Preimplantation genetic diagnosis (PGD) refers to the testing of embryos produced through in vitro fertilization (IVF) in order to identify those unaffected by a specific genetic disorder or chromosomal abnormality. In this study, different methodologies were examined and developed for performance of PGD. Investigation of various whole genome amplification (WGA) methods identified multiple displacement amplification as a reliable method for genotyping single cells. Furthermore, this technology was shown to be compatible with subsequent analysis using single nucleotide polymorphism (SNP) microarrays. Compared to conventional methods used in this study to perform single cell diagnosis (e.g. multiplex PCR), WGA techniques were found to be advantageous since they streamline the development of PGD protocols for couples at high risk of transmitting an inherited disorder and simultaneously offer the possibility of comprehensive chromosome screening (CCS). This study also aimed to develop a widely applicable protocol for accurate typing of the human leukocyte antigen (HLA) region with the purpose of identifying embryos that will be HLA-identical to an existing sibling affected by a disorder that requires haematopoietic stem cell transplantation. Additionally, a novel microarray platform was developed that, apart from accurate CCS, was capable of reliably determining the relative quantity of mitochondrial DNA in polar bodies removed from oocytes and single cells biopsied from embryos. Mitochondria are known to play an important role in oogenesis and preimplantation embryogenesis and their measurement may therefore be of clinical relevance. Moreover, real-time PCR was used for development of protocols for CCS, DNA fingerprinting of sperm samples and embryos and the relative quantitation of telomere length in embryos (since shortened telomeres might be associated with reduced viability). As well as considering the role of genetics in terms of oocyte and embryo viability assessment and the diagnosis of inherited genetic disorders, attention was given to a specific gene (Phospholipase C zeta) of relevance to male infertility. A novel mutation affecting the function of the resulting protein was discovered highlighting the growing importance of DNA sequence variants in the diagnosis and treatment of infertility.

Bovine tuberculosis (TB) is a contagious neglected zoonosis of cattle that is prevalent but under-investigated in Cameroon, hence this study was designed to assess the epidemiology of bovine TB in cattle, risks for M. bovis infection in cattle and humans; and public health implications of zoonotic bovine TB in the highlands of Cameroon. A retrospective study of meat inspection records (1994 – 2010) was done to estimate the prevalence of TB lesions in slaughtered cattle in the North West region. The prevalence of bovine TB and anti-bovine TB antibodies in live cattle based on tuberculin skin tests (2 surveys) and immune-chromatographic assays respectively were carried out in the Western and Adamawa highlands of Cameroon. The performance of the tuberculin tests for bovine TB diagnosis in cattle using various tuberculin skin test cut-off points against the detection of anti-bovine TB antibodies (hypothesised risks of exposure) was compared. Suspected TB lesions from slaughtered cattle and infected human sputa were cultured on Lowentein – Jesen and Middlebrook 7H9 media to isolate mycobacteria agents for molecular genotyping using genomic deletion analysis and spoligotyping. Risk factors for exposure and transmission of zoonotic bovine TB infection of cattle and cattle professionals, and its public health significance were determined using structured questionnaires. Seventeen years of meat inspection record revealed that suspect TB lesions were identified in 599 of 129,165 slaughtered cattle at the Bamenda abattoir. The lungs and associated lymph nodes (over 60%) were the most affected tissues. Other results showed that the prevalence of anti-bovine TB antibodies in cattle in the study regions was 37.17%. Chi square statistics revealed that irrespective of the tuberculin test cut-off value (P<0.05; χ2>48), strong associations existed between the detection of anti-bovine TB antibodies and disease status. A 95% confidence interval analysis of the comparative cervical tuberculin tests revealed that the prevalence rates were 4.67% – 7.15%, 12.02% – 15.67% and 20.56% – 24.98% at the ≥ 4mm, ≥ 3mm and ≥ 2mm cut-off points, respectively. Overall, the best test performance was realised at ≥ 3-mm, though the ≥ 2-mm cut-off point predicted more positive reactors. Age, sex, breed and husbandry practices served as significant (P<0.05) risks to the prevalence and exposure of bovine TB in cattle. The feedbacks from cattle professionals suggested that there was high possibility of cattle to cattle and cattle to human transmission of bovine TB such as intimate and repeated animal / animal and animal / human interactions, consuming unpasteurised milk and eating raw meat. Genomic deletion analysis of cultured isolates showed evidence of M. tuberculosis from cattle and M. bovis from human while spoligotyping identified five cattle M. bovis strains; and four spoligotype patterns that had not been previously described anywhere. The study has important epidemiological and public health implications requiring prompt and decisive actions from the Cameroonian authority towards controlling zoonotic bovine TB in both humans and animals. A multidisciplinary approach is needed for further collaborative research and effective control strategies such as enhancing the awareness of people to this deadly disease through continuous education, proper food handling and personal hygiene, healthy husbandry practices and maintenance of the environment.

