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Détection et quantification d’acides nucléiques par l’intercalation de sondes électro-actives appliquée à l’intégration d’approches d’amplifications isothermes in vitro. Détecter et quantifier la présence d’agents pathogènes à travers leur ADN représente un enjeu majeur dans de nombreux secteurs comme l’analyse biomédicale, l’agroalimentaire ou l’environnement. La technique de PCR optique en temps réel, couplant la réaction d’amplification génique in vitro dite PCR à une détection par fluorescence constitue la méthode standard pour réaliser ces analyses. Cependant, elle nécessite l’utilisation d’un thermocycleur et l’intégration d’une instrumentation optique rendant l’équipement onéreux et encombrant. De plus, les échantillons troubles ou colorés ne peuvent pas être traités. Pour remédier aux problèmes posés par l’optique, une méthode de suivi électrochimique de la PCR au moyen de sondes électroactives interagissant préférentiellement avec l’ADN double-brin a été développée au LEM. Un démonstrateur préindustriel réalisé en partenariat avec la start-up Easy Life Science permet la parallélisation des mesures de PCR électrochimique en temps réel dans les 48 cellules d’un consommable. Dans ce travail, la problématique de la régulation thermique est adressée en remplaçant la PCR par deux méthodes d’amplifications isothermes de l’ADN. La première d’entre elles, la LAMP (Loop Mediated Isothermal Amplification), particulièrement efficace et spécifique, est combinée à une détection électrochimique via l’utilisation d’un panel de sondes redox afin d’obtenir des performances analytiques rivalisant avec les méthodes optiques standards. Une seconde approche d’amplification isotherme présentant un schéma réactionnel plus simple que la LAMP, basée sur le recyclage des produits d’une HCR (Hybridization Chain Reaction), est également proposée. Enfin, un nouveau consommable miniaturisé intégrant, à ce jour, cent cellules électrochimiques de faible volume (≤ μL) ainsi que le potentiostat dédié sont présentés. L’originalité de ce consommable repose sur son processus de fabrication à très bas coût n’utilisant que des empilements de supports sérigraphiés. Fonctionnel à température ambiante, l’objectif à terme sera de l’utiliser pour des réactions d’amplifications isothermes dites « digitalisées » / Nucleic acids detection and quantification via electro-active probes intercalation applied to the integration of in-vitro isothermal amplification reactions. Detection and quantification of pathogenic agents through their DNA has become increasingly important in applications such as molecular diagnostics and food safety control to environmental monitoring. Routinely, these analyses are performed using optical quantitative PCR method by coupling the in vitro DNA amplification reaction PCR with fluorescent detection. However, it requires the use of a thermocycler as well as the integration of optical instrumentation leading to a bulky and expensive apparatus, unable to process turbid or colored samples. In order to circumvent these limitations, an alternative electrochemical monitoring method for PCR, based on the use of electro-active probes preferentially binding to double stranded DNA, has been proposed and developed at the LEM. A pre-industrial demonstrator developed in partnership with the start-up Easy Life Science allows the parallelization of real time electrochemical PCR measurements in a custom-made 48 wells plate. This work deals with the thermic regulation problem by replacing PCR by two alternatives isothermal DNA amplification methods. First, the LAMP (Loop Mediated Isothermal Amplification), known to be highly efficient and specific, is combined to an electrochemical detection through the use of a panel of redox probes in order to obtain analytical performances competing with the standard optical techniques. Secondly an isothermal amplification method with a simpler mechanism than LAMP, based on the recycling of the products of an HCR (Hybridization Chain Reaction), is also proposed. Finally, a new miniaturized plate integrating, to date, one hundred low volume (≤ μL) electrochemical cells as well as the corresponding potentiostat are presented. The novelty of this plate relies on its very low cost fabrication process based on the simple assembly of screen-printed films. Although the results presented were at room temperature, in the long term, it is envisioned to monitor isothermal amplification reactions in a “digital” manner

Conventionally, metal halide lamps were struck by voltages higher than those required for breaking down the electrodes to ensure successful ignition. These high ignition voltages may hurt the electrodes to some extent, leading to a shorter lamp lifecycle. In practice, the breakdown voltage can be affected by the dark current which occurs when a voltage is applied on lamp before the electrodes have been broken down. A lamp model to account for the dark current is derived from the test results. Three ignition schemes with single-pulse, multiple pulses and step voltage are used for describing the effect of the dark current on the breakdown voltage. Experimental results exhibit that the breakdown voltage can be lowered by applying a higher dark current or allotting more times of dark current to the lamp. The investigation provides useful information for the design of the ignition circuit.

