In der molekularen Diagnostik besteht ein Bedarf an schnellen und spezifischen Testsystemen, die entweder für die Labordiagnostik oder in Point of Care-Umgebungen eingesetzt werden können. Um dieses Ziel zu erreichen, stehen die Miniaturisierung und Parallelisierung im Mittelpunkt des Forschungsinteresses. Die führende Methode im Bereich der DNA-Analytik ist derzeit die Realtime-PCR. Dieser Technologie sind hinsichtlich der Multiplexfähigkeit technologischen Hürden gesetzt, da derzeit nur eine Analyse von maximal vier Parametern parallel in einem Versuchsansatz erfolgen kann. Microarrays stellen hingegen die benötigten Voraussetzungen zur Verfügung, um als Werkzeuge für die Multiparameteranalyse in verschiedensten Anwendungsbereichen zu dienen. Ein Schwerpunkt dieser Arbeit war es, Multiplex-PCRs und diagnostische Microarrays zu entwickeln, die für analytische Fragestellungen eine schnelle und zuverlässige Multiparameteranalytik ermöglichen, um die bisherigen Einschränkungen aktueller Nachweisverfahren zu vermeiden. Als Anwendungen wurden zum einen ein Nachweissystem für acht relevante Geflügelpathogene zur Überwachung in der Geflügelzucht, zum anderen ein Nachweissystem zur Identifikation potentiell allergener Lebensmittelinhaltstoffe entwickelt. Neben der Entwicklung geeigneter PCR und Multiplex-PCR-Verfahren sowie spezifischer Microarrays für die Detektion der gesuchten Zielsequenzen stand auch die weiterführende Integration von DNA-Amplifikation und Microarray-Technologie im Fokus dieser Arbeit. Die OnChip-Amplifikation stellt eine Möglichkeit dar, um DNA-Analytik und Detektion in einem Reaktionsschritt zu integrieren. Entsprechend wurden die in der Arbeit entwickelten PCR- und Multiplex-PCR-Verfahren zum Nachweis potentieller allergener Lebensmittelinhaltsstoffe für die OnChip-Amplifikation adaptiert und Reaktionsbedingungen getestet, die eine Multiparameteranalyse auf dem Chip ermöglichen. Die entwickelten OnChip-PCR-Verfahren zeigten eine hohe Spezifität sowohl in Single- als auch in der Multiplex-OnChip-PCR. Eine Sensitivität von 10 Kopien bzw. <10ppm konnte in Single-OnChip-PCRs für den Nachweis allergener Lebensmittelinhaltsstoffe gezeigt werden. In Multiplex-OnChip-PCRs konnten 10-100ppm allergene Verunreinigungen spezifisch in unterschiedlichen Lebensmitteln nachgewiesen werden. Ein weiterer Schritt in Richtung einer möglichen Verwendung im Point of Care-Bereich stellt der Einsatz eines isothermalen Amplifikationsverfahrens dar. Vorteil eines solchen Verfahrens ist die Möglichkeit, auf das ansonsten benötigte Thermocycling zu verzichten. Dies vereinfacht eine Integration der OnChip-Amplifikation in mobile Analysegeräte oder Lab on Chip-Systeme und qualifiziert das Verfahren für den Einsatz in Point of Care-Umgebungen. In dieser Arbeit wurde eine noch junge isothermale Amplifikationsmethode, die helikase-abhängige Amplifikation (HDA), hinsichtlich ihrer Eignung für die Integration auf einem Microarray getestet. Hierfür konnte die bislang erste OnChip-HDA für Einzel- und Duplex-Nachweise von Pathogenen entwickelt werden. / In molecular diagnostics there is a need for fast and specific assay systems that could be used in the clinics and in point of care settings alike. Therefore miniaturisation and parallelisation are in the main focus of current assay development researches. The current gold standard for DNA analytics is the realtime PCR. However, this technology has its restraints in context to multiplex analysis. With the currently available technology an efficient multiplexing is only possible for four different targets per analysed sample. Microarrays in contrast offer the needed multiplex capabilities and have advanced to capable tools used in multiple fields of application. One focus of this work was the integration of Multiplex PCR and microarray technology, developing a microarray capable of analysing multiple parameters in one given sample, circumventing the problems and restraints of the exsisting technologies. As an example microarray assays for two different application fields were developed. One microarray assay for the detection of pathogens in poultry and another microarray assay for the detection of potentially allergenic food ingredients. Single- and Multiplex OnChip-PCR assays for both applications were developed and tested. OnChip-PCRs developed in this work showed high specificity in Single- and Multiplex-OnChip amplifications. The sensitivity was in the range of 10 DNA copies or 10ppm respectively for Single-OnChip-PCR in experiments for the detection of allergenic food contaminations. In Multiplex-OnChip-PCR experiments 100 DNA copies or 100ppm of food contaminents could be detected in different food matrices. A further focus of this work was the adaption of the OnChip amplification for the use in Point of Care settings. Isothermal amplification is a promising approach having the advantage of avoiding the thermocycling needed in the PCR. This opens up certain opportunities for the development of smaller, more flexible mobile diagnostic analysis devices. In this work we have evaluated the helicase dependent amplification (HDA) in terms of usability in OnChip amplification. In this work it was shown for the first time that HDA could be used for the detection of different pathogens in an Duplex-OnChip-PCR, showing the potential of this technology for integration in Point of Care settings.