This thesis provides an in-depth analysis of wh-question formation in Jordanian Arabic (JA) and presents a uniform approach that can accommodate all of its various wh-constructions. JA makes use of five different wh-constructions, four of which involve clause-initial wh-phrases and the fifth is a typical in-situ wh-construction. Although wh-phrases surface clause-initially in four different wh-constructions in JA, I propose that bona fide wh-movement to [Spec, CP] does not occur in any of these constructions, whether overtly in syntax or covertly at LF. I abandon the classification of JA as a wh-movement language (Abdel Razaq 2011) and focus instead on identifying the syntactic role that wh-phrases realize and the underlying structures that feed each wh-construction. I propose that the clause-initial position of the wh-phrase results either from the syntactic function that the wh-phrase serves or from other syntactic operations that are independently attested in JA. There are three clause-initial positions that the wh-phrase can occupy: it surfaces in [Spec, TP] when functioning as the subject of a verbal or verbless structure, in [Spec, TopP] when functioning as a clitic-left-dislocated element (as in CLLD questions and ʔilli-interrogatives involving PRON), or in [Spec, FocP] when undergoing focus fronting. Thus, all instances of clause-initial wh-phrases in JA constitute what I refer to as “pseudo wh-fronting”, as the clause-initial position of the wh-phrase arises from mechanisms other than canonical wh-movement to [Spec, CP]. To account for the interpretation of wh-phrases in JA, I adopt a binding approach in which a null interrogative morpheme (Baker 1970; Pesetsky 1987; Chomsky 1995) unselectively binds the wh-phrase regardless of its surface position, whether clause-initial or clause-internal (in-situ). A major implication of this analysis is that JA is a concealed wh-in-situ language of the Chinese type although it looks at a cursory glance as though it were a wh-movement language of the English type. A broader typological implication of my analysis is the convergence of Cheng’s (1991) Clausal Typing Hypothesis to which JA previously appeared to constitute a counterexample. The recognition of the null interrogative particle, or its optional overt realization as the Q-particle huwweh, as the locus of interrogative clause typing in all JA wh-questions entails that JA employs just one unique strategy to type a clause as a wh-question, as predicted by Cheng’s Clausal Typing Hypothesis, regardless of whether the wh-phrase surfaces clause-initially or clause-internally. / February 2017

Le genre Pneumocystis désigne un groupe de champignons opportunistes présentant une étroite spécificité d'hôte. Il détermine lors d'immunodépression sévère une infection pulmonaire grave, la pneumonie à Pneumocystis (PPC). La transmission de Pneumocystis par voie aérienne d'un hôte développant une PPC à un hôte susceptible a été démontrée à l'aide des modèles murins. Les travaux menés chez la souris ont montré également que des sujets immunocompétents colonisés par Pneumocystis murina peuvent transmettre le champignon à des souris immunodéprimées qui développeront une PPC ultérieurement. Les individus colonisés par Pneumocystis sp., ainsi que ceux développant une PPC, participeraient au réservoir du champignon. La survenue de cas groupés de PPC en milieu hospitalier est en faveur de la transmission interindividuelle de Pneumocystis jirovecii (P.jirovecii) chez l’homme. La détection de l'ADN de P.jirovecii dans l'air exhalé par les patients développant une PPC suggère que cette transmission se fait par voie aérienne. La caractérisation des populations infectées par P.jirovecii et la caractérisation génotypique du champignon au sein de son réservoir humain constituent la base de ce travail de recherche. Nous avons montré que la prévalence de la colonisation par P.jirovecii est faible chez les patients atteints de mucoviscidose et suivis dans notre CHU. La participation de ces patients au réservoir de P.jirovecii à Brest serait donc marginale. Cette faible prévalence pourrait être le reflet d'une faible circulation du champignon dans les communautés humaines dans notre région. Nous avons évalué le dosage du ß-1,3-D glucane sérique pour dépister les populations infectées. Ce dosage couplé à la détection de P.jirovecii dans les prélèvements respiratoires par la microscopie et la PCR, permet de différencier les patients développant une PPC et les patients présentant une colonisation pulmonaire par P.jirovecii. De plus, les premières données sur le ß-1,3-D glucane au cours de la primo-infection chez le nourrisson ont été obtenues.En termes de caractérisation de P.jirovecii dans notre région, l'analyse du locus dihydropteroate synthase (DHPS) a montré que: i) le lieu habituel de résidence plutôt que le lieu de diagnostic de l’infection à P.jirovecii serait un facteur prédictif d’infection par un mutant, ii) P.jirovecii pourrait circuler en France d’une région à une autre via des voyageurs infectés, iii) la prévalence de mutants potentiellement résistants chez les patients vivant effectivement à Brest était de 0%. L'analyse des séquences des "internal transcribed spacers" (ITS) 1 et 2 de P. jirovecii conforte l'hypothèse que les patients développant une PPC et les patients colonisés sont infectés par des populations fongiques présentant des caractéristiques identiques. Tous les patients, quelle que soit la présentation clinique de leur infection, constitueraient un réservoir unique et commun de P.jirovecii. Les travaux de génotypage ont constitué l'étape préalable nécessaire à l'analyse de cas groupés d'infections à P.jirovecii survenus chez des patients transplantés rénaux au CHU de Brest. Nous avons apporté des données originales sur le rôle des patients colonisés en tant que source potentielle de P. jirovecii dans un contexte d'acquisition et de transmission nosocomiales du champignon. Par ailleurs, la concordance partielle ou complète des génotypes ITS et DHPS dans les couples "prélèvements d'air–LBA" réalisés chez des patients développant une PPC est compatible avec l’exhalation du champignon et sa diffusion aérienne dans l’environnement hospitalier. Ces données apportent des arguments pour l'application de mesures de prévention des infections nosocomiales à P. jirovecii. Les précautions "gouttelettes" recommandées par la Société Française d'Hygiène Hospitalière devraient être appliquées a minima aux patients développant une PPC. Nous proposons leur extension aux patients colonisés par le champignon. / The genus Pneumocystis represents a group of opportunistic fungi that show strong host