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Chisty, L. T. "PcrA function in plasmid replication." Thesis, University College London (University of London), 2014. http://discovery.ucl.ac.uk/1427879/.
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PcrA is a DNA helicase involved in unwinding plasmi ds as a part of a complex in asymmetric rolling - circle replication of certain plasmids carrying antibiotic resistance genes. PcrA translocates on single stranded DNA by coupling ATP hydrolysis to movement on DNA. Initiator protein, RepD is required to nick supercoiled plasmid site - specifically and open an ssDNA stretch that PcrA can bind. The presence of RepD is needed throughout plasmid unwinding to maintain processivity. Using fluorescent - based techniques, PcrA helicase mechanistic functions and interactions with different components of the replication complex were examined at the ensemble and single molecule level. The development of ensemble techniques included labelling of PcrA with environment sensitive fluorophores such as the coumarin derivative, MDCC. Using MDCC - PcrA(K138C) a transloc ation assay was developed, which determined that PcrA translocates two times faster on dC (500 bases s -1) than on dT oligos (240 bases s -1). MDCC - PcrA(E449C) was used to investigate the PcrA and RepD interaction. The signal observed with this and other labelled PcrA mutants indicated that the 2B subdomain of PcrA is likely to be interaction site between PcrA and RepD . MDCC - PcrA(E449C) enabled the determination of PcrA kin etics with the initiation complex and showed that ATP - PcrA binds differently to PcrA to RepD - DNA complex as compared to apo PcrA , possibly indicating tha t ATP binding stabilises PcrA in a specific conformation when binding to RepD - DNA. A single - molecule a ssay was developed to study individual surface - bound PcrA helicases unwinding full length plasmids by total internal reflection fluorescence microscop y . PcrA unwinds plasmid with the average rate of 40 - 50 bp s - 1 showing 10 - fold variation between the indivi dual helicases. The experiments indicated that the unwinding starts almost immediately after addition of ATP . The single molecule experiments with fluorophore - labelled biotinylated PcrA so clear indication that PcrA unwinds plasmids as a monomer with proce ssivity over 3000 base pairs. The comparison of the ssDNA translocation rate and dsDN A unwinding rates show ed a significant difference between the two forms of translocation, which indicate d PcrA to be a passive helicase. This means that PcrA is n o t active ly destabilising the hydrogen bonding between DNA bases but taking advantage of the thermal fluctuation between the base pairs and so leading to separation of component DNA strands.
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Cerqueira, Lucas Saar. "PCRA - um protocolo cooperativo de acesso ao meio para redes de sensores aquáticas." Universidade Federal de Juiz de Fora (UFJF), 2018. https://repositorio.ufjf.br/jspui/handle/ufjf/7214.
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Previous issue date: 2018-03-27<br>CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior<br>O monitoramento de ambientes aquáticos ainda é uma tarefa difícil e dispendiosa. De fato, em ambientes aquáticos, ondas eletromagnéticas e ópticas sofrem alta atenuação e, mesmo a comunicação acústica apresenta baixa vazão e alta taxa de erro de bits. A maioria das abordagens existentes para melhorar o desempenho da comunicação subaquática se baseia no desenvolvimento de modems acústicos, acesso múltiplo ao canal de comunicação e roteamento de dados. Neste trabalho apresentamos PCRA: um Protocolo Cooperativo para Redes de Sensores Aquáticas. O PCRA funciona de forma síncrona/assíncrona sobre o método TDMA combinado com um esquema ARQ baseado em Selective Repeat. Cada nó que não possui dados para transmitir pode se tornar um cooperador e retransmitir mensagens para auxiliar os nós vizinhos. Ele usa os nós sensores ociosos como nós retransmissores, aumentando a diversidade do espaço de comunicação. Nossas simulações mostram que, quando comparado a um protocolo não cooperativo, o PCRA reduz a taxa de erro de pacotes em 65% e aumenta o goodput em 16% enquanto gasta menos de 1% a mais de energia.<br>Monitoring underwater environments is still a hard and costly task. Indeed, electromagnetic and optical waves suffer high attenuation, being absorbed in a few meters and even acoustic communication presents low throughput and high bit error rate. Most of the existing approaches to enhance underwater communication performance relies on developing acoustic modems, multiple access of the communication channel, and data routing. In this paper we present PCRA: a Cooperative Protocol for Underwater Sensor Networks. PCRA synchronously/asynchronously works on top of TDMA method combined with an ARQ scheme based on selective repeat technique. Each node that has no data to transmit can become a cooperator and retransmit messages to assist neighboring nodes. It uses idle sensor nodes as relay nodes, enhancing communication space diversity. Our simulations show that, when compared to a non-cooperative protocol, PCRA enhances overall network performance metrics. For instance, it reduces packet error rate by 65% and increases goodput by 16% while spending less than 1% more energy.
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Lynch, Gerard Patrick. "Molecular characterization of the interaction between the plasmid initiator protein RepD and PcrA helicase." Thesis, University of Leeds, 2010. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.536100.
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Dillingham, Mark Simon. "Biochemical studies on DNA helicases." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312245.
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Cravello, Laëtitia. "Etudes structurales des protéines par spectrométrie de masse couplée aux échanges hydrogène/deuterium et à la réticulation chimique." Université Joseph Fourier (Grenoble), 2005. https://tel.archives-ouvertes.fr/tel-00009698.
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Les protéines sont impliquées dans de nombreux processus biologiques. Il est nécessaire pour comprendre en détail leur fonction et leur mode d’action afin d’obtenir des informations sur leur structure et sur leurs interactions éventuelles avec leurs partenaires. Le travail réalisé durant cette thèse a consisté à développer deux méthodes innovantes utilisant la spectrométrie de masse pour étudier la structure des protéines et à appliquer ces méthodes à une problématique biologique. Nous avons optimisé une méthode associant les échanges H/D et la spectrométrie de masse sur une protéine modèle, la protéine PBP-2X. L’utilisation combinée de trois protéases nous a permis d’obtenir un meilleur recouvrement de séquence de la protéine étudiée et une plus grande résolution spatiale dans la localisation des zones d’intérêt. Une méthode associant la réticulation chimique et la spectrométrie de masse a été mise au point sur une protéine modèle : le cytochrome c. Les contraintes de distances ainsi obtenues vont intervenir dans une démarche bioinformatique visant à déterminer la famille de repliement d’une protéine de structure inconnue. Enfin, ces deux méthodes ont été appliquées avec succès sur des protéines du système de sécrétion de type III de Pseudomonas aeruginosa : PcrV et PcrG. Nos résultats expérimentaux sur PcrV corrèlent à la structure modélisée de PcrV et la protéine PcrG est globalement peu structurée. L’interaction PcrV-PcrG a été caractérisée, elle met en jeu les domaines « coiled-coil » de chacune des deux protéines. La formation du complexe induit un changement de la conformation de PcrV qui pourrait avoir pour conséquence la stabilisation de PcrG<br>Proteins are involved in many biological processes. They might be targets for medical treatments as well as therapeutic agents. A detailed knowledge of protein structure and a characterization of protein complexes are important to understand protein functions in a cell. In this study, we developed two new methods, which use mass spectrometry, to elucidate protein structure. These methods were then applied with success on a biological study. We improved a method that combines H/D exchange experiments with mass spectrometry on a model protein: PBP-2X. We show that the combination of three proteases increases the sequence coverage of the protein and the spatial resolution in the determination of interest areas. We developed a method, which associates intramolecular cross-linking and mass spectrometry on a model protein: the cytochrome c. Distance constraints determined by this way will be included in a bioinformatic project, that could give the folding family of a protein of which the tri-dimensional structure is unknown. We applied these two methods on proteins which are involved in type III protein secretion system from Pseudomonas aeruginosa: PcrV and PcrG. Experimental data (accessibility, secondary structures and distance constraints) are in agreement with the structural predictions on PcrV. PcrG is mainly unstructured. PcrG and PcrV are in interaction through their coiled-coil domains. Complexation between the two proteins induces conformational changes on PcrV, which could stabilize PcrG
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Caye-Eude, Aurélie. "Génétique et architecture clonale des leucémies myélomonocytaires juvéniles sporadiques et syndromiques." Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCC173/document.
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La LMMJ est un syndrome myéloprolifératif et myélodysplasique rare du jeune enfant, initiée par des mutations classiquement décrites comme mutuellement exclusives de RAS (NRAS, KRAS) ou de régulateurs de la voie RAS (PTPN11, NF1 ou CBL). Ces mutations, somatiques ou constitutionnelles, entraînent l’hyperactivation de cette voie de signalisation et une hypersensibilité spécifique au GM-CSF. La LMMJ est une hémopathie sévère dont le seul traitement est l’allogreffe de moelle osseuse. Cependant sa présentation et son évolution sont particulièrement hétérogènes puisqu’une transformation en leucémie aiguë myéloide survient chez un tiers des patients quand d’autres présentent des formes plus indolentes, voire des rémissions spontanées en l’absence de greffe. Cette hétérogénéité n’est que partiellement liée à la mutation initiatrice et pourrait s’expliquer par la présence de mutations additionnelles et/ou par une variabilité dans la cellule initiatrice de la leucémie, qui n’a jamais été précisément caractérisée.La caractérisation génétique de 118 LMMJ nous a permis de montrer que les anomalies génétiques additionnelles sont peu nombreuses dans les LMMJ sporadiques, et exceptionnelles dans les LMMJ syndromiques sauf en cas de neurofibromatose de type-1. Ces anomalies se concentrent sur deux grands systèmes, la voie RAS et le PRC2, et leur présence s’accompagne d’un pronostic défavorable (particulièrement en cas de mutations multiples de la voie RAS). L’absence d’anomalie additionnelle permet à l’inverse de distinguer un sous-groupe de patients qui présentent une forte probabilité de survie à long terme sans greffe et pour lesquels une soultion attentiste serait à privilégier. Une collaboration avec l’équipe de D Bonnet (Crick Institute) nous a ensuite permis d’établir un modèle murin de xénotransplantation dans des souris immunodéficientes de type NSG ou NSG-S et de montrer que la capacité de propagation de la leucémie est bien portée par la fraction souche, mais s’étend aussi chez certains patients à des fractions plus différenciées. Le profil génétique des 15 xénogreffes étudiées reproduit fidèlement l’architecture clonale des LMMJ natives, tant dans les souris NSG que NSG-S. L’architecture clonale des LMMJ est dans la majorité des cas compatible avec une acquisition linéaire des altérations, mais une architecture complexe est parfois observée, avec coexistence de clones distincts, dont les plus faiblement représentés sont susceptibles de devenir dominants lors de la rechute. Le séquençage de sous-populations isolées a montré que l’ensemble des mutations (initiatrice et additionnelles) est présent dès les fractions les plus immatures (HSC/MPP/MLP). Le séquençage de colonies obtenues par culture des progéniteurs en méthylcellulose révèle que les mutations coexistent dans les mêmes cellules, sans qu’il soit possible de hiérarchiser leur ordre de survenue, témoignant d’un avantage sélectif majeur de leur association dès la cellule souche. Au total, nos résultats remettent en cause le dogme de l’exclusivité mutuelle des mutations activant RAS dans les LMMJ, confirment le rôle central et initiateur de cette voie oncogénique dans la leucémogénèse et suggérent un effet-dose de l’activation de RAS, en particulier en cas de mutation de NRAS. La présence d’altérations multiples ciblant la voie RAS marque des LMMJ agressives et rapidement évolutives. La mise en évidence d’une fréquente dérégulation du PRC2 offre de nouvelles perspectives therapeutiques (comme l’utilisation des inhibiteurs de bromodomaine). La mise en place d’un modèle de souris xénotransplantée devrait de plus faciliter les études biologiques et la mise en place d’évaluations précliniques<br>JMML is a rare myeloproliferative and myelodysplastic neoplasm of early childhood, initiated by mutations classically described as mutually exclusive of RAS (NRAS, KRAS) or RAS pathway regulators (PTPN11, NF1 or CBL). These mutations, either germline or somatically aquired, lead to an hyperactivation of the RAS signalling pathway and a to a specific hypersensitivity to GM-CSF. JMML is a severe hemopathy, and the only curative treatment is allogenic bone marrow transplantation. However, its presentation and evolution are particularly heterogeneous since transformation into acute myeloid leukaemia occurs in about one third of patients, when others present more indolent forms, or even spontaneous remissions in the absence of transplantation. This heterogeneity is only partially accounted for by the initiating mutation and could be related to the presence of additional mutations, or some variability in the leukemia initiating cell, which has never been precisely characterized so far.Establishing the genetic landscape of 118 LMMJ allowed us to show that additional genetic abnormalities are scarse in sporadic JMML and exceptional in syndromic JMML, except in the case of type-1 neurofibromatosis. These additional abnormalities mainly target two major biologic components, the RAS pathway and the PRC2, and their presence is associated with an unfavourable prognosis (particularly in the case of multiple mutations targeting the RAS pathway). On the other hand, the absence of any additional abnormality allows to delineate a subgroup of patients who have a high probability of long-term survival in the absence of bone marrow transplantation, and for whom a wait-and-see approach would be preferable. A collaboration with D. Bonnet’s group (Crick Institute) allowed us to establish a mouse model of xenotransplantation in immunodeficient NSG or NSG-S mice and to demonstrate that the leukemia propagating cell is present in the stem cell fractions (HSC, CD34+/CD38-…) but also extends in certain patients to more differentiated fractions, such as CMP. The genetic profile of xenografts established from 15 JMML faithfully reproduced the clonal architecture of the native leukemia, either in NSG or NSG-S mice. The clonal architecture of JMML is linear in the great majority of cases, with linear acquisition of alterations, but a complex architecture is sometimes observed, with coexistence of distinct clones, the weakest of which being susceptible to become dominant at relapse. Sequencing of sorted cell populations showed that all mutations (initiating and additional) are present in the most immature fractions (HSC/MPP/MLP). The sequencing of colonies obtained by culturing progenitors into methylcellulose revealed that mutations coexist in the same cells, their order of appearance being often impossible to determine, showing a major selective advantage of their association from the most immature compartment. In conclusion, our findings confirm the central role of RAS activation in JMML leukemogenesis. The identification of multiple alterations targeting the RAS pathway challenges the dogma of the mutual exclusivity of these mutations and defines a subset of aggressive and rapidly evolving JMML, suggesting a dose-effect of RAS activation, particularly in case with NRAS mutation. Recurrent deregulation of PRC2 in JMML may offer new therapeutic approaches, such as bromodomain inhibitors. The implementation of a xenotransplanted mouse model should also facilitate biological studies and the implementation of preclinical evaluations
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Salgado, Vanessa Riesz. "Desenvolvimento de Reações em Cadeia pela Polimerase (PCRs) para o diagnóstico diferencial das principais espécies de Brucella." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-11102012-115215/.
