotróficas. Foi realizada a detecção de Listeria sp. por imunoensaio (Oxoid Listeria Rapid Test) e de Salmonella por método convencional. Alíquotas congeladas de amostras positivas para L. monocytogenes e Salmonella sp. foram também avaliadas quantitativamente empregando-se métodos convencionais. Para quantificação de L. monocytogenes foi utilizado o método da Reação de Polimerase em Cadeia (PCR) em tempo real, com primers de DNA baseados em 16S rRNA. Para a comparação do método de PCR em tempo real e método convencional de cultivo, foram inoculadas seis amostras de hortaliças minimamente processadas (alface, cheiro-verde, couve, almeirão, repolho e acelga) com três níveis de inoculação. Dentre as 162 amostras de hortaliças analisadas, L. monocytogenes foi detectada em uma amostra de couve e uma de cheiro-verde, com populações de 1,5 x 103 e 0,43 NMP/g, respectivamente. Salmonella sp. foi isolada de duas amostras de almeirão, com populações de 0,09 e 4,6 NMP/g. Contaminação por coliformes totais foi detectada em 132 (81,5%) amostras e por coliformes termotolerantes em 107 (66%) amostras. Escherichia coli foi confirmada em 86 (53,1%) amostras, enquanto que 158 (96,7%) amostras apresentaram população de bactérias psicrotróficas maior que 5 log UFC/g. Os resultados obtidos entre os dois métodos desenvolvidos apresentaram pouca variação e todos os resultados foram concordantes considerando-se o intervalo de confiança de 95% da técnica do NMP (SWANSON; PETRAN; HANLIN, 2001). A PCR em tempo real foi otimizada com a preparação comercial ABSOLUTETM QPCR SYBR® Green Mix (ABgene, Reino Unido) e os primers utilizados mostraram ser específicos para L. monocytogenes. As populações de microrganismos encontradas nestes alimentos indicaram a baixa qualidade microbiológica e evidenciaram a importância da implantação de programas para garantia da inocuidade de alimentos na cadeia produtiva das hortaliças minimamente processadas. A PCR em tempo real mostrou ser de fácil execução e diminuiu o tempo de obtenção de resultados para pouco mais que 48 horas em comparação com o tempo gasto (7 dias) quando as hortaliças foram analisadas por meio do método convencional de cultivo. / Modern lifestyles have deeply changed eating habits worldwide, with an increasing demand for ready-to-eat foods, including minimally processed fruits and leafy greens. Packaging and storage conditions of these products may favor the growth of psychrotrophic bacteria, such as the pathogen Listeria monocytogenes. For microbiological risk assessment, it is crucial to generate quantitative data on foodborne pathogens, but the enumeration methods currently available are labor and time consuming. There is great interest in the development of reliable and fast methods for enumeration of L. monocytogenes, in order to shorten the time needed to obtain quantitative results. In this study 162, samples of leafy vegetables minimally processed were acquired at retail market in Ribeirão Preto from September, 2007 to August 2008 and they were analysed by conventional enumerating methods for total and thermophilic coliforms, Escherichia coli and psychrotrophic aerobic bacteria. Presence or absence of Listeria spp. was evaluated by immunoassay (Oxoid Listeria Rapid test) and detection of Salmonella was performed by conventional methods. Frozen aliquots of Salmonella spp. and L. monocytogenes positive samples were quantitatively evaluated by conventional methods and L. monocytogenes was also enumerated by real-time Polymerase Chain Reaction (PCR) with DNA primers based on 16S rRNA. Real-time PCR method was applied to analyze six artificially contaminated vegetables and the results obtained with this methodology were similar to the ones obtained with conventional most probably number (MPN) technique with a confidence interval of 95% (SWANSON; PETRAN; HANLIN, 2001). Of the 162 samples analyzed, L. monocytogenes was detected in one sample of collard green and in one mixed bunch of parsley plus spring onion (1.5x103 and 0.43 MPN/g, respectively). Salmonella sp was detected in two samples of common chicory with populations of 0.09 and 4.6 MPN/g. One hundred and thirty two samples (81.5%) showed contamination by total coliforms and 107 (66%) by thermophilics coliforms. Escherichia coli was confirmed in 86 (53.1%) samples while 156 (96.7%) showed populations of psychrotrophic bacteria above of 5 log CFU/g. PCR was optimized with a commercial preparation ABSOLUTE TM QPCR SYBRÒ Green Mix (ABgene, UK) and the primers utilized were specific for L. monocytogenes. These results showed the low microbiologic quality of minimally processed vegetables samples studied and indicate an urge for implementation of quality programs from producer to processors of these ready-to-eat foods. Realtime In comparison with the conventional culture method, real-time PCR was more easily performed and the time required to obtain the results was reduced to ca. 48 hours, in comparison with 7 days for conventional method.

