Tesis sobre el tema "Real-Time PCR technique"

The incidence of Squamous Cell Carcinoma is growing in certain populations to the extent that it is now the most common skin lesion in young men and women in high ultraviolet exposure regions such as Queensland. In terms of incidence up to 45% of the Australian population over 40 years of age is thought to possess the precancerous Solar Keratosis lesion and with a small but significant chance of progression into SCC, understanding the genetic events that play a role in this process is essential. The major aims of this study were to analyse whole blood derived samples for DNA aberrations in genes associated with tumour development and cellular maintenance, with the ultimate aim of identifying genes associated with non-melanoma skin cancer development. This study had an explicit emphasis on the mitochondrial genome and nuclear genes that encode for subunits in the mitochondrial regulated energy transducing oxidative phosphorylation pathways. More specifically the first aim of this project was to analyse the NDUFA8, PTCH, NDUFAS, SMOH, SDHD, MMPI2, NDUFV1, EMSI, COXVIIc, and RASAI genes via non-specific fluorophoric Real-Time PCR for genetic aberrations in an affected Solar Keratosis and control cohort. The second aim was to analyse two specific genes, SDHD and MMPI2, for copy number aberrations via Dual-Labelled Probe Real-Time PCR in the same affected Solar Keratosis and control cohort. The third aim was to analyse Mitochondrial DNA Depletion syndrome (MDS) in a chemically exposed RAAF personnel cohort via Dual-Labelled Probe Real-Time PCR. The significance of these studies is in their contribution to the knowledge of the genetic pathways that are malformed in the progression and development of the pre-cancerous skin lesion Solar Keratosis. Furthermore, it would determine whether the genes analysed in this study exist in greater prevalence in the affected Solar Keratosis population compared to the control cohort. With regard to the MDS component, identifying the presence of this disease in these individuals was initially undertaken as part of a study to provide evidence in compensation claims. The diagnosis may assist in their medical therapy, insofar as some of them were now suffering from liver malfunctions and atypical male breast cancer. Another application of this effective and low cost method of diagnosing MDS is in populations with high HTV incidences. This is due to the fact that the most common drug used to treat this disease can give rise to the expression of MDS, thus further complicating the health status of HIV infected individuals. The analysis of this research was accomplished via the Real-Time PCR technique, with a non-specific fluorophore component in addition to specific Dual-Labelled Probe components, to ascertain the general nature of any aberration identified in the sample cohort. This project also employed additional methods of analysis such as DHPLC and DNA sequencing to assist in determining the veracity of its aims, particularly in terms of the precise detection of genetic aberrations via Real-Time PCR. Patients exhibiting male breast cancer and liver malftinctions were also analysed via Dual-Labelled Probe RealTime PCR to ascertain the presence of Mitochondrial DNA Depletion syndrome, a disorder characterised by lactic acidosis, liver failure, seizures, and congestive heart failure. Determining the presence of this syndrome in these patients would assist in their medical treatment, and contribute to the analytical methods available to diagnose this syndrome, which is known to occur in HIV sufferers due to the nucleoside drugs used to combat the disease. Real-Time PCR can adequately gauge the integrity of a genetic area in terms of amplicon malformities (non-specific-fluorophoric) and DNA copy number aberrations (Dual-Labelled Probe) via fluorophore signal differentials compared to wild-type samples and housekeeper profiles. The results of the first component of this project, namely the analysis of five gene pairs by non-specific fluorophoric Real-Time PCR, highlighted that a significantly higher incidence of putative aberrants is evident in the affected population when compared to the control cohort. The genes analysed were NDUFA8, PTCH, NDUFA5, SMOH, SDHD, MMP 12, NDUFVI, EMS 1, COXVIIc, and RASA 1. These ten genes were subdivided into five pairs; one of the pair being a gene associated with the development of a non-melanotic skin cancer (NMSC), the other a gene encoding for a subunit of the Electron Transport Chain (ETC). Each of these pairs exists in close proximity to one another on a particular chromosomal locale. Differences were highlighted in the single gene triplicate run population. The ETC genes (NDUFA8, NDUFA5, SDHD, NIDUFVI, COXVIIc) exhibited 10 / 720 (1.37%) as being putative mutants in the control population, compared to 117 / 675 (17.3%) for the affected population (p value less than 0.0001). The NMSC gene analysis (PTCH, SMOH, MMPI2, EMSI, RASA1) produced a 16 / 720 (2.22%) ratio for the control population, with the affected population having an incidence of 97 / 675 (14.4 %) for putative mutants (p value less than 0.0001). The observance of putative aberrants in the NDUFVI (p less than 0.018), EMS1 (p less than 0.003), COXVTIc (p less than 0.001), and RASA I (p less than 0.009) genes in the affected Solar Keratosis (SK) population was significantly higher than that observed in the control population. The majority of aberrations detected via the non-specific fluorophoric Real-Time PCR technique were small nucleotide base insertions and deletions. The analysis of the SK affected and control cohort via Real-Time PCR proved a cost-effective and reliable method in identifying the presence of DNA aberrations such as non-instructional sites. The results of the second component extended the findings of the non-specific fluorophoric analysis. The SDHD and MMPI 2 genes were analysed for copy number aberrations via Dual-Labelled Probe Real-Time PCR for genetic aberrations the same affected and control Solar Keratosis cohort. It was found that 12 of 279 samples had identifiable copy-number aberrations in either the SDHD or MMPI2 gene (this means that a genetic section of either of these two genes is aberrantly amplified or deleted), with five of the samples exhibiting aberrations in both genes. The MMPI2 gene also had nine samples identified as possessing an intronic heterozygous base-pair substitution anomaly via DNA sequencing. The NDUFA8 gene had 12 samples identified as anomalous via the DHPLC technique, 11 of which were identified via non-specific fluorophoric Real-Time PCR, with the analysis performed to verify the accuracy of the Real-Time technique in identifying DNA aberrations. This study identified DNA aberrations in an affected Solar Keratosis and control cohort and ascertained several particular genomic abnomialities in the SDHD, MMPI2 and NDUFA8 genes, with an emphasis on copy-number aberrations and amplicon abnormalities. In the third component of this study, namely the analysis of Mitochondrial DNA Depletion syndrome (MDS) in a jet-fuel exposed RAAF personnel cohort via Dual-Labelled Probe Real-Time PCR, the results indicated that four of the seven patients were expressing MDS. Of the four patients who exhibited a reduction in mitochondrial copy-number the average decrease was of a four-fold level, or approximately a depletion of mitochondrial copies from 200 plus to ~ 54 (74 % reduction in MtDNA). The patients who contributed DNA for investigation into the presence of MDS were suffering from liver malfunction and atypical male breast cancer. The Dual-Labelled Probe technique proved a reliable and cost effective method in identifying the presence of MDS in these patients, with the DNA extracted from fresh white blood cells that had been isolated using the Ficoll-Hypaque method. The importance of this is that accurate levels of Mitochondrial DNA copy numbers can be ascertained in white blood cells as it removes the presence of platelets, which also contain mitochondria but no nucleus. The analysis of ETC and NMSC associated genes in addition to mitochondrial copy number integrity means that this study investigated two aspects of the carcinogenetic pathway i.e. abnormal energy regulation and the regulation of micromolecular and macromolecular cellular homeostatic mechanisms. The mechanism of programmed cell death or apoptosis is regulated by the mitochondria and the ability of a genetically damaged cell to evade the apoptotic process is directly linked to a cell becoming cancerous. It is only after the evasion of apoptosis and the replication of the damaged cells' DNA into daughter cells that neoplastic events can occur. Thus, this study contributed to the understanding of how neo-plastic lesions may develop and progress into invasive tumours. It additionally assisted in proving the effectiveness of the RealTime PCR technique in detecting DNA aberrations and mitochondrial copy number anomalies.