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A brucelose é uma doença altamente contagiosa, responsável por grandes prejuízos econômicos e de saúde pública. É causada por bactérias do gênero Brucella, cujas espécies e seus biovares costumam ser caracterizados pelo isolamento e identificação de características fenotípicas da colônia. Dificuldades como, o perigo na manipulação dos microrganismos, processos laboriosos de tipificação, demora na obtenção de resultados e a instabilidade de características fenotípicas ou isolamento de linhagens atípicas dificultam a tipificação e encorajaram a busca de técnicas mais sensíveis e específicas, como a PCR, que resolveria as dificuldades e facilitaria a investigação epidemiológica dos casos humanos e animais. Diversas análises e o sequenciamento de determinados genes e do genoma completo de algumas espécies, demonstraram a existência de polimorfismos únicos no DNA das brucelas, que podem ser utilizados na sua identificação. Baseado nas dificuldades de identificação e na descoberta de polimorfismos únicos no DNA bacteriano das espécies, nosso objetivo foi desenvolver primers específicos para identificação de seis espécies do gênero B. abortus, B. melitensis, B. suis, B. canis, B. ovis e B. neotomae, e padronizar PCRs que permitissem identificá-las com maior sensibilidade e rapidez. Tentamos caracterizar marcadores moleculares para o desenho de primers espécie-específicos, através da amplificação randômica e clonagem dos fragmentos específicos, sem resultados satisfatórios. Apenas um primer para B. abortus foi conseguido quando foram analisados os polimorfismos já descritos na literatura. Assim, realizou-se o alinhamento múltiplo das sequências dos cromossomos I e II das espécies de Brucella, que permitiu a identificação de vários eventos polimórficos específicos para cada espécie, dos quais foram escolhidas regiões potenciais para o desenho de sete primers (dois para B. canis, B. melitensis e B. ovis, e outro para B. canis/B. suis) que tiveram sua especificidade analítica verificada com o programa Primer BLAST e testada nas 18 cepas de referência de Brucella, compreendendo a B. abortus, B. melitensis, B. suis e seus biovares, além da <iB. canis, B. ovis e B. neotomae, e em 231 isolados de campo, incluindo B. abortus, B. canis e B. suis. Os testes de especificidade dos primers resultaram na amplificação do fragmento esperado de quase todas as cepas de referência e de campo, exceto para o primer de B. canis e o de B. canis/B. suis. Estes resultados sugerem que os marcadores desenhados são promissores na diferenciação das espécies.<br>Brucellosis is responsible for great economic losses and serious impact on public health. This infectious disease is caused by bacteria of the genus Brucella, whose species and their respective biovars are often characterized by isolation and identification of differences in phenotypic tests. The complex and laborious process of Brucella typing, comprising the danger in handling of microorganisms, delay in obtaining results and instability of phenotypic characteristics or isolation of atypical strains, stimulated the search for more sensitive and specific techniques such as PCR. This technique would facilitate the epidemiological investigation of human and animal cases. Several analyses even as sequencing of certain genes and the complete genome of some species, demonstrated the existence of polymorphisms in the DNA of Brucella, which can be used to identify them. Due to typing difficulties and discovery of single polymorphisms in DNA bacterial species, our goals were to develop specific primers for identification of six species of the genus B. abortus, B. melitensis, B. suis, B. canis, B. ovis and B. neotomae and standardize PCRs to identify them with greater sensitivity and speed. We tried to characterize species-specific molecular markers using random amplification and cloning of specific fragments to design primers, without satisfactory results. Only one primer based on polymorphisms already described in the literature was successful for B. abortus specie differentiation. Thus, we performed the multiple alignment of the complete sequences of chromosomes I and II of Brucella species. This approach allowed the identification of several specie-specific polymorphic events, from which potential regions were chosen for the design of seven primers (two for B. canis, B. melitensis and B. ovis, and one for B. canis / B. suis). The analytical specificity of all primers was verified with the Primer BLAST software. Tests with specific primers were performed on 18 reference strains of Brucella, including all the six species of the genus Brucella and 231 field strains of B. abortus, B. canis and B. suis. The PCRs showed the expected fragment amplification in almost all reference and field strains, except for the B. canis and the B. canis / B. suis primers. Ours results suggest that these PCRs are able for Brucella species differentiation.
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Voigt, Kristina. "Einfluss der patientenkontrollierten epiduralen Analgesie versus der patientenkontrollierten intravenösen Analgesie auf immunologische Parameter nach großen Wirbelsäulenoperationen." Doctoral thesis, Humboldt-Universität zu Berlin, Medizinische Fakultät - Universitätsklinikum Charité, 2007. http://dx.doi.org/10.18452/15589.
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Operationen mit großem Gewebetrauma können mit starken postoperativen Schmerzen und ausgeprägten perioperativen Homöostasestörungen einhergehen. Dabei werden sowohl hyperinflammatorische als auch immunparalytische Reaktionen beobachtet, die sich negativ auf den postoperativen Verlauf auswirken können. Um eine effektive und sichere Analgesie zu gewähren, werden alternativ zu der intravenösen Therapie mit Opioiden zunehmend epidurale Verfahren eingesetzt. In dieser prospektiven, randomisierten und doppelblinden Studie wurde die patientenkontrollierte epidurale Analgesie mit der patientenkontrollierten intra-venösen Schmerztherapie hinsichtlich der analgetischen Effektivität und der Beeinflussung der postoperativen Immunkompetenz verglichen. 54 Patienten erhielten bis zum Morgen des 4. postoperativen Tages entweder über einen intraoperativ gelegten epiduralen Katheter (PDK) Ropivacain und Sufentanil (PCEA-Gruppe) oder intravenös Morphin (PCIA-Gruppe). Cortisol, Leukozytenpopulationen, lymphozytäre Subpopulationen, monozytäre Oberflächenmarker und die löslichen Mediatoren TNF alpha, MCP-1, MIF, IL-8, IL-6 und IL-10 wurden perioperativ gemessen. Zudem wurde die Schmerzempfindung der Patienten in Ruhe und bei Mobilisation erhoben. Im Vergleich zur PCIA-Gruppe profitierten die Patienten der PCEA-Gruppe von einer deutlich besseren Analgesie. Cortisol wies postoperativ in beiden Studiengruppen einen ähnlich leichten Anstieg auf. Die monozytären Oberflächenmarker (HLA-DR, CD86) fielen im Verlauf deutlich ab mit einem Minimum am 1. postoperativen Tag, erholten sich bis zum 7. postoperativen Tag nahezu vollständig und zeigten keine signifikanten Gruppenunterschiede. Dagegen wurde der postoperative Abfall der CD4+ T-Lymphozyten, CD4/CD8 T-Zellratio, CD3+ Lymphozyten und CD19+ Lymphozyten bei den Patienten, die eine Epiduralanalgesie erhielten, signifikant vermindert. Hinsichtlich der löslichen Mediatoren gab es keine signifikanten Gruppenunterschiede. Somit scheint die epidurale Schmerztherapie die T-Zellkompetenz während der postoperativen Phase besser zu erhalten, während sich bei den monozytären Oberflächenmarkern und dem Stresshormon Cortisol kein Unterschied zwischen den beiden Analgesie-verfahren zeigte.<br>Surgeries accompanied by an extensive tissue trauma are associated with intense postsurgical pain and major perioperative homeostatic disorders. Both hyper-inflammatory and immuneparalytic reactions can be observed, what can negatively effect the postoperative course. To realise an effective and safe analgesia, epidural procedures are used to an increasing degree as an alternative method to the therapy with intravenous opioids. In this prospective, randomized, double-blinded trial we compared the patient-controlled epidural analgesia and the patient-controlled intravenous analgesia with respect to the analgesic efficiency and the influence on the postoperative immune competence. 54 patients received until the morning of the fourth postoperative day either ropivacaine plus sufentanil through an intraoperatively placed epidural catheter (PCEA-group) or intravenous morphine (PCIA-group). Cortisol, populations of leukocytes and lymphocytes, cell-surface molecules of monocytes and the soluble mediators TNF-alpha, MCP-1, MIF, IL-8, IL-6 and IL-10 were measured perioperatively. Additionally we determined the subjective pain scores of the patients in rest and with mobilisation. Patients of the PCEA-group had a better pain control compared to the patients of the PCIA-group. Cortisol showed a similar slight increase in both study-groups. The monocyte cell-surface molecules (HLA-DR, CD86) decreased in the observed period with a minimum on the first postoperative day and recovered until the seventh postoperative day without a significant difference between both groups. In contrast, the postoperative decrease in CD4+ T-lymphocytes, CD4+/CD8+ T-cell ratio, CD3+ lymphocytes and CD19+ lymphocytes was significantly reduced in patients receiving epidural analgesia. No group differences were found in soluble mediators. This implicates a better postoperative competence of T-cells induced by epidural analgesia, whereas no differences between both analgetic methods were found in cell-surface markers of monocytes and the stress hormone cortisol.
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Minnella, Walter Settimo Leonardo. "Development of microfluidic tools for biological applications." Thesis, Bordeaux, 2017. http://www.theses.fr/2017BORD0664.