Beta lactam resistance producing isolates of Enterobacteriacea and non-Enterobacteriacea have emerged since more than seventy years ago (Abraham and Chain, 1940). They are known to cause both community and hospital-acquired infections. Resistance against carbapenem is primarily mediated by the production of enzymes that destroy the beta lactam antimicrobials, which are produced by these isolates involving the expression of serine and metalobetalactamase genes KPC, IMP, VIM, NDM-1 and OXA-48. Quinolone resistance is predominanty mediated by mutations in the qnrA, qnrB, qnrS, and aac-6-Ib genes. Carbapenemase-producing organisms especially Klebsiella pneumoniae carbapenemases (KPCs) emerged as important pathogens especially among critically ill patients causing significant morbidity and mortality. This study aims to determine the prevalence and types of 15 quinolone resistance and carbapenemases genes among different isolates from patients admitted to the University Hospital of Leipzig over a period of ten months. During the period from January 2011 through October 2011, a total of 50 carbapenemases isolates were recovered from patients of the University Hospital of Leipzig/ Germany. The isolates were identified by biochemical tests and their susceptibility to antimicrobials was determined by the microbroth dilution method according to ISO standard. The KPC, IMP, VIM, OXA-48, NDM-1, and aac-6-Ib genes as well as qnrA, qnrB, and qnrS genes were detected by multiplex PCR, respectively. Results showed that KPC gene was detected in 82% of the isolates while 8% were KPC negative. The qnrA, qnrS, IMP, NDM-1, and OXA-48 genes were not detected in any of the isolates while qnrB and VIM genes were found in 2%. On the other hand, aac-6-Ib gene was the most prevalent gene among the study isolates and composed a percentage of 96%. Results also showed that KPC, and aac-6-Ib genes were detected in isolates collected from urine, blood, wounds, swabs, sputum, tracheal secretions, biopsies, and anal smears, while VIM gene was detected in one isolate collected from blood. The qnrB gene was found in one isolate collected from urine specimen. The wide spread of carbapenem and quinolone resistance-producing organisms is a critical problem that complicates the treatment of infections resulting from these organisms. Necessary measures must, therefore, be taken to limit their spread, which include appropriate antibiotic treatment, control of hospital infections, observe of personal hygiene, and the use of appropriate methods of sterilization and disinfection to prevent the dissemination of these organisms. Keywords: Resistance, carbapenemases, QRDR, multiplex PCR, antimicrobials

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Novas metodologias de análise molecular voltadas para estudos populacionais, clínicos, evolutivos, da biodiversidade e identificação forense foram desenvolvidas com base em marcadores microssátelites ou STR Short Tandem Repeats. Os marcadores STR, que estão amplamente espalhados nos genomas e se caracterizam por apresentar alto grau de polimorfismo, podem ser analisados a partir da amplificação por PCR (Reação em Cadeia da polimerase). A análise foi facilitada a partir do desenvolvimento de sistemas de amplificação simultânea de múltiplos STR (multiplex STR) e com a detecção automatizada dos produtos de amplificação marcados por fluorescência. Recentemente, o uso de marcadores STR do cromossomo X (X-STR) tornou-se significativo na prática forense. Devido ao seu modo de transmissão, os X-STR são úteis em situações particulares de investigação de relações de parentesco, apresentando vantagens sobre o uso de STR autossômicos. Este estudo teve como principal objetivo o desenvolvimento e validação de sistema multiplex, denominado LDD (X-STR) Decaplex, capaz de amplificar dez loci X-STR (DXS7133, DXS7424, DXS8378, DXS6807, DXS7132, DXS10074, DXS7423, DXS8377, GATA172D05 e DXS10101) para aplicação em genética populacional, identificação e análises forenses. Utilizando o LDD (X-STR) Decaplex 170 indivíduos autodenominados afrodescendentes, não aparentados geneticamente, foram genotipados. As