Submitted by Luiz Felipe Barbosa (luiz.fbabreu2@ufpe.br) on 2015-04-17T13:58:12Z No. of bitstreams: 2 Tese Ana Paula da Costa.pdf: 11703201 bytes, checksum: 21beb28c4cce405340195e0c8ba78a09 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) / Made available in DSpace on 2015-04-17T13:58:12Z (GMT). No. of bitstreams: 2 Tese Ana Paula da Costa.pdf: 11703201 bytes, checksum: 21beb28c4cce405340195e0c8ba78a09 (MD5) license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) Previous issue date: 2013-02-28 / FACEPE / Listeria monocytogenes é um bacilo gram-positivo, intracelular facultativo, transmitido por alimentos, que pode causar doença, a listeriose, em indivíduos susceptíveis. Embora rara, a listeriose tem grande importância para a saúde pública pela sua alta letalidade. Este trabalho teve como objetivo, identificar marcadores moleculares para utilização no desenvolvimento de nova técnica de diagnóstico molecular da listeriose. Os estudos envolveram sorotipagem, tipagem molecular pela amplificação das sequências 23S, 16S-23S e RAPD, pesquisa de plasmídios e de genes de virulência (inlA, inlB, inlC, inlJ, hly, iap, plcA, actA) em 135 cepas de L. monocytogenes de casos humanos e alimentos isoladas no Brasil no período de 1975 a 2001; infecção experimental em camundongos com cepas portando ausência ou alterações em genes de virulência; e a caracterização de isolados de um caso de endocardite atendido numa unidade de emergência cardiológica em Recife, PE, em 2010. Nenhuma relação foi encontrada entre a origem das cepas, sorotipos, perfis genéticos, conteúdo plasmidial e distribuição dos genes de virulência. Os ensaios de infecção experimental em camundongos também não permitiram estabelecer uma relação entre a presença dos marcadores de virulência considerados no estudo e as respostas dos animais inoculados. Seis isolados de hemocultura de um caso de endocardite com identificação presuntiva de Listeria spp. foram classificados como L. monocytogenes sorotipo 4b pela amplificação por PCR dos genes 23S, lmo2243 e ORF2210 específicos do gênero Listeria, espécie L. monocytogenes e sorotipo b respectivamente. Todos os genes de virulência pesquisados foram detectados por PCR nas amostras e a análise do perfil de amplificação da sequência 16S-23S revelou que os seis isolados são uma mesma cepa. Embora esses estudos não tenham revelado nenhum marcador molecular de patogenicidade, a alta frequência de genes de virulência observada entre cepas dos sorotipos mais patogênicos (1/2a, 1/2b, 4b) evidencia o potencial patogênico nas cepas brasileiras de L. monocytogenes e a necessidade de incrementar a vigilância e diagnóstico dessa bactéria. Considerando a prevalência dos sorotipos 1/2a, 4b nas amostras clinicas e dos sorotipos 1/2a, 1/2b, 4b em alimentos, desenvolvemos e padronizamos um procedimento baseado em LAMP (loop-mediated isothermal amplification) para identificação desses sorotipos. A técnica é rápida e de fácil operacionalização, utiliza um bloco de aquecimento ou banho-maria e o produto da reação pode ser visualizado a olho nu mediante adição de reagentes fluorescentes. O procedimento padronizado mostrou-se sensível, capaz de detectar 100pg de DNA ou 104 UFC, e espécie - específica, capaz de diferenciar L. monocytogenes de espécies intimamente relacionadas geneticamente. Outros sorotipos intimamente relacionados (3a, 3b, 4e e 4d) foram amplificados sem detrimento da técnica porque são raramente encontrados em amostras humanas e animais. A técnica LAMP se apresenta como uma alternativa fácil e rápida para diagnóstico e tipagem de L. monocytogenes especialmente para os programas de vigilância e investigação epidemiológica.