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Cette thèse traite le développement de dispositifs, basés sur la technologie "laboratoire sur puce"(LOC) qui visent à contrôler l'environnement des systèmes biologiques pour des applications macro et microbiologiques. En effet, les caractéristiques de la microfluidique permettent de manipuler l'environnement cellulaire à un niveau supérieur à celui du degré de contrôle atteignable avec les techniques ordinaires. Dans ce travail de thèse sera explorée la possibilité de profiter de ces fonctions afin de développer des outils de diagnostic peu coûteux et pourtant efficaces. En particulier, on rapporte le développement de systèmes microfluidiques permettant une perfusion des médias fluide et rapide, ainsi qu'une plateforme LOC capable de réaliser des PCRq hautement multiplexes. Au sujet des systèmes de perfusion, le but était d'obtenir une substitution du médium entourant les particules afin d'augmenter les capacités de séparation des modules de tri microfluidiques couplés. L'efficacité de notre approche a été validée par les hauts taux de séparation obtenus (>90%) avec l'utilisation de notre système de perfusion microfluidique couplé à une puce d'acoustophorèse. De plus, nous avons conçu et développé un système de thermalisation microfluidique capable d'opérer des changements de température en moins de 1s. Plus spécifiquement, cette plateforme exploite l'échange de chaleur entre un liquide de thermalisation qui circule dans une puce microfluidique et l'échantillon. Ces performances de thermalisation, et le rapport surface/volume élevé typique des appareils microfluidiques, ont permis d'effectuer 50 cycles de PCRq et l'analyse de courbe de fusion en moins de dix minutes<br>The topic of this manuscript is the development of microdevices, based on "lab on chip" (LOC) technology, aimed to the environmental control and regulation of biological systems for macro and microbiological applications. Indeed, microfluidics possesses some inherent features which allow the manipulation of the environment at the cell and sub-cell level which are superior than the degree of control achievable with standard techniques. In this thesis work the possibility to leverage these features to develop inexpensive yet effective diagnostic tools is explored. In particular, we report the development of microfluidic systems which allow seamless and fast media perfusion and a novel LOC platform capable of performing highly multiplexed real-time PCR assays. Concerning the microfluidic perfusion systems, the aim was to achieve in-flow substitution of the particles' surrounding media in order to enhance the separation capabilities of the coupled microfluidic sorting modules. The effectiveness of our approach was validated by obtaining high separation purities (>90%) using our microfluidic perfusion system coupled with an acoustophoresis chip to discern two population of micro-sized beads. Moreover, we conceived and developed a microfluidic thermalisation system capable of sub-second temperature switches. Specifically, this platform relies on conductive heat exchange between a thermalisation liquid flowing inside a microfluidic chip and the biological sample. These thermalisation performances, and the high surface to volume ratio typical of microfluidic devices, allowed to perform 50 qPCR cycles and subsequent melting curve analysis in less than ten minutes
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Ohlsson, Staffan. "Modellering och styrning av flis till en sulfatkokare." Thesis, Linköping University, Department of Electrical Engineering, 2005. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-2941.
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<p>At the Skoghall pappermill, sulphatepaper pulp is produced in a continuous digester originally from 1969. To be able to maintain a high level of production there is a need for a process with few disturbances. Variations in how well the wooden chips are packed in the digester is one form of disturbance. Today there are no available measurements on how well the chips are packed. Instead this is regarded as being constant. </p><p>The variation in the so called bulk density of the chips is mainly due to variations in the percentage with small dimensions. Chips are classified in relation to their size and one of the smallest classes is referred to as pin chips. These are believed to have a big impact on the bulk density. The amount of pin chips fluctuate more then the other classes, there by causing disturbances. </p><p>The Skoghall pappermill has invested in a ScanChip. This is an instrument that measures the dimensions of the chips optically. ScanChip presents figures on chip quality, including a measurement of the bulk density. However, it has been shown that this measurement is not valid for the Skoghall pappermill. By using data from ScanChip a model that predicts how well the chips are packed has been devised. This value is the bulk density divided by the basic density. The model has proved to yield good results, despite a relatively small amount of data. </p><p>A theoretical value of the amount of produced pulp has been computed based on the revolutions of the production screw that feeds chips into the digester. This value takes in consideration how well the chips are packed. The value has shown great similarities with the empirical measurements that are used today. A simulation during one month has shown that differences in the mixture of chips have effected the measurement of produced pulp with up to 7 ton/h. </p><p>Chips are stored in open pile storages before they are being used in the process of transforming them into pulp. Four screws are used to move chips from the piles to conveyer belts. It has been shown in work done previously, that the movement of the screws contributes to variations in the amount of pin chips measured by ScanChip. </p><p>During the work with this master’s thesis I have found that there are variations in the piles that make it difficult to predict the amount of pin chips accordingly. However by filtering the measurements of pin chips to remove these variations, the results are improved. A new way of controlling the movements of the screws was operational on the 10 of March and this improved the results. </p><p>The direction in which the screws are moving influence the speed of the screws, mainly in the pile with the so called sawmill chips. By changing the amount of chips that each screw puts out, the differences in speed have been reduced. The mixtures found in the two piles are not completely homogenous. There are a greater amount of pin chips in the northern parts compared with the southern parts. This could be an effect of the wind direction, and will still cause variations.</p>
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Rozales, Franciéli Pedrotti. "Real time-pcr e nested-pcr no diagnóstico da tuberculose pulmonar." reponame:Biblioteca Digital de Teses e Dissertações da UFRGS, 2013. http://hdl.handle.net/10183/72990.
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A tuberculose (TB) é um importante problema de saúde pública, portanto, é necessário o desenvolvimento de novas ferramentas para a detecção rápida e confiável do Mycobacterium tuberculosis, prevenindo a transmissão da TB. O objetivo deste estudo foi avaliar dois testes moleculares para detecção de bactérias do Complexo Mycobacterium tuberculosis (MTBC) diretamente de amostras clínicas. Foi realizado um estudo transversal, no qual foram selecionadas 124 amostras respiratórias. As amostras foram avaliadas por dois testes moleculares “in house” para detecção do MTBC: NESTED-PCR (NPCR) e Real Time PCR (RT-PCR) ambos com primers para o IS6110. As amostras também foram avaliadas por um teste direto (pesquisa de BAAR). Os resultados foram comparados com a cultura para M.tuberculosis e também com os resultados da cultura juntamente com dados clínicos. Uma amostra comercial com quantificação de DNA conhecida foi utilizada para determinação do Limite de Detecção (LOD) dos testes moleculares. O LOD foi de 1 cópia/μL para o RT-PCR e de 25 cópias/μL para o NPCR. O BAAR apresentou baixa sensibilidade - SE - (40%) e alta especificidade - SP - (94%). Ambos os ensaios moleculares, apresentaram elevada SE e SP (RT-PCR 98% e 91%, NPCR 86% e 93%, respectivamente) em relação à cultura. Quando comparamos os resultados frente a cultura juntamente com os dados clínicos, a SE e a SP foram de 90% e 97% para a RT-PCR e de 80% e 99% para a NPCR, respectivamente. Houve um pequeno decréscimo da SE dos métodos moleculares, quando comparados com a cultura mais os dados clínicos em relação à cultura isoladamente, no entanto, a SP foi consideravelmente elevada para os três métodos avaliados. Avaliamos o custo dos insumos para os ensaios moleculares: o custo do NPCR foi de $ 17.77/teste enquanto que o custo do RT-PCR foi de $ 15.76/teste. Em relação ao tempo de execução dos testes o RT-PCR foi mais rápido (2 horas) do que o NPCR (4 horas). Este estudo confirma que técnicas de PCRs podem ser muito úteis para o diagnóstico rápido da TB respiratória, com altas taxas de SP. Ele também pode ser muito importante para a exclusão de diagnóstico, considerando o alto VPN encontrados no nosso estudo. Os resultados demonstraram que os ensaios moleculares visando IS6110 do M. tuberculosis podem ajudar a melhorar o diagnóstico da TB pulmonar, com muitos potenciais efeitos positivos para a gestão clínica e de controle da doença.<br>Tuberculosis (TB) remains as an important public health problem worldwide. Therefore, the rapid detection of M. tuberculosis is of primary importance to effectively reduce transmission among patients. The aims of this study were to evaluate two molecular tests to detect M. tuberculosis complex (MTBC) directly from clinical samples. The study included 124 respiratory samples which were evaluated by two in house molecular assays for MTBC detection: Nested PCR (NPCR) and Real Time PCR (RT-PCR). The respiratory samples were also evaluated by the direct test (AFB assay). The results were compared with the results of culture and also compared with the culture results plus clinical data of patients. We used a commercial DNA sample with known quantification to establish the Limit of Detection (LOD). The LOD was 1 copy/μL for RT-PCR and 25 copies/μL for NPCR. The AFB assay presented low sensitivity – SE - (40%) and a high specificity - SP – (94%). Both molecular assays, RT-PCR and NPCR presented high SE and SP (RT-PCR 98% and 91%, NPCR 86% and 93%, respectively) compared to culture. When the results of the molecular tests were compared to the culture plus clinical data the SE and SP were 90,20% and 97,26% for RT-PCR and 80,39% and 98,63% for the NPCR, respectively. It was possible to observe a slight decrease of SE of the molecular methods in comparison to culture plus clinical data in relation to culture; however, the SP was increased, since many cases of TB could not be confirmed by culture. Furthermore we evaluated the cost of molecular assays: the NPCR cost was $17.77/test while the RT-PCR cost was $15.76/test. The RT-PCR test was faster (2 hours) than the NPCR (4 hours) to be performed. Our study confirms that PCRs may be useful for rapid diagnosis of respiratory TB, with high SP rates. It may also be very important to exclude such diagnosis, considering the high NPV found in our study. In summary, PCRs targeting IS6110 of MTB improve the accuracy of the diagnosis of pulmonary TB, with many potential positive effects for clinical management and control of the disease.
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Ziebell, Kim. "Evaluation of PCR and PCR-RFLP protocols to identify the Shiga toxins." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0026/MQ51107.pdf.
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SPÍNDULA, FILHO José Vieira de. "Detecção de HPV e avaliação do índice de proliferação celular entre carcinomas espinocelulares e carcinomas verrucosos de boca." Universidade Federal de Goiás, 2006. http://repositorio.bc.ufg.br/tede/handle/tde/1364.