freqüências alélicas e genotípicas não apresentaram desvio do equilíbrio de Hardy-Weinberg e estão em concordância com aquelas observadas em outros estudos. Os haplótipos observados foram únicos em indivíduos de amostra masculina. A análise de desequilíbrio de ligação não revelou associação entre os marcadores X-STR. A diversidade genética foi elevada, variando entre 0,6218 para o locus DXS7133 a 0,9327 para o locus DXS8377. Os parâmetros de Probabilidade de Vinculação (PV), Índice de Vinculação (IV), Poder de Exclusão (PE), Poder de Discriminação e Razão de Verossimilhança foram também elevados, demonstraram que os dez X-STRs são altamente polimórficos e discriminativos na população estudada. A concentração mínima de DNA para a amplificação dos loci do LDD (X-STR) Decaplex é de 0,5 ng e verificamos que amplificação por PCR pode ser afetada quando são adicionados mais de 5 ng de DNA nas reações. Os percentuais de bandas stutter foram elevados para os loci DXS7132 e DXS8377. No teste de reprodutibilidade observamos consistência entre as tipagem de diferentes amostras biológicas, incluindo as de restos mortais. No teste de mistura a proporção limite em que observamos a coexistência de duas espécies biológicas foi de 2,5:1ng (feminino-masculino). Os resultados evidenciaram que os loci do LDD (X-STR) Decaplex são altamente informativos, consistindo, em conjunto, uma ferramenta importante em estudos de identificação humana e de relações de parentesco. / New molecular analysis technologies used to evolving clinic and population studies of biodiversity and forensic identification have been developed based on microsatellite markers or STR Short Tandem Repeats. These STR markers, which are widely spread on genomes and characterized by their high degree of polymorphism, can be analyzed by PCR (Polymerase Chain Reaction) amplification technology. This analysis was facilitated from the development of simultaneous amplification system of multiples STR (multiplex STR) and automatic detection of amplification products by fluorescence. Lately, the use of STR markers of chromosome X (X-STR) has become significant in forensic practice. Due to their transmission ways, X-STR are useful at especial kinship investigation, showing advantages in relation to the use of STR autosomal. This study main focus was the development and validation of the multiplex system called LDD (X-STR) Decaplex, which was able to amplify ten X-STR loci (DXS7133, DXS7424, DXS8378, DXS6807, DXS7132, DXS10074, DXS7423, DXS8377, GATA172D05 and DXS10101) in order to be used in population genetics application, identification and forensic analysis. By using LDD (X-STR) Decaplex 170, individuals, who were self-appointed Africandescendants and genetically unrelated, were genotyped. Allele and genotype frequencies have not shown Hardy-Weinberg equilibrium deviation and are in agreement with the ones observed in other studies. The observed haplotypes were unique in male individual samples. The linkage disequilibrium analysis has not shown any association among the X-STR markers. The genetic diversity was high, ranging from 0.6218 (DXS7133 locus) to 0.9327 (DXS8377 locus). The parameters of Probability of Relatedness (PR), Avuncular Index (AI), Power of Exclusion (PE), Power of Discrimination and Likelihood Ratios were also high, showing that these ten X-STRs are highly polymorphous and discriminating on the studied population. The minimum DNA concentration for the amplification of the LDD (X-STR) Decaplex loci is 0.5 ng and it was also verified that the PCR amplification can be affected when more than 5 ng of DNA are added to the reactions. The "stutter" bands percentages were high to DXS7132 and DXS8377 loci. At the reproducing test, consistency among typing of different biological samples was observed, including the ones from remains. At the mixing test, the limit proportion in which two biological species coexisted was 2.5:1 ng (female-male). The results point out that LDD (X-STR) Decaplex loci are able to provide many important piece of information which, as a whole, are a very important tool at human identification and of kinship studies.

