The RNA polymerase T7 is utilized as a component of motor complexes in DNA nanotechnology due to its high promotor specificity, the lack of external transcription factors and its very high processivity, but there is no experience of its application on small double stranded DNA circles. Circular templates from 210 to 126 bp in circumference sharing a common promotor termination motif were synthesized and transcription was monitored at end point on gel and in real time with a 2’ O methyl RNA molecular beacon. The RNAP T7 was found to be able to utilize circular dsDNA templates down to 126 bp with moderate impact on transcription rate for saturated systems and rolling circle transcription products were evident with denaturizing PAGE gel electrophoresis for templates down to 167 bp.

The management of equipment based on Reliability-Centered Maintenance (RCM) has been widely applied with great success in several industries such as military, commodity production, civil airliners, nuclear power plant and the petroleum industry since the reprint of RCM-II written by John Moubray in 1997.The application of RCM not only increases the reliability of the equipment and the assets of a company but also promotes the overall equipment effectiveness (OEE), which includes the excellent ok yield to order and the better productivity of the equipment. As a methodology, RCM differs from the traditional equipment maintenance in the aspect of emphasis of maintaining the ¡§function¡¨ of the equipment instead of the equipment itself. Therefore, the need of equipment maintenance results from the speed and the severity of function loss, which determines the strategy and interval of equipment maintenance. This is the major difference between RCM and time based maintenance (TBM). This article discusses the critical success factors that drive the application of RCM in steel plant. The result reveals that the application of RCM could promote the reliability of equipment, cut the cost of maintenance and increase compatibility of the steel plant. In order to facilitates the implementation of RCM in the steel mill, the basic skills of the management includes the construction of vision¡Bconsensus¡Bthe set up of regulation and the cultivation of innovating culture. so as to build an effective managing teamwork features in strong execution. As for the equipment management both the standardization and the strict deviation of equipment function(6£m) are vital to sustain the excellent equipment reliability. The Integration of Maintenance Management System(IMMS) consists of three components, CMMS(Computerized Maintenance Management System) and RTMS(Real Time Monitoring System) link by RCM. RCM acts as a bridge for the information flow of maintenance management in the IMMS and promotes the effectiveness of maintenance management.

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This thesis encompasses the development and testing of an interstitial photodynamic therapy (iPDT) system for the treatment of prostate cancer. It begins with the optical characterization of a novel photosensitizer (SL-052) followed by a study of tissue optics as it applies to iPDT. The design and integration of a time-fractionated light delivery system with real-time spectral detection is then examined. An optical phantom test medium is formulated and in vitro system operation and testing is performed. Finally, in vivo experiments are performed on animal models with a focus on canine prostate iPDT. Unique optical results with dosimetric relevance are discovered and investigated. This includes metrics for optically measuring local in vivo SL-052 concentrations in real-time as well as novel oscillatory drug photobleaching and recovery behavior during time-fractionated light delivery. / Photonics and Plasmas

Development of the advanced, real-time methods for monitoring of bioaerosols is becoming increasingly important. At present, the Ultraviolet Aerodynamic Particle Sizer (UVAPS, Model 3312, TSI, St. Paul., MN) is the only commercially available method for in-situ, continuous measurements of viable airborne microorganisms. Research included in this thesis aimed towards comprehensive evaluation of the method over a wide range of operating conditions, linking the experimental results to the theoretical basis of its design and operation, and to developing a scientific basis for its application to real-time monitoring of bioaerosols. Specifically, due to a growing concern in the general community about the environmental and health aspects of biological aerosols originated from various types of agricultural operations including animal farming, this research was focussed on developing a research methodology/strategy for applying the method to the investigation of bioaerosols in the swine confinement buildings (SCB). Investigations under controlled laboratory conditions were primarily concerned with selectivity, sensitivity, counting efficiency, and detection limits of the spectrometer. This study also examined the effect of physiological state (metabolic activity) of bacteria on the performance characteristics of the method. The practical implications of the research findings are discussed in this thesis. Further field investigations undertaken on a pig farm advanced understanding of the UVAPS performance in the real-life environmental settings. The research also provided a new insight on the particle size distribution and the effect of on-farm-activities on aerosol load inside the SCBs, for both biological and non-biological aerosols. This study has proved that the UVAPS is a powerful tool for investigation of viable bioaerosols in the environment. However, this method is limited to detection of active metabolising bacteria that excludes dormant bacterial spores. In addition, the method is very sensitive to physiological state of bacteria and to the effect of adverse environmental conditions on metabolic activity of airborne bacteria, which may decrease the amount of the intrinsic fluorophores in the cells below sensitivity level iv of the monitor. Possible limitations of this technology include also the lack of selectivity and thus interferences from the non-microbial organic components of airborne particles. In addition, the sensitivity of the method is insufficient for monitoring viable bacteria in the environments with relatively low concentrations of bioaerosols. In order to increase sensitivity of the method, it would be desirable to concentrate the bioaerosols into a smaller volume with the aim of high-volume virtual impactors (aerosol concentrators) prior to the monitoring. Therefore, in the indoor environments where an application of the concentrator is not feasible, the utilisation of the UVAPS may be problematic. Due to the intrinsic limitations, the method is not recommended for the direct measurements of viable bioaerosols and should be used in conjunction with the conventional biosamplers for obtaining more realistic insights into the microbial air quality. Nevertheless, the UVAPS has been found to be an adequate method for the investigation of the dynamics of biological aerosols in real-time. Overall, this thesis contributes to the advancing of the understanding of the method and may assist in developing new, more advanced technologies for the real-time monitoring of viable bioaerosols, as well as in developing sampling strategies for the application of the method to various bioaerosol studies.