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Previous issue date: 2006-11-27<br>Squamous cell carcinoma (SCC) is the most common malignant neoplasm of the bucal cavity, and one of its variants is verrucous carcinoma (VC), of low degree malignancy. The diagnosis of VC is difficult from the clinical as well as from the histopathological point of view, and an effective diagnosis is vital when deciding on the treatment and prognosis of this tumor. The aim of this research was to evaluate cell proliferation and investigate the presence
of HPV in spindle cell carcinoma of the mouth so as to check for possible differences in the aetiopathogenesis and biological behavior of these lesions. Forty-seven samples were selected
and divided as follows: 39 SCCs, 8 VCs and 9 control (CT). Cell proliferation was qualitatively evaluated according to the location of the expression of the immunomarker in the cell and epithelium layers and by quantitatively considering the percentage of positive cells expressed. The analysis of HPV+ carcinomas was undertaken by means of the polymerase chain reaction (PCR), having GP5+/6+ as primers for identification of the virus. The qualitative analysis showed that the immunomarking in the VC as well as in the control
group was concentrated mainly in the basal and parabasal layers and the counting of the positive cells at the base of the epithelium showed a significant statistical difference in the
expressions of all three markers (p<0,05). The quantitative analysis of the cell proliferation markers was calculated by means of the Mann-Whitney and Kruskal Wallis tests and through the Pearson and Spermans correlation. They pointed to differences between the SCC and VC groups for the PCNA and cyclin B1 markers (p<0,05). On considering the three groups, it was proved that there was a positive correlation between Ki67 and the cyclin B1 (r=0,56) but not between the PCNA and the Ki67. The PCNA immunomarking was greater in the control group (average=100%), and the Ki67 showed itself to be effective as a proliferation cell marker although it showed no significant difference between the carcinoma variants. Whereas the cyclin B1 showed a significant difference in the comparison between the SCC and the VC
groups (p<0,05), and a positive correlation to the extent that the histological grading of the malignancy (WHO model) of the carcinomas increased (r=0,44). All tumor samples were
negative for HPV. Although the lesions showed different biological behaviors, the cell proliferation index in both types of mouth carcinoma was higher than in the control group, as
shown by the analysis of the Ki67 and cyclin B1 markers. On considering the total sample of carcinomas, independently of the tumor variety, cyclin B1 showed a positive correlation with
the histological degree of malignancy according to WHO. There is a need for further study to be carried out in the field of cell proliferation and detection of HPV especially with regard to VC, because it is a rare variant of SCC.<br>O carcinoma espinocelular (CEC) é a neoplasia maligna mais comum na cavidade bucal, e uma de suas variantes é o carcinoma verrucoso (CV), considerado de baixo grau de
malignidade. O diagnóstico do CV é difícil, tanto do ponto de vista clínico quanto histopatológico e um efetivo diagnóstico é fundamental para estabelecer o tratamento e o prognóstico desse tumor. Neste estudo foi avaliada a proliferação celular e investigada a presença de HPV em carcinomas espinocelulares de boca com intuito de verificar possíveis diferenças na etiopatogênese e comportamento biológico destas lesões. Foram selecionadas 47 amostras de CEC assim distribuídas: 39 CECs, 8 CVs e 9 controles (CT). A proliferação celular foi avaliada qualitativamente de acordo com a localização da expressão do imunomarcador na célula e nas camadas do epitélio e quantitativamente considerando o percentual de células positivas expressas. A análise de carcinomas HPV+ foi realizada por meio da reação em cadeia da polimerase (PCR), tendo como primers GP5+/6+ na identificação do vírus. A análise qualitativa revelou que a imunomarcação tanto no CV como no controle concentrava se principalmente nas camadas basal e parabasal e a contagem das células positivas na base do epitélio mostraram diferença estatisticamente significativa na expressão dos três marcadores (p<0,05). A análise quantitativa dos marcadores de proliferação celular foi calculada pelos testes estatísticos Mann-Whitney, Kruskal Wallis, correlação de Pearson e Spermans, que revelaram diferenças entre o grupo CEC e CV para os marcadores PCNA e ciclina B1 (p<0,05). Considerando os três grupos, verificou-se correlação positiva entre Ki67 e a ciclina B1 (r=0,56) e inexistência de correlação entre o PCNA e Ki67. A imunomarcação do PCNA foi maior no grupo controle (média=100%), e o Ki67, mostrou-se efetivo como marcador de proliferação celular, entretanto, não mostrou diferença significativa entre as variantes de carcinomas. Já a ciclina B1 apresentou diferença significativa na comparação entre o grupo CEC e o grupo CV (p<0,05) e correlação positiva
na medida em que a gradação histológica de malignidade (padrão OMS) dos carcinomas aumentava (r=0,44). Todas as amostras de tumores foram negativas para o HPV. Embora as
lesões apresentem comportamento biológico diferente, o índice de proliferação celular nos dois tipos de carcinomas de boca mostrou ser superior ao do grupo controle, por meio da
análise dos marcadores Ki67 e ciclina B1. Quando considerada a amostra total de carcinomas, independente da variante tumoral, a ciclina B1 mostrou correlação positiva com o grau
histológico de malignidade segundo a OMS. Há necessidade que mais estudos possam ser empreendidos na área de proliferação celular e detecção de HPV em especial com relação ao CV, por se tratar de uma variante rara do CEC.
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Hill, Philip John. "PCR based gene engineering." Thesis, University of Nottingham, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.317040.
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Costa, Luciana Fachini da [UNESP]. "Avaliação comparativa entre PCR gênero-específica, PCR espécie-específica e nested PCR espécie-específica no diagnóstico da infecção por Brucella ovis." Universidade Estadual Paulista (UNESP), 2010. http://hdl.handle.net/11449/94670.
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costa_lf_me_botfmvz.pdf: 594547 bytes, checksum: 7d609573704d70bd41e6633e86629767 (MD5)<br>Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)<br>Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)<br>O diagnóstico da infecção por Brucella ovis em ovinos usualmente é realizado por meio de exame clínico, testes sorológicos e bacteriologia. Devido às limitações apresentadas pelas técnicas, o diagnóstico é geralmente obtido mediante aplicação de duas ou mais técnicas para obtenção de um resultado conclusivo. A Reação em Cadeia pela Polimerase (PCR) tem sido utilizada como ferramenta diagnóstica aplicável, pois é sensível, pouco dispendiosa, rápida, simples de ser realizada e permite o diagnóstico específico do agente infectante. Neste trabalho foram realizados dois experimentos. No primeiro compararam-se os percentuais de positividade em duas técnicas de PCR padronizadas, sendo a primeira gênero-específica e a seguinte espécie-específica, em 191 amostras de sêmen e 214 de urina provenientes de ovinos inoculados experimentalmente com a cepa de B. ovis REO 198. Posteriormente, desenvolveu-se a nested PCR a partir do produto amplificado da reação espécie-específica, e o percentual de positividade obtido pela nested PCR foi comparado aos obtidos pelas PCRs gênero-específica e espécie-específica. Foi observada diferença significativa no percentual de positividade (P<0,05) entre PCR gênero-específica e PCR espécie-específica (24,08% e 15,18%, respectivamente) para amostras de sêmen de ovinos e concordância moderada entre os resultados destas técnicas (kappa de 0,623); para amostras de urina, não houve diferença significativa entre as positividades obtidas pela PCR gênero-específica e a espécie-específica (10,28% e 7,011%, respectivamente), e a concordância entre os resultados foi moderada (kappa de 0,6167). A nested PCR espécie-específica apresentou percentual de positividade significativamente maior (P<0,001) quando comparada às PCRs gênero-específica e espécie-específica em amostras de sêmen (53,93%) e de urina (49,07%)...<br>Diagnosis of Brucella ovis infection in rams is routinely performed by clinical examination, serology and bacteriology. Due to limitations presented by each technique, the diagnosis is usually made by two or more techniques to obtain a conclusive result. The Polymerase Chain Reaction (PCR) has been used as a diagnostic tool applicable because it is sensitive, inexpensive, rapid, simple to perform and allows the specific diagnosis of infectious agents. In this study, the positivity percentages of two techniques of PCR previous described, one genus-specific and other species-specific, were measured in 191 semen and 214 urine samples from sheep experimentally infected with strain of B. ovis REO 198. Then, a species-specific nested PCR was developed from amplified products of the species-specific PCR, and the percentage of positivity obtained by nested PCR was compared to those obtained by genus and species-specific PCRs. Significant difference was observed when comparing the percentage of positivity (P <0.05) between genus-specific and species-specific PCR (24.08% and 15.18%, respectively) in semen samples from sheep, and there was moderate agreement between the results of these techniques ( kappa 0.623). In urine, no significant difference between the percentages of positives samples obtained by genus and species-specific PCR was observed (10.28% and 7.011% respectively) and the concordance between the results was moderate (kappa 0.6167). The species-specific nested PCR showed significantly higher percentage of positivity (P <0.001) when compared to genus-specific and species-specific PCRs in semen (53.93%) and urine (49.07%). Thus, according to the results obtained in this experiment, the species-specific PCR showed the lower percentage of positivity when compared to genus-specific PCR, but the implementation of the species-specific nested PCR showed highly significant increase... (Complete abstract click electronic access below)
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Costa, Luciana Fachini da. "Avaliação comparativa entre PCR gênero-específica, PCR espécie-específica e nested PCR espécie-específica no diagnóstico da infecção por Brucella ovis /." Botucatu : [s.n.], 2010. http://hdl.handle.net/11449/94670.
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Resumo: O diagnóstico da infecção por Brucella ovis em ovinos usualmente é realizado por meio de exame clínico, testes sorológicos e bacteriologia. Devido às limitações apresentadas pelas técnicas, o diagnóstico é geralmente obtido mediante aplicação de duas ou mais técnicas para obtenção de um resultado conclusivo. A Reação em Cadeia pela Polimerase (PCR) tem sido utilizada como ferramenta diagnóstica aplicável, pois é sensível, pouco dispendiosa, rápida, simples de ser realizada e permite o diagnóstico específico do agente infectante. Neste trabalho foram realizados dois experimentos. No primeiro compararam-se os percentuais de positividade em duas técnicas de PCR padronizadas, sendo a primeira gênero-específica e a seguinte espécie-específica, em 191 amostras de sêmen e 214 de urina provenientes de ovinos inoculados experimentalmente com a cepa de B. ovis REO 198. Posteriormente, desenvolveu-se a nested PCR a partir do produto amplificado da reação espécie-específica, e o percentual de positividade obtido pela nested PCR foi comparado aos obtidos pelas PCRs gênero-específica e espécie-específica. Foi observada diferença significativa no percentual de positividade (P<0,05) entre PCR gênero-específica e PCR espécie-específica (24,08% e 15,18%, respectivamente) para amostras de sêmen de ovinos e concordância moderada entre os resultados destas técnicas (kappa de 0,623); para amostras de urina, não houve diferença significativa entre as positividades obtidas pela PCR gênero-específica e a espécie-específica (10,28% e 7,011%, respectivamente), e a concordância entre os resultados foi moderada (kappa de 0,6167). A nested PCR espécie-específica apresentou percentual de positividade significativamente maior (P<0,001) quando comparada às PCRs gênero-específica e espécie-específica em amostras de sêmen (53,93%) e de urina (49,07%)... (Resumo completo, clicar acesso eletrônico abaixo)<br>Abstract: Diagnosis of Brucella ovis infection in rams is routinely performed by clinical examination, serology and bacteriology. Due to limitations presented by each technique, the diagnosis is usually made by two or more techniques to obtain a conclusive result. The Polymerase Chain Reaction (PCR) has been used as a diagnostic tool applicable because it is sensitive, inexpensive, rapid, simple to perform and allows the specific diagnosis of infectious agents. In this study, the positivity percentages of two techniques of PCR previous described, one genus-specific and other species-specific, were measured in 191 semen and 214 urine samples from sheep experimentally infected with strain of B. ovis REO 198. Then, a species-specific nested PCR was developed from amplified products of the species-specific PCR, and the percentage of positivity obtained by nested PCR was compared to those obtained by genus and species-specific PCRs. Significant difference was observed when comparing the percentage of positivity (P <0.05) between genus-specific and species-specific PCR (24.08% and 15.18%, respectively) in semen samples from sheep, and there was moderate agreement between the results of these techniques ( kappa 0.623). In urine, no significant difference between the percentages of positives samples obtained by genus and species-specific PCR was observed (10.28% and 7.011% respectively) and the concordance between the results was moderate (kappa 0.6167). The species-specific nested PCR showed significantly higher percentage of positivity (P <0.001) when compared to genus-specific and species-specific PCRs in semen (53.93%) and urine (49.07%). Thus, according to the results obtained in this experiment, the species-specific PCR showed the lower percentage of positivity when compared to genus-specific PCR, but the implementation of the species-specific nested PCR showed highly significant increase... (Complete abstract click electronic access below)<br>Orientador: Jane Megid<br>Coorientador: Renato de Lima Santos<br>Banca: Sony Dimas Bicudo<br>Banca: Luis antônio Mathias<br>Mestre
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Nyström, Stefan. "Evaluation of a New Method for Extraction of Drift-Stable Information from Electronic Tongue Measurements." Thesis, Linköping University, Department of Electrical Engineering, 2003. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-1615.
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<p>This thesis is a part of a project where a new method, the base descriptor approach, is studied. The purpose of this method is to reduce drift and extract vital information from electronic tongue measurements. Reference solutions, called descriptors, are measured and the measurements are used to find base descriptors. A base descriptor is, in this thesis, a regression vector for prediction of the property that the descriptor represent. The property is in this case the concentration of a chemical substance in the descriptor solution. Measurements from test samples, in this case fruit juices, are projected onto the base descriptors to extract vital and drift-stable information from the test samples. </p><p>The base descriptors are used to determine the concentrations of the descriptors'chemical substances in the juices and thereby also to classify the different juices. It is assumed that the measurements of samples of juices and descriptors drift the same way. This assumption has to be true in order for the base descriptor approach to work. The base descriptors are calculated by multivariate regression methods like partial least squares regression (PLSR) and principal component regression (PCR). </p><p>Only two of the descriptors tested in this thesis worked as basis for base descriptors. The base descriptors'predictions of the concentrations of chemical substances in the juices are hard to evaluate since the true concentrations are unknown. Comparing the projections of juice measurements onto the base descriptors with a classification model on the juice measurements performed by principal component analysis (PCA), there is no significant difference in drift of the juice measurements in the results of the two methods. The base descriptors, however, separates the juices for classification somewhat better than the classification of juices performed by PCA.</p>
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Jakobsson, Sanna. "Quantitative analysis of BCR-ABL1 fusion gene by Droplet Digital PCR and qRT-PCR." Thesis, Umeå universitet, Biomedicinsk laboratorievetenskap, 2015. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-103957.