The incidence of Squamous Cell Carcinoma is growing in certain populations to the extent that it is now the most common skin lesion in young men and women in high ultraviolet exposure regions such as Queensland. In terms of incidence up to 45% of the Australian population over 40 years of age is thought to possess the precancerous Solar Keratosis lesion and with a small but significant chance of progression into SCC, understanding the genetic events that play a role in this process is essential. The major aims of this study were to analyse whole blood derived samples for DNA aberrations in genes associated with tumour development and cellular maintenance, with the ultimate aim of identifying genes associated with non-melanoma skin cancer development. This study had an explicit emphasis on the mitochondrial genome and nuclear genes that encode for subunits in the mitochondrial regulated energy transducing oxidative phosphorylation pathways. More specifically the first aim of this project was to analyse the NDUFA8, PTCH, NDUFAS, SMOH, SDHD, MMPI2, NDUFV1, EMSI, COXVIIc, and RASAI genes via non-specific fluorophoric Real-Time PCR for genetic aberrations in an affected Solar Keratosis and control cohort. The second aim was to analyse two specific genes, SDHD and MMPI2, for copy number aberrations via Dual-Labelled Probe Real-Time PCR in the same affected Solar Keratosis and control cohort. The third aim was to analyse Mitochondrial DNA Depletion syndrome (MDS) in a chemically exposed RAAF personnel cohort via Dual-Labelled Probe Real-Time PCR. The significance of these studies is in their contribution to the knowledge of the genetic pathways that are malformed in the progression and development of the pre-cancerous skin lesion Solar Keratosis. Furthermore, it would determine whether the genes analysed in this study exist in greater prevalence in the affected Solar Keratosis population compared to the control cohort. With regard to the MDS component, identifying the presence of this disease in these individuals was initially undertaken as part of a study to provide evidence in compensation claims. The diagnosis may assist in their medical therapy, insofar as some of them were now suffering from liver malfunctions and atypical male breast cancer. Another application of this effective and low cost method of diagnosing MDS is in populations with high HTV incidences. This is due to the fact that the most common drug used to treat this disease can give rise to the expression of MDS, thus further complicating the health status of HIV infected individuals. The analysis of this research was accomplished via the Real-Time PCR technique, with a non-specific fluorophore component in addition to specific Dual-Labelled Probe components, to ascertain the general nature of any aberration identified in the sample cohort. This project also employed additional methods of analysis such as DHPLC and DNA sequencing to assist in determining the veracity of its aims, particularly in terms of the precise detection of genetic aberrations via Real-Time PCR. Patients exhibiting male breast cancer and liver malftinctions were also analysed via Dual-Labelled Probe RealTime PCR to ascertain the presence of Mitochondrial DNA Depletion syndrome, a disorder characterised by lactic acidosis, liver failure, seizures, and congestive heart failure. Determining the presence of this syndrome in these patients would assist in their medical treatment, and contribute to the analytical methods available to diagnose this syndrome, which is known to occur in HIV sufferers due to the nucleoside drugs used to combat the disease. Real-Time PCR can adequately gauge the integrity of a genetic area in terms of amplicon malformities (non-specific-fluorophoric) and DNA copy number aberrations (Dual-Labelled Probe) via fluorophore signal differentials compared to wild-type samples and housekeeper profiles. The results of the first component of this project, namely the analysis of five gene pairs by non-specific fluorophoric Real-Time PCR, highlighted that a significantly higher incidence of putative aberrants is evident in the affected population when compared to the control cohort. The genes analysed were NDUFA8, PTCH, NDUFA5, SMOH, SDHD, MMP 12, NDUFVI, EMS 1, COXVIIc, and RASA 1. These ten genes were subdivided into five pairs; one of the pair being a gene associated with the development of a non-melanotic skin cancer (NMSC), the other a gene encoding for a subunit of the Electron Transport Chain (ETC). Each of these pairs exists in close proximity to one another on a particular chromosomal locale. Differences were highlighted in the single gene triplicate run population. The ETC genes (NDUFA8, NDUFA5, SDHD, NIDUFVI, COXVIIc) exhibited 10 / 720 (1.37%) as being putative mutants in the control population, compared to 117 / 675 (17.3%) for the affected population (p value less than 0.0001). The NMSC gene analysis (PTCH, SMOH, MMPI2, EMSI, RASA1) produced a 16 / 720 (2.22%) ratio for the control population, with the affected population having an incidence of 97 / 675 (14.4 %) for putative mutants (p value less than 0.0001). The observance of putative aberrants in the NDUFVI (p less than 0.018), EMS1 (p less than 0.003), COXVTIc (p less than 0.001), and RASA I (p less than 0.009) genes in the affected Solar Keratosis (SK) population was significantly higher than that observed in the control population. The majority of aberrations detected via the non-specific fluorophoric Real-Time PCR technique were small nucleotide base insertions and deletions. The analysis of the SK affected and control cohort via Real-Time PCR proved a cost-effective and reliable method in identifying the presence of DNA aberrations such as non-instructional sites. The results of the second component extended the findings of the non-specific fluorophoric analysis. The SDHD and MMPI 2 genes were analysed for copy number aberrations via Dual-Labelled Probe Real-Time PCR for genetic aberrations the same affected and control Solar Keratosis cohort. It was found that 12 of 279 samples had identifiable copy-number aberrations in either the SDHD or MMPI2 gene (this means that a genetic section of either of these two genes is aberrantly amplified or deleted), with five of the samples exhibiting aberrations in both genes. The MMPI2 gene also had nine samples identified as possessing an intronic heterozygous base-pair substitution anomaly via DNA sequencing. The NDUFA8 gene had 12 samples identified as anomalous via the DHPLC technique, 11 of which were identified via non-specific fluorophoric Real-Time PCR, with the analysis performed to verify the accuracy of the Real-Time technique in identifying DNA aberrations. This study identified DNA aberrations in an affected Solar Keratosis and control cohort and ascertained several particular genomic abnomialities in the SDHD, MMPI2 and NDUFA8 genes, with an emphasis on copy-number aberrations and amplicon abnormalities. In the third component of this study, namely the analysis of Mitochondrial DNA Depletion syndrome (MDS) in a jet-fuel exposed RAAF personnel cohort via Dual-Labelled Probe Real-Time PCR, the results indicated that four of the seven patients were expressing MDS. Of the four patients who exhibited a reduction in mitochondrial copy-number the average decrease was of a four-fold level, or approximately a depletion of mitochondrial copies from 200 plus to ~ 54 (74 % reduction in MtDNA). The patients who contributed DNA for investigation into the presence of MDS were suffering from liver malfunction and atypical male breast cancer. The Dual-Labelled Probe technique proved a reliable and cost effective method in identifying the presence of MDS in these patients, with the DNA extracted from fresh white blood cells that had been isolated using the Ficoll-Hypaque method. The importance of this is that accurate levels of Mitochondrial DNA copy numbers can be ascertained in white blood cells as it removes the presence of platelets, which also contain mitochondria but no nucleus. The analysis of ETC and NMSC associated genes in addition to mitochondrial copy number integrity means that this study investigated two aspects of the carcinogenetic pathway i.e. abnormal energy regulation and the regulation of micromolecular and macromolecular cellular homeostatic mechanisms. The mechanism of programmed cell death or apoptosis is regulated by the mitochondria and the ability of a genetically damaged cell to evade the apoptotic process is directly linked to a cell becoming cancerous. It is only after the evasion of apoptosis and the replication of the damaged cells' DNA into daughter cells that neoplastic events can occur. Thus, this study contributed to the understanding of how neo-plastic lesions may develop and progress into invasive tumours. It additionally assisted in proving the effectiveness of the RealTime PCR technique in detecting DNA aberrations and mitochondrial copy number anomalies.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Biomolecular and Biomedical Sciences
Full Text

The transmission of human pathogens by faecally contaminated fruit and vegetables is well established, but the burden of disease caused by foodborne pathogens is unknown. Fresh produce can be contaminated through the use of polluted irrigation water or by the handling of the produce by infected individuals either pre- or post harvest. There is very little known regarding the extent of viral contamination of irrigation water and fresh produce in South Africa. Noroviruses (NoV) and hepatitis A virus (HAV) are recognized as leading causes of foodborne viral disease. These viruses are transmitted predominantly via the faecal–oral route, primarily person-to-person by direct contact with an infected person, or indirectly by ingestion of contaminated food and water. The detection of enteric viruses in food or water is problematical and complex as many foodborne viruses, including HAV and NoV, cannot be readily isolated in cell culture. The aim of this investigation was to develop and optimise simple and efficient methods for the concentration and detection of NoV GII and HAV in irrigation water and fresh produce. These methods would then be applied to field samples of irrigation water and fresh produce to try and establish a link between viral contamination detected in irrigation water and that on associated irrigated fresh produce. The efficiency of different commercial real-time reverse transcriptase-polymerase chain reaction amplification kits for the realtime detection of HAV, NoV GI and NoV GII was assessed, and standard curves for the quantitative detection of these viruses were constructed using the most appropriate kit. Using two types of fresh produce, three different elution buffers, each at two pHs, with two different elution times were compared to establish which buffer was the most efficient for the extraction of viruses from the fresh produce. The tris-glycine beef extract buffer (pH 9.5) with an elution time of 20 minutes most efficient for the extraction of the selected enteric viruses from fresh produce. From April 2008 to November 2009, 86 irrigation water and 72 fresh produce samples were collected from commercial and subsistence farms, street vendors and commercial outlets. All the irrigation water and fresh produce samples were analysed for HAV, NoV GI and NoV GII. Overall, 16.3 % (13/86) and 12.5 % (9/72) of irrigation water and fresh produce samples tested positive for one or more human pathogenic viruses, namely NoV GII and HAV, respectively. Nucleotide sequence and phylogenetic analysis of the HAV and NoV GII strains identified clinically relevant viruses in the irrigation water and on the fresh produce. A direct link between contaminated irrigation water and contamination of fresh produce could not be established, but irrigation water was identified as a possible source of contamination of the fresh produce. The results also suggested that food handlers contributed significantly to the viral contamination of the fresh produce. This study highlights the potential health risk posed by fresh produce to consumers in South Africa and highlights the need for further in depth studies to quantify the risk to consumers. This study represents new data on the occurrence of enteric viruses in food and water in South Africa and is crucial for the development of effective intervention and control strategies for food safety in South Africa. Copyright
Dissertation (MSc)--University of Pretoria, 2011.
Medical Virology
unrestricted

There is a significant need to identify approaches for classifying chemical sensor array data with high success rates that would enhance sensor detection capabilities. The present study attempts to fill this need by investigating six machine learning methods to classify a dataset collected using a chemical sensor array: K-Nearest Neighbor (KNN), Support Vector Machine (SVM), Classification and Regression Trees (CART), Random Forest (RF), Naïve Bayes Classifier (NB), and Principal Component Regression (PCR). A total of 10 predictors that are associated with the response from 10 sensor channels are used to train and test the classifiers. A training dataset of 4 classes containing 136 samples is used to build the classifiers, and a dataset of 4 classes with 56 samples is used for testing. The results generated with the six different methods are compared and discussed. The RF, CART, and KNN are found to have success rates greater than 90%, and to outperform the other methods.

Thesis (Ph. D.)--Ohio State University, 2005.
Title from first page of PDF file. Document formatted into pages; contains xviii, 131 p.; also includes graphics. Includes bibliographical references (p. 120-131). Available online via OhioLINK's ETD Center

Salmonellae are enteric bacteria infecting animals and humans. Large animal clinics and Veterinary Teaching Hospitals are greatly affected by Salmonella outbreaks and nosocomial infection. The risk of environmental contamination and spread of infection is increased when animals are confined in close contact with each other and subjected to increased stress factors. This study was designed to compare double-enrichment culture techniques with Gel-based and Real-time PCR assays in the quest for improved diagnostic methods for detecting Salmonella in equine fecal samples. 120 fecal samples submitted to the Clinical Microbiology Laboratory of the Veterinary Medical Teaching Hospital at Texas A&M University (CML, VMTH, TAMU) were tested for Salmonella using all three techniques. Double-enrichment bacterial culture detected 29 positive results (24%), Real-time PCR detected 33 positive results (27.5%), and Gel-based PCR detected 73 positives results (60.8%). While culture and real-time PCR methods had similar results, the gel-based PCR method detected many more positive results, indicating probable amplicon contamination. Real-time PCR can be completed as soon as the day after submission while culture techniques may take 2 to 5 days to complete. However, viable bacterial cells are needed for antimicrobial susceptibility testing and serotyping: both important for epidemiological studies. Therefore, double-enrichment bacterial culture performed concurrently with real-time PCR methods could be efficient in clinical settings where both accurate and expedient results are required.