specificity. It is the cause of severe pneumonia (Pneumocystis Pneumonia [PCP]) in immunocompromised subjects. Pneumocystis transmission from a host with PCP to another susceptible host via the airborne route has been demonstrated in rodent models. Moreover, it has been established that Pneumocystis murina can be transmitted from immunocompetent mice, transiently colonized by the fungus, to immunocompromised susceptible mice that subsequently develop PCP. Colonized subjects and those developing PCP may be part of the fungus reservoir. Reports of PCP case cluster in hospital strongly suggest that Pneumocystis jirovecii (P.jirovecii) transmission in humans may also occur. P.jirovecii DNA detection in the air surrounding PCP patients is consistent with the transmission of P.jirovecii via the airborne route.Our goals were to characterize human populations infected with P.jirovecii and to characterize P.jirovecii within its human reservoir. We showed that P.jirovecii was rarely involved in pulmonary colonization in patients with cystic fibrosis monitored in the Brest Hospital. Thus this patient population was not part of the human reservoir of the fungus in our region (Brittany, Western France). This low prevalence of colonization may reflect a low level of P.jirovecii circulation within human communities in Brittany. In order to improve the identification of patients infected with P.jirovecii, we evaluated ß-1,3-D glucan detection in serum samples. We showed that serum ß-1,3-D glucan levels combined with P.jirovecii detection in pulmonary samples using microscopic examination and a PCR assay make it possible to distinguish between PCP and pulmonary colonization. Moreover the first data on ß-1,3-D glucan levels during primary infection were obtained.In order to characterize P.jirovecii in our region, we performed the typing of P.jirovecii isolates from infected patients monitored at Brest hospital, using the dihydropteroate synthase (DHPS) and the internal transcribed spacer (ITS) 1 and 2 locus analysis. DHPS typing showed that i) the usual city of patient residence rather that the city in which the diagnosis of P.jirovecii infection has been made is a predictor of mutants, ii) mutants can be imported from one region to another through infected visitors, iii) the prevalence of mutants potentially resistant to sulfonamides was 0% in patients who effectively lived in the Brest geographic area. Results of ITS analysis in PCP patients and colonized patients are consistent with the hypothesis that these 2 patient groups are infected with similar P.jirovecii populations. All infected patients, whatever their clinical presentation, may be part of a common and unique reservoir of the fungus. We investigated an outbreak of P.jirovecii infections in 18 renal transplant recipients using the same typing method combined with patient encounter analysis. The results provided evidence of the role of colonized patients as potential sources of P.jirovecii. The same typing method was applied to pairs of pulmonary samples and room air samples of PCP patients. Full or partial matches of P.jirovecii types in pulmonary and air sample pairs were observed. These results are consistent with P.jirovecii exhalation by PCP patients in their close environment. These data support arguments for applying droplet precautions, at least to PCP patients, to prevent P.jirovecii transmission, as recommended by the "Société française d'hygiène hospitalière". We suggest extending droplet precautions to colonized patients to achieve the prevention of P.jirovecii nosocomial infections.

The present study aimed to evaluate bacterial isolates and the microbiome of dairies. The specific aims were: to characterize Staphylococcus spp. isolated from mastitic milk, to evaluate the presence of Lactococcus in mastitic milk as a potential causative agent of mastitis, to evaluate the association between microbiome and milk quality parameters, and to characterize Staphylococcus spp. isolated from production lines of Minas Frescal cheese. The detection of genes encoding virulence factors (enterotoxins (sea, seb, sec, sed, see, seg, seh, sei, selj, selk, sell, selm, seln, selo, selp, seIq, ser, ses, set, selu, selv, and selx), hemolysins (hla, hlb, hld, hlg, and hlgv), exfoliative toxins (eta, etb, and etd), Panton-Valentine leukocidin (pvl), and toxic shock syndrome toxin (tst)), genes encoding antibiotic resistance (resistance to tetracycline (tetK, tetL, and tetM), erythromycin (ermA, ermB, and ermC), methicillin (mecA and mecC), and tobramycin (ant(4\')-Ia)), molecular typing (spa, SCCmec, and agr types), and phenotyping regarding antibiotic resistance were performed in staphylococci isolates from mastitic milk, and from cheese processing plant samples. Staphylococcus aureus was identified in the majority of isolates from both origins. Several virulence factor genes were detected. The distribution of genes encoding staphylococcal enterotoxins (85.0% - 85.7% of isolates were positive for one or more enterotoxin gene) was highlighted and the gene related to H toxin was the most prevalent. Methicillin-resistant Staphylococcus aureus were identified in isolates from mastitic milk (4.1%) and cheese processing (6.0%); the genotyping and phenotyping of these isolates were described. t605 had the highest frequency in the S. aureus population studied. In mastitic milk, Lactococcus was suggested as the causative agent of an outbreak of mastitis in a dairy farm. Using next generation sequencing, the abundance of Lactococcus was observed in microbiome samples. Bacterial isolation and DNA sequencing confirmed the presence of Lactococcus lactis and Lactococcus garvieae. The microbiome of environmental samples and bulk tank milk from the dairy farm showed the Lactococcus genus among the most common bacterial taxa, suggesting other sources of this genus. Regarding milk quality parameters, the microbiome of bulk tank milk from several dairy farms was associated with somatic cell count and bacterial count. The core microbiome was described and many genera of importance were identified. Among the associations performed between microbiome and milk quality parameters, the identification of Streptococcus in samples classified with high somatic cell count and high bacterial count was highlighted. Several bacterial taxa with relative abundance significantly higher in samples classified as high and low cell count and bacterial count were shown. Real-time polymerase chain reaction was also performed associated with bacterial diversity, bacterial taxa, and bacterial count. These findings highlight the need to control and prevent bacterial contamination