Les bioprocédés industriels mettant en œuvre la culture de cellules animales sont devenus incontournables pour la production d’anticorps monoclonaux (AcM). Cependant, l'état physiologique des cellules et la qualité des AcM produits, en particulier leur glycosylation, sont tributaires des variations intervenant au cours du procédé. Il en découle des risques d'altération de l'efficacité et de la sûreté des AcM. C'est pourquoi, depuis quelques années, l'initiative Process Analytical Technology (PAT) encourage le développement du suivi en ligne de ces procédés, avec l'objectif de mieux les maîtriser et d'assurer la qualité finale des produits. Dans ce contexte, cette thèse propose des approches innovantes pour le suivi en ligne de procédés de culture de cellules CHO (Chinese Hamster Ovary) en bioréacteur, basées sur l'utilisation de trois types de spectroscopies in situ (diélectrique, Raman, proche infrarouge(NIR)). Le premier chapitre présente une nouvelle démarche permettant de prédire en temps réel l'état physiologique des cellules, au travers de la vitesse spécifique de croissance cellulaire (μ). A partir de la mesure en ligne de la permittivité grâce à la spectroscopie diélectrique, la µ a été calculée en temps réel, permettant de détecter le moment critique correspondant au moment où μ diminue. Comparée à une démarche hors ligne, l'utilisation de cette méthode pour le pilotage de cultures en mode recharge-récolte, permet d’assurer à la fois la quantité et la qualité de glycosylation des AcM. Le second chapitre aborde l'utilisation des spectroscopies NIR et Raman in situ combinées à des méthodes chimiométriques. Les performances de ces deux spectroscopies ont été comparées en parallèle. Des modèles en ligne ont été développés pour prédire la concentration de différents paramètres (cellules viables, glucose, lactate, glutamine, ions ammonium, anticorps). L'évaluation de ces modèles par facteurs de mérite (FOM), a révélé certains avantages de la spectroscopie Raman. La combinaison de ces deux spectroscopies par diverses stratégies de fusion de données a été également évaluée. Dans le troisième chapitre, l'intérêt de la spectroscopie Raman a été démontré pour le suivi en ligne, non seulement, de la concentration, mais aussi, de la glycosylation des AcM. Des modèles ont été développés pour la prédiction en ligne, à la fois, de la macro-hétérogénéité (sites de glycosylation), et de la micro-hétérogénéité (structures glycanniques) de la glycosylation des AcM dans le cas de cultures en mode discontinu et recharge-récolte. Le dernier chapitre a utilisé les spectroscopies NIR et diélectrique, en les intégrant à un « capteur logiciel » combinant des équations de bilans de matière. Ce « capteur logiciel », mis en œuvre au cours d'une culture en mode semi-continu pour le contrôle automatique du débit d'alimentation, a conduit à une augmentation de la productivité du procédé ainsi qu'à une meilleure glycosylation des AcM produits / Bioprocesses of mammalian cell culture have become essential for the production of therapeutic recombinant proteins, such as monoclonal antibodies (mAb). However, the physiological state of the cells and the quality of the mAb produced, in particular their glycosylation, may vary during the process, and may lead to the alteration of the safety and efficacy of the final product. Consequently, the Process Analytical Technology (PAT) initiative has encouraged the development of online monitoring techniques, with the aim to better control the process and ensure the quality of the final product. In this context, this thesis proposes innovative approaches for online monitoring of CHO (Chinese Hamster Ovary) cells bioreactor cultures, by using three types of in situ spectroscopic measurements (dielectric, Raman, near infrared (NIR)). The first chapter presents a novel approach to predict in real-time one of the major cell physiological state parameters, the specific growth rate (µ). Based on online permittivity measured by in situ dielectric spectroscopy, the cell concentration was estimated and µ was calculated in real-time, making possible to detect the critical moment when µ begins to decrease significantly. Compared to an offline approach, this online approach allowed to maintain the cells in a stable physiological state, ensuring the glycosylation of the mAb produced in feed-harvest cultures. The second chapter shows the use of in situ NIR and Raman spectroscopies combined with chemometric methods. For the first time, the performances of these two spectroscopies were compared