Células tronco mesenquimais de tecido adiposo, são uma promissora ferramenta para aplicações clínicas em terapias celular e regenerativa, em vista da facilidade de sua extração e da maior quantidade de células por unidade de massa de tecido quando comparado a outras fontes clássicas de células mesenquimais como medula óssea. O protocolo clássico de extração e purificação dessas células, depende de sua adesão em plástico e xeno-materiais demandando muito tempo para ser utilizado por médicos para auxiliar pacientes em procedimentos de emergência. Estas células são capazes se diferenciar em diversos tipos celulares, o que as torna boas candidatas para terapia celular, embora sua capacidade de transdiferenciação para fenótipos neuronais seja ainda discutida. Neste trabalho demonstramos um novo processo para isolar essas células na base de epitopos específicos expressos (assinatura molecular de superfície) utilizando aptâmeros como ligantes de alta afinidade para estes sitios. Aptâmeros, moléculas de DNA simples fita identificadas a partir de uma biblioteca combinatória de sequencias de DNA simples-fita foram identificados por ciclos reiterativos de seleção in vitro (SELEX) utilizando células tronco do lipoaspirado como alvo. Dois aptâmeros isolados, denominados APT9 e APT11, foram capazes de identificar subpopulações (15,8 e 23,7% respectivamente) dentre as células tronco mesenquimais (classicamente CD29+/CD90+/CD45-) e separá-las usando nano-partículas magnéticas acopladas aos aptâmeros. Além disso, seguindo uma indução para diferenciação neuronal, as células tronco mesenquimais passam a apresentar morfologia neuronal e apresentam expressão e atividade de diversos receptores de neurotransmissores, avaliados por PCR real-time e imageamento de variações da concentração de cálcio intracelular ápos stimulação com vários agonistas de receptores metatrópicos e ionotrópicos. Ao longo da diferenciação, os níveis transcricionais de mRNA de receptores de cininas (B1 e B2), nicotínicos (alfa 7), muscarínicos (M1, M3 e M4), glutamatérgicos (AMPA2 e mGluR2), purinérgicos (P2Y1 e P2Y4) e GABAergicos (GABA-A, subunidade 3) e da óxido nítrico sintase neural aumentaram quando comparados aos níveis das células não diferenciadas, enquanto que os níveis de expressão de outros receptores incluindo purinérgicos P2X1, P3X4, P2X7 e P2Y6 e muscarínico M5 diminuíram. Os níveis de atividade das classes dos receptores estudados, por imageamento de variações da concentração de cálcio intrac, aumentaram para a maioria dos agonistas analisados durante a diferenciação neuronal com exceção para respostas induzidas por glutamato e NMDA. Células diferenciadas expressavam altos níveis de antígenos específicos de neurônios como &#946;3-tubulina, NF-H, NeuN e MAP-2 indicando uma diferenciação em fenótipo neuronal bem sucedida. Desta maneira, esta tese, ao identificar aptâmeros, prove uma inovadora solução para médicos usarem as células tronco mesenquimais dentro de uma sala de cirurgia, através de um método que é capaz de purificar essas células em um tempo clínico viável, com pureza e sem contato