Made available in DSpace on 2014-06-11T19:27:22Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-10-26Bitstream added on 2014-06-13T18:56:02Z : No. of bitstreams: 1 guastalli_eal_me_jabo.pdf: 288811 bytes, checksum: 03b7643862d7f270422c422ff91b53b9 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Foram isoladas 90 estirpes de E. coli de fígados e de intestinos, de pintainhas de postura comercial, com sete dias de idade. Com o objetivo de caracterizar as estirpes isoladas, a patogenicidade das mesmas foi determinada, em inoculação “in vivo”. O teste revelou 44 estirpes de alta ou de intermediária patogenicidade, que foram analisadas por PCR multiplex quanto á presença de oito genes de virulência (astA, iss, iucD, irp2, papC, tsh, vat e cva/cvi) e tiveram os sorotipos O:H identificados. Os resultados demonstraram que todas as estirpes analisadas continham pelo menos um dos oito genes pesquisados e que a maioria (93,20%) possuíam o gene iss. Foram detectados 17 perfis genéticos diferentes, sendo 15 deles com combinações de dois ou mais genes, representando 70,45% do total de estirpes analisadas. Onze sorogrupos e onze antígenos “H” foram identificados, sendo O8 (15,89%) e o H17 (23,8%) os mais frequentes. Com o objetivo de verificar a susceptibilidade das estirpes aos antimicrobianos: ampicilina, enrofloxacina, eritromicina, espectinomicina, estreptomicina, fosfomicina, kanamicina, lincomicina, norfloxacina, sulfa+trimetoprim e tetraciclina, comumente utilizados na avicultura, todas as estirpes de E. coli isoladas foram analisadas. O antimicrobiano que apresentou maior atividade antibacteriana foi a espectinomicina (92,2%) e o de menor atividade foi a lincomicina. Nenhuma das estirpes foi sensível a todos os antimicrobianos testados. Os resultados demonstraram uma diversidade de sorotipos e de genes de virulência envolvidos no quadro clínico de colibacilose estudado, como também sorogrupos que não haviam sido relatados em APEC e a alta incidência de resistência antimicrobiana / A total of 90 strains of E. coli were isolated from the livers and intestines of seven-day-old commercial layer chicks. With the objective of characterising the isolated strains, their pathogenicity levels were determined by in vivo inoculation. These tests identified 44 strains with high or intermediate levels of pathogenicity, which were then analysed by multiplex PCR for the presence of eight virulence genes (astA, iss, iucD, irp2, papC, tsh, vat e cvi/cva) and the serotypes O:H were identified. The results demonstrated that these isolated strains contained at least one of the eight genes of interest, and the majority (93.20%) possessed the iss gene. Seventeen different genetic patterns were detected, 15 of which had combinations of two or more genes, representing 70.45% of all analysed strains. Eleven serogroups and eleven antigens “H” were identified, O8 (15.89%) and H17 (23.80%) were most frequent. Aiming to verify the susceptibility of strains to antimicrobial agents: ampicillin, enrofloxacin, erythromycin, spectinomycin, streptomycin, fosfomycin, kanamycin, lincomycin, norfloxacin, trimethoprim sulfa and tetracycline, commonly used in poultry, all strains of E. coli isolates were analyzed. The antibiotics that showed the highest antibacterial activity was spectinomycin (92.2%) and less activity was the lincomycin (100%) strains were resistant. None of the strains were sensitive to all antibiotics tested. The results showed a diversity of serotypes and virulence genes involved in the clinical study of colibacillosis, as well as serogroups that had not been reported in APEC and the high incidence of antimicrobial resistance

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Novas metodologias de análise molecular voltadas para estudos populacionais, clínicos, evolutivos, da biodiversidade e identificação forense foram desenvolvidas com base em marcadores microssátelites ou STR Short Tandem Repeats. Os marcadores STR, que estão amplamente espalhados nos genomas e se caracterizam por apresentar alto grau de polimorfismo, podem ser analisados a partir da amplificação por PCR (Reação em Cadeia da polimerase). A análise foi facilitada a partir do desenvolvimento de sistemas de amplificação simultânea de múltiplos STR (multiplex STR) e com a detecção automatizada dos produtos de amplificação marcados por fluorescência. Recentemente, o uso de marcadores STR do cromossomo X (X-STR) tornou-se significativo na prática forense. Devido ao seu modo de transmissão, os X-STR são úteis em situações particulares de investigação de relações de parentesco, apresentando vantagens sobre o uso de STR autossômicos. Este estudo teve como principal objetivo o desenvolvimento e validação de sistema multiplex, denominado LDD (X-STR) Decaplex, capaz de amplificar dez loci X-STR (DXS7133, DXS7424, DXS8378, DXS6807, DXS7132, DXS10074, DXS7423, DXS8377, GATA172D05 e DXS10101) para aplicação em genética populacional, identificação e análises forenses. Utilizando o LDD (X-STR) Decaplex 170 indivíduos autodenominados afrodescendentes, não aparentados geneticamente, foram genotipados. As freqüências alélicas e genotípicas não apresentaram