Made available in DSpace on 2014-06-12T18:07:54Z (GMT). No. of bitstreams: 2 arquivo894_1.pdf: 10228754 bytes, checksum: eeb46b108153af421d159eebe9f86d6a (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2010 / Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco / A vacinação com o vírus atenuado 17D/17DD é o principal método de prevenção da Febre Amarela. Apesar do sucesso desta vacina em todo o mundo, reações adversas, aumento da severidade dos sintomas e até casos fatais têm sido reportados, o que estimula o desenvolvimento de uma vacina de DNA codificando seqüências específicas do vírus da Febre Amarela (VFA). Entretanto antígenos codificados por vacinas de DNA são expressos intracelularmente e preferencialmente apresentados ao sistema imune através de moléculas do Complexo Maior de Histocompatibilidade de classe I (MHCI). O aumento da eficiência destas vacinas é possível através da fusão de seus antígenos com a Proteína de Associação à Membrana Lisossomal humana (hLAMP-1), direcionando-os para o compartimento do Complexo Maior de Histocompatibilidade de classe II (MHCII). A via do MHCII é responsável pela ativação de linfócitos T CD4+, importantes para sustentar a resposta celular de linfócitos T CD8+ e para o desenvolvimento de memória, mudança de classe de anticorpos e expansão clonal de linfócitos B antígeno-específicos. As quimeras antígeno/LAMP apresentam uma maior indução da resposta imune quando comparadas aos antígenos nãofusionados à LAMP. Neste trabalho, apresentamos a análise da expressão e localização intracelular das proteínas não-estruturais NS1 e NS3 do VFA, nas suas formas fusionadas e não-fusionadas à LAMP. Para tanto, as seqüências de DNA das proteínas NS1 e NS3 foram selecionadas no banco de dados do NCBI (National Center for Biotechnology Information) e otimizadas através do algoritmo genético do programa LETO 1.0 (Entelechon®), de acordo com parâmetros como codon usage, estrutura secundária do mRNA, distribuição do conteúdo GC, motivos repetitivos de DNA, sítios crípticos de splicing, dentre outros, com o intuito de aumentar a expressão antigênica. As seqüências de DNA otimizadas foram enviadas para síntese comercial (Geneart®) e clonadas em vetores de expressão eucarióticos, nas formas fusionadas e não-fusionadas à LAMP. As construções vacinais obtidas foram enão utilizadas na transfecção de células eucarióticas cujos extratos foram analisados quanto à expressão protéica através de ensaios de Western-blot e imunofluorescência, utilizando anticorpos policlonais específicos produzidos através da imunização de coelhos com proteínas NS1 e NS3 recombinantes. Nestes ensaios, todas as construções vacinais apresentaram expressão eficiente e distribuição intracelular adequada. Enquanto as proteínas nativas apresentaram a distribuição reticular característica, os antígenos fusionados à LAMP apresentaram uma distribuição lisossomal típica do LAMP endógeno. As respostas imunes geradas contra as construções vacinais de NS1 foram avaliadas em camundongos BALB/c. Ambas as construções, fusionada e não-fusionada à LAMP, foram capazes de induzir uma forte resposta celular contra os mesmos epítopos induzidos pela vacina convencional 17DD. A resposta gerada pela construção fusionada a LAMP, entretanto, apresentou a melhor performance. Os resultados obtidos neste trabalho serão integrados a dados previamente obtidos em nosso laboratório em estudos com proteínas estruturais para o desenvolvimento de vacinas de DNA capazes de neutralizar infecções pelo VFA