com contaminantes. Além disso, nós mostramos aqui que com um protocolo como o proposto para diferenciação neuronal, nós poderíamos induzir essas células para se diferenciar em neurônios, através da ativação de fatores de transcrição específicos, levando às células tronco mesenquimais a serem possivelmente utilizadas em terapias celulares de reparo neuronal. / Adipose mesenchymal stem cells are promising tools for clinical applications in cellular and regeneration therapies, in view of easiness of extraction and higher amount of isolated stem cells per mass of tissue when compared to other classical mesenchymal stem cell sources including bone marrow. The classical protocol to extract and purify these cells, depending on plastic adherence and xeno-materials, is too time consuming to be used by physicians to help patients at emergency procedures. These cells are able to differentiate into various cell types, making them good candidates for cell therapy, however their capability for transdifferentiation into neural phenotypes is yet discussed. Here we show a novel process to isolate these cells using their surface molecular signature and aptamers, ssDNA molecules identified through the SELEX technique, denominated APT9 and APT11 that are able to identify subpopulations (15,8 and 23,7% respectively) within the mesenchymal stem cells (classically CD29+/CD90+/CD45-) and separate them using magnetic nano-particles attached to the aptamers. Moreover, following induction to neural differentiation, mesenchymal cells presents neuronal morphology and present expression and activity of several neurotransmitter receptors, as evaluated by real-time PCR and calcium imaging. During this process, mRNA transcription levels of bradykinin (B1 and B2), cholinergic (alpha 7), muscarinic (M1, M3 and M4), glutamatergic (AMPA2 and mGlu2), purinergic (P2Y1 and P2Y4) and GABAergic (GABA-A, subunit 3) receptors and neuronal nitric oxide synthase were augmented when compared to levels of undifferentiated cells, while the expression levels of other receptors including purinergic P2X1, P2X4, P2X7 and P2Y6 and muscarinic M5 receptors were down-regulated. Activity levels of the studied receptor classes, as studied by calcium imaging, increased for most of the agonists analyzed during the neuronal differentiation with the exception for glutamate- and NMDA-induced receptor responses. Differentiated cells expressed high levels of neuron-specific antigens such as &#946;3-tubulin, NF-H, NeuN and MAP-2, indicating a successful differentiation into neuronal phenotypes. This thesis, by identifying aptamers, provides a novel solution for physicians to use mesenchymal stem cells inside a surgery room, by using a method that are able to purify the cells in a clinical viable time, with purity and no contact with contaminats. Furthermore, we show here that with a protocol as provided for neuronal differentiation, we could induce these cells to differentiate into neurons, by activating specific transcription factors,making mesenchymal stem cells to possibly be used in neuronal repair cell therapies.