L'installation de systèmes de mesure est d'une utilisation cruciale pour la gestion des réseaux d'assainissement ou des canaux d 'irrigation. La plupart des structures gouvernementales ou privées ainsi que les agglomérations s'équipent de systèmes de mesure de débit afin de se conformer avec la législation européenne. La plupart des débitmètres fournissent des données en temps réel i.e. l'information est transmise en permanence. aux centrales d'acquisition pour une gestion de l'architecture du système de canaux. La mesure en canaux ouverts est souvent ultrasonore. L'objectif de cette thèse est de proposer une méthode en temps réel afin de corréler les vitesses locales en une vitesse moyenne dans les conditions observables par les utilisateurs en canaux ouverts. Les thématiques impliquées à cette étude sont multiples: les techniques de mesure, l'hydrodynamique en canaux ouverts représentée par la turbulence (ici plus particulièrement les courants secondaires), les lois de paroi, le nombre de Froude ... l'ensemble de ces thématiques doit être investi en canaux pleinement développés où les conditions sont stables dans l'espace mais aussi pour des conditions perturbées telles que les structures hétérogènes ou transitoires.La technique de mesure est un point clé: quelle est la technique la plus applicable aux conditions de mesure i.e. les canaux étroits? Les canaux étroits varient très rapidement en tem1es de taux de remplissage : la technique la plus adaptée est le profileur ultrasonique.La compréhension des effets hydrodynamiques est essentielle afin de développer un modèle de conversion. Les canaux droits sont influencés par l'hydrodynamique des écoulements, la géométrie mais aussi et principalement par leurs interactions. En canaux droits, les courants secondaires sont primordiaux même s'ils se traduisent par un effet le plus observable : le dip-phénomène, i.e. la présence d'un maximum de vitesse non pas à la surface d'eau mais en dessous pour les canaux étroits. Ces courants secondaires sont fortement sensibles au rapport d'aspect, la géométrie et la variabilité de la rugosité le long de la paroi, passablement sensible à la rugosité et indépendant du nombre de Froude .Les perturbations, à l'aval desquelles sont installés les débitmètres ultrasonores, sont majoritairement représentées par les coudes et les jonctions. Dans les coudes, les tourbillons sont liés aux forces centrifuges (gros tourbillon) et la turbulence (petit tourbillon). Pour les jonctions, les tourbillons diffèrent des deux précédentes configurations avec la présence à l'aval de la jonction de 3 tourbillons (due à un étirement des tourbillons par l'arrivée latérale). Les capteurs ne sont pas installés directement au niveau de la perturbation mais à l'aval. Dans la littérature, les distances requises pour retrouver des conditions proches de l'écoulement pleinement développé devraient excéder environ 50 hauteurs d'eau. En pratique, ces distances sont plus proches de5-10 fois la largeur du canal ou du tirant d'eau. L'application de modèle basée sur l'écoulement pleinement développé corrélé à un capteur n'est pas recommandable
The installation of flow rate measurement systems is an important factor in regard to the management of sewer and irrigation networks. Most cities and infrastructure succeed in obtaining sufficient flow measurements to satisfy European Regulation rules. Most flow meters comprise real time systems; this means that the information is permanently transferred to a data base for the management and optimization of the particular network. The measurement technology deployed is typically ultrasound based. Within the number of measurement points a high percentage are often deficient and create specific difficulties (>75% of Venturi flumes are inaccurate according to Anglian Water, a UK water and wastewater company). The study presented here focuses on flow meters which calculate discharge using measurement of level, cross sectional area and the correlation of local velocity to generate a mean value. The aim of this thesis is to propose a real time method to enable determination of this “conversion” under realistic configurations which Users find in open channels. The synthesis of measurement points through an understanding of hydraulic conditions (Bonakdari, 2006) provides a method to create flow data allowing local point velocities to be converted into an overall mean value. The approach has limitations and may fail in industrial situations but can be used for very complex configurations. It also requires specialists with knowledge of the technique who are rarely available to Users. What is proposed here is an alternative method to Bonakdari for simpler configurations. The aim is to evaluate the flow rate with acceptable accuracy using these technics and to establish a relationship between local velocities and the mean velocity according to Regulatory requirements (8% are required in UK, 5 to 8% in Germany depending on area). The individual components are here: the measurement techniques; the hydrodynamics represented with the turbulence (secondary currents in open channels); the wall / roughness effects; the Froude number … for fully developed conditions where conditions become stable in space but for disturbed conditions, as well such as heterogeneous structures or transition conditions

Brazil is the largest citrus producer in the world and has a large global citrus market share. However, several diseases affect the crop, being postbloom fruit drop (PFD) one of them. PFD has gained importance in São Paulo State for the displacement of citrus areas to regions with weather conditions more favorable for this disease. The accurate identification of the causal agent of the PFD has been performed and it was renamed as Colletotrichum abscissum. The origin of the initial inoculum is still an enigma for PFD epidemics and the hypotheses that the initial inoculum could be present in propagation material have been discussed but it has never been demonstrated. The objective of this work was to detect and quantify Colletotrichum abscissum from citrus leaves of budwood increase block and citrus nursery plants by qPCR. Four commercial citrus farms from São Paulo State, Brazil with budwood increase block and citrus nursery plants of Pera and Valencia sweet orange varieties were used for this work. C. abscissum was detected in budwood increase block and in nursery plant in both varieties (Valencia and Pera) at the four farms sampled. Out of 122 budwood increase block samples, 89 (73%) were positive for C. absicissum. From nursery plants, out of 175 samples, 129 (73%) were detected with the pathogen. The majority of the positive samples of budwood increase blocks and nursery plants contained 10 to 200 and 10 to 400 conidia of C. absicissum, respectively. With the methods used was not possible to isolate the fungus from vegetative material. This finding suggests a new long distances dispersion type of C. abscissum in the cycle of postbloom fruit drop by propagation material. Confirmation of C. abscissum in budwood increase block and nursery plants would lead to update regulations for the production of certified citrus nursery trees and searching for new control strategies of the pathogen.
O Brasil é o maior produtor de citros do mundo e possui uma grande participação no mercado global de citros. No entanto, várias doenças afetam a cultura, sendo uma delas a podridão floral dos citros (PFC). PFC ganhou importância no Estado de São Paulo pelo deslocamento de áreas de citros para regiões com condições climáticas mais favoráveis para a doença. A identificação precisa do agente causal do PFC foi realizada, tendo sido renomeado como Colletotrichum abscissum. A origem do inóculo inicial ainda é um enigma para as epidemias de PFC e as hipóteses do que o inóculo inicial poderia estar presente no material de propagação já foram discutidas, mas nunca foram demonstradas. O objetivo deste trabalho foi detectar e quantificar Colletotrichum abscissum em folhas de borbulheiras e mudas de citros por meio de qPCR. Neste trabalho, foram utilizadas quatro fazendas comerciais de citros do Estado de São Paulo, Brasil, com borbulheiras e viveiros de mudas de citros das variedades laranja Pera e Valência. C. abscissum foi detectado em borbulheiras e em mudas em ambas as variedades (Valência e Pêra) nas quatro fazendas amostradas. Das 122 amostras de folhas de borbulheiras, 89 (73%) foram positivas para C. absicissum. Das 175 amostras de folhas de mudas de citros, 129 (73%) foram detectadas com o patógeno. A maioria das amostras positivas de borbulheiras e mudas de citros continham 10 a 200 e 10 a 400 conídios de C. absicissum, respectivamente. Com os métodos utilizados, não foi possível isolar o fungo do material vegetativo. Esta descoberta sugere um novo tipo de dispersão a longas distâncias de C. abscissum no ciclo de podridão floral dos citros por meio do material de propagação. A confirmação de C. abscissum nas borbulheiras e mudas de citros levaria à atualização da regulamentação para a produção de mudas de citros certificadas e à busca de novas estratégias de controle do patógeno.

Infectious myonecrosis, caused by infectious myonecrosis virus (IMNV), is an important disease of shrimp that has adversely affected the production of cultured Litopenaeus vannamei. The studies reported here were centered on development and/or validation of alternative diagnostic methods for detection of IMNV. Hence, two manuscripts were published in the Journal of Aquaculture and one manuscript was published in the Journal of Fish Diseases. Chapter 2 describes the development of a real-time reverse transcription polymerase chain reaction (real-time RT-PCR) method using TaqMan probe. The results showed that this real-time RT-PCR assay can detect as little as 10 IMNV copies/μl RNA, while the nested RT-PCR can detect 1000 copies/μl RNA. These findings suggest that the TaqMan real-time RT-PCR is “the gold standard” for screening shrimp to protect aquaculture production systems from losses caused by IMNV, because it provides quantification, higher sensitivity and specificity, and because it is less time consuming and less prone to contamination compared to conventional gel-based RT-PCR. In Chapter 3 I evaluated if prolonged storage of infectious myonecrosis virus (IMNV) infected shrimp in Davidson’s AFA fixative can degrade its double-stranded RNA genome resulting in false negative ISH reactions. Shrimp were collected at Day 12 post-injection and fixed in Davidson’s AFA for five different preservation times (1, 2, 4, 7 and 10 days). Hence, in the present report it was found that the length of time (up to 10 days) in Davidson’s AFA did not have a deleterious effect on the ISH reaction for IMNV. The Chapter 4 describes the development of a reverse transcription loop-mediated isothermal amplification and nucleic acid lateral flow (RT-LAMP-NALF) for detection of IMNV. The RT-LAMP-NALF method combines simplified nucleic acid extraction, a reverse-transcription loop-mediated isothermal amplification platform, and one-step visual colorimetric confirmation of the IMNV amplified sequences using a generic NALF qualitative detection test strip. The RT-LAMP-NALF was found to be 100 and 10 times more sensitive than one-step RT-PCR and RT-LAMP (two primer pairs), respectively. These results clearly demonstrate that the RT-LAMP-NALF method is specific, sensitive, can shorten the time for analysis, and has potential application for IMNV diagnosis in resource-poor diagnostic settings.