DNA polymerases play a fundamental role in the transmission and maintenance of genetic information and have become an important in vitro diagnostic and analytical tool. The Loop-mediated isothermal DNA amplification (LAMP) method has major applications for disease and pathogen detection and utilises the unique strand-displacement activity of a small group of thermostable DNA polymerases. The Large (Klenow-like) Fragment of Geobacillus stearothermophilus DNA polymerase I (B.st LF Pol I) currently serves as the enzyme of choice for the majority of these isothermal reactions, with few alternatives commercially available. An increasing need for point-of-care nucleic acid diagnostics is now shifting detection methods away from traditional laboratory based chemistries, such as the polymerase chain reaction (PCR), in favour of faster, and often simpler, isothermal methods. It was recognised that in order to facilitate these rapid isothermal reactions there was a requirement for alternative thermostable, strand-displacing DNA polymerases and this was the basis of this thesis. This thesis reports the successful identification of polymerases from Family A, chosen for their inherent strand-displacement activity, which is essential for the removal of RNA primers of Okazaki fragments during lagging-strand DNA synthesis in vivo. Twelve thermophilic organisms, with growth temperature ranges between 50oC and 80oC, were identified and the genomic DNA extracted. Where DNA sequences were unavailable, a gene-walking technique revealed the polA sequences, enabling the Large Fragment Pol I to be cloned and the recombinant protein over-expressed in Escherichia coli. A three-stage column chromatography purification permitted the characterisation of ten newly identified Pol I enzymes suitable for use in LAMP. Thermodesulfatator indicus (T.in) Pol I proved to be the most interesting enzyme isolated. Demonstrating strong strand-displacement activity and thermostability to 98oC, T.in Pol I is uniquely suitable to a newly termed heat-denaturing LAMP (HD-LAMP) reaction offering many potential advantages over the existing LAMP protocol. The current understanding of strand-displacement activity of Pol I is poorly understood. This thesis recognised the need to identify the exact regions and motifs responsible for this activity of the enzyme, enabling potential enhancements to be made. Enzyme engineering using site-directed mutagenesis and the formation of chimeras confirmed the importance of specific subdomains in strand-separation activity. With this knowledge, a unique Thermus aquaticus (T.aq) Pol I mutant demonstrated sufficient strand-displacement activity to permit its use in LAMP for the first time. The fusion of Cren7, a double-stranded DNA binding protein, to Pol I for use in LAMP is also reported. Although the fusion construct was found to reduce amplification speed, enhancements were observed in the presence of increased salt concentrations and it is suggested here as a means for future enzyme development.

This thesis investigates the relevance between the acoustic resonance and power harmonics on a metal halide lamp. First, a sinusoidal current ranging from 20 kHz to 400 kHz is used to drive a 70 W metal halide lamp. Second, a hybrid-current test circuit is designed to generate a current waveform consisting of a low-frequency square-wave and a high-frequency sinusoidal wave. Both of the frequency and the amplitude can be adjusted independently. The test lamp is deliberately driven at its acoustic-resonance eigen-frequencies to observe the effect of the power spectrum on the degree of the acoustic resonance. The experimental results indicate that the occurrence of acoustic resonance is indeed affected by the DC level and related power harmonics. The power harmonic spectrum that elucidates the initiation of acoustic resonance is deduced from the observations. It is found that the power harmonics that excites acoustic resonance can be divided into three categories. The first is independent of the average lamp power; it excites acoustic resonance only if the magnitude of its power exceeds a specific level. The thresholds of power harmonics belong to the second category are proportional to their DC powers. One can also find those remaining power harmonics belong to the third category. The power harmonic spectrum of the acoustic resonance is demonstrated by driving the test lamp with quasi-square-wave and triangle-wave currents. This work helps advance the understanding of the phenomena and mechanism of acoustic resonance in a metal halide lamp.

Conventionally, metal halide lamps were ignited by striking the lamp electrodes several times with high voltage pulses. Such a starting scenario causes uncomfortable light strobes to users. To solve this problem, this thesis attempts to ignite small-wattage metal halide lamps with a single pulse strike. At first, the forms of the high voltage pulses required for breaking down the electrodes are investigated. After being broken down, a continuous current is critical for sustaining the lamp arc. With conventional electronic ballasts, however, the lamp current tends to resonate to zero resulting in break of the lamp arc. This problem can be solved by adding an extra switch to remove the capacitor of the output filter during the ignition stage. An electronic ballast is designed and tested on 70 W metal halide lamps with an associated switch for single pulse striking. Experiments have demonstrated that the proposed ignition criteria can start up the lamps successfully with a single-pulse high voltage.

This study investigates the starting characteristics of metal halide lamps. A laboratory electronic ballast was built to drive metal halide lamps with a programmable low-frequency square-wave current. The lamp current at each stage of the starting transient can be independently adjusted. Experiments were conducted on 150-W metal halide lamps. By examining the waveforms of transient voltage, current and power, the starting period can be classified into four stages, breakdown, glow discharging, glow-to-arc transition, and thermal equilibrium. In addition, the stable operation is defined by observing the variations of the lamp arc, lighting spectrum and luminous output. Based on the investigation results, four starting scenarios are presented and examined to learn the different acceleration schemes. Experimental evidence shows that the starting time of a metal halide lamp can be effectively shortened by increasing the lamp current during the start-up transition. More importantly, a specifically-regulated operating power enables the lamp to further enhance the luminance producing, and hence to greatly reduce the starting transient period.