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Metivier, Romain. "Ecologie microbienne de produits végétaux : Adaptation de traitements assainissants pour la valorisation de ces produits." Thesis, Bordeaux, 2015. http://www.theses.fr/2015BORD0419/document.
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L’utilisation de coproduits en tant que matière première provenant d’une autre voie industrielle, fait qu’il n’est plus considéré comme « déchet ». Leur valorisation est donc un axe de développement pour les entreprises agronomiques et agroalimentaires. Cependant, leur nouveau statut de « matière première » entraîne des contraintes pour les industriels.Celles-ci sont diverses selon les voies de destination : sanitaires, toxicologiques …Ce travail s’intéresse à deux coproduits issus de filières différentes de transformation végétale :(1) l’épiderme de pomme, comme source d’antioxydants. Leur valorisation passe par l’emploi de matières premières peu traitées d’un point de vue phytosanitaire qui pourront être a priori plus contaminées par des flores diverses.(2) Les broyats de végétaux issus de la culture céréalière comme matière première de produits biosourcés. Ils présentent naturellement de fortes contaminations en microorganismes sporulés.La valorisation de ces deux coproduits nécessite donc des traitements assainissants adaptés.Ainsi, il était indispensable de déterminer la nature, la variabilité et l’évolution des écologies microbiennes présentes sur ces coproduits par des techniques rapides de dénombrement ainsi que d’identification par biologie moléculaire. L’étude de différents procédés assainissants a également été réalisé pour combiner l’efficacité de désinfection à la préservation des qualités nutritionnelles (pomme) ou des propriétés physiques (broyats)<br>The use of byproduct as raw material from another industrial sector, facts that it is not considered any more as "waste". Their valuation is thus an axis of development for the agronomic and food-processing industry. However, their new consideration of "raw material" entails constraints for the industrialists. These constraints are diverse according to the destination ways of the byproduct: sanitary, toxicological… This work focus on two byproducts resulting from different vegetable process: (1) apple peels, as antioxidant source. Their valuation needs to use raw materials with low phytosanitary treatment, so these materials may be more contaminated by different floras. (2) Crushed vegetable matter stemming from cereal crop as raw material of biosourced products. They occur naturally a strong microbial spore contamination. The valuation of these two byproducts requires adapted cleaning treatments. So, it was the main thing to determine nature, variability and evolution of the present microbial ecologies of these byproducts by fast techniques of enumeration and identification by molecular biology. The study of different cleaning process was also realized to combine efficiency of disinfection with the preservation of nutritional qualities (apple) or physical properties (crushed vegetable matter)
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Ertongur, Sabahat Işin. "Funktionen und Regulation der PCNA-Ubiquitinierung in Vertebraten." kostenfrei, 2009. http://edoc.ub.uni-muenchen.de/11549/.
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Ertongur, Sabahat Isin. "Funktionen und Regulation der PCNA-Ubiquitinierung in Vertebraten." Diss., lmu, 2009. http://nbn-resolving.de/urn:nbn:de:bvb:19-115497.
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Smith, Jane Showler. "PCNA and P53 expression in mouse liver tumours /." [S.l : s.n.], 1995. http://www.ub.unibe.ch/content/bibliotheken_sammlungen/sondersammlungen/dissen_bestellformular/index_ger.html.
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Ohayon, Delphine. "Rôle de PCNA cytoplasmique dans la survie cellulaire." Thesis, Sorbonne Paris Cité, 2016. http://www.theses.fr/2016USPCC324.
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Notre laboratoire a mis en évidence la présence de la protéine Proliferating Cell Nuclear Antigen (PCNA) localisée exclusivement dans le cytosol du neutrophile exerçant une activité anti-apoptotique. C'est une protéine dite "scaffolding" qui s'associe à de nombreuses protéines partenaires pour assumer ses fonctions. Dans des conditions physiologiques, la relocalisation du noyau vers le cytosol de PCNA s'effectue à la fin de différenciation granulocytaire. Mon projet de thèse a permis d'identifier que cette relocalisation cytoplasmique était dérégulée dans les cellules leucémiques contribuant au phénomène de survie exagérée et à la résistance à la chimiothérapie. Nous avons montré qu'il y avait une augmentation très significative de PCNA cytoplasmique dans le cytosol des cellules HL-60 rendues résistantes à la daunorubicine (HL-60R) résultant d'un export nucléaire actif, en comparaison aux cellules HL-60 sensibles (HL-60S). Dans ces cellules HL-60R, PCNA cytoplasmique interagit avec NAMPT, une protéine qui a un rôle clé dans la voie de la glycolyse leur conférant un avantage de survie des cellules.Enfin, dans le neutrophile, nous avons mis en évidence pour la première fois une association structurale et fonctionnelle entre la protéine cytosolique p47Phox de la NADPH oxydase et PCNA suggérant que ce dernier contrôle à la fois la survie et l'état de repos du neutrophile.PCNA est donc un facteur clé cytoplasmique dans la survie de plusieurs types cellulaires. Identifier ses mécanismes d'action dans le but de moduler ses partenaires s'avère être un axe de recherche très utile pour développer de nouveaux traitements thérapeutiques<br>Cytosolic proliferating cell nuclear antigen (PCNA), a scaffolding protein involved in DNI replication, has been described as a key element in survival of neutrophil, a non-proliferating cell. Without enzymatic activity this main function is to build a protein scaffold through the binding and functional coordination of its different partners. This relocation of PCNA from the nucleus into the cytoplasm occurs at the end of granulocytic differentiation. From our present findings, we propose new paradigm in which cytosolic PCNA builds a protein scaffold that dictates Acute Myeloid Leukemia (AML) cell survival by enhancing their glycolytic metabolism and in turn conferrinl chemotherapy resistance. We have demonstrated that daunorubicin-resistant HL-60 cells (HL-60R have a prominent cytosolic PCNA localization due to increased nuclear export compared to their sensitive counterpart. By interacting with nicotinamide phosphoribosyltransferase (NAMPT), protein involved in the NAD biosynthesis, PCNA coordinates the glycolysis pathway and survival especially in HL-60R cells.In neutrophil, we have also demonstrated a functional and structural interaction between a protein p47Phox: a cytosolic subunit of NADPH oxidase and PCNA which suggested that PCNA control the survival and maintain the resting state of neutrophils. PCNA is a key element involved in survival of different types of cells. Decipher the molecular mechanisms of PCNA to modulate its partners represent a promising avenue of research
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Saukkoriipi, A. (Annika). "Detection of pneumococcus by PCR." Doctoral thesis, University of Oulu, 2003. http://urn.fi/urn:isbn:9514272110.
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Abstract
New rapid methods for sensitive and specific detection of pneumococci are not only needed to improve the diagnosis of pneumococcal disease but are also essential for vaccine and carriage studies. The purpose of this study was to develop sensitive PCR methods for the detection and quantification of S. pneumoniae and to study the applicability of these methods to detecting pneumococci in clinical samples.
A previously described PCR method was first developed further by introducing a Europium-labelled hybridisation probe for the detection of amplification products. The hybridisation method was easy to use and improved the specificity of the PCR assay. The developed PCR assay was established as a sensitive method for detecting pneumococcal DNA when the presence of pneumococcal DNA in over 2500 middle ear fluid (MEF) samples of children with acute otitis media (AOM) was studied by using the method. Pneumococcal findings increased by 76% when using PCR detection in addition to culture, compared to using culture alone. However, the PCR-positive, culture-negative AOM events represented a less severe type of disease compared to the culture-positive events. A positive PCR finding seems to indicate the presence of viable, although often non-culturable pneumococci within the middle ear cleft.
To be able to rapidly detect and quantify the initial numbers of pneumococcal genome copies in clinical samples, a real-time PCR method for the detection and quantification of pneumococcal DNA was developed. In real-time PCR, amplification and detection of amplification products occur simultaneously, which makes it possible to monitor the phase of the reaction at a particular stage or continuously. The method developed here was applied to the analysis of MEF samples and to investigating the nasopharyngeal carriage of pneumococcus. The sensitivities of bacterial culture and real-time PCR in detecting pneumococci were also compared. The real-time PCR assay was found to be rapid and sensitive and to provide information about the differences between the numbers of bacteria in samples. However, the quantitative results were shown to be dependent on the DNA extraction method applied. The real-time PCR method developed appears to be a good aid in research where an accurate and sensitive pneumococcal diagnosis is needed.
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Beauchamps, Patrick. "Contribution de l'amplification génique (PCR) au diagnostic de la toxoplasmose : intérêts de la PCR quantitative." Lille 1, 1999. https://pepite-depot.univ-lille.fr/LIBRE/Th_Num/1999/50376-1999-413.pdf.
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La toxoplasmose est une parasitose ubiquitaire occupant une large place en médecine humaine et vétérinaire. Cette affection parasitaire très fréquente en France est le plus souvent bénigne voire asymptomatique. Pourtant, elle reste redoutable en situation de transmission congénitale. De même, sa survenue chez les malades immunodéprimés a radicalement changé la conception de cette maladie. Pour compenser les insuffisances du sérodiagnostic et du diagnostic parasitologique, nous avons mis au point un diagnostic permettant un dépistage très précoce face à la suspicion d'une toxoplasmose congénitale ou cérébrale. Ce diagnostic repose sur l'amplification spécifique d'un gène répété 35 fois de Toxoplasma gondii (gène B1) associé à un système de révélation par chimioluminescence. A partir de prélèvements comparables aux liquides amniotiques humains, nous sommes capables de détecter spécifiquement un tachyzoïte. Nous avons également mis au point un modèle de PCR quantitative sur ADN utilisant un standard interne compétitif et spécifique des gènes SAGl, B1 et ARN 18S. Ce modèle est applicable à tout modèle d'étude expérimental où la quantification d'une charge parasitaire est nécessaire. Nos premiers essais ont porté sur la quantification d'une charge parasitaire cérébrale (kyste, souche Prugniaud) dans un modèle murin (BALB/C et C57 BI/6) d'encéphalite toxoplasmique. Afin de documenter une éventuelle corrélation entre l'expression des gènes de granules denses et la virulence des différentes souches du parasite, nous avons élaboré l'ensemble des outils techniques d'un modèle de RT-PCR quantitative pour le gène GRA2 (gène reconnu être impliqué dans le pouvoir pathogène). Ce système utilise une molécule standard interne compétitive, différente de 4 paires de bases comparée à la cible génomique. Les produits PCR marqués à la fluorescéine par nested-PCR, seront quantifiés à l'aide d'un analyseur automatique d'ADN. Les variations de rendement d'amplification seront quant à elles, corrigées par la quantification d'un gène rapporteur (bêta-tubuline).
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Chandramoulee, Swaran Yuvaneswari. "Evaluation of direct PCR for forensic DNA profiling and the development of a direct PCR multiplex." Thesis, University of Strathclyde, 2012. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=18935.