In recent years the increase of global plant trade and human movement has promoted the risk of introduction of invasive plants and exotic pathogens. Biological invasions operate globally and are considered to be the second cause of biodiversity loss after direct habitat alteration and destruction. In this context, Phytophthora is one of the most important and aggressive plant pathogen in agriculture and forestry. Early detection and identification of its pathways are of high importance to minimize the threat that they pose to natural ecosystems. Different molecular-based methods, including real-time PCR and Next Generation Sequencing, have been developed and applied for the detection of plant pathogens in environmental samples. These methods allow fast and accurate pathogen detection and identification even when the inoculum amount is low. Therefore, the main objective of this thesis was the development of a new method for Phytophthora detection in environmental samples starting from extraction of environmental DNA (eDNA) from different sources (soil, roots and water) and different ecosystems. Different studies have applied High Throughput Sequencing (HTS) for the detection of Phytophthora species in soil samples, but not, to date, for water. In the Chapter 3, genus-specific primers were adapted to assess Phytophthora species diversity in natural ecosystems using high-throughput amplicon pyrosequencing of eDNA from soil and water environments, based in the polymorphic and widely accepted barcoding target Internal Transcribed Spacer 1 (ITS1). The assay was validated with a control reaction with DNA of pure cultures. The objectives raised and developed of this study were: a) as main objective, development and application of HTS (High Throughput Sequencing) of Phytophthora-specific PCR amplicons to investigate the presence of Phytophthora in soil samples from different plant communities in natural forests, plantations and aquatic environments in the north of Spain; b) optimization of the conditions for emPCR amplification in order to obtain the best results in the pyrosequencing run; c) development of a bioinformatics pipeline for NGS data, focusing in the optimization of a barcoding threshold value to separate Molecular Operational Taxonomic Units (MOTUs). Different score coverage threshold values were tested for optimal Phytophthora species separation in the bioinformatics analyses. Clustering at 99 % was the best criteria to separate most of the Phytophthora species. Multiple Molecular Operational Taxonomic Units (MOTUs) corresponding to 36 distinct Phytophthora species were amplified in the environmental samples. Pyrosequencing of amplicons from soil samples revealed low Phytophthora diversity (13 species) in comparison with the 35 species detected in water samples. Thirteen of the MOTUs detected in rivers and streams did not show significant matches to sequences in international sequence databases, revealing that eDNA pyrosequencing is a useful strategy to assess Phytophthora species diversity in natural ecosystems. Once the technique was developed and validated, another objective was proposed in Chapter 2, focused on the oak decline. The evergreen holm oak (Quercus ilex) is the most representative tree species in the Iberian Peninsula and the main tree in oak-rangeland ecosystems (dehesas). Oak decline in non-calcareous soils in south-western Spain has been associated with Phytophthora cinnamomi for decades. However, other Phytophthora species such as P. quercina and P. psychrophila have been associated with Quercus decline in the eastern part of Spain where calcareous soils are predominant. With the aim of investigating the involvement of Phytophthora spp. in oak decline in eastern Spain, two forests in different geographical areas (Alcoi and Vallivana) were selected as sampling sites. Soil and root samples were analysed in parallel by amplicon pyrosequencing and real-time PCR. Metabarcoding analyses showed Phytophthora
En los últimos años, el aumento del comercio mundial de plantas y el movimiento humano ha promovido el riesgo de introducción de plantas invasoras y patógenos exóticos. Las invasiones biológicas operan a nivel mundial y se consideran como la segunda causa de pérdida de biodiversidad después de la alteración y destrucción directas del hábitat. En este contexto, Phytophthora es uno de los patógenos vegetales más importantes y agresivos en la agricultura y la silvicultura. La detección temprana y la identificación de sus vías son de gran importancia para minimizar la amenaza que representan para los ecosistemas naturales. Se han desarrollado y aplicado diferentes métodos moleculares para la detección de patógenos de plantas en muestras ambientales. Estos métodos permiten una detección e identificación de patógenos rápida y precisa incluso cuando la cantidad de inóculo es baja. Por lo tanto, se propone un nuevo método mejorado para su detección en muestras ambientales a partir de la extracción de ADN ambiental (eDNA) de diferentes fuentes (suelo, raíces y agua) y diferentes ecosistemas. El objetivo del primer capítulo fue aplicar HTS (High Throughput Sequencing) para investigar la presencia de Phytophthora en diferentes comunidades de plantas en bosques naturales, plantaciones y ambientes acuáticos en el norte de España. El eDNA se extrajo del suelo y del agua de los ríos y arroyos de los bosques de Fagus sylvatica y Abies alba y de plantaciones de Chamaecyparis lawsoniana y Pseudotsuga menziesii en el norte de España (bosque de Irati y Villanúa). Se diseñó y aplicó un ensayo específico para la detección de Phytophthora mediante la secuenciación masiva de amplicones basado en la región ITS1. Diferentes valores de threshold se analizaron para la separación óptima de especies de Phytophthora en los análisis bioinformáticos. El agrupamiento al 99% fue el mejor criterio para separar la mayor parte de las especies de Phytophthora. Múltiples Unidades Operacionales Taxonómicas Moleculares (MOTU) correspondientes a 36 especies distintas de Phytophthora se amplificaron en las muestras ambientales. La pirosequenciación de amplicones de muestras de suelo reveló una diversidad baja de Phytophthora (13 especies) en comparación con las 35 especies detectadas en muestras de agua. Trece de los MOTU detectados en los ríos y arroyos no mostraron homología con secuencias depositadas en las bases de datos, lo que revela que la pirosequenciación del ADN ambiental es una estrategia útil para evaluar la diversidad de especies Phytophthora en los ecosistemas naturales. Una vez que la técnica fue desarrollada y validada, se propuso otro objetivo enfocado en el decaimiento de la carrasca. La carrasca (Quercus ilex) es la especie arbórea más representativa de la Península Ibérica y el árbol principal de las dehesas. El decaimiento de la carrasca en suelos no calcáreos en el suroeste de España se ha asociado con Phytophthora cinnamomi durante décadas. Sin embargo, otras especies de Phytophthora como P. quercina y P. psychrophila se han asociado con el declive de Quercus en la parte oriental de España donde predominan los suelos calcáreos. Con el objetivo de investigar la implicación de Phytophthora spp. en el declive de la carrasca en el este de España, se seleccionaron dos bosques en diferentes zonas geográficas (Alcoi y Vallivana) como lugares de muestreo. Las muestras de suelo y raíz se analizaron por pirosequenciación de amplicones. Los resultados de la secuenciación masiva mostraron la diversidad de especies de Phytophthora, y reveló que un taxón nunca aislado de Phytophthora, llamado provisional Phytophthora taxon ballota, fue la especie predominante en ambas áreas. Además, se desarrolló un ensayo de PCR a tiempo real, basado en los resultados de la pirosequenciación, para la detección de este taxón de Phytophthora nunca aislado, y también para la detección de P. quercina
En els últims anys, l'augment del comerç mundial de plantes i el moviment humà ha promogut el risc d'introducció de plantes invasores i patògens exòtics. Les invasions biològiques operen a nivell mundial i es consideren de com la segona causa de pèrdua de biodiversitat després de l'alteració i destrucció directa de l'hàbitat. En aquest context, Phytophthora és un dels mes importants patògens vegetals i agressius en l'agricultura i la silvicultura. La detecció primerenca i la identificació de les seves vies resulten de gran importància per a minimitzar l'amenaça que representen per als ecosistemes naturals. S'han desenvolupat i aplicat diferents mètodes moleculars per a la detecció de patògens de plantes en mostres ambientals. Aquests mètodes permeten una detecció i identificació de patògens ràpida i precisa fins i tot quan la quantitat d'inòcul és baixa. Per tant, es proposa un nou mètode millorat per a la seva detecció en mostres ambientals a partir de l'extracció d'ADN ambiental (eDNA) de diferents fonts (sòl, arrels i aigua) i diferents ecosistemes. L'objectiu del primer capítol va ser aplicar HTS (High Throughput Sequencing) per investigar la presència de Phytophthora en diferents comunitats de plantes en boscos naturals, plantacions i ambients aquàtics al nord d'Espanya. L'eDNA es va extraure del sòl i de l'aigua dels rius i rierols dels boscos de Fagus sylvatica i Abies alba i de plantacions de Chamaecyparis lawsoniana i Pseudotsuga menziesii al nord d'Espanya (bosc d'Irati i Villanúa). Es va dissenyar i va aplicar un assaig específic per a la detecció de Phytophthora mitjançant la seqüenciació massiva de amplicons basat en la regió ITS1. Diferents valors de threshold es van analitzar per a la separació òptima d'espècies de Phytophthora en les anàlisis bioinformàtics. L'agrupament al 99% va ser el millor criteri per separar la major part de les espècies de Phytophthora. Múltiples Unitats Operacionals Taxonòmiques Moleculars (MOTU) corresponents a 36 espècies diferents de Phytophthora es van amplificar en les mostres ambientals. La piroseqüenciació d'amplicons de mostres de sòl va revelar una diversitat baixa de Phytophthora (13 espècies) en comparació amb les 35 espècies detectades en mostres d'aigua. Tretze dels MOTU detectats en els rius i rierols no van mostrar homologia amb seqüències dipositades en les bases de dades, el que revela que la pirosequenciació de l'ADN ambiental és una estratègia útil per avaluar la diversitat d'espècies de Phytophthora en els ecosistemes naturals. Una vegada que la tècnica va ser desenvolupada i validada, es va proposar un altre objectiu enfocat en el decaïment de la carrasca. La carrasca (Quercus ilex) és l'espècie arbòria més representativa de la Península Ibèrica i l'arbre principal de les deveses. El decaïment de la carrasca en sòls no calcaris al sud-oest d'Espanya s'ha associat amb Phytophthora cinnamomi durant dècades. No obstant això, altres espècies de Phytophthora com P. quercina i P. psychrophila s'han associat amb el declivi de Quercus a la part oriental d'Espanya on predominen els sòls calcaris. Amb l'objectiu d'investigar la implicació de Phytophthora spp. en el declivi de la carrasca a l'est d'Espanya, es van seleccionar dos boscos en diferents zones geogràfiques (Alcoi i Vallivana) com a llocs de mostreig. Les mostres de sòl i arrel es van analitzar per piroseqüenciació d'amplicons. Els resultats de la seqüenciació massiva van mostrar la diversitat d'espècies de Phytophthora, i va revelar que un taxó mai aïllat de Phytophthora, anomenat de forma provisional Phytophthora taxon ballota, va ser l'espècie predominant en les dues àrees. A més, es va desenvolupar un assaig de PCR a temps real, basat en els resultats de la piroseqüenciació, per a la detecció d'aquest taxó de Phytophthora mai aïllat, i també per a la detecció de P. quercina. Els assajos de qPCR es van aplicar en mo
Catalá García, S. (2017). Development of DNA massive sequencing techniques and Real-Time PCR for the detection, identification and quantitation of Phytophthora spp. in environmental samples [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/90644
TESIS