desvio do equilíbrio de Hardy-Weinberg e estão em concordância com aquelas observadas em outros estudos. Os haplótipos observados foram únicos em indivíduos de amostra masculina. A análise de desequilíbrio de ligação não revelou associação entre os marcadores X-STR. A diversidade genética foi elevada, variando entre 0,6218 para o locus DXS7133 a 0,9327 para o locus DXS8377. Os parâmetros de Probabilidade de Vinculação (PV), Índice de Vinculação (IV), Poder de Exclusão (PE), Poder de Discriminação e Razão de Verossimilhança foram também elevados, demonstraram que os dez X-STRs são altamente polimórficos e discriminativos na população estudada. A concentração mínima de DNA para a amplificação dos loci do LDD (X-STR) Decaplex é de 0,5 ng e verificamos que amplificação por PCR pode ser afetada quando são adicionados mais de 5 ng de DNA nas reações. Os percentuais de bandas stutter foram elevados para os loci DXS7132 e DXS8377. No teste de reprodutibilidade observamos consistência entre as tipagem de diferentes amostras biológicas, incluindo as de restos mortais. No teste de mistura a proporção limite em que observamos a coexistência de duas espécies biológicas foi de 2,5:1ng (feminino-masculino). Os resultados evidenciaram que os loci do LDD (X-STR) Decaplex são altamente informativos, consistindo, em conjunto, uma ferramenta importante em estudos de identificação humana e de relações de parentesco. / New molecular analysis technologies used to evolving clinic and population studies of biodiversity and forensic identification have been developed based on microsatellite markers or STR Short Tandem Repeats. These STR markers, which are widely spread on genomes and characterized by their high degree of polymorphism, can be analyzed by PCR (Polymerase Chain Reaction) amplification technology. This analysis was facilitated from the development of simultaneous amplification system of multiples STR (multiplex STR) and automatic detection of amplification products by fluorescence. Lately, the use of STR markers of chromosome X (X-STR) has become significant in forensic practice. Due to their transmission ways, X-STR are useful at especial kinship investigation, showing advantages in relation to the use of STR autosomal. This study main focus was the development and validation of the multiplex system called LDD (X-STR) Decaplex, which was able to amplify ten X-STR loci (DXS7133, DXS7424, DXS8378, DXS6807, DXS7132, DXS10074, DXS7423, DXS8377, GATA172D05 and DXS10101) in order to be used in population genetics application, identification and forensic analysis. By using LDD (X-STR) Decaplex 170, individuals, who were self-appointed Africandescendants and genetically unrelated, were genotyped. Allele and genotype frequencies have not shown Hardy-Weinberg equilibrium deviation and are in agreement with the ones observed in other studies. The observed haplotypes were unique in male individual samples. The linkage disequilibrium analysis has not shown any association among the X-STR markers. The genetic diversity was high, ranging from 0.6218 (DXS7133 locus) to 0.9327 (DXS8377 locus). The parameters of Probability of Relatedness (PR), Avuncular Index (AI), Power of Exclusion (PE), Power of Discrimination and Likelihood Ratios were also high, showing that these ten X-STRs are highly polymorphous and discriminating on the studied population. The minimum DNA concentration for the amplification of the LDD (X-STR) Decaplex loci is 0.5 ng and it was also verified that the PCR amplification can be affected when more than 5 ng of DNA are added to the reactions. The "stutter" bands percentages were high to DXS7132 and DXS8377 loci. At the reproducing test, consistency among typing of different biological samples was observed, including the ones from remains. At the mixing test, the limit proportion in which two biological species coexisted was 2.5:1 ng (female-male). The results point out that LDD (X-STR) Decaplex loci are able to provide many important piece of information which, as a whole, are a very important tool at human identification and of kinship studies.

S. pneumoniae coloniza a nasofaringe e é um dos principais agente de otite média, pneumonia, bacteremia e meningite com altas taxas de morbidade e mortalidade. Estima-se que 1,6 milhões de pessoas morram de doença pneumocócica por ano, a maioria crianças menores de cinco anos de idade, principalmente em países em desenvolvimento. A cápsula polissarídica antifagocitária é o principal fator de virulência deste microrganismo e determina os 93 sorotipos conhecidos, sendo o alvo de vacinas pneumocócicas. No presente trabalho foi padronizada a tipagem molecular por Multiplex PCR de S. pneumoniae, que compreende 30 pares de iniciadores agrupados em seis reações sequenciais. Foram tipadas 270 cepas de pneumococo isoladas entre janeiro de 2005 a setembro de 2011, proveniente de líquor (13%), sangue (76%) e líquido pleural (11%) de 232 pacientes atendidos no Hospital Universitário da USP. Além disso, a caraterização dessas amostras quanto ao perfil de sensibilidade aos antimicrobianos e à diversidade foi realizada, segundo o CLSI 2011 e a genotipagem molecular pelas