in the dairy industry, from herd to consumers. / O presente estudo apresentou como objetivo avaliar isolados bacterianos e microbioma de lácteos. Os objetivos específicos foram: caracterizar Staphylococcus spp. isolados de leite de vacas com mastite, avaliar a presença de Lactococcus em leite de vacas com mastite como um potencial agente causador de mastite, avaliar a associação entre microbioma de leite de tanque e parâmetros da qualidade de leite, e caracterizar Staphylococcus spp. isolados de linhas de processamento de queijo Minas frescal. A detecção de genes codificadores de fatores de virulência (enterotoxinas (sea, seb, sec, sed, see, seg, seh, sei, selj, selk, sell, selm, seln, selo, selp, seIq, ser, ses, set, selu, selv, e selx), hemolisinas (hla, hlb, hld, hlg, e hlgv), toxinas exfoliativas (eta, etb e etd), leucocidina de Panton-Valentine (pvl), toxina da síndrome do choque tóxico (tst)), genes codificadores de resistência a antibióticos (resistência a tetraciclina (tetK, tetL e tetM), eritromicina (ermA, ermB e ermC), meticilina (mecA e mecC) e tobramicina (ant(4\')-Ia)), tipagem molecular (spa, SCCmec e agr types), e fenotipagem quanto à resistência a antibióticos foram realizadas em estafilococos isolados de leite de vacas com mastite e de amostras de planta de processamento de queijo. Staphylococcus aureus foi identificado na maioria dos isolados de ambas as origens. Diversos genes de fatores de virulência foram detectados, com destaque para a distribuição de genes codificadores de enterotoxinas estafilocócicas (85,0%-85,7% dos isolados foram positivos para um ou mais genes codificadores de enterotoxinas), sendo o gene relacionado com a toxina H o mais frequente. Staphylococcus aureus meticilina resistente foram identificados em isolados de leite de vacas com mastite (4.1%) e em processamento de queijo (6.0%); o perfil genotípico e fenotípico destes isolados foram descritos. t605 foi o mais freqüente na população de S. aureus estudada. Em leite de vacas com mastite, Lactococcus foi sugerido como o agente causador de um surto de mastite numa fazenda leiteira. Usando sequenciamento de nova geração, a abundância de Lactococcus foi observada no microbioma das amostras. O isolamento e sequenciamento de DNA confirmaram a presença de Lactococcus lactis e Lactococcus garvieae. O microbioma de amostras ambientais e de leite de tanque da fazenda mostrou o gênero Lactococcus entre os mais comuns, sugerindo outras fontes deste gênero. Contemplando parâmetros da qualidade de leite, o microbioma de leite de tanque de várias fazendas leiteiras foi relacionado com contagem de células somáticas e contagem bacteriana. O core microbiome foi descrito e muitos gêneros bacterianos de importância foram identificados. Dentre as análises realizadas associando microbioma com parâmetros da qualidade de leite, foi destacada a identificação de Streptococcus em amostras classificadas com alta contagem de células somáticas e alta contagem bacteriana. Diversos táxons bacterianos com abundância relativa significativamente maior em amostras classificadas com alta e baixa contagem de células somáticas e contagem bacteriana foram mostrados. Reação em cadeia da polimerase em tempo real também foi realizada e associada com diversidade bacteriana, táxons bacterianos e contagem bacteriana. Estes levantamentos confirmam a necessidade de controlar e prevenir a contaminação bacteriana na indústria de lácteos, do rebanho leiteiro até os consumidores.

A presente pesquisa teve por objetivo avaliar as fontes de contaminação por Listeria monocytogenes em uma linha de processamento de \"nuggets\" congelados de frango. Linhagens de L. monocytogenes isoladas de diferentes pontos de uma usina de processamento industrial nas diversas etapas do processamento foram avaliadas quanto à sua diversidade genética. A técnica empregada foi o RAPO com metodologia modificada da Organização Mundial da Saúde (OMS/WHO). Os perfis RAPO gerados com os \"primers\" M13 e UBC155 foram agrupados, combinados e analisados quanto à sua similaridade. As cepas foram também sorotipadas e 189 pertenciam ao sorogrupo 1 e 63 ao sorogrupo 4. A correlação entre a diversidade genética e a distribuição do microrganismo na linha de processamento foi estabelecida. As 252 L. monocytogenes estudadas puderam ser divididas em dois grandes \"clusters\" cada qual dividido em dois grupos. Os resultados da análise de \"clusters\" foram relacionados aos da sorologia, determinando sete subtipos. Verificou-se que três subtipos são introduzidos no ambiente de processamento juntamente com a matériaprima. Um deles foi encontrado somente na matéria-prima e os outros dois também foram detectados em superfícies de equipamentos e no ambiente de processamento. Outros subtipos encontrados em superfícies de equipamentos e no ambiente foram encontrados no produto em etapas subseqüentes. A contaminação da matéria-prima por cepas diferentes daquelas encontradas no ambiente de processamento mostra a sua importância como fonte de contaminação. Formas de controle da presença de L. monocytogenes na matéria-prima devem ser buscadas assim como o controle da contaminação ambiente. / This research was carried out in order to evaluate the sources of Listeria monocytogenes contamination in a frozen chicken nugget processing line. Strains of L. monocytogenes from different origins and isolated from different steps of the processing line were analysed for their genetic diversity. RAPO methodology modified from a WHO protocol was used. The RAPO profiles generated by primers UBC155 and M13 were grouped, combined and the similarities analysed. The strains were also serotyped and 189 belonged to serogroup 1 and 63 to serogroup 4. The correlation between genetic diversity and the strain distribution along the processing line was established. The 252 L. monocytogenes strains analysed were divided in two clusters, each of them containing 2 groups. Seven subtypes could be determined when the results of RAPO and serotyping were combined. It could be established that from the three sub-types of L. monocytogenes that belonged to the raw material, two could establish themselves in the processing line. These sub-types were detected latter in the environmental samples (food contact and non-food contact surfaces). On the other hand, other sub-types found initially in environmental samples were detected in the product in subsequent steps. The introduction of L. monocytogenes into the plant by raw material highlights its importance as a contamination source. Measures must be taken to control the presence of L. monocytogenes in the raw material as well as in the processing environment.