La presencia de microorganismos patógenos en alimentos es uno de los problemas esenciales en salud pública, y las enfermedades producidas por los mismos es una de las causas más importantes de enfermedad. Por tanto, la aplicación de controles microbiológicos dentro de los programas de aseguramiento de la calidad es una premisa para minimizar el riesgo de infección de los consumidores. Los métodos microbiológicos clásicos requieren, en general, el uso de pre-enriquecimientos no-selectivos,
enriquecimientos selectivos, aislamiento en medios selectivos y la confirmación posterior usando pruebas basadas en la morfología, bioquímica y serología propias de cada uno de los microorganismos objeto de estudio. Por lo tanto, estos métodos son laboriosos, requieren un largo proceso para obtener resultados definitivos y, además, no siempre pueden realizarse. Para solucionar estos inconvenientes se han desarrollado diversas metodologías alternativas para la detección identificación y cuantificación de microorganismos patógenos de origen alimentario, entre las que destacan los métodos
inmunológicos y moleculares. En esta última categoría, la técnica basada en la reacción en cadena de la polimerasa (PCR) se ha convertido en la técnica diagnóstica más popular en microbiología, y recientemente, la introducción de una mejora de ésta, la PCR a tiempo real, ha producido una segunda revolución en la metodología diagnóstica molecular, como pude observarse por el número creciente de publicaciones científicas y la aparición continua de nuevos kits comerciales. La PCR a tiempo real es una
técnica altamente sensible -detección de hasta una molécula- que permite la cuantificación exacta de secuencias de ADN específicas de microorganismos patógenos de origen alimentario. Además, otras ventajas que favorecen su implantación potencial en laboratorios de análisis de alimentos son su rapidez, sencillez y el formato en tubo cerrado que puede evitar contaminaciones post-PCR y favorece la automatización y un alto rendimiento.
En este trabajo se han desarrollado técnicas moleculares (PCR y NASBA) sensibles y fiables para la detección, identificación y cuantificación de bacterias patogénicas de origen alimentario (Listeria spp., Mycobacterium avium subsp. paratuberculosis y Salmonella spp.). En concreto, se han diseñado y optimizado métodos basados en la técnica de PCR a tiempo real para cada uno de estos agentes: L. monocytogenes, L. innocua, Listeria spp. M. avium subsp. paratuberculosis, y también se ha optimizado y
evaluado en diferentes centros un método previamente desarrollado para Salmonella spp. Además, se ha diseñado y optimizado un método basado en la técnica NASBA para la detección específica de M. avium subsp. paratuberculosis. También se evaluó la aplicación potencial de la técnica NASBA para la detección específica de formas viables de este microorganismo.
Todos los métodos presentaron una especificidad del 100 % con una sensibilidad adecuada para su aplicación potencial a muestras reales de alimentos. Además, se han desarrollado y evaluado procedimientos de preparación de las muestras en productos cárnicos, productos pesqueros, leche y agua. De esta manera se han desarrollado métodos basados en la PCR a tiempo real totalmente específicos y altamente sensibles para la determinación cuantitativa de L. monocytogenes en productos
cárnicos y en salmón y productos derivados como el salmón ahumado y de M. avium subsp. paratuberculosis en muestras de agua y leche. Además este último método ha sido también aplicado para evaluar la presencia de este microorganismo en el intestino de pacientes con la enfermedad de Crohn's, a partir de biopsias obtenidas de colonoscopia de voluntarios afectados.
En conclusión, este estudio presenta ensayos moleculares selectivos y sensibles para la detección de patógenos en alimentos (Listeria spp., Mycobacterium avium subsp. paratuberculosis) y para una rápida e inambigua identificación de Salmonella spp. La exactitud relativa de los ensayos ha sido excelente, si se comparan con los métodos microbiológicos de referencia y pueden serusados para la cuantificación de tanto ADN genómico como de suspensiones celulares. Por otro lado, la combinación con tratamientos de preamplificación ha resultado ser de gran eficiencia para el análisis de las bacterias objeto de estudio. Por tanto, pueden constituir una estrategia útil para la detección rápida y sensible de patógenos en alimentos y deberían ser una herramienta adicional al rango de herramientas diagnósticas disponibles para el estudio de patógenos de origen alimentario.
The presence of pathogens in foods is among the most serious public health concerns, and the diseases produced by them are a major cause of morbidity. Consequently, the application of microbiological control within the quality assessment programs in the food industry is a premise to minimize the risk of infection for the consumer. Classical microbiological methods involve, in general, the use of a non-selective pre-enrichment, selective enrichment, isolation on selective media, and subsequent confirmation using morphological, biochemical and/or serological tests. Thus, they are laborious, time consuming and not always reliable (e.g. in viable but non-culturable VBNC forms). A number of alternative, rapid and sensitive methods for the detection, identification and quantification of foodborne pathogens have been developed to overcome these drawbacks. PCR has become the most popular microbiological diagnostic method, and recently, the introduction of a development of this technique, RTi-PCR, has produced a second revolution in the molecular diagnostic methodology in microbiology. RTi-PCR is highly sensitive and specific. Moreover, it allows accurate quantification of the bacterial target DNA. Main advantages of RTi-PCR for its application in diagnostic laboratories include quickness, simplicity, the closed-tube format that avoids risks of carryover contaminations and the possibility of high throughput and automation.
In this work, specific, sensitive and reliable analytical methods based on molecular techniques (PCR and NASBA) were developed for the detection, identification and quantification of foodborne pathogens (Listeria spp., Mycobacterium avium subsp. paratuberculosis and Salmonella spp.). Real-time PCR based methods were designed and optimised for each one of these target bacteria: L. monocytogenes, L. innocua, Listeria spp. M. avium subsp. paratuberculosis, and also a real-time PCR based
method previously described for Salmonella spp. was optimised and multicenter evaluated. In addition, an NASBA-based method was designed and optimised for the specific detection of M. avium subsp. paratuberculosis. The potential application of the NASBA technique for specific detection of viable M. avium subsp. paratuberculosis cells was also evaluated.
All the amplification-based methods were 100 % specific and the sensitivity achieved proved to be fully suitable for further application in real food samples. Furthermore, specific pre-amplification procedures were developed and evaluated on meat
products, seafood products, milk and water samples. Thus, fully specific and highly sensitive real-time PCR-based methods were developed for quantitative detection of L. monocytogenes on meat and meat products and on salmon and cold smoked salmon products; and for quantitative detection of M. avium subsp. paratuberculosis on water and milk samples. The M. avium subsp. paratuberculosis-specific real-time PCR-based method was also applied to evaluate the presence of this bacterium in the bowel
of Crohn's disease patients using colonic biopsy specimens form affected and unaffected volunteers. In addition, fully specific and highly sensitive real-time NASBA-based methods were developed for detection of M. avium subsp. paratuberculosis on water and milk samples.
In conclusion, this study reports selective and sensitive amplification-based assays for the quantitative detection of foodborne pathogens (Listeria spp., Mycobacterium avium subsp. paratuberculosis and) and for a quick and unambiguously identification of Salmonella spp. The assays had an excellent relative accuracy compared to microbiological reference methods and can be used for quantification of genomic DNA and also cell suspensions. Besides, in combination with sample pre-amplification treatments,
they work with high efficiency for the quantitative analysis of the target bacteria. Thus, they could be a useful strategy for a quick and sensitive detection of foodborne pathogens in food products and which should be a useful addition to the range of diagnostic tools available for the study of these pathogens.

Numerical simulation, the use of computers to run a program which implements a mathematical model for a physical system, is an important part of today technological world. It is required in many scientific and engineering fields to study the behaviour of systems whose mathematical models are too complex to provide analytical solutions and it makes virtual evaluation of systems responses possible (virtual twins). This drastically reduces the number of experimental tests for accurate designs of the real system that the numerical model represents. However these virtual twins, based on classical methods which make use of a rich representations of the system (ex. finite element method), rarely allows real-time feedback, even when considering high performance computing, operating on powerful platforms. In these circumstances, the real-time performance required in some applications are compromised. Indeed the virtual twins are static, that is, they are used in the design of complex systems and their components, but they are not expected to accommodate or assimilate data so as to define dynamic data-driven application systems. Moreover significant deviations between the observed response and the one predicted by the model are usually noticed due to inaccuracy in the employed models, in the determination of the model parameters or in their time evolution. In this thesis we propose different methods to solve these handicaps in order to perform real-time monitoring and control. In the first part Model Order Reduction (MOR) techniques are used to accommodate real-time constraints; they compute a good approximation of the solution by simplifying the solution procedure instead of the model. The accuracy of the predicted solution is not compromised and efficient simulations can be performed (digital twins). In the second part Data-Driven modelling are employed to fill the gap between the parametric solution computed by using non-intrusive MOR techniques and the measured fields, in order to make dynamic data-driven application systems, DDDAS, possible (Hybrid Twins).
La simulación numérica, el uso de ordenadores para ejecutar un programa que implementa un modelo matemático de un sistema físico, es una parte importante del mundo tecnológico actual. En muchos campos de la ciencia y la ingeniería es necesario estudiar el comportamiento de sistemas cuyos modelos matemáticos son demasiado complejos para proporcionar soluciones analíticas, haciendo posible la evaluación virtual de las respuestas de los sistemas (gemelos virtuales). Esto reduce drásticamente el número de pruebas experimentales para los diseños precisos del sistema real que el modelo numérico representa. Sin embargo, estos gemelos virtuales, basados en métodos clásicos que hacen uso de una rica representación del sistema (por ejemplo, el método de elementos finitos), rara vez permiten la retroalimentación en tiempo real, incluso cuando se considera la computación en plataformas de alto rendimiento. En estas circunstancias, el rendimiento en tiempo real requerido en algunas aplicaciones se ve comprometido. En efecto, los gemelos virtuales son estáticos, es decir, se utilizan en el diseño de sistemas complejos y sus componentes, pero no se espera que acomoden o asimilen los datos para definir sistemas de aplicación dinámicos basados en datos. Además, se suelen apreciar desviaciones significativas entre la respuesta observada y la predicha por el modelo, debido a inexactitudes en los modelos empleados, en la determinación de los parámetros del modelo o en su evolución temporal. En esta tesis se proponen diferentes métodos para resolver estas limitaciones con el fin de realizar un seguimiento y un control en tiempo real. En la primera parte se utilizan técnicas de Reducción de Modelos para satisfacer las restricciones en tiempo real; estas técnicas calculan una buena aproximación de la solución simplificando el procedimiento de resolución en lugar del modelo. La precisión de la solución no se ve comprometida y se pueden realizar simulaciones efficientes (gemelos digitales). En la segunda parte se emplea la modelización basada en datos para llenar el vacío entre la solución paramétrica, calculada utilizando técnicas de reducción de modelos no intrusivas, y los campos medidos, con el fin de hacer posibles los sistemas de aplicación dinámicos basados en datos (gemelos híbridos).
La simulation numérique, c'est-à-dire l'utilisation des ordinateurs pour exécuter un programme qui met en oeuvre un modèle mathématique d'un système physique, est une partie importante du monde technologique actuel. Elle est nécessaire dans de nombreux domaines scientifiques et techniques pour étudier le comportement de systèmes dont les modèles mathématiques sont trop complexes pour fournir des solutions analytiques et elle rend possible l'évaluation virtuelle des réponses des systèmes (jumeaux virtuels). Cela réduit considérablement le nombre de tests expérimentaux nécessaires à la conception précise du système réel que le modèle numérique représente. Cependant, ces jumeaux virtuels, basés sur des méthodes classiques qui utilisent une représentation fine du système (ex. méthode des éléments finis), permettent rarement une rétroaction en temps réel, même dans un contexte de calcul haute performance, fonctionnant sur des plates-formes puissantes. Dans ces circonstances, les performances en temps réel requises dans certaines applications sont compromises. En effet, les jumeaux virtuels sont statiques, c'est-à-dire qu'ils sont utilisés dans la conception de systèmes complexes et de leurs composants, mais on ne s'attend pas à ce qu'ils prennent en compte ou assimilent des données afin de définir des systèmes d'application dynamiques pilotés par les données. De plus, des écarts significatifs entre la réponse observée et celle prévue par le modèle sont généralement constatés en raison de l'imprécision des modèles employés, de la détermination des paramètres du modèle ou de leur évolution dans le temps. Dans cette thèse, nous proposons di érentes méthodes pour résoudre ces handicaps afin d'effectuer une surveillance et un contrôle en temps réel. Dans la première partie, les techniques de Réduction de Modèles sont utilisées pour tenir compte des contraintes en temps réel ; elles calculent une bonne approximation de la solution en simplifiant la procédure de résolution plutôt que le modèle. La précision de la solution n'est pas compromise et des simulations e caces peuvent être réalisées (jumeaux numériquex). Dans la deuxième partie, la modélisation pilotée par les données est utilisée pour combler l'écart entre la solution paramétrique calculée, en utilisant des techniques de réduction de modèles non intrusives, et les champs mesurés, afin de rendre possibles des systèmes d'application dynamiques basés sur les données (jumeaux hybrides).