técnicas de Multilocus Sequencie Typing Scheme (MLST) e Pulsed Field Eletrophoresis Gel (PFGE), respectivamente. A tipagem por Multiplex PCR detectou 24 sorotipos/sorogrupos diferentes, que foram: 14 (22%), 5 (12%), 12F/A (11%), 6A/B/C (10%), 7F/A (5%), 1 (4%), 3 (4%), 10A (4%), 19A (4%), 18 A/B/C/F (3%), 4 (3%), 8 (3%), 23F (3%), 19F (3%) e outros (9%) (9V/A, 9N/L, 15A, 22F, 11A/D, 31, 38, 34, 16F, 17F e não tipável). Este método apresentou 100% de especificidade e 98% de sensibilidade para determinação de sorogrupos e 66% para sorotipos. Os sorotipos 14, 6B, 5 e 19F foram significativamente mais comuns em criança até dois anos, já entre adultos, os sorotipos 5 e 12F foram os predominantes. O perfil de sensibilidade em infecções não meníngeas foi de 99% de sensibilidade e 1% de resistência intermediária para penicilina e ceftriaxona. Para infecções meníngeas os resultados mostraram 73% de sensibilidade e 27% de resistência para penicilina e 88% de sensibilidade e 12% de resistência intermediária para ceftriaxona. A resistência aos beta-lactâmicos está ligada principalmente ao sorotipo 14 que foi o sorotipo mais isolado com 52 cepas e dessas foram realizados MLST e PFGE. No MLST encontramos 51 cepas pertencentes ao clone Spain9V-3 (ST 156) que é predominante na região sul e sudeste do Brasil e uma cepa com um tipo de sequência ainda não depositada. Pela técnica de PFGE foram detectados três clusters e quatro amostras não relacionadas, o cluster A foi predominante com 41(79%) cepas com 81,7% de similaridade entre elas. A técnica de Multiplex PCR demonstrou ser excelente ferramenta para a detecção dos sorotipos/sorogrupos de S. pneumoniae. Não foi detectada resistência plena à penicilina e ceftriaxona em infecções não meníngeas consolidando a importância do uso da penicilina no tratamento da doença pneumocócica não meníngea. Houve grande similaridade genética entre cepas de S. pneumoniae sorotipo 14. / S.pneumoniae colonizes the nasopharynx and is a major agent of otitis media, pneumonia, bacteremia and meningitis with high morbidity and mortality. It is estimated that 1.6 million people die of pneumococcal disease every year, mostly children under five years old, mainly in developing countries. The antiphagocytic polissarídica capsule is the main virulence factor of this organism and determine the 93 serotypes known for being the target of pneumococcal vaccines. In the present study was standardized molecular typing by Multiplex PCR molecular typing, which comprises 30 primer pairs grouped into six sequential reactions. We performed antimicrobial susceptibility profile, according to the CLSI 2011 and the most frequent serotype was made by molecular genotyping techniques Multilocus Sequence Typing (MLST) and pulsed-field gel Eletrophoresis (PFGE). We studied 270 pneumococcal strains isolated from 2005 to September 2011, from CSF (13%), blood (76%) and pleural fluid (11%) of 232 patients attended at University Hospital of USP. Typing by Multiplex PCR detected 24 serotypes / serogroups different, which were: 14 (22%), 5 (12%), 12F / A (11%), 6A/B/C (10%), 7F / A (5 %), 1 (4%), 3 (4%), 10A (4%), 19A (4%), 18 A / B / C / F (3%), 4 (3%), 8 (3% ), 23F (3%), 19F (3%) and others (9%) (9V / A, 9N / L, 15A, 22F, 11A / D, 31, 38, 34, 16F, 17F and nontypable). This method showed 100% specificity and 98% sensitivity for the determination of 66% for serogroups and serotypes. Serotypes significantly more common in children under two years were: 14, 6B, 5 and 19F among adults serotypes 5 e12F were predominant. The sensitivity profile in non-meningeal infections was 99% sensitivity and 1% penicillin intermediate resistance to ceftriaxone. For meningeal infections the results showed 73% sensitivity and 27% resistance to penicillin and 88% sensitivity and 12% intermediate resistance to ceftriaxone. Resistance to beta-lactams is linked mainly to serotype 14 was the serotype most isolated, and of these 52 strains were performed MLST and PFGE. MLST found in 51 strains belonging to clone Spain9V-3 (ST 156) which is prevalent in south and southeastern Brazil and a strain with a type of sequence is not deposited. The technique of PFGE found three clusters and four non-related samples, cluster A predominated with 41 (79%) strains with 81.7% similarity between them. Multiplex PCR technique proved to be an excellent tool for the detection of serotypes/serogroups of S. pneumoniae. We did not detect full resistance to penicillin and ceftriaxone in non-meningeal infections showing the importance of use of penicillin in the treatment of pneumococcal non-meningeal disease. There was great genetic similarity among strains of S. pneumoniae serotype 14.