Salmonella é um dos principais agentes de enfermidades transmitidas por alimentos em diversos países, sendo a carne de frango um dos principais veículos envolvidos em surtos. O Brasil vem se destacando como um dos maiores exportadores mundiais deste alimento. O ambiente de criação das aves é apontado como um importante foco de infecção das aves e o ambiente industrial de abate e processamento é importante na disseminação deste ·microrganismo. Na busca pela produção de alimentos seguros do ponto de vista microbiológico, uma das ferramentas utilizadas é a subtipagem de microrganismos isolados ao longo da cadeia de produção, que permite determinar rotas de contaminação do produto final. Os objetivos deste trabalho são: o estudo da disseminação dos subtipos de Salmonella Enteritidis nas várias etapas de uma cadeia de produção industrial de carne de frango, empregando-se diversos métodos de subtipagem e a determinação da resistência a antimicrobianos destas cepas. 108 isolados de Salmonella Enteritidis dos fagotipos PT1, PT4 e PT7a foram obtidos nos anos de 2002 e 2003, a partir de amostras ambientais e de frango relativas a sete sub-regiões de uma cadeia produtiva industrial avícola. Os perfis de resistência destes isolados foram determinados frente a antimicrobianos de uso humano e veterinário e eles foram submetidos a subtipagem por PFGE, RAPO, ribotipagem e PCR-ribotipagem. Foram detectados 21 perfis de resistência diferentes, com 6,5% das cepas sensíveis a todas as drogas, 33,3% resistentes a um ou dois antimicrobianos e 83,3% apresentando resistência intermediária a até quatro deles. Os níveis relativamente elevados de resistência são preocupantes e a diminuição da pressão seletiva deve ser um objetivo para os produtores de aves. De modo geral, a subtipagem permitiu separar as cepas em 13 genótipos, com elevada similaridade entre si. Porém, a maior parte das cepas (69,4%) pertenceu a apenas três deles, que foram encontrados ao longo de toda a cadeia produtiva. A ribotipagem foi o método que apresentou o melhor poder discriminatório (D = 0,701), porém nem todas as cepas foram tipáveis por este método. Não foram encontradas correlações entre os perfis de resistência a antimicrobianos e fagotipos, nem entre genótipos e fagotipos. Porém, dois genótipos proximamente correlacionados e predominantemente encontrados em uma sub-região reuniram apenas cepas com resistência intermediária ou resistentes exclusivamente à furazolidona. A similaridade elevada entre os genótipos evidencia a origem clonal das cepas. / Salmonella is one of the most important foodborne disease agents all over the world, and chicken is recognized as an important vehicle of the infection. Chicken production in Brazil has increased in the last couple of years and the country is now ranked 2nd as producer/exporter of this commodity. For this reason there is an increased concern over the safety of these goods. This study deals with the dissemination, antimicrobial resistance, and genetic characterization of S. Enteritidis strains isolated from an industrial chicken production chain. 108 isolates, phagetypes PT1, PT4 and PT7a, were obtained at different steps of the commercial production from farm to frozen cuts, and the broilers were from different producers supplying the same processing plant. Tests for susceptibility to 12 human and veterinary antimicrobial agents were performed. The strains were also typed by PFGE, RAPO, ribotyping, and PCR-ribotyping. 6.5% of the strains were susceptible to the 12 drugs tested and 33.3% were resistant to 1 or 2 of them. Intermediate resistance to up to 4 agents was observed in 83.3% of the isolates. Combining all the typing methods allowed the division of the strains in 13 genotypes with elevated degree of similarity. However, 69.4% of the strains belonged to 3 main phagetypes spread along the production chain. There was no correlation between phagetypes and genotypes, or phagetypes and resistance profiles. However, most strains from one sub-region were from 2 genotypes and showed intermediate resistance to, or were resistant to furazolidone. The high degree of similarity amongst the genotypes indicates the clonal origin of the strains. The relatively high resistance to antimicrobial agents is a cause of concern and trying to diminish the selective pressure has to be a goal for broiler producers.

Infecções invasivas por Trichosporon spp. ocorrem com maior frequência em pacientes neutropênicos, principalmente portadores de doenças hematológicas malignas, e estão associadas a elevados índices de mortalidade devido às dificuldades na identificação do patógeno e à resistência aos fármacos mais empregados na terapêutica antifúngica. A identificação das espécies de Trichosporon é importante tanto para estudos epidemiológicos, como para associar aspectos clínicos com as espécies causadoras das infecções. Além disso, auxilia no tratamento da enfermidade, uma vez que a suscetibilidades aos fármacos antifúngicos pode variar de acordo com a espécie. Além disso, as leveduras do gênero Trichosporon sintetizam a glucuronoxilomanana (GXM) em sua parede celular, que pode estar envolvida no mecanismo de virulência do patógeno. Este estudo teve como objetivo determinar, por identificação fenotípica e molecular, espécies isoladas de pacientes internados em unidades hospitalares, comparando os resultados obtidos por ambos os métodos; avaliar diferenças na distribuição dessas espécies em relação às formas invasivas e não invasivas da infecção; determinar o perfil de suscetibilidade dessas leveduras aos antifúngicos, empregando um método de micro-diluição de referência e um método comercial; e avaliar a presença de GXM na parede celular dos isolados. Foram avaliados 74 isolados obtidos de amostras clínicas de pacientes do Hospital das Clinicas da FMUSP e de outras unidades hospitalares do Estado de São Paulo, no período de 2003 a 2011. Dezenove amostras foram isoladas de sítios estéreis do organismo (infecções invasivas) e 55 foram isoladas de urina e cateter (isolados não invasivos). Para a identificação das espécies, os isolados foram submetidos a análises fenotípicas, que incluíram estudo macro e micromorfológico, provas fisiológicas e avaliação do perfil bioquímico por sistema