Several genera of bacteria residing in the sea surface microlayer and in the near-surface layer of the ocean have been found to be involved in the production and decay of surfactants. Under low wind speed conditions, surfactants can suppress short gravity capillary waves at the sea surface and form natural sea slicks. These features can be observed with both airborne and satellite-based synthetic aperture radar (SAR). Using a new microlayer sampling method, a series of experiments have been conducted in the Straits of Florida and the Gulf of Mexico in 2013 to establish a connection between the presence of surfactant-associated bacteria in the upper layer of the ocean and sea slicks. In a number of cases, sampling coincided with TerraSAR-X and RADARSAT-2 satellite overpasses to obtain SAR images of each study site. Samples collected from slick and non slick conditions have been analyzed using real time PCR techniques to determine Bacillus relative abundance in each area sampled. Previous work has shown that the sea surface microlayer plays a role in air-sea gas exchange, sea surface temperature, climate-active aerosol production, biochemical cycling, as well as the dampening of ocean capillary waves. Determining the effect of surfactant-associated bacteria on the state of the sea surface may help provide a more complete global picture of biophysical processes at the air-sea interface and uptake of greenhouse gases by the ocean.

Prenatal assessment of fetal health is routinely offered throughout pregnancy to ensure that the most effective management can be provided to maintain fetal and maternal well-being. Currently, invasive testing is used for definitive diagnosis of fetal aneuploidy, which is associated with a 1% risk of iatrogenic fetal loss. Developing non-invasive prenatal testing (NIPT) is a key area of research and methods to increase the level of cell-free fetal DNA (cffDNA) within the maternal circulation have been discussed to improve accuracy of such tests. In this study, three strategies; co-amplification at lower denaturation temperature polymerase chain reaction (COLD-PCR), inverse-PCR and Pippin Prep™ gel electrophoresis, were analysed to identify a novel approach to selectively enrich shorter cffDNA fragments from larger maternal cell-free DNA (cfDNA). The sensitivity of droplet digital PCR (ddPCR) against real-time PCR (qPCR) was compared for fetal sex and RHD genotyping. In addition RHD zygosity testing was carried out for non-maternal samples. Consequently, Pippin Prep™ gel electrophoresis was combined with ddPCR analysis for the NIPD of Down Syndrome (DS) in pseudo-maternal samples. The results revealed that the Pippin Prep™ gel electrophoresis enrichment approach successfully demonstrated 2-fold to 5-fold increases in the cffDNA fraction. However, further optimisation assays of COLD-PCR and inverse-PCR using actual maternal samples were required. The spike experiments for DS detection revealed that with the present assay IV overrepresentation of the chromosome 21 target could be significantly detected for samples with ≥15% ‘cffDNA fraction’. In conjunction with the Pippin Prep™ enrichment method, this would have enabled assessment of all 10 maternal samples. Alternatively, fetal sex and RHD genotyping results determined that ddPCR provides a more sensitive platform compared to qPCR approaches, particularly for samples that express low cffDNA fractions (<2%). The ddPCR platform also proved to be a rapid and accurate system for the determination of RHD zygosity. This study highlights that ddPCR could be used as opposed to qPCR for accurate determination of fetal sex and RHD status. While sequencing approaches currently provide the most sensitive platforms for NIPT of fetal aneuploidy, high costs (>£400) prevent universal application. The combination of cffDNA enrichment with ddPCR analysis could provide a cheaper and more widely available platform for NIPD. However, further large scale validation studies using actual maternal samples are required.

La previz on-set est une étape de prévisualisation qui a lieu directement pendant la phase de tournage d’un film à effets spéciaux. Cette proposition de prévisualisation consiste à montrer au réalisateur une vue assemblée du plan final en temps réel. Le travail présenté dans cette thèse s’intéresse à une étape spécifique de la prévisualisation : le compositing. Cette étape consiste à mélanger plusieurs sources d’images pour composer un plan unique et cohérent. Dans notre cas, il s’agit de mélanger une image de synthèse avec une image issue de la caméra présente sur le plateau de tournage. Les effets spéciaux numériques sont ainsi ajoutés à la prise de vue réelle. L’objectif de cette thèse consiste donc à proposer un système permettant l’ajustement automatique du mélange entre les deux images. La méthode proposée nécessite la mesure de la géométrie de la scène filmée. Pour cette raison, un capteur de profondeur est ajouté à la caméra de tournage. Les données sont relayées à l’ordinateur qui exécute un algorithme permettant de fusionner les données du capteur de profondeur et de la caméra de tournage. Par le biais d’un démonstrateur matériel, nous avons formalisé une solution intégrée dans un moteur de jeux vidéo. Les expérimentations menées montrent dans un premier temps des résultats encourageants pour le compositing en temps réel. Nous avons observé une amélioration des résultats suite à l’introduction de la méthode de segmentation conjointe. La principale force de ce travail réside dans la mise en place du démonstrateur qui nous a permis d’obtenir des algorithmes efficaces dans le domaine de la previz on-set
Previz on-set is a preview step that takes place directly during the shootingphase of a film with special effects. The aim of previz on-set is to show to the film director anassembled view of the final plan in realtime. The work presented in this thesis focuses on aspecific step of the previz : the compositing. This step consists in mixing multiple images tocompose a single and coherent one. In our case, it is to mix computer graphics with an imagefrom the main camera. The objective of this thesis is to propose a system for automaticadjustment of the compositing. The method requires the measurement of the geometry ofthe scene filmed. For this reason, a depth sensor is added to the main camera. The data issent to the computer that executes an algorithm to merge data from depth sensor and themain camera. Through a hardware demonstrator, we formalized an integrated solution in avideo game engine. The experiments gives encouraging results for compositing in real time.Improved results were observed with the introduction of a joint segmentation method usingdepth and color information. The main strength of this work lies in the development of ademonstrator that allowed us to obtain effective algorithms in the field of previz on-set