A Esclerose Lateral Amiotrófica (ELA) é uma doença crônica, progressiva e neurodegenerativa causada pela morte dos neurônios motores. O diagnóstico destes pacientes pode levar até 12 meses para acontecer, sendo que estes vão à óbito entre 3-5 anos do início dos sintomas. Há, porém, grande variabilidade de quadro clínico, com alguns pacientes falecendo com menos de 1 ano do início dos primeiros sinais, e outros que sobrevivem por décadas. A identificação da ELA8, causada por uma mutação missense no gene VAPB (c.C166T, p.P56S), tem contribuído significativamente com o conhecimento dos mecanismos moleculares por trás da ELA. A literatura recente tem evidenciado que a diminuição dos níveis de VAPB está presente em modelos celulares e murinos da doença, e também em amostras de pacientes, sugerindo que esta proteína teria papel central na doença e uma contribuição significativa para a morte dos neurônios motores. O presente trabalho buscou três objetivos principais: (1) o diagnóstico molecular através de um painel de sequenciamento de nova geração que inclui os genes SOD1, FUS, TARDBP, SETX, SPG11, FIG4 e VAPB; (2) a avaliação dos níveis de RNAm de VAPA, VAPB e EPHA4 em pacientes de ELA8, controles familiares e outros pacientes de ELA, com o intuito de investigar possíveis papéis destes genes na doença; e por fim, (3) o desenvolvimento de um ensaio quantitativo para as proteínas VAPA, VAPB e VAPC baseado em cromatografia líquida acoplada à espectrometria de massas em tandem (LC-MS/MS), para a posterior avaliação de VAPB como possível biomarcador em ELA, e de suas isoformas VAPA e VAPC como modificadores da doença. Para a análise genômica, foram avaliados 67 pacientes, sendo que 31 (ou 46%) apresentaram a mutação c.C166T em VAPB; 4 pacientes (6%) em SOD1, sendo que um destes apresentou uma mutação também em FIG4; 1 paciente (1.5%) foi identificado uma mutação patogênica em FUS; outro, duas mutações deletérias em trans em SPG11. Os níveis de RNAm de VAPB, VAPA e EPHA4 não são estatisticamente distintos entre pacientes e controles; porém, os níveis de EPHA4 estavam significativamente elevados em dois pacientes de início bulbar da doença. Para o desenvolvimento do método quantitativo por LC-MS/MS, foram escolhidos 8 peptídeos inequívocos para análise, estabelecidos dos parâmetros de corrida, e desenvolvidos dois padrões internos (linhagens SILAC e VAPB recombinante) para a quantificação. Esta ferramenta desenvolvida poderá auxiliar não apenas os estudos moleculares que envolvem os mecanismos por trás ELA8, responsável por uma elevada taxa dos casos familiais brasileiros, mas também poderá determinar o potencial de VAPB como biomarcador para Esclerose Lateral Amiotrófica / Amyotrophic Lateral Sclerosis is a chronic, progressive neurodegenerative disorder caused by the death of motor neurons. Diagnosis can take up to 12 months, with no molecular marker to expedite this process. In this scenario, patients die within 3 to 5 years of symptom onset, although a large clinical variability is seen, with severe patients dying less than one year after onset, and others surviving for decades. The identification of ALS8, caused by a missense mutation in the VAPB gene (c.C166T; p.P56S), has contributed significantly to the knowledge of molecular mechanisms behind ALS. Recent literature has evidenced that the decrease of VAPB levels is present in cellular and murine models, and also in patient samples, suggesting a central role in motor neuron death in ALS. The present work sought three main objectives: (1) a molecular diagnosis through a NGS sequencing panel including the SOD1, FUS, TARDBP, SETX, SPG11, FIG4 and VAPB genes; (2) analyze the expression levels of VAPA, VAPB and EPHA4 in patients, family controls and other forms of ALS, in order to investigate their possible roles in ALS8; and (3) the development of a targeted quantitative mass spectrometry based assay, gold standard in protein quantification due to its precision and sensitivity, for the VAPA, VAPB and VAPC proteins, seeking the analysis of VAPB as a potential biomarker in ALS and of its isoform\'s potential roles as modifiers in the disease. The genomic analyses revealed that out of 67 patients, 31 presented the ALS8 mutation in VAPB, 4 patients (6%) presented a mutation in SOD1, with one patient carrying a second mutation in FIG4; 1 (1.5%) patient was identified with a pathogenic mutation in FUS; and another presented two pathogenic mutations in trans in the SPG11 gene. Thus, we were able to diagnose over half of the patients included in this study with a panel of only 7 genes. VAPB, VAPA and EPHA4 mRNA levels are not statistically different between patients and controls; however, EPHA4 was shown to be highly elevated in two bulbar-onset non-ALS8 patients. For the development of the LC-MS/MS targeted assay, 8 surrogate peptides were chosen for analysis, run parameters were established, and two internal standards for quantification were developed (SILAC cell lines and recombinant VAPB). This tool will prove to be useful not only towards elucidating the molecular mechanisms behind ALS8, one of the most prevalent forms of familial ALS in Brazil, but also to determine VAPB\'s potential as a biomarker for ALS