automatizado VITEK 2. A identificação molecular foi realizada pelo sequenciamento das regiões IGS e D1/D2 do DNA ribossomal. O perfil de suscetibilidade dos 74 isolados foi analisado pelo método de micro-diluição EUCAST (referência) com os fármacos fluconazol (FCZ), itraconazol (ITZ), voriconazol (VCZ), cetoconazol (CTZ), anfotericina B (AMB) e 5-fluocitosina (5FC); e pelo método de micro-diluição comercial Sensititre YeastOne, com os mesmos fármacos empregados no EUCAST, acrescidos do posaconazol (POS) e caspofungina (CAS). Os valores das concentrações inibitórias mínimas (CIM), erros categórico e essencial, bem como outros parâmetros foram comparados entre os dois métodos. A presença de GXM na parede celular dos 74 isolados foi determinada por citometria de fluxo, empregando anticorpo monoclonal anti-GXM. Os resultados dos estudos morfológicos e fisiológicos foram insuficientes para definir as espécies dos 74 isolados. Pela assimilação de carboidratos analisada pelo sistema VITEK 2, verificou-se que 71 isolados foram identificados como T. asahii (17 de infecção invasiva e 54 não invasivos), um isolado como T. mucoides (invasivo), e para dois isolados (um invasivo e um não invasivo), a identificação não foi conclusiva. Para estes últimos foi realizado o auxanograma (método manual), e a identificação permaneceu inconclusiva, pois pelo perfil de assimilação, os isolados poderiam ser identificados como T. asahii ou T. faecale. Pela técnica de sequenciamento, 62 dos 74 isolados foram identificados como T. asahii, demonstrando 82,4% de concordância com o sistema VITEK 2. Onze isolados com identificações discordantes pertenciam às espécies T. inkin (8), T. faecale (2) e T. dermatis (1), como determinado por sequenciamento. Dos dois isolados com identificação inconclusiva pelo VITEK 2, um foi identificado pela técnica molecular como T. asahii, enquanto para o outro isolado não foi possível definir a espécie. Portanto, dos 74 isolados do estudo, 62 foram identificados como T. asahii, 8 como T. inkin, 2 como T. faecale e 1 T. dermatis; dois isolados permaneceram sem identificação conclusiva. Os resultados dos testes de suscetibilidade in vitro mostraram que, em ambos os métodos, VCZ apresentou a melhor atividade antifúngica. Pelo método EUCAST, foram obtidos valores elevados de CIM para AMB, enquanto o mesmo não foi observado no teste comercial. Neste último, foram observados valores elevados de CIM para FCZ, POS e CAS. Em relação à 5FC, os valores de CIM 90% por ambos os testes foram elevados (16mg/L). Diferenças significantes foram observadas entre os valores de CIM obtidas pelos dois métodos, e percentuais relativamente elevados de erros categóricos graves quando o método comercial foi comparado ao de referência. Não houve diferença estatística significante de valores de CIM entre isolados de infecção invasiva e não invasiva, exceto para ITZ e 5FC. Cerca de 30% dos isolados obtidos de casos de infecção invasiva e não invasivos apresentaram resistência cruzada entre os azóis FCZ e VCZ, e uma pequena porcentagem apresentou multirresistência. Para a análise de GXM na parede celular dos 74 isolados do estudo, foi avaliada a intensidade de fluorescência emitida pela citometria de fluxo, não tendo sido observada diferença estatística significante entre isolados invasivos e não invasivos. O estudo permitiu concluir que T. asahii foi a espécie mais isolada das amostras clínicas obtidas de sítios estéreis e não estéreis. A metodologia clássica de identificação fenotípica não foi suficiente para definir as espécies do gênero Trichosporon, e o sistema VITEK 2 apresentou discordância quando comparado à técnica molecular para as espécies não T. asahii. Em relação aos testes de suscetibilidade in vitro, VCZ apresentou-se mais adequado para a inibição das leveduras, enquanto os fármacos AMB, FCZ e POS não foram eficazes para a maior parte dos isolados. As discordâncias encontradas entre o método de referência e o comercial sugerem que, para o segundo, são necessárias mais avaliações para seu emprego em rotina laboratorial para o gênero Trichosporon. A detecção de GXM não resultou em diferenças entre os isolados de ambos os grupos; no entanto, para se determinar o efeito protetor do polissacarídeo contra a ação de macrófagos, ensaios de fagocitose devem ser realizados / Invasive Trichosporon spp. infections occur more frequently in neutropenic patients, especially those with hematologic malignancies, and are associated with high mortality rates due to difficulties in identifying the pathogen and treating patients with drugs most currently employed in antifungal therapy. Trichosporon species identification is important for epidemiological studies and to better define eventual species-specific clinical association. Additionally, antifungal susceptibility may vary according to the species. Furthermore, glucuronoxylomannan (GXM) is a cell wall-associated polysaccharide produced by genus Trichosporon, which may be involved in virulence mechanisms of this pathogen. This study aimed (i) to identify Trichosporon species isolated from hospitalized patients by both phenotypic and molecular methods, comparing results; (ii) to verify the distribution of these species in invasive and non-invasive infection episodes; (iii) to determine the in vitro activities of various antifungals agents against the Trichosporon spp. isolates, employing a reference micro-dilution method and a commercial system; (iv) and to analyze the surface expression of GXM. Seventy-four Trichosporon spp. isolates obtained from clinical specimens of patients admitted to the Hospital das Clínicas-FMUSP and to other hospitals in the state of São Paulo, from 2003 to 2011, were included in the study. Nineteen samples were isolated from sterile deep sites (invasive infections) and 55 were isolated from catheter and urine samples (non-invasive isolates). All isolates were submitted to phenotypic analysis, which consisted in morphological features observation, physiological tests and determination of the biochemical profile by VITEK 2 system. Molecular identification was performed by sequencing of IGS1 and D1/D2 regions from the ribosomal DNA. The susceptibility antifungal profiles of the 74 isolates were analyzed by both