Durant les quinze dernières années, une attention particulière a été portée aux effets potentiels sur le vivant des champs radiofréquence (RF) des communications sans fil. Malgré l’intensité des efforts de recherche sur les effets biologiques et sanitaires potentiels des RF, nos connaissances en bioélectromagnétisme n’ont pu suivre l’évolution rapide des technologies.[…] La capacité des RF à provoquer un échauffement des tissus est parfaitement caractérisée. Des recommandations et des normes ont été définies afin de protéger les populations des risques associés, sachant qu’aucun échauffement n’est provoqué par l’exposition aux dispositifs de communications sans fil en raison des très faibles niveaux correspondant. Il est donc capital de savoir si l’on peut totalement exclure que des effets non-thermiques des RF de faible niveau existent au niveau moléculaire au sein de la matière vivante. L’objectif de cette thèse est d’évaluer en temps réel et sur cellules vivantes, les effets de l'exposition aux champs radiofréquences (CW, GSM-1800, UMTS, Wi-Fi, WiMax, LTE), soit au niveau moléculaire en ciblant l’activité du canal ionique TRPV1 qui est l’un des thermorécepteurs de notre organisme, soit au niveau cellulaire en étudiant le comportement général de cellules exposées aux RF à l’aide d’une technique dite « sans marquage », l’impédancemétrie. Le suivi de l’activité du canal TRPV1 sous exposition RF a été réalisé à l’aide de la technique du transfert d’énergie en résonance de bioluminescence (BRET), une technique spectroscopique qui permet l’analyse des interactions protéines-protéines ou des changements de conformation des protéines en temps réel et sur cellules vivantes. La mise en place de cette technique a demandé la construction et la caractérisation de sondes BRET ciblant les canaux TRP ainsi que la mise au point d’un dispositif de mesure déporté des spectres de BRET à l’aide d’une fibre optique, afin de pouvoir exposer les échantillons aux champs RF. La conclusion de ce volet de la thèse est que les RF sont capables d’activer le canal TRPV1 en produisant un échauffement diélectrique, mais qu’en absence d’augmentation de la température il n’y a aucun effet des RF sur le niveau d’activité basal du canal TRPV1 ou sur l’efficacité de la Capsaïcine, un agoniste, à activer TRPV1. L’analyse du comportement global de cellules en culture sous exposition RF a été réalisée à l’aide d’un système xCELLigence modifié afin de pouvoir à la fois suivre le comportement cellulaire par impédancemétrie tout en utilisant le réseau d’électrodes des plaques de mesure pour exposer les cellules mises en culture aux RF. À l’aide de ce dispositif, nous avons pu réaliser des expositions de cellules SH-SY5Y avec un DAS de 24 W/Kg sans provoquer d’échauffement dans le milieu de culture ou dans les cellules. Aucun effet des RF sur le comportement de la lignée de neuroblastome SH-SY5Y n’a cependant pu être mis en évidence, que ce soit en absence ou en présence d’une co-stimulation par un agent chimique. La conclusion de cette étude est que dans des conditions où la température reste stable, nous n’avons pas pu mettre en évidence de modification du fonctionnement du vivant que ce soit au niveau moléculaire ou au niveau cellulaire. Les outils développés dans ce travail de thèse ouvrent, de plus, d’importantes perspectives tant dans le domaine du criblage de médicaments candidats à l’aide du BRET spectral, que pour de futures études en bioélectromagnétisme
The biological and health effects of radiofrequency (RF) electromagnetic fields (EMF) exposure have been very actively studied in the past two decades, mainly triggered by concerns about potential health effects of wireless communication systems. This physical agent is among the most common, fastest-growing environmental factors, triggering concerns in the population, as even a minor effect of EMF exposure on health could have a major public health impact. While the effects of extremely low frequency electromagnetic fields (ELF EMF) on the excitation of nerve and muscle cells have been well-characterized, the only well-described effects of radiofrequency electromagnetic fields (RF EMF) on biological systems are caused by dielectric-relaxation heating. In contrast, “nonthermal” RF EMF effects refer to other potential biological effects that are not caused by temperature elevation of living tissue or cell culture medium. The investigation of such mechanisms has been hampered by the absence of robust, reliable and repeatable effects occurring as a consequence of low-level exposures, for which temperature elevation is minimal. Moreover, no plausible mechanistic hypotheses have been given concerning thermal or nonthermal effects of low-level RF EMF exposures, making difficult to draw conclusions on the basis of available experimental results. Nonetheless, in 2011, the International Agency for Research on Cancer (IARC) classified RF emitted by cell phones as “possibly carcinogenic to humans” (Class 2B). The characterization of nonthermal biological RF EMF effects is therefore of primary importance for setting safety limits since guidelines and standards have so far been set to protect from the known health risks associated only with the thermal effects of RF EMF exposures. The aim of this basic science thesis work is to characterize the effects of environmental RF EMF signals on living matter at the cellular and molecular level. In this work, we took advantage of modern and innovative methods to observe the behavior of living matter under RF EMF exposure in real time at various specific absorption rates (SAR). In particular, we have studied: (i) Specific RF EMF effects on the ionic channel TRPV1, a major thermoreceptor in our body. TRPV1 activation under RF EMF exposure was studied using the bioluminescence resonance energy transfer (BRET) technique. The implementation of this technique called for the construction and characterization of BRET probes targeting TRP channels as well as the development of a device for the remote measurement of BRET spectra, using an optical fiber. The conclusion of this part of the thesis is that RFs are able to activate the TRPV1 channel by producing a dielectric heating but in the absence of temperature increase there is no RF effect on the basal activation state of TRPV1 and no change of capsaicin maximal efficacy to activate TRPV1. (ii) The analysis of the global behavior of cells in culture under RF exposure was carried out using a modified xCELLigence system where the array of electrodes of the measuring plates were also used to expose the cells to RF EMF. Using this device, we were able to perform SH-SY5Y cell exposures with a SAR of 24 W/kg without causing heating in the culture medium or in the cell culture. No effect of RF EMF on the behavior of the neuroblastoma SH-SY5Y line could however be demonstrated, either in the absence or in the presence of a co-stimulation by a chemical agent. The conclusion of this study is that under conditions where the temperature remains stable, we have not been able to demonstrate any changes in the functioning of living cells, ether at the molecular level or at the cellular level. The tools developed in this thesis work offer important prospects both in the field of drug screening using spectral BRET, and pave the ways for future studies in bioelectromagnetics

Introdução: A meningite tuberculosa (MTB) é a forma mais grave e fatal de tuberculose. O diagnóstico oportuno e o tratamento adequado e precoce são os principais fatores associados com o bom prognóstico. Os métodos utilizados na prática médica diária - achados clínicos, exames de imagem e análise de líquido cefalorraquidiano (LCR) - têm baixa acurácia. A pesquisa do DNA do Mycobacterium tuberculosis no LCR através da reação em cadeia da polimerase (PCR, do inglês polimerase chain reaction) com a metodologia nested é promissora, especialmente quando associada à praticidade da amplificação do DNA em tempo real. Objetivo: Avaliar o valor diagnóstico da nested PCR em tempo real (nRT-PCR, do inglês nested real-time PCR) na investigação de pacientes com MTB. Métodos: Estudo observacional realizado em duas fases: uma prospectiva e outra retrospectiva. Na fase prospectiva, foram incluídos pacientes com suspeita de MTB internados no Instituto de Infectologia Emílio Ribas (IIER). Informações clínicas, laboratoriais e radiológicas foram coletadas, assim como amostra de LCR de todos os pacientes. A partir de critérios internacionais padronizados, os pacientes foram categorizados como \"MTB Definitiva\", \"MTB Provável\", \"MTB Possível\" e \"Não MTB\". A nRT-PCR, utilizando o gene alvo mpt64, foi realizada em todas as amostras de LCR no Laboratório de Meningites Bacterianas do Instituto Adolfo Lutz. Sensibilidade, especificidade e intervalos de confiança (IC 95%) da nRT-PCR foram calculados com base no padrão-ouro (cultura positiva para M. tuberculosis ou isolamento de BAAR no sistema nervoso central) e nos pacientes com outros diagnósticos estabelecidos (Não MTB). Também foi calculada a proporção de pacientes com a nRT-PCR positiva em cada categoria clínica. Na fase retrospectiva, foi realizada uma revisão de prontuários de pacientes que tiveram a nRT-PCR solicitada no IIER e no Centro de Referência e Treinamento em DST/AIDS. Os mesmos procedimentos de categorização diagnóstica, cálculos de sensibilidade e especificidade foram adotados. Resultados: Na fase prospectiva, foram incluídos 102 pacientes, sendo 92 deles infectados por HIV. Nove deles tiveram o padrão-ouro positivo e foram classificados como \"MTB Definitiva\" e 81 deles tiveram outros diagnósticos estabelecidos (\"Não MTB\"). A sensibilidade e a especificidade da nRT-PCR foi 100% (IC95%:70-100 e 95-100, respectivamente). A positividade da nRT-PCR na categoria \"MTB Provável\" foi 50% (4/8 pacientes) e 25% na \"MTB Possível\" (1/4). Na fase retrospectiva, 56 pacientes foram incluídos, sendo 48 infectados por HIV. A nRT-PCR teve sensibilidade de 83% (5/6) e especificidade de 100% (0/45). A positividade na categoria \"MTB Provável\" foi 60% (3/5) e não houve pacientes classificados como \"MTB Possível\". Conclusão: A nRT-PCR apresentou boa sensibilidade e ótima especificidade, demonstrando seu valor diagnóstico na identificação oportuna de casos de MTB
Background: Tuberculous meningitis (TBM) is the most serious and lethal presentation of tuberculosis. Timely diagnosis and appropriated treatment are the main factors associated with good outcome. Methods used in the daily medical practice - clinical, radiological and cerebrospinal fluid (CSF) findings - have low accuracy. Search for Mycobacterium tuberculosis DNA in the CSF by polymerase chain reaction (PCR) using the nested methodology is promising, especially when combined with the practical approach of the real time DNA amplification. Objective: To evaluate the diagnostic value of a nested real-time PCR (nRT-PCR) in the investigation of patients with TBM. Methods: A two-phase observational study was carried out: prospective and retrospective. In the prospective phase, patients with suspected TBM hospitalized at \"Instituto de Infectologia Emílio Ribas\" (IIER) were included. Clinical, laboratory and radiological data were collected, as well as CSF samples of all patients. According to international standard criteria, patients were categorized as \"TBM Definite\", \"TBM Probable\", \"TBM Possible\" and \"Not TBM\". The nRT-PCR, using the mpt64 gene, was performed on all CSF sample in the Laboratory of Bacterial Meningitis, Adolfo Lutz Institute. Sensitivity, specificity and confidence intervals (95% CI) of the nRT-PCR were calculated based on the gold standard (culture positive for M. tuberculosis or AFB isolation on the central nervous system) and on patients with other established diagnoses (\"Not TBM\"). The proportion of patients with a positive nRT-PCR in each clinical category was also calculated. In the retrospective phase, medical chart review was performed in those patients who had the nRT-PCR requested in IIER and in the \"Centro de Referência e Treinamento em DST/AIDS\". The same diagnostic categorization and calculations of sensitivity and specificity were adopted. Results: 102 patients were included in the prospective phase, 92 of them HIV-infected. Nine of them had the gold standard positive and were classified as \"TBM Definite\" and 81 of them had other diagnoses established (\"Not TBM\"). The sensitivity and specificity of the nRT-PCR were 100% (95%CI: 70-100 and 95-100, respectively). The nRT-PCR positivity in category \"TBM Probable\" was 50% (4/8 patients) and 25% in \"TBM Possible\" (1/4). In retrospective phase, the nRT-PCR had a sensitivity of 83% (5/6) and specificity of 100% (0/45), among the 56 included patients (48 of them HIV infected). Positivity in \"TBM Probable\" category was 60% (3/5) and no patients were classified as \"TBM Possible\". Conclusion: The nRT-PCR showed good sensitivity and excellent specificity, showing its diagnostic value in the timely identification of TBM