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The use of direct PCR with different types of sample was explored in this study. Genomic DNA preparations at various concentrations and buccal cell counts were deposited on commonly encountered substrates, recovered and amplified using direct PCR before subjecting them to capillary electrophoresis. The electropherograms obtained were compared to those obtained using the standard DNA profiling protocol which involves extraction and amplification prior to capillary electrophoresis. Direct PCR was found to be better than the standard DNA profiling protocol in both studies and was further tested with fingerprints, touch DNA on fabric and blood and semen stains on fabrics. All these tests were successful with direct PCR indicating that this technique has the potential to be incorporated into routine forensic DNA testing. Supplementary tests were also carried out to compare the efficiency of the swabbing technique utilised and the effect different substrates had on DNA recovery. Four non -porous substrates, which were glass, stainless steel, plastic and ceramic, and four types of dyed fabrics, which were white cotton, light blue denim, nylon and brown cotton, were used to deposit DNA and the resulting DNA profiles were evaluated. Of the non-porous substrates tested, the highest recovery of DNA was observed with plastic while the lowest was observed with stainless steel. DNA deposited on fabric on the other hand gave variable results which we believe is dependent on the dye used to stain the fabric and the thickness of the fibres used. The results in this experiment indicated that the substrate DNA is deposited on plays an important role in determining the resulting DNA profiles. Finally, a novel multiplex consisting of five autosomal and two Y-chromosomal STRs which also provides the inhibitor status of the sample was developed. This multiplex also addresses the issues concerning sensitivity and robustness that was encountered with other commercially available multiplexes. The multiplex was developed, validated and tested with various mock crime scene samples successfully. Allelic ladder, panels and bins were created to be used with this multiplex to aid in sample designation when subjected to capillary electrophoresis.
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Moldovan, George-Lucian. "Regulation of replication-linked functions by PCNA and SUMO." Diss., lmu, 2006. http://nbn-resolving.de/urn:nbn:de:bvb:19-63450.
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Ola, Ayodele Oluronke. "A functional analysis of proliferating cell nuclear antigen (PCNA)." Thesis, King's College London (University of London), 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.391441.
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Shetty, Shiphali. "In vivo replication dynamics : analysis of PCNA-interacting proteins." Thesis, University of Cambridge, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607734.
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Cloux, Boccoz Stéphanie. "Développement de PCRs multiplexes pour le diagnostic : microarrays analytiques." Thesis, Lyon 1, 2015. http://www.theses.fr/2015LYO10282/document.
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Les travaux présentés dans cette thèse font suite à celle de Melle LE GOFF. Ils se concentrent sur la technologie HIFI brevetée et développée pendant ses travaux. Une première partie du travail présenté dans ce manuscrit concerne le test HIFI Blood 96™ et plus particulièrement les améliorations et les évolutions apportées au test afin d'en faire un véritable outil de génotypage, multiparamétrique et haut-débit pouvant être installé dans les banques de sang dans le but de constituer des inventaires de sang génotypé de façon étendue, participant ainsi à améliorer la sécurité transfusionnelle. Il permet de caractériser 96 échantillons sur 15 polymorphismes (divisés en deux panels) associés aux groupes sanguins en approximativement 4h30. Cette plateforme a fait l'objet d'une étude de validation à moyenne échelle sur 583 donneurs pour le panel 1 et 190 donneurs pour le panel 2. La deuxième partie des travaux décrit l'adaptation de la technologie HIFI appliquée au diagnostic des pathologies respiratoires, avec le développement d'une autre plateforme, ReSynPlex, en partenariat avec 3 équipes de recherche de Grenoble<br>The work reported in this thesis follows the one undertaken by Ms LE GOFF. It is focused on HIFI technology, which is patented and developed during her thesis. The first part of this work concerns the HIFI Blood 96™ test, and particularly the improvements and developments adduced to the test to make it a real diagnostic tool, multiparametric and high-throughput which can be implemented in blood banks in order to constitute negative antigen inventories, thus contributing to improve blood safety. It allows to characterize 96 samples on 15 polymorphisms (divided in two panels) associated to blood group systems in approximately 4.5 hours. A mesoscale validation study has been conducted on 583 samples for panel 1 and 190 samples for panel 2. The second part of this work describes the adaptation of HIFI technology applied to diagnosis of respiratory tract infections, with the development of another platform, ReSynPlex, in partnership with 3 research teams in Grenoble
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Bischoff, Erik. "Schnellnachweis von bierschädlichen Bakterien mit PCR." [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=964420333.
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Lien, Tonje Gulbrandsen. "Statistical Analysis of Quantitative PCR Data." Thesis, Norges teknisk-naturvitenskapelige universitet, Institutt for matematiske fag, 2011. http://urn.kb.se/resolve?urn=urn:nbn:no:ntnu:diva-13094.
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This thesis seeks to develop a better understanding of the analysis of gene expression to find the amount of transcript in a sample. The mainstream method used is called Polymerase Chain Reaction (PCR) and it exploits the DNA's ability to replicate. The comparative CT method estimate the starting fluorescence level f0 by assuming constant amplification in each PCR cycle, and it uses the fluorescence level which has risen above a certain threshold. We present a generalization of this method, where different threshold values can be used. The main aim of this thesis is to evaluate a new method called the Enzymological method. It estimates f0 by considering a cycle dependent amplification and uses a larger part of the fluorescence curves, than the two CT methods. All methods are tested on dilution series, where the dilution factors are known. In one of the datasets studied, the Clusterin dilution-dataset, we get better estimates from the Enzymological method compared to the two CT methods.
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Karlsson, Magdalena, and Emilia Semberg. "Tracing probiotics in salami using PCR." Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2011. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-157177.
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Starter cultures of different bacteria strains like lactic acid producing bacteria, Staphylococcus and Kocuria are used when making salami. Starter cultures give the sausage specific flavours and improve the quality and ripening of the final product. Probiotic strains can also be added during the production of salami. Studies have shown that probiotics are good for health and are therefore added to food, such as fermented sausages. In order to work as a probiotic strain, the bacteria have to survive during the production process, storage and through the whole human gastrointestinal tract. The aim of this study was to trace the probiotic strains Lactobacillus casei and Lactobacillus paracasei in salami samples to see if they had survived the production process. Methods used were DNA extraction, PCR, colony PCR and gel electrophoresis. Out of 100 samples in duplicate run in PCR, probiotics were found in only 3 of them. To see if screening of probiotics directly from plates was possible, a colony PCR was done. Colony PCR was made on colonies of two different strains of Lactobacillus casei, Lactobacillus paracasei and Lactobacillus sakei. From each bacteria strain, 5 colonies were analysed. Result showed that colony PCR, to screen for probiotic is a possible method.
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Renkjumnong, Wasuta. "SVD and PCA in Image Processing." Digital Archive @ GSU, 2007. http://digitalarchive.gsu.edu/math_theses/31.
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The Singular Value Decomposition is one of the most useful matrix factorizations in applied linear algebra, the Principal Component Analysis has been called one of the most valuable results of applied linear algebra. How and why principal component analysis is intimately related to the technique of singular value decomposition is shown. Their properties and applications are described. Assumptions behind this techniques as well as possible extensions to overcome these limitations are considered. This understanding leads to the real world applications, in particular, image processing of neurons. Noise reduction, and edge detection of neuron images are investigated.
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Halpern, Micah. "Immuno-PCR detection of Lyme borreliosis." Doctoral diss., University of Central Florida, 2013. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/6286.
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Lyme borreliosis, more commonly referred to as Lyme disease, is the fastest growing zoonotic disease in North America with approximately 30,000 confirmed cases and 300,000 estimated infections per year. In nature, the causative agent of Lyme disease, the bacterium Borrelia burgdorferi, cycles between Ixodes sp. ticks and small mammals. Humans become infected with Lyme disease after being bitten by an infected tick. The primary indicator of a Borrelia burgdorferi infection is a bull's eye rash typically followed by flu-like symptoms with treatment consisting of a 2-4 week course of antibiotics. If not treated, later stages of the disease can result in arthritis, cardiovascular and neurological symptoms. Diagnosis of Lyme disease is challenging and currently requires a complex laboratory diagnostic using indirect detection of host-generated antibodies by a two-tiered approach consisting of an enzyme linked immunosorbent assay (ELISA) followed by IgM and IgG immunoblots. Although two-tier testing has provided an adequate approach for Lyme disease diagnosis, it has weaknesses including subjective analysis, complex protocols and lack of reagent standardization. Immuno-PCR (iPCR) is a method that combines ELISA-based detection specificity with the sensitivity of PCR signal amplification and has demonstrated increased sensitivity for many applications such as detection of disease biomarkers but has yet to be applied for diagnosis of Lyme disease.
Herein, using iPCR and recombinant B. burgdorferi antigens, an assay for both the direct and the indirect detection of Lyme disease was developed and demonstrated improved sensitivity for detection of B. burgdorferi antibodies using a murine model. Moreover, we present evidence using human Lyme disease patient serum samples that iPCR using both multiple antigens and a unique single hybrid antigen is capable of achieving increased sensitivity and specificity compared to existing methodology. These data represent the first demonstration of iPCR for Lyme disease diagnosis and support the replacement of two-tier testing with a more simplified and objective approach.<br>Ph.D.<br>Doctorate<br>Molecular Biology and Microbiology<br>Medicine<br>Biomedical Sciences
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Bennett, Marissa A. "Improving Model Performance with Robust PCA." Digital WPI, 2020. https://digitalcommons.wpi.edu/etd-theses/1366.
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As machine learning becomes an increasingly relevant field being incorporated into everyday life, so does the need for consistently high performing models. With these high expectations, along with potentially restrictive data sets, it is crucial to be able to use techniques for machine learning that increase the likelihood of success. Robust Principal Component Analysis (RPCA) not only extracts anomalous data, but also finds correlations among the given features in a data set, in which these correlations can themselves be used as features. By taking a novel approach to utilizing the output from RPCA, we address how our method effects the performance of such models. We take into account the efficiency of our approach, and use projectors to enable our method to have a 99.79% faster run time. We apply our method primarily to cyber security data sets, though we also investigate the effects on data sets from other fields (e.g. medical).
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Liu, Peng. "Adaptive Mixture Estimation and Subsampling PCA." Case Western Reserve University School of Graduate Studies / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1220644686.
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Feugeas, Olivier. "Pcr (polymerase chain reaction) et vih." Lille 2, 1990. http://www.theses.fr/1990LIL2M264.
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Moravčíková, Simona. "Stanovenie hodnoty firmy PCA Slovakia, s.r.o." Master's thesis, Vysoká škola ekonomická v Praze, 2015. http://www.nusl.cz/ntk/nusl-206699.
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The aim of this thesis is to estimate the value of the company PCA Slovakia, s.r.o. to the 31st December 2015. The thesis is divided into two parts, in the concrete the theoretical and practical parts. The theoretical part describes the basic concepts necessary for the valuation of the company and it is kind of the point for the practical part. On the other hand, the practical part is focused on the introduction of the company and the application of strategic analysis and financial analysis, the prognosis of revenue and other value drivers, financial plan and finally the actual valuation of the company. There was used the DCF method of valuation in the term of FCFF and EVA for the valuation of the company.
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Šuranská, Hana. "Identifikace vinných kvasinek metodou PCR-RFLP." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2009. http://www.nusl.cz/ntk/nusl-216494.
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This thesis deals with identification of the wine yeasts by applying the PCR-RFLP method. The identification and characteristic of the yeasts has gone through substantial changes in recent years. There have been introduced new methods of taxonomic classifying based on the molecular methods, which are oriented to easy and fast identification. One of these methods is the PCR-RFLP method. The amplification of the 5•8S-ITS rDNA sequence by the polymerase chain reaction with use of the primers ITS1 and ITS4 leads to the amplification of the specific sequence of DNA. Such multiplied DNA is after repurifying by the ethanol and drying submitted to the restriction analysis. With use of the restriction endonuklases DNA is chopped into the specific segments typical for the particular genus. The chopped fragments can be separated in the electric field in the agarose gel and subsequently evaluated. In this thesis together 63 type yeasts were used. These yeasts were analysed by applying of the seven restriction endonuklases – HaeIII, HhaI, HinfI, HpaII, TaqI, AluI a MseI. The final image of type yeasts splitting was compared to the results of splitting of already identified wine yeasts and these yeasts were subsequently taxonomically classified. Evaluation of genetic similarity was conducted by program BioNumerics and as the results the dendrograms that were created with use of Jaccard‘s coefficients are obtained.
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Pelikánová, Veronika. "Automatická genotypizace bakterií metodou rep-PCR." Master's thesis, Vysoké učení technické v Brně. Fakulta elektrotechniky a komunikačních technologií, 2018. http://www.nusl.cz/ntk/nusl-378029.