the EUCAST micro-dilution method (reference) employing fluconazole (FCZ), itraconazole (ITZ), voriconazole (VCZ), ketoconazole (CTZ), amphotericin B (AMB) and 5 - flucytosine (5FC), and the commercial micro-dilution test Sensititre YeastOne, with the same drugs employed in EUCAST plus posaconazole (POS) and caspofungin (CAS). The minimum inhibitory concentration values (MIC), categorical and essential errors as well as other susceptibility parameters were compared between both methods. The cell wall expression of GXM of all isolates was measured by flow cytometry employing an anti-GXM monoclonal antibody. The morphological and physiological features of the Trichosporon spp. isolates were insufficientto define species. The carbohydrate assimilation analysis, performed by VITEK 2 system, has resulted in 71 isolates identified as T. asahii (17 from invasive infections and 54 non-invasive isolates) and one isolate as T. mucoides (invasive). The species identification for the two remaining isolates (one invasive and one non-invasive) was inconclusive. For this reason, a manual auxanogram was performed with these isolates, resulting again in non-conclusive species identification. By the automated sequencing method, 62 of the 74 isolates were identified as T. asahii, showing 82.4% of agreement with the VITEK 2 identification. Eleven isolates were identified by sequencing as T. inkin (8), T. faecale (2) and T. dermatis (1), showing disagreement identification with the VITEK 2 system. Regarding the two isolates with inconclusive results by the carbohydrate assimilation, the molecular technique identified one as T. asahii, whereas for the other isolate the sequencing was also unable to define species. Therefore, among the 74 studied isolates, 62 were identified as T. asahii, eight as T. inkin, two as T. faecale and one as T. dermatis; and two isolates remained with unconclusive identification. Almost all Trichosporon spp. isolates displayed susceptibility to VCZ with both methods. By the EUCAST method, high values of MIC were observed for AMB, while by the commercial test especially the invasive isolates showed susceptibility to this drug. Additionally, the Sensititre kit provided elevated MIC values for FCZ, POS and CAS. In regards to 5FC, the MIC 90% values were consistently high (16 mg/L) in both methodologies. The MIC values obtained by both EUCAST and commercial methods were compared, resulting in significant differences of MIC values for all tested antifungal drugs; major categorical errors occurred at relatively high percentage with the commercial method. No statistically significant differences in MIC values were verified when invasive and non-invasive isolates were compared. Around 30% of both invasive and non-invasive isolates showed cross-resistance to FCZ and VCZ, while a small number of isolates was multiresistant. The GXM analysis by cytometry demonstrated no significant differences between invasive and non- invasive isolates. This study demonstrated that T. asahii was the most frequently isolated species from both deep and non-sterile sites of the patients. The classical phenotypic methodology was not able to define Trichosporon species, and the VITEK 2 system identification showed disagreement with the sequencing technique for the non-T. asahii species. Regarding the in vitro susceptibility tests, VCZ was the most effective drug against the isolates, whereas most of them appear to be less susceptible to AMB, FCZ and POS. The discrepancies in the Trichosporon spp. susceptibility results between the reference and commercial methods suggest that the latter requires further evaluation tests before it can be used in routine laboratory. Although the GXM expression seemed to be equal in both invasive and non-invasive Trichosporon spp. isolates, phagocytic assays should be performed in order to determine the protective effect of the polysaccharide against phagocytosis

MRSA é um importante agente de colonização e infecção em pacientes hepatopatas e transplantados de fígado. Este estudo tem como objetivo avaliar a clonalidade e a virulência de isolados MRSA de pacientes hepatopatas atendidos no Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo. De agosto de 2010 a janeiro de 2012, foram coletados swabs nasais e inguinais de 190 pacientes (126 pré-transplante e 64 de pós-transplante). Isolados de MRSA foram identificados fenotipicamente e foi realizada detecção de genes de virulência, caracterização do tipo de SCCmec, análise de polimorfismo genômico por PFGE e técnica de microarranjo. Além disso, determinou-se a CIM para dez antimicrobianos pelo método de microdiluição em caldo. MRSA foi detectado em 20% dos pacientes pelo método de cultura e em 82% por PCRs. Apenas três pacientes colonizados desenvolveram infecção após o transplante. Entre os 69 isolados de MRSA, 42,0% (29/69) apresentaram SCCmec tipo II, 20,3% (14/69) SCCmec tipo I, 20,3% (14/69) SCCmec tipo III, 13,0% (9/69) SCCmec tipo IVa, 2,9% (2/69) SCCmec tipo IV e 1,5% (1/69) SCCmec tipo V. O gene tst foi detectado em 5,8% (4/69) dos isolados MRSA e todos eles foram definidos como SCCmec tipo I. Outros genes identificados por PCR foram: lukD (89,9%; 62/69), lukE (89,9%; 62/69), clf (91,3%; 63/69) e fnbA (89,9%; 62/69). A análise por PFGE dos 69 isolados mostrou a presença de um clone predominante chamado cluster A em 36,2% (25/69) e este cluster apresentou 84,6% de similaridade com o clone NewYork/Japan (BK2464). O dendrograma demonstrou também a presença de um cluster relacionado com BEC (Clone Endêmico Brasileiro) HSJ216. Atualmente o tipo de SCCmec mais prevalente em nosso hospital é o tipo II. Neste estudo, observou-se a presença de isolados virulentos tanto em pacientes hepatopatas como em pacientes transplantados. Nossos resultados mostraram que o clone predominante (cluster A) apresentou diferentes genes de virulência (genes fnbA, clf e lukD-lukE) e foi resistente a pelo menos seis diferentes drogas, além de ser caracterizado como HA-MRSA SCCmec tipo II. Em conclusão, a técnica de microarranjo permite a genotipagem e detecção de genes estafilocócicos clinicamente relevantes, e pode, na maioria dos casos, ser utilizada como uma importante ferramenta para a triagem da virulência e resistência