in parallel in the same cultures. Online models were developed to predict in real-time the concentration of different parameters (viable cells, glucose, lactate, glutamine, ammonium ions and antibodies). The evaluation of these models by the multivariate Figures of Merit (FOM) revealed some of the advantages of Raman spectroscopy. The combination of the two spectroscopies by various data fusion strategies has also been evaluated. In the third chapter, the interest of Raman spectroscopy for the online monitoring of both the quantity and the glycosylation of the mAb was demonstrated. Models were developed for online prediction of both macroheterogeneity (glycosylation site occupancy) and microheterogeneity (glycan structures) of mAb glycosylation in batch and feed-harvest cultures. The last chapter used models previously developed for NIR and dielectric spectroscopies, to integrate into a “soft sensor” by combining with cell metabolic and mass balance equations. This “soft sensor”, implemented in a fed-batch cell culture for the automatic control of the feed rate, leads to an increased mAb productivity and better mAb glycosylation

Détection et quantification de bactéries à faible taux par SPRi Résumé: Le protocole standard pour le diagnostic des bactéries dans le domaine médical et agro-alimentaire reste la culture microbienne, qui prend plusieurs jours pour identifier l'agent pathogène. Cependant, ces temps de latence ne sont pas compatibles avec l'urgence d'analyser rapidement la présence de bactéries pathogènes. Il ya un besoin croissant de vouloir développer de nouveaux outils pour identifier les bactéries pathogènes en un temps plus court. Afin atteindre cet objectif, la technologie de l'imagerie par Résonance des Plasmons de Surface (SPRi) a été utilisée avec succès pour la détection spécifique durant leur croissance des populations bactériennes en utilisant des protéines et/ou des anticorps comme molécule de bio reconnaissance des surfaces bactériennes. Ainsi, la détection spécifique de un à plusieurs milliers de pathogènes/ml peut être réalisé en quelques heures seulement. Dans le présent travail, plusieurs souches bactériennes ont été utilisées comme modèle : Streptococcus pneumoniae R6, Escherichia coli K12 ; ainsi que des agents pathogènes réels (Salmonella enteritidis) apportant la preuve que cette méthode peut être élargie à d'autres souches. Les résultats peuvent être quantitatifs par l'établissement d'une courbe d'étalonnage, permettant ainsi de remonter à la concentration initiale d'un échantillon contaminé. La stratégie développée au cours de ce travail se base sur le couplage simultané de la culture bactérienne et de la détection/identification spécifique, en utilisant l'imagerie SPR. Mots clés en français : Imagerie par résonance plasmonique de surface (SPRi), Streptococcus pneumoniae, E.coli K12, Salmonella enteritidis, croissance bactérienne, interactions, détection, capture, diagnostic, agro-alimentaire, pathogènes. / Low Level Bacteria Detection and Quantification using SPRi Abstract: The standard protocol for bacterial diagnosis in the medical and agronomic fields remains microbial culture, which takes several days to identify the pathogen. However, these time lags are not compatible with the urgency to rapidly analyse the presence of pathogenic bacteria. There is a strong need to develop new tools for identifying pathogenic bacteria in a shorter time. To achieve this goal, Surface Plasmon Resonance imaging (SPRi) technology has been successfully used for the specific detection of bacterial populations during their growth, using proteins and/or antibodies as bio-recognition molecules targeting bacterial surfaces. Thus, the specific detection of one to thousand pathogens/ml could be achieved within few hours. Several bacterial strains were used as models in this work: Streptococcus pneumoniae R6, Escherichia coli K12, as well as a real pathogen (Salmonella enteritidis) providing the proof that this method can be enlarged to other strains. The results can be quantitative by establishing a calibration curve, allowing extrapolation of the initial concentration of a contaminated sample. The strategy developed in this work, is based on the simultaneous coupling of the bacterial enrichment with the specific detection and identification, using the SPR imaging technique. Mots clés en anglais : Surface Plasmon Resonance imaging (SPRi), Streptococcus pneumoniae, E.coli K12, Salmonella enteritidis, bacterial growth, interactions, detection, capture, diagnosis, agri-food, pathogens.