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2015-12-14T08:11:41Z No. of bitstreams: 1 Dissertação - ítalo de Araújo Castro - 2015.pdf: 2281966 bytes, checksum: fa56d97881ce8b52bd310d3446c61552 (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2015-12-14T08:16:03Z (GMT) No. of bitstreams: 1 Dissertação - ítalo de Araújo Castro - 2015.pdf: 2281966 bytes, checksum: fa56d97881ce8b52bd310d3446c61552 (MD5) / Made available in DSpace on 2015-12-14T08:16:03Z (GMT). No. of bitstreams: 1 Dissertação - ítalo de Araújo Castro - 2015.pdf: 2281966 bytes, checksum: fa56d97881ce8b52bd310d3446c61552 (MD5) Previous issue date: 2015-04-23 / Fundação de Amparo à Pesquisa do Estado de Goiás - FAPEG / Acute respiratory infection (ARI) is a major cause of morbity and mortality worldwide, particularly among children, and most of these infections are caused by viruses. Respiratory viral infections can cause symptoms ranging cough, coryza, sneezing, fever and airflow obstruction. Furthermore, the infection poses as an important trigger of asthma exacerbation, frequent clinical condition in children, and its prevalence has been rising in the last years. There are few epidemiologic studies analyzing the relationship between ARIs and asthma in Brazil. Based on this background, the aim of the study was investigate the occurrence of viral respiratory infections in pediatric patients with and without asthma in Goiânia – Goiás. Between august, 2012 and august, 2013 225 nasal aspirates and/or nasal swab samples were obtained from children with four to 14 years old. The samples were screened by Multiplex Nested-PCR for detection of 16 common respiratory viruses. From 225 samples, 42 had at least one virus detected. Samples from four different patients had more than one virus detected. The viral detection rate in ARI patients (25%), exacerbated asthma (16.3%) and stable asthma (14.8%) showed no significant difference. The most frequent viruses detected were Rhinovirus (28.6%), FLUA (11.9%), Adenovirus (11.9%), HBoV (11.9%) and RSVA (9.5%). The monthly detection rate was higher during the rainy season, period marked by great rainfall and high relative air humidity. Among the positive samples, RSV were detected during the great rainfall months and high air humidity, while the FLU and HBoV were detected during the winter months, period with low air humidity in the Mid-West region. The seasonal profile from the other viruses was unclear. The obtained results reinforces the importance of the viral pathogens in the pediatric population. Although the viral detection rate was not statistically significant, the presence of these pathogens in children is an important matter for consideration, especially to delineate control and prevention measures concerning ARIs and its impact on preexistent asthma. Hence, this study is the first of the kind in the region, and the data provided tried to fill the knowledge gaps about seasonality and circulation of these pathogens. / Infecções agudas do trato respiratório (ITR) representam importante causa de morbidade e mortalidade entre crianças e os vírus constituem os principais agentes etiológicos responsáveis por essas infecções. Além da capacidade de causar doenças com sintomas comuns desde a tosse, coriza, espirros, febre e obstrução nasal, estão frequentemente associados à exacerbação de quadros de asma, condição clínica crônica mais frequente em crianças cuja prevalência vem aumentando nos últimos anos. Considerando a população de asmáticos, os dados epidemiológicos relacionando infecções respiratórias virais e asma no Brasil são escassos. Neste contexto, o presente estudo objetivou investigar a ocorrência de infecções por vírus respiratórios em população pediátrica asmática e não asmática da cidade de Goiânia – Goiás. Para isso, entre agosto de 2012 e agosto de 2013 foram coletadas 225 amostras de aspirado nasofaríngeo e/ou swab nasal de crianças entre quatro e 14 anos de idade, as quais foram submetidas à triagem molecular para detecção de 16 vírus respiratórios, por meio de três protocolos de Multiplex Nested-PCR. Das 225 amostras, 42 apresentaram positividade para pelo menos um vírus. Amostras de quatro pacientes apresentaram mais de um vírus detectado. Os índices de detecção viral encontrados para pacientes apresentando ITR (25%), asma exacerbada (16,3%) e asma estável (14,8%) não apresentaram diferenças significativas. Os vírus mais frequentemente detectados foram os Rinovírus (28,6%), Influenza A (FLUA) (11,9%), Adenovírus (11,9%), Bocavírus Humanos (HBoV) (11,9%) e Vírus Sincicial Respiratório A (RSV) (9,5%). Maiores índices de detecção viral foram observados durante a estação chuvosa da região, período de maior precipitação pluviométrica e umidade relativa do ar (UR). Dentre as amostras positivas, os RSV foram detectados nos meses de maior precipitação pluviométrica e UR, enquanto os FLU e HBoV foram detectados durante os meses de inverno coincidindo com a estação seca. Os demais vírus não apresentaram perfil sazonal definido. Os dados obtidos ressaltam a importância dos agentes virais na população pediátrica. Embora o índice de detecção viral não tenha sido significativo entre os pacientes apresentando asma exacerbada e asma estável, a presença desses agentes infecciosos em crianças constitui um fator a ser considerado ao traçar-se estratégias de prevenção e controle dos quadros de ITR, e seu impacto em quadros preexistentes de asma. Além disso, os resultados agregam conhecimento sobre a circulação e sazonalidade desses patógenos, uma vez que este é o primeiro estudo visando a detecção molecular de vírus respiratórios em crianças na região Centro-Oeste.