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This thesis deals with automatic bacteria genotyping by rep-PCR method. Its theoretical part presents various methods of DNA typing, basic information on electrophoresis and modern electrophoretic approaches, including their problems, misleading data distortion. In order to automate typing, there has been introduced a program for phylogenetic sample classification from rep-PCR, also applicable for data from chip capillary electrophoresis. The program consists of three main parts: digitization, bandmatching and clustering apparatus to bacterial type classification. The result of the algorithm is a phylogenetic tree, which indicates the cluster of sapmles according to bacterial type. The program has a graphical user interface for possible use in the Children's Hospital. Finally, the program is tested with data from the Children´s Hospital.
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Reis, Levi Eduardo Soares. "Detecção de Leishmania por PCR e suas variações (seminested PCR e PCR em tempo real), em fragmentos de pele e de baço de cães com leishmaniose visceral." Programa de Pós-Graduação em Ciências Farmacêuticas. CIPHARMA, Escola de Farmácia, Universidade Federal de Ouro Preto, 2013. http://www.repositorio.ufop.br/handle/123456789/3364.
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Previous issue date: 2013<br>A detecção do DNA de Leishmania spp, pela Reação em Cadeia da Polimerase (PCR) e suas variações, surgem como alternativas para o diagnóstico da LVC, por serem métodos altamente sensíveis e específicos. O objetivo deste trabalho foi comparar a PCR e suas variações (Seminested PCR e PCR em tempo real) utilizando amostras de pele e de baço de 60 cães soropositivos (RIFI e ELISA). Os animais foram agrupados considerando a forma clínica, sendo classificados como assintomáticos (CA; n=20), oligossintomáticos (CO; n= 22) e sintomáticos (CS; n= 18). Como controle negativo, foram utilizados fragmentos de três cães não infectados provenientes do canil da UFOP. O diagnóstico parasitológico, utilizado como padrão ouro, foi realizado por meio de duas metodologias: cultivo de aspirado medular em meio de cultura NNN/LIT e pela visualização direta de formas amastigotas do parasito em lâminas com imprints de pele e de baço. As análises moleculares foram realizadas utilizando iniciadores direcionados para a região conservada do minicírculo do kDNA de Leishmania – L150/L152 e LINR4/LIN17/LIN19 para as técnicas de PCRc e snPCR, respectivamente. Na qPCR foram utilizados iniciadores que amplificam o gene da DNA polimerase (DNA pol α) de L. infantum. De acordo com os testes parasitológicos 61,7% das amostras foram positivas. Nos fragmentos de pele, a sensibilidade da PCRc foi de 89,2%, já para a snPCR e qPCR foi de 86,5% e 97,3% respectivamente. VPP para a PCRc foi de 36,0%, snPCR de 35,3% e da qPCR foi 38,1%. O VPN foi de 93,6%, 92,1% e 98,3% pelas técnicas de PCRc, snPCR e qPCR respectivamente. Em amostras de baço, a sensibilidade da PCRc foi 81,1%, snPCR de 94,6% e de 100,0% pela qPCR. O VPP para a PCRc foi 33,9%, snPCR 37,4% e qPCR 38,7%. O VPN da PCRc foi de 89,3%, snPCR 96,7% e qPCR 100,0%. A positividade nos testes moleculares aumentou de acordo com a gravidade dos sinais clínicos. Foi observado que a qPCR apresentou os melhores resultados na pele e no baço devido a maior sensibilidade, VPP e VPN, em comparação as outras técnicas moleculares. Sendo assim, concluímos que a melhor técnica e tecido para o diagnóstico molecular da LVC é a qPCR de pele, devido à elevada sensibilidade e fácil obtenção da amostra biológica. ____________________________________________________________________________________<br>ABSTRACT: The detection of Leishmania DNA based on polymerase chain reaction (PCR) and its variations represent alternatives for CVL diagnosis with highly sensitive and specific methods. The aim of this work was to compare the PCR and its variations (Seminested PCR and quantitative PCR) in samples of skin and spleen of 60 seropositive dogs (IFAT and ELISA). The animals were divided according to their clinical presentation and were classified as asymptomatic (CA, n = 20), oligosymptomatic (CO, n = 22) and symptomatic (CS, n = 18). As a negative control, we used fragments of three uninfected dogs from the kennel of UFOP. The parasitological diagnosis used as gold standard was performed by two methods: culture of bone marrow aspirate in culture medium NNN/LIT and direct visualization of amastigotes of the parasite on slides with imprints of skin and spleen. The primers L150/L152 and LINR4/LIN17/LIN19 were used to amplify the conserved region of the Leishmania kDNA minicircle in the cPCR and snPCR and qPCR were performed out using the DNA polymerase gene (DNA pol α) primers from L. infantum. According to the parasitological test 61.7% the samples were positive. In skin samples, the sensitivity of cPCR was 89.2%, snPCR was 86.5% and qPCR was 97.3%. PPV for cPCR was 36.0%, snPCR was 35.3% and qPCR was 38.1%. The NPV was 93.6%, 92.1% and 98.3% by the techniques of cPCR, snPCR and qPCR. In spleen samples, the sensitivity of cPCR was 81.1%, snPCR was 94.6% and qPCR was 100.0%. PPV for cPCR was 33.9%, snPCR was 37.4% and qPCR was 38.7%. NPV for cPCR was 89.3%, snPCR was 96.7% and qPCR was 100.0%. Positivity in molecular tests increased with the progression of clinical signs. It was observed that the qPCR showed the best results in skin and spleen due to higher sensitivity, PPV and NPV compared to other molecular techniques. Thus, we conclude that the best technique and tissue for molecular diagnosis of CVL is the qPCR skin due to the high sensitivity and easy obtaining the biological sample.
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Noll, Lance. "Escherichia coli O157: detection and quantification in cattle feces by quantitative PCR, conventional PCR, and culture methods." Thesis, Kansas State University, 2015. http://hdl.handle.net/2097/18923.
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Master of Science<br>Department of Diagnostic Medicine/Pathobiology<br>T. G. Nagaraja<br>Shiga toxin-producing E. coli O157 is a major foodborne pathogen. The organism colonizes the hindgut of cattle and is shed in the feces, which serves as a source of contamination of food. Generally, cattle shed E. coli O157 at low concentrations (≤ 10[superscript]2 CFU/g), but a subset of cattle, known as “super-shedders”, shed high concentrations (>10[superscript]3 CFU/g) and are responsible for increased transmission between animals and subsequent hide and carcass contamination. Therefore, concentration data are an important component of quantitative microbial risk assessment. A four-plex quantitative PCR (mqPCR) targeting rfbE[subscript]O157, stx1, stx2 and eae was developed and validated to detect and quantify E. coli O157 in cattle feces. Additionally, the applicability of the assay to detect E. coli O157 was compared to conventional PCR (cPCR) targeting the same four genes, and a culture method. Specificity of the assay to differentially detect the four genes was confirmed. In cattle feces spiked with pure cultures, detection limits were 2.8 x 10[superscript]4 and 2.8 x 10[superscript]0 CFU/g before and after enrichment, respectively. Detection of E. coli O157 in feedlot cattle fecal samples (n=278) was compared between mqPCR, cPCR, and a culture method. Of the 100 samples that were randomly picked from the 136 mqPCR-positive samples, 35 and 48 tested positive by cPCR and culture method, respectively. Of the 100 samples randomly chosen from the 142 mqPCR-negative samples, all were negative by cPCR, but 21 samples tested positive by the culture method. McNemar’s chi-square tests indicated significant disagreement between the proportions of positive samples detected by the three methods. Applicability of the assay to quantify E. coli O157 was determined with feedlot cattle fecal samples (n=576) and compared to spiral plate method. Fecal samples that were quantifiable for O157 by mqPCR (62/576; 10.8%) were at concentrations of ≥ 10[superscript]4 CFU/g of feces. Only 4.5% (26/576) of samples were positive by spiral plate method, with the majority (17/26; 65.4%) at below 10[superscript]3 CFU/g. In conclusion, the mqPCR assay that targets four genes is a novel and more sensitive method than the cPCR or culture method to detect and quantify E. coli O157 in cattle feces.
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Lima, Ana Carolina Stocco de. "Diagnóstico molecular de Leishmaniose Tegumentar Americana: identificação de espécies de Leishmania por SSUrDNA PCR e G6PD PCR." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/5/5160/tde-20092010-162811/.
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A Leishmaniose Tegumentar Americana (LTA) representa um sério problema de saúde pública. No Brasil muitas espécies são reconhecidas como patogênicas para o homem, portanto o diagnóstico diferencial é necessário para compreender o perfil epimemiológico da LTA em áreas endêmicas. Com o objetivo de identificar espécies de Leishmania utilizando ferramentas moleculares, cinquenta e três biópsias de pele de pacientes com LTA, fixadas em formalina e incluídas em parafina dos Estados do Pará (N=33) e Maranhão (20) foram submetidos a diferentes protocolos da Reação em Cadeia da Polimerase (PCR) para a identificação dos agentes causadores. Biopsias foram desparafinizadas e o DNA foi extraído usando o protocolo de fenol-clorofórmio, quantificado e submetido a reações de PCR, com base na sequência de nucleotídeos codificadora do RNA que compõe a subunidade menor do ribossomo (SSUrDNA) e da enzima Glicose 6-Fosfato Desidrogenase (G6PD). O alvo G6PD foi utilizado tanto em reações de PCR convencional (cPCR), como de PCR quantitativo (qPCR). As reações de cPCR e Nested PCR SSUrDNA apresentaram resultado positivo para o gênero Leishmania em 40 (83,3%) das amostras submetidas. Vinte e sete desses produtos de PCR foram sequenciados, sendo 17 identificados como L.(L.) amazonensis e 10 como L.(Viannia) sp. A cPCR G6PD identificou 9 amostras como L.(V.) braziliensis , sendo 7 do Maranhão (36%) e 2 do Pará (6%). O DNA de L.(Viannia) sp. foi quantificado em quatro amostras do Maranhão através da reação de qPCR G6PD, mesmo esse alvo sendo de cópia única. Esses resultados indicam que sequências especificas de Leishmania sp. presentes em múltiplas cópias devem ser escolhidas para a aplicação de cPCR em DNA provindo de amostras parafinadas,uma vez que é freqüente casos de LTA com baixo parasitismo e consequentemente, pequenas concentrações de DNA. E ainda a cPCR SSUrDNA pode ser um bom alvo para estudos diagnósticos e epidemiológicos.A qPCR G6PD permitiu a detecção, identificação e quantificação de L. (Viannia) sp. em uma única etapa de amplificação em quatro amostras que presentaram resultados positivos somente na Nested ou Semi-Nested PCR, demonstrando uma maior sensibilidade oferecida pela q PCR<br>American Cutaneous Leishmaniasis (ACL) presents a serious problem of public healthy. In Brazil many species are recognized as pathogenic to humans, therefore differential diagnostic is necessary to understand the epidemiological profile of ACL in endemic areas. Fifty-three paraffinembedded skin biopsies of ACL patients from Pará (N=33) and Maranhão (20) States, were submitted to different protocols of polymerase chain reaction (PCR) for identification of their causative agents. Biopsies were deparaffinized and DNA were extracted using phenol-chloroform, quantified and submitted to PCR reaction, using small subunit coding sequence (SSUrDNA) and enzyme glucose-6-phosphate dehydrogenase (G6PD). The target of G6PD was used both in conventional PCR reactions (cPCR) and quantitative PCR (qPCR). The reactions of cPCR and Nested PCR SSUrDNA showed positive result for the genus Leishmania in 40 (83.3%) of the samples. Twenty-seven positive samples were submitted to sequencing and 10 were identified as L. (Viannia) sp. and 17 as L. (L.) amazonensis. The G6PD PCR identified 9 samples as L. (V.) braziliensis, 2 from Pará (6%) and 7 from Maranhão (35%).Four samples were quantified in G6PD qPCR, even this is a single copy. These results indicate that specific sequences from Leishmania sp. present in multiple copies should be chosen in relation to those from unique copies in paraffin-embedded tissues, once is frequent cases of ACL with low parasitism, consequently small DNA concentrations and that SSUrDNA can be a good target to diagnostic and epidemiologic studies of ACL. The qPCR allowed the detection and identification of L. (Viannia) sp. in a single round of amplification in four samples that when showed positive results only in the Nested or Semi Nested cPCR suggesting a higher sensitivity offered by qPCR
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SPACOV, Isabel Cristina Guerra. "Utilização de marcadores de rDNA-PCR e tDNA-PCR para tipagem de isolados clínicos de Pseudomonas aeruginosa." Universidade Federal de Pernambuco, 2005. https://repositorio.ufpe.br/handle/123456789/6640.