a antimicrobianos em isolados de MRSA / MRSA is an important agent of colonization and infection in patients with liver disease and liver transplant. This study aims to evaluate clonality and virulence of MRSA isolates from liver diseases patients treated at Hospital of Clinics Faculty of Medicine from University of Sao Paulo. From August 2010 to January 2012, we collected nasal and groin swabs from 190 patients (126 pre-liver and 64 post-liver). MRSA isolates were identified phenotypically and the detection of virulence genes, characterization of SCCmec type, microarray and genomic polymorphism analysis by PFGE were done. In addition, it was determined the MIC for ten antibiotics by broth microdillution method. MRSA was detected in 20% patients by culture method and 82% by PCR. Only three patients colonized developed infection post-transplantation. Among the 69 MRSA isolates, 42.0% (29/69) had type II SCCmec, 20.3% (14/69) SCCmec type I, 20.3% (14/69) SCCmec type III, 13.0% (9/69) SCCmec type IVa, 2.9% (2/69) SCCmec type IV and 1.5% (1/69) SCCmec type V. The tst gene was detected in 5.8% (4/69) of MRSA isolates and all of them were defined as SCCmec type I. Other genes were identified by PCR: lukD (89.9%; 62/69), lukE (89.9%; 62/69), clf (91.3%; 63/69) and fnbA (89.9%; 62/69). The PFGE analysis of 69 isolates showed the presence of a predominant cluster named cluster A in 36.2% (25/69) and this cluster had 84.6% similarity with New York/Japan clone (BK2464). Dendrogram also demonstrated presence of one cluster related with BEC (Brazilian Endemic Clone) HSJ216. Currently the most prevalent SCCmec type in our hospital is type II. In this study, we observed virulent isolates in pre and post-transplantation patients. Our results showed that the predominant clone (cluster A) had different virulence genes (genes fnbA, clf and lukD-lukE) and was resistant to at least six different drugs, in addition to being characterized as HA-MRSA SCCmec type II. In conclusion, microarray profiling allows genotyping and detection of clinically relevant staphylococcal genes, and can, in most cases, be used as an important tool to screening virulence and antibiotic resistance genes in MRSA isolates

As technology evolves, new devices and interaction techniques are developed. These transformations create several challenges in terms of usability and user experience. Our research faces some challenges for data input or content exploration in scenarios with restrictions. It is not our intention to investigate all possible scenarios, but we deeply explore a broad range of devices and restrictions. We start with a discussion about the use of an interactive coffee table for exploration of personal photos and videos, also considering a TV set as an additional screen. In a second scenario, we present an architecture that offers to interactive digital TV (iDTV) applications the possibility of receiving multimodal data from multiple devices. Our third scenario concentrates on supporting text input for iDTV applications using a remote control, and presents an interface model based on multiple input modes as a solution. In the last two scenarios, we continued investigating better ways to provide text entry; however, our restriction becomes not using the hands, which is the kind of challenge faced by severely motor-disabled individuals. First, we present a text entry method based on two input symbols and an interaction technique based on detecting internal and external heel rotations using an accelerometer, for those who keep at least a partial movement of a leg and a foot. In the following scenario, only the eyes are required. We present an eye-typing technique that recognizes the intended word by weighting length and frequency of all possible words formed by filtering extra letters from the sequence of letters gazed by the user. The exploration of each scenario in depth was important to achieve the relevant results and contributions. On the other hand, the wide scope of this dissertation allowed the student to learn about several technologies and techniques. / Com a evolução da tecnologia, novos dispositivos e técnicas de interação são desenvolvidas. Essas transformações criam desafios em termos de usabilidade e experiência do usuário. Essa pesquisa enfrenta alguns desafios para a entrada de dados e exploração de conteúdo em cenários com restrições. Não foi intenção da pesquisa investigar todos os possíveis cenários, mas sim a exploração em profundidade de uma ampla gama de dispositivos e restrições. Ao todo cinco cenários são investigados. Primeiramente é apresentada uma discussão sobre o uso de uma mesa de centro interativa para a exploração de fotos e vídeos pessoais, a qual também considera um aparelho de TV como tela adicional. Com base no segundo cenário, uma arquitetura que oferece a aplicações de TV digital interativa (TVDI) a possibilidade de receber dados multimodais de múltiplos dispositivos é apresentada. O terceiro cenário se concentra no suporte a entrada de texto para aplicações de TVDI usando o controle remoto, resultando na apresentação de um modelo de interface baseado em múltiplos modos de entrada como solução. Os dois últimos cenários permitem continuar a investigação por melhores formas de entrada de texto, porém, a restrição se torna a impossibilidade de usar as mãos, um dos desafios enfrentados por indivíduos com deficiência motora severa. No primeiro deles, são apresentados um método de entrada de texto baseado em dois símbolos de entrada e uma técnica de interação baseada na detecção de rotações do pé apoiado sobre o calcanhar usando acelerômetro, para aqueles que mantêm pelo menos um movimento parcial de uma perna e um pé. No senário seguinte, apenas os movimentos dos olhos são exigidos. Foi apresentada uma técnica de escrita com o olho que reconhece a palavra desejada ponderando o comprimento de a frequência de ocorrência de todas as palavras que podem ser formadas filtrando letras excedentes da lista de letras olhadas pelo usuário. A exploração de cada cenário em profundidade foi importante para a obtenção de resultados e contribuições relevantes. Por outro lado, o amplo escopo da dissertação permitiu ao estudante o aprendizado de diversas técnicas e tecnologias.