The thesis focuses on the optical access networks and deals with the issue of transmission parameters of the physical layer, their negative impact on the quality of the services parameters and the possibilities of reducing their effect using the polarization multiplexing. The first and second chapter describe the theoretical background necessary for understanding the subsequent parts. The third chapter contains the practical part of the thesis. At first the optical routes in the Czech Republic in terms of polarization mode dispersion, which is together with insertion loss the main limiting parameter of high-speed optical networks, are evaluated. In the following part the measuring of the temperature changes effects on the polarization mode dispersion of the passive components and the effect of temperature changes to the polarization multiplexed signal are evaluated. To double the bandwidth and reduce the influence of polarization mode dispersion on the quality of services, the two simulation models of optical access networks using polarization multiplexing were carried out. These theoretical outputs were verified by the practical measurements in the laboratory and subsequently on the real optical route. Finally, a draft of the broadband optical access network based on wavelength and polarization multiplexing was designed based on obtained knowledge.

Conventional methods of body fluid identification use labor-intensive, technologically diverse techniques that are performed in a series, not parallel, manner and are costly in terms of time and sample. Furthermore, for some frequently encountered body fluids, such as saliva or vaginal secretions, no confirmatory technique exists. Terminally differentiated cells, such as blood lymphocytes or epithelial cells lining the oral cavity, have a unique pattern of gene expression, which is evinced by the presence and relative abundance of specific mRNA species. If the type and abundance of mRNAs can be determined in a stain or tissue sample recovered at the crime scene, it would be possible to definitively identify the tissue or body fluid in question. Advantages of an mRNA-based approach, compared to conventional biochemical analysis, include greater specificity, simultaneous and semi-automated analysis though a common assay format, improved timeliness, decreased sample consumption and compatibility with DNA extraction methodologies. In this report, we demonstrate that RNA is stable in biological stains and can be recovered in sufficient quantity and quality for analysis using reverse transcriptasepolymerase chain reaction assay (RT-PCR). We have identified sets of candidate tissuespecific genes for body fluids and tissues of forensic interest, namely blood, saliva, semen, vaginal secretions, menstrual blood, urine, skin, muscle, adipose, and brain. We also report the identification of a new housekeeping gene for use in mRNA based assays. Select body fluid-specific genes have been incorporated into multiplex PCR and real-time PCR assays. These assays allow for the positive identification of blood, saliva, semen,vaginal secretions, and/or menstrual blood in a stain. The final task of this work was the molecular characterization of mRNA degradation patterns in biological stains, which not only has fundamental importance in possibly revealing mRNA degradation pathways in dried biological stains, but may ultimately lead to better assay design strategies for mRNA markers for forensic use. An mRNA-based approach described in this report could allow the facile identification of the tissue components present in a body fluid stain and could conceivably supplant the battery of serological and biochemical tests currently employed in the forensic serology laboratory.

Complex network theory has been well established as one of the main tools for understanding and analyzing the behavior of the natural systems that surround us. Social networks, genetic and protein interaction networks, airline and road traffic networks, brain connectivity networks and web graphs are only some of the examples. As network theory evolves it becomes more apparent that these complex systems are often composed of multiple types of interactions, each carrying a different piece of information, and therefore are commonly represented in the form of multiplex networks, where each layer represents a different type of interaction among nodes. In addition to the interactions among the nodes of the networks, these systems also present correlations among the various types of interactions, as represented by the intrinsic structure and the associations of the various layers of the graph. For example, in social sciences, a network with a large overlap between two layers that represent two distinct types of people interactions i.e. friendship and professional ties might indicate that there is an interconnection between the two in the given network. In another example, in transportation networks, where nodes represent airports connected by flights operated by specific airlines (each airline representing a layer of the graph), the structure of the layers can provide information about the airline: traditional airlines such as Lufthansa tend to have a large overlap in activity pattern with other airlines, whereas low-cost airlines such as easyJet tend to avoid such overlaps. Due to their ability to represent such complex entity interactions, multiplex networks have lately been the focus of a large part of the research community, studying a variety of aspects, such as structural measures, node communities detection, layer reducibility, network generative models, and information spreading. In this work we focus on techniques for the exploration of the intrinsic structure of multiplex networks, and contemplate ways of addressing common challenges of learning from multiplex networks. In particular, our work focuses on three main directions: structured regression, graph summarization and graph similarity. We analyze and discuss the main challenges of each of these research directions, and then we propose novel methods to address them. For each problem, we utilize artificial data to study their effectiveness, understand their intrinsic properties and evaluate their behavior under a controlled network structure. Then, we report applications on real-world data sets, from variety of domains, and compare our proposed methods with state-of-the-art and well established baseline methods. Through this work, we aim to offer proof that the networks' intrinsic structure, when utilized, can increase the informative power of network theory models and allow researchers to build more educated algorithms. / Computer and Information Science