A avicultura brasileira é atualmente uma atividade de grande sucesso. A utilização de sistemas de planejamento associados a novas tecnologias, reflete-se no extraordinário crescimento da atividade. A produção brasileira de frango ultrapassou a marca anual de 10 milhões de toneladas, em 2007. O Brasil está entre os três maiores produtores de frango no ranking mundial, junto com Estados Unidos e China. Haja vista a importância que a avicultura representa para o país, pela geração de benefícios sociais e econômicos, o risco que a Doença de Newcastle (DNC) constitui para a avicultura brasileira é enorme. Um surto desta doença em um centro de produção avícola representaria um risco à economia e incidiria de forma negativa nos níveis de consumo de proteína de qualidade e economicamente acessível à população. A fim de estabelecermos um monitoramento do vírus da Doença de Newcastle (NDV) em aves selvagens, livres ou de cativeiro, e aves domésticas não vacinadas, residentes em regiões de elevada confluência migratória aviária no Brasil e em pingüins ao redor da Estação Antártica Comandante Ferraz (EACF), coletamos amostras de swabs orais e cloacais para a posterior análise por PCR em Tempo Real (qPCR), além de sangue para testes sorológicos, tendo como objetivos maiores, contribuir para o fortalecimento dos serviços de defesa sanitária animal, aumentar a capacidade de investigação, e finalmente, atualizar e harmonizar normas e procedimentos para a prevenção e controle da DNC, referenciando-se nas recomendações da Organização Mundial de Sanidade Animal (Office International des Epizooties - OIE). Das 1072 aves amostradas em diferentes regiões do Brasil, 8 (0,75%) apresentaram resultado positivo para o NDV por qPCR, sendo 5 (62,5% das positivas) delas provenientes da região Norte, 2 (25% das positivas) do Nordeste e 1 (12,5% das positivas) da região Sul do Brasil. Na Antártica, dos 100 pingüins estudados, 2 apresentaram resultado positivo para o NDV por qPCR e em cerca de 33,3% dos soros testados foi detectada a presença de anticorpo pelo teste de Inibição da Hemaglutinação (HI). Todas as amostras positivas foram re-analisadas por qPCR específico para cepas mesogênicas e/ou velogênicas, resultando negatividade, corroborando os dados que certificam o Brasil como sendo livre da Doença de Newcastle. / Brazilian chicken meat exports ended 2007 with shipments of 3,3 million tons, which represented a 21% increase in comparison with 2006. These results were the best in the history of the poultry sector in Brazil. The production reached 10.2 million tons, a result that kept the country as the worlds largest chicken meat exporter and the third largest producer, only behind USA and China. Thus the risk of an introduction of the Newcastle disease virus (NDV) into domestic poultry is enormous and will play severe consequences for the economy and poltry industries. In order to provide the surveillance of the NDV in wild, free or captive, and non vaccinated domestic birds from some regions of migratory birds confluence in Brazil and in penguins around the brasilian Antarctic Station Comandante Ferraz, we collected oral and cloacal swabs, for the viral detection by Real Time PCR (qPCR), and blood for the serological test (hemaglutination inhibition test HI). A total of 1072 birds were sampled in diferent regions of Brazil, where 8 (0.75%) shown positive results to NDV, in which 5 (62.5% of positive) were from North region, 2 (25% of positive) were from Northeast and 1 (12,5% of positive) was from South. In the Antarctic 100 penguins were studied, in which 2 were detected the NDV (2% of total). HI test showed that 33.3% of penguins were seropositives for NDV, indicating their previous contact with the pathogen. All the positive samples by qPCR were repeated with primers projected to detect only virulent strains and no sample was positive, indicating the absence of velogenic strains in Brazil. The epidemiological profile was richer with the isolament of positive samples in embrioned chiken eggs specific patogen free and latter nucleotidic genomic sequencing of isolate.