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Previous issue date: 2005<br>Pseudomonas aeruginosa é uma bacteria Gram-negativa ubíqua e oportunista. Na rotina
hospitalar, os marcadores fenotípicos nem sempre revelam a diversidade das bactérias
distribuídas nos diversos setores, assim, aplicamos três métodos moleculares baseados na
amplificação por PCR do locus de rDNA e tDNA para caracterizar a diversidade genética de
linhagens de P. aeruginosa isoladas em um hospital público em Recife-PE, Brasil. O rDNA-PCR
detectou 15% de variabilidade genética, contra 23% do tDNA-PCR e 23% do Duplex-PCR. O
setor com maior diversidade genética foi a Unidade de Tratamento Intensivo do hospital, o qual
apresentou quatro genótipos bacterianos diferentes. A ocorrência de linhagens de P. aeruginosa
pertencentes ao mesmo genótipo e mesmo perfil de resistência a múltiplas drogas (MDR), em
diferentes setores do hospital, sugere que há infecção cruzada entre pacientes. Os dados
apresentados pelo rDNA-PCR, tDNA-PCR e Duplex-PCR, em associação ao perfil de
susceptibilidade antimicrobiana provêem valiosas informações epidemiológicas para o controle
de infecções hospitalares causadas por P. aeruginosa
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Ferreira, Karin Correa Scheffer. "Detecção do vírus da raiva em órgãos de morcegos do gênero Artibeus (Leach, 1821) por meio de RT-PCR, Hemi-Nested RT-PCR e Real Time RT-PCR." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/10/10134/tde-19102012-132944/.
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Este estudo teve como objetivo detectar a presença do vírus da raiva em diferentes órgãos de morcegos do gênero Artibeus empregando as técnicas moleculares como RT-PCR, hnRT-PCR e Real Time RT-PCR. De aproximadamente 4000 espécimes de morcegos recebidas no Instituto Pasteur para o diagnóstico da raiva, foram selecionados 30 morcegos do gênero Artibeus, com resultados positivos para raiva pelas técnicas tradicionais de IFD e inoculação em células N2A utilizando suspensões feitas a partir do SNC. Para as técnicas moleculares, foram retirados glândulas salivares, bexigas urinárias, rins, pulmões e conteúdos fecais e ainda foram lavadas as calotas cranianas dos espécimes. Os órgãos e conteúdos fecais foram diluídos a 1:10 (P/V) e as bexigas urinárias a 1:20 (P/V). As suspensões foram inoculadas em células N2A para o isolamento viral. Foi realizada a extração do RNA total usando o TRIzol®, foram realizadas a transcrição reversa seguida da PCR e hnRT-PCR com utilização de primers específicos para o gene codificante da proteína N. A partir do produto da transcrição reversa foi realizada a técnica de Real Time RT-PCR, utilizando primers e sonda específicos para variante antigênica 3. Das 30 suspensões de lavado cerebral, 28 (93,33%) resultaram positivos, na inoculação em cultura de células, seguido de glândulas salivares (36,67%), bexigas (16,67%) e conteúdos fecais (3,33%). Os resultados encontrados da sensibilidade nas técnicas de RT-PCR, hnRT-PCR e Real Time RT-PCR foram 56,25%, 82,57% e 82,19% quando avaliadas as 180 amostras analisadas. A comparação das técnicas de hnRT-PCR e Real Time RT-PCR feita pelo teste exato de Fisher quanto a proporção de positivos detectados mostrou que para o lavado cerebral, órgãos e conteúdos fecais a proporção foi igual (P>0,05). Em relação à positividade os resultados encontrados nas técnicas de hnRT-PCR e Real Time RT-PCR foram 100% em lavado cerebral; 90% e 93,33% em glândulas salivares; 83,33% e 90% em bexigas; 80% e 93,33% em rins; 76,67% e 50% em pulmões e 43,33% em ambas as técnicas em conteúdos fecais. Esses resultados sugerem que tanto as técnicas de hnRT-PCR como Real Time RT-PCR podem ser utilizadas como métodos complementares para o diagnóstico da raiva e são sensíveis o bastante para o uso em estudos de patogênese. A técnica de Real Time RT-PCR realizada neste estudo se mostrou eficiente em detectar o RABV em diferentes órgãos e tecidos extraneurais com a vantagem de ser uma técnica mais rápida e sensível.<br>This study was aimed to detect the presence of rabies virus in different organs of the genus Artibeus bats using molecular techniques such as RT-PCR, hnRT-PCR, and the Real Time RT-PCR. From about 4,000 specimens of bats received for rabies diagnosis at the Pasteur Institute, 30 bats of the genus Artibeus were then selected. The selected bats presented positive results by the traditional DFA and N2A-cells inoculation test using brain tissue suspensions. Samples of salivary glands, urinary bladders, kidneys, lungs, and fecal contents and washings of the skulls were collected for the molecular techniques testing. The organs and the fecal contents were diluted at 1:10 (w/v) and the urinary bladder, at 1:20 (w/v) and these suspensions were inoculated into N2A cells for viral isolation. The extraction of the total RNA was performed by using TRIzol® and followed by the reverse transcription and the PCR and the hnRT-PCR were performed by using specific primers for the gene encoding the protein N. The product obtained by the reverse transcription technique was submitted to the Real Time RT-PCR technique, using primers and probe specific for antigenic variant 3 of the rabies virus. Of the 30 suspensions of the brain washings, 28 (93.33%) were positive in N2A cell culture inoculation, followed by the suspensions of the salivary glands (36.67%), bladders (16.67%) and fecal contents (3.33%). For the 180 samples evaluated, the results of sensitivity found for the RT-PCR, hnRT-PCR and Real Time RT-PCR techniques were 56.25%, 82.57%, and 82.19%, respectively. A comparison of hnRT-PCR and Real Time RT-PCR techniques performed by Fisher\'s exact test showed that the proportion of positives detected by the brain washings, organs and of the fecal content was non-significant (P> 0.05). Regarding the results found in hnRT-PCR and Real Time RT-PCR techniques, 100% positives were in brain washing, 90% and 93.33% in salivary glands, 83.33% and 90% in bladders, 80% and 93.33% in kidneys, 76.67% and 50% in lungs and 43.33% for both techniques on fecal contents. These results suggest that both hnRT-PCR and Real-Time PCR techniques can be used as complementary methods for the diagnosis of rabies and are sensitive enough for use in pathogenesis studies. The Real Time RT-PCR technique performed in this study proved to be faster and more sensitive and effective in detecting RABV in different organs and extra neural tissues of bats.
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Pfander, Boris. "Regulation der Genomstabilität durch SUMO- und Ubiquitin-Modifikation von PCNA." Diss., lmu, 2005. http://nbn-resolving.de/urn:nbn:de:bvb:19-44792.
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Tomi, Nils-Sebastian. "Funktionelle Analyse der PCNA-Polyubiquitinierung und der E3-Ligase SHPRH." Diss., Ludwig-Maximilians-Universität München, 2015. http://nbn-resolving.de/urn:nbn:de:bvb:19-182348.
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Ludwig, Cornelia. "Structural and biochemical studies of PCNA and its molecular interactions." Thesis, University of Edinburgh, 2008. http://hdl.handle.net/1842/12483.
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Proliferating cell nuclear antigen (PCNA) plays a key role in DNA replication and repair. In this thesis I have determined the crystal structure of <i>S. pombe </i>PCNA on its own and in complex with a peptide derived from the natural inhibitor of PCNA in humans, p21. Using the p21 peptide as a template, chemical libraries were screened for small drug-like molecules that are able to mimic the protein-protein interaction between human PCNA and p21 and tested for binding using various fluorescence-based methods. Binding of selected compounds could not be observed, however, which may have been due to poor solubility of the compounds as well as a lack of assays sensitive enough to pick up on ligands with low affinity. Not all direct PCNA binding proteins bind to the above mentioned pocket and PCNA:protein co-crystal structures of these examples without the PIP-box motif are not available so far, so information on the mode of binding in those cases is not accessible yet. Therefore, I tried to narrow down and characterize the PCNA binding site of Gadd45. This protein does not contain a PIP-motif, but previously was shown using yeast-two hybrid technology to bind directly to PCNA <i>via </i>its C-terminus. <i>In vitro </i>binding of the two full-length proteins could not be confirmed, due to the inherent instability of Gadd45, which also has been reported by others. GST-tagged truncations of the C-terminus of Gadd45 were expressed and purified and binding to PCNA was studied using SPR. These results are at odds with the published binding data and suggest that either the interaction between PCNA and Gadd45 is not direct and needs to mediated by a third factor or Gadd45 binds to PCNA <i>via </i>a different part than the C-termination (as also suggested in the literature).
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Lamb, Vanessa Martins. "Arqueologia hist?rica eg?pcia do per?odo de Amarna." Pontif?cia Universidade Cat?lica do Rio Grande do Sul, 2008. http://tede2.pucrs.br/tede2/handle/tede/2255.
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Previous issue date: 2008-06-25<br>Com a ascen??o de Akhenaton ao trono egipcio, inicia-se um per?odo que traz in?meras transforma??es ? sociedade do per?odo. Forem objetos de pesquisa as teorias que apontam para uma poss?vel co-reg?ncia entre novo rei e seu pai, Amen?fis III; a substitui??o do antigo pante?o por um ?nico Deus, Aton, e todas as implica??es que essa transforma??o trouxe ? sociedade eg?pcia; al?m da transfer?ncia da capital de Tebas para Akhetaton e a constru??o dessa nova capital. O novo fara? acaba com o culto aos antigos deuses e imp?e o culto a Aton, Deus que estava em segundo plano at? ent?o, acompanhado de outras a??es do rei. Ap?s substituir seu nome Anen?fis IV por Akhenaton, transfere a capital do Egito de Tebas para sua pr?pria cidade, Akhetaton. Seu espa?o ambiental, as semelhan?as e diferen?as entre a casa dos nobres e a dos oper?rios; o Grande Templo de Aton; a casa do Fara?; a aldeia dos trabalhadores; os recursos e atividades econ?micas que mantiam a cidade; e o que aconteceu com a nova capital ap?s a morte de Akhenaton e o fim do culto a Aton foram assuntos focados neste trabalho. Ao redor da nova capital, foram escavados t?mulos nas montanhas rochosas, usados para o sepultamentos dos dignat?rios da Fara?: os de pedra que cercavam Akhetaton; o misterioso t?mulo 55; os dos maiores homens de confian?a do rei, Ay e Horemheb; e a maior descoberta da arqueologia eg?pcia, o t?mulo de Tutanc?mon. Atrav?s de dados arquel?gicos, obtidos nas escava??es realizados no s?tio da cidade e nas tumbas de pedra, ? poss?vel realizar uma caracteriza??o na vida na cidade. A arte do per?odo, chamada "arte amarniana", traz transforma??es est?ticas ?nicas, novas formas e representa??es: a nova est?tica utilizada; o questionamento acerca das representa??es dos rei e da fam?lia real e os temas que nesse per?odo passaram a ser representados. Objetos de uso cotidiano e de uso da fam?lia real tamb?m possibilitam o estabelecimento de caracter?sticas da vida, da religi?o e da arte de Akhenaton. Objetos encontrados no sitio de Akhetaton e nos t?mulos do per?odo puderam nos dar indica??es de como era a vida, a religi?o e a arte do per?odo. Ressaltamos que objetos de uso da fam?lia real poderiam indicar o seu uso pela popula??o da nova capital.