Made available in DSpace on 2016-06-02T19:29:41Z (GMT). No. of bitstreams: 1 3958.pdf: 6641801 bytes, checksum: a9bfe381c8418a0f649ea728b37c8c2d (MD5) Previous issue date: 2011-08-29 / Financiadora de Estudos e Projetos / Madeira River is the fourth major river of the planet considering its discharge, only behind the Amazonas, Congo and Orinoco rivers. It is the main affluent contributing with half of the suspended solids transported by the Amazon River. In the present work a limnological research was carried out in a 300 Km stretch of Middle Madeira River. Two different approaches were adopted in order to characterize physically and chemically the system: A real time monitoring of physical and chemical variables at short time intervals and trimestrally punctual surveys. Seven field expeditions were carried out corresponding to different hydrological periods: decreasing water level, low water, rising water level and high waters. Eight sampling stations were distributed along the 300 km of River Madeira stretch for measurements of physical and chemical variables, plus water and plankton samples collection. Some variables were measured in situ by the Yellowsprings multiparameter probe and water was taken for laboratory suspended matter, nutrient and ion cocentration measurements. In the real time monitoring the measurements were taken each 30 minutes along 423 days in the period between 23/01/2010 and 22/03/2011. The results obtained evidenced the strong influence of the hydrological cycle on the water turbidity, electrical conductivity, dissolved oxygen concentration and pH. Short time pulses starting with abrupt changes in the water level and discharge of River Madeira greatly influenced water turbidity and electrical conductivity. The zooplankton community was composed by 101 taxa, being Rotifera the group of highest species richness (45 species). Copepoda was a group with noticeably high richness of species, being represented by fifteen species, among which some Amazon endemic taxa. Total diversity is moderate compared to other fluvial systems in Brazil. Protozoa was the group that contributed most to the zooplankton biomass particularly in the periods of decreasing water and low water levels of the year 2010. Mean density of zooplankton was 9,714 ind. m-3 and mean biomass was relatively low with a mean value of 14.22 x 103 μg.m-3. The zooplankton community is probably limited by the low primary productivity of this white water river and primarily responds to the wide fluctuaions of the main driving forces of the system, both the seasonal and interannual changes in the river water level. / O rio Madeira gracas as caracteristicas geomorfologicas e climaticas de sua bacia hidrografica apresenta uma intensa atividade hidrologica e biogeoquimica sendo o 4o maior rio do planeta em termos de vazao. Em adicao a sua reconhecida relevancia ecologica e social, o rio Madeira ganhou tambem proeminencia economica a partir dos empreendimentos hidreletricos planejados para o aproveitamento dessas elevadas vazoes. Foram utilizadas duas abordagens distintas para a caracterizacao limnologica espacial e temporal do rio Madeira: (i) o monitoramento sistematico de um conjunto de 51 variaveis fisicas e quimicas e zooplancton amostrados trimestralmente e, (ii) o monitoramento de seis variaveis fisicas e quimicas em tempo real e curtos intervalos de tempo. Para o monitoramento sistematico foram realizadas sete campanhas de campo trimestrais representativas dos periodos de vazante, aguas baixas, enchente, aguas altas em oito estacoes distribuidas ao longo de 300 Km no rio Madeira com amostragem de variaveis fisicas, quimicas e da comunidade zooplanctonica para posterior analise em laboratorio. No monitoramento em tempo real as medidas foram realizadas a cada 30 minutos durante 423 dias, no periodo de 23/01/10 a 22/03/11, preenchendo uma importante lacuna no conhecimento limnologico da dinamica do rio Madeira combinando medicoes continuas e acuradas em curtos intervalos de tempo durante um periodo superior a um ciclo sazonal completo. Os resultados obtidos evidenciaram a forte influencia da sazonalidade e do ciclo hidrologico sobre a turbidez, condutividade, oxigenio dissolvido, pH e solidos dissolvidos. Pulsos de curta duracao iniciados por oscilacoes repentinas nas vazoes e niveis do rio Madeira determinaram variacoes rapidas e expressivas na turbidez, condutividade e solidos dissolvidos. A comunidade zooplanctonica foi constituida por 101 taxons, sendo o grupo Rotifera o de maior riqueza (45 especies). Os Copepoda foram representados por 15 especies entre as quais se destacaram especies endemicas amazonicas. A densidade media do zooplancton foi de 9.714 ind.m-3 e a biomassa apresentou valor medio de 14,22 x 103 μg. m-3, considerado baixo. A comunidade zooplanctonica e provavelmente limitada pelas altas vazoes e baixa produtividade primaria, e assim como outros parametros analisados, responde com amplas flutuacoes a principal funcao de forca do sistema, as variacoes sazonais e interanuais no nivel de agua do rio.

In the global and dynamic business environment of today, Industry 4.0 and the development of digital networking and real-time connection are pushing manufacturing companies to introduce new technologies into their manufacturing processes. IIoT is seen as a key element of Industry 4.0 that enable the virtualization of manufacturing processes that can ensure smooth operations across the supply chain through real-time information collection. While researchers and practitioners alike agree that there are potential advantages of implementing real-time monitoring through IIoT they also uphold that there are critical challenges for adoption. Research shows that few companies have developed the structured strategies necessary to attain a consistent competitive advantage while implementing IIoT into their manufacturing processes. A large reason contributing to this is that there is no established industry standard for how to ensure interoperability, i.e the ability of different computerized products or systems to connect and exchange information with one another. To bridge the gap between theoretical knowledge of IIoT and the practical challenges of its implementation in a manufacturing process, the purpose of the study is to present a framework of the potential challenges and enablers of implementing IIoT for real-time monitoring. To meet the purpose of this study a multiple case study was performed at Volvo Construction Equipment sites in different stages of IIoT implementation. As a result, four categories related to potential challengers and enablers of implementing IIoT for real-time monitoring were identified. These categories were: external environments, IT infrastructure, technology, and human factors. Four project phases of implementing an IIoT platform were also identified, which lays the foundation for the proposed framework. The framework provides a general understanding of how manufacturing companies can implement IIoT to achieve real-time monitoring in practice by highlighting potential challenges and enablers in each project phase. Contributing to current research, this thesis considers the holistic perspective of the research area, i.e a combination of both technical and social aspects, where the resulting framework provides guidance on how to implement IIoT for real-time monitoring. Future research may include confirmation of the framework in practice and performing a quantitative case study to achieve generalized results. / I dagens globala och dynamiska affärsmiljö driver Industri 4.0 och utvecklingen av digitala nätverk och realtidsanslutning tillverkande företag till att introducera ny teknik i sina tillverkningsprocesser. IIoT ses som ett nyckelelement i Industri 4.0 som möjliggör virtualisering av tillverkningsprocesser som kan säkerställa smidig drift över hela försörjningskedjan genom informationsinsamling i realtid. Även om både forskare och praktiker är överens om att det finns potentiella fördelar med att implementera realtidsövervakning genom IIoT, hävdar de också att det finns kritiska utmaningar för adoption. Forskning visar att få företag har utvecklat de strukturerade strategier som krävs för att uppnå en konsekvent konkurrensfördel samtidigt som de implementerar IIoT i sina tillverkningsprocesser. En stor anledning till detta är att det inte finns någon etablerad branschstandard för hur man säkerställer interoperabilitet, det vill säga förmågan hos olika datoriserade produkter eller system att koppla ihop och utbyta information med varandra. dess implementering i en tillverkningsprocess, är syftet med studien att presentera ett ramverk för de potentiella utmaningarna och möjliggörarna för att implementera IIoT för realtidsövervakning. För att uppfylla syftet med denna studie utfördes en multipel fallstudie på Volvo Construction Equipment-anläggningar i olika stadier av IIoT-implementering. Som ett resultat identifierades fyra kategorier relaterade till potentiella utmanare och möjliggörare för implementering av IIoT för realtidsövervakning. Dessa kategorier var: externa faktorer, IT-infrastruktur, teknik och mänskliga faktorer. Fyra projektfaser för att implementera en IIoT-plattform identifierades också, vilket lägger grunden för det föreslagna ramverket. Ramverket ger en allmän förståelse för hur tillverkningsföretag kan implementera IIoT för att uppnå realtidsövervakning i praktiken genom att lyfta fram potentiella utmaningar och möjliggörare i varje projektfas. Som ett bidrag till aktuell forskning tar denna avhandling hänsyn till forskningsområdets helhetsperspektiv, det vill säga en kombination av både tekniska och sociala aspekter, där det resulterande ramverket ger vägledning om hur man implementerar IIoT för realtidsövervakning. Framtida forskning kan innefatta bekräftelse av ramverket i praktiken och utförande av en kvantitativ fallstudie för att uppnå generaliserade resultat.

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