Dans le cadre industriel et académique, les méthodologies de développement logiciel exploitent de plus en plus le concept de “modèle” afin d’appréhender la complexité des systèmes temps réel critiques. En particulier, celles-ci définissent une étape dans laquelle un modèle fonctionnel, conçu comme un graphe de blocs fonctionnels communiquant via des échanges de signaux de données, est déployé sur un modèle de plateforme d’exécution matérielle et un modèle de plateforme d’exécution logicielle composé de tâches et de messages. Cette étape appelée étape de déploiement, permet d’établir une architecture opérationnelle du système nécessitant une validation des propriétés temporelles du système. Dans le contexte des systèmes temps réel dirigés par les évènements, la vérification des propriétés temporelles est réalisée à l’aide de l’analyse d’ordonnançabilité basée sur l’analyse des temps de réponse. Chaque choix de déploiement effectué a un impact essentiel sur la validité et la qualité du système. Néanmoins, les méthodologies existantes n’offrent pas de support permettant de guider le concepteur d’applications durant l’exploration de l’espace des architectures possibles. L’objectif de ces travaux de thèse consiste à mettre en place des techniques d’analyse et de synthèse automatiques permettant de guider le concepteur vers une architecture opérationnelle valide et optimisée par rapport aux performances du système. Notre proposition est dédiée à l’exploration de l’espace des architectures en tenant compte à la fois des quatre degrés de liberté déterminés durant la phase de déploiement, à savoir (j) le placement des éléments fonctionnels sur les éléments de calcul et de communication de la plateforme d’exécution, (ii) le partitionnement des éléments fonctionnels en tâches temps réel et des signaux de données en messages, (iii) l’affectation de priorités d’exécution aux tâches et aux messages du système et (iv) l’attribution du mécanisme de protection des données partagées pour les systèmes temps réel périodiques. Nous nous intéressons principalement à la satisfaction des contraintes temporelles et celles liées aux capacités des ressources de la plateforme cible. De plus, nous considérons l’optimisation des latences de bout-en-bout et la consommation mémoire. Les approches d’exploration architecturale présentées dans cette thèse sont basées sur la technique d’optimisation PLNE (programmation linéaire en nombres entiers) et concernent à la fois les applications activées périodiquement et celles dont l’activation est pilotée par les données. Contrairement à de nombreuses approches antérieures fournissant une solution partielle au problème de déploiement, les méthodes proposées considèrent l’ensemble du problème de déploiement. Les approches proposées dans cette thèse sont évaluées à l’aide d’applications génériques et industrielles
Modern development methodologies from the industry and the academia exploit more and more the ”model” concept to address the complexity of critical real-time systems. These methodologies define a key stage in which the functional model, designed as a network of function blocks communicating through exchanged data signals, is deployed onto a hardware execution platform model and implemented in a software model consisting of a set of tasks and messages. This stage so-called deployment stage allows establishment of an operational architecture of the system, thus it requires evaluation and validation of the temporal properties of the system. In the context of event-driven real-time systems, the verification of temporal properties is performed using the schedulability analysis based on the response time analysis. Each deployment choice has an essential impact on the validity and the quality of the system. However, the existing methodologies do not provide supportto guide the designer of applications in the exploration of the operational architectures space. The objective of this thesis is to develop techniques for analysis and automatic synthesis of a valid operational architecture optimized with respect to the system performances. Our proposition is dedicated to the exploration of architectures space considering at the same time the four degrees of freedom determined during the deployment phase, (i) the placement of functional elements on the computing and communication resources of the execution platform, (ii) the partitioning of function elements into real time tasks and data signals into messages, (iii) the priority assignment to system tasks and messages and (iv) the assignment of shared data protection mechanism for periodic real-time systems. We are mainly interested in meeting temporal constraints and memory capacity of the target platform. In addition, we are focusing on the optimization of end-to-end latency and memory consumption. The design space exploration approaches presented in this thesis are based on the MILP (Mixed Integer Linear programming) optimization technique and concern at the same time time-driven and data-driven applications. Unlike many earlier approaches providing a partial solution to the deployment problem, our methods consider the whole deployment problem. The proposed approaches in this thesis are evaluated using both synthetic and industrial applications

Les techniques de biologie moléculaire ont pris au cours des 20 dernières années une place importante dans le diagnostic direct des pathogènes viraux. Notre travail a porté sur la mise en place et le développement d’une plate-forme de biologie moléculaire, au sein du laboratoire de virologie de l’hôpital de la Timone, pour répondre aux demandes et contraintes du diagnostic en milieu hospitalier. L’organisation de cette plate-forme a nécessité plusieurs étapes : la prévention des risques de contamination, l’aliquotage et le stockage des réactifs, l’automatisation des techniques d’extraction des acides nucléiques, la mise au point de témoins positifs synthétiques et de témoins internes et l’optimisation des protocoles de PCR. Cette approche optimisée du diagnostic moléculaire des infections virales a été appliqué notamment à la détection de la grippe pandémique A/H1N1v dans les laboratoires de routine hospitalière et d’urgence « Point Of Care ». La mise en place de cette plate-forme a fait progresser de manière considérable le diagnostic moléculaire du laboratoire. Elle nous permet actuellement de détecter un grand nombre de pathogènes (>80) et de réaliser des tests dans un format à haut débit (≈40 000 tests/an). Au total, cette plateforme est au coeur de la capacité du laboratoire pour réagir de manière rapide aux évènements d'émergence en mettant en place rapidement des procédures diagnostiques standardisées. Ces techniques ont été transférées à de nombreux autres laboratoires de virologie partenaires nationaux et internationaux. Nous envisageons maintenant son utilisation dans une approche syndromique avec notamment, le développement du diagnostic des virus respiratoires
Molecular biology techniques have taken an important role in the direct diagnosis of viral pathogens over the last 20 years. Our work focused on establishing and developing a platform for molecular diagnosis in the laboratory of Virology (Timone Hospital) to meet the demands and constraints of diagnosis in hospitals. The organization of this platform required several steps: prevention of contamination risks, aliquoting and storage of reagents, automation techniques of nucleic acid extraction, development of synthetic positive controls and internal controls and optimization of PCR protocols. This optimized approach of the molecular diagnosis of viral infections has particularly been applied to the detection of pandemic influenza A/H1N1v in hospital laboratories for routine and emergency "Point Of Care." The implementation of this platform has significantly improved molecular diagnosis in our laboratory. It currently allows us to detect a large number of pathogens (> 80) and perform tests in a high-throughput (≈ 40,000 tests per year). In total, this platform is at the heart of the laboratory capacity to react quickly to emerging events by rapidly implementing standardized procedures. These techniques have been transferred to many other partners’ laboratories nationally and internationally. We are now considering its use in a syndromic approach including the development of the diagnosis of respiratory viruses

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

Dans son ensemble, les techniques de pointe actuelles procurent l'information nécessaire à une analyse approfondie de la cellule, ce qui nécessite cependant l’utilisation d’instruments et de plateformes analytiques différentes. Les biopuces à cellule permettent l’analyse des cellules vivantes en temps réel et constituent donc un outil important pour de nombreuses applications dans la recherche biomédicale telles que la toxicologie et la pharmaceutique.En effet, le suivi en temps réel de la réponse non-seulement physique mais aussi chimique des cellules, obtenue suite à des stimuli externes spécifiques et en utilisant un système d'imagerie cellulaire, peut fournir une meilleure compréhension des mécanismes et des voies de signalisation impliquées dans la réaction toxicologique.Le développement de tels dispositifs multianalytiques pour l'analyse biologique repose essentiellement sur la capacité de produire des surfaces fonctionnelles de pointe permettant une interaction et organisation contrôlée des cellules et d'autres entités telles que par exemple des anticorps ou des nanoparticules. Par conséquent, un grand effort technologique repose sur le développement des techniques permettant la création de motifs fonctionnels sur une surface de nature souvent inerte. Dans cette thèse, nous proposons deux techniques de micro- et nanofabrication permettant la création de motifs de cellules et d’anticorps sur un revêtement non-adhésif composé de poly (oxyde d'éthylène) (« PEO-like ») déposé par plasma. La première approche consiste à immobiliser par physisorption un micro-réseau de molécules adhésives de la matrice extracellulaire (par exemple la fibronectine) en utilisant des techniques d’impression par microcontact et par non-contact. La deuxième approche permet la création de motifs adhésifs sur la surface constitués de nanoparticules d'or (Au NPs) en utilisant des techniques d’impression similaire. L'immobilisation des Au NPs sur le revêtement « PEO-like » ne nécessite pas de modifications chimiques et est réalisé par une technique d'autoassemblage simple et irréversible. Ces surfaces d'or nanostructurées ont été testées pour l’analyse du phénomène de reconnaissance biomoléculaire et en tant que plateforme de culture cellulaire. Finalement, cette plateforme a été intégrée à un microscope plasmonique qui a permis, de façon préliminaire, la surveillance et la visualisation de la motilité d’une cellule unique, cela en temps réel et sans marquage, ainsi que la détection spécifique et sensible de protéines tests
State of the art techniques give as a whole the required information needed for the complete cell analysis but require different instruments and different types of platforms. The concept of cells on-a-chip allowing real-time analysis of living cells is, therefore, an important tool for many biomedical research applications such as toxicology and drug discovery. Monitoring in real-time the physical but also chemical response of live cells to specific external stimuli using live-cell imaging can provide a better understanding of the mechanisms and pathways involved in the toxicological reaction. The development of such multianalytical devices for biological analysis relies essentially on the ability to design advanced functional surfaces enabling a controlled interaction and organisation of cells and other nanostructures (e.g antibodies and nanoparticles). Therefore, a large technological effort is related on the development of advanced patterning techniques. In this thesis, we propose two simple and direct micro- and nano-fabrication techniques enabling the creation of cellular and sensing patterns on a non-adhesive and cell repellent plasma-deposited poly (ethyleneoxide) (PEO-like) coating. The first approach consists in immobilising a microarray of ECM molecules (cell-adhesive proteins, e.g fibronectin) on the cell repellent PEO-like surface by physisorption using microspotting or microcontact printing techniques. The second approach enables the creation of Gold nanoparticles (Au NPs) adhesive patterns on the surface using similar spotting techniques. The immobilization of Au NPs on PEO-like coatings does not require any prior chemical modifications and is achieved by a straightforward and irreversible self-assembly technique. These gold nanostructured surfaces have been tested for protein bio-recognition analysis and as a cell culture platform. Ultimately, this platform was integrated to a novel plasmonic microscope which enabled, preliminarily, the label-free monitoring and visualisation of a single cell attachment and detachment in real time, as well as the specific and sensitive detection of test proteins in a cell-free environment

L'isolement de Mycobacterium tuberculosis par culture est la méthode de référence pour le diagnostic de la tuberculose. Le but de notre travail était d'améliorer et de faciliter le diagnostic par culture de la tuberculose. Dans un premier temps, nous avons produit une revue bibliographique en comparant les différentes techniques ou protocoles utilisés pour le diagnostic de la tuberculose. Ce travail nous a permis d'actualiser notre protocole de diagnostic, avec la mise en place d’un "kit-tuberculose" contenant des containers imprégnés de chlorhexidine pour la récupération et la décontamination directe d’échantillons cliniques non- invasifs, suivi par la culture sur un milieu solide à base d'oeuf, et détection des colonies par microscope inversé ou par un système d'imagerie en temps réel. Nous avons mis en place une méthode de décontamination par 0,7%-chlorhexidine et avons montré que cette méthode était plus efficace que la méthode de référence NALC-NaOH. Ensuite, nous avons développé un milieu de culture à base de sérum animal, le MOD9 dont nous avons montré par une étude comparative qu'il était supérieur au milieu solide LJ de référence. Une deuxième étude comparant un protocole de décontamination par la chlorhexidine et culture sur milieu MOD9 au protocole standard, NALC-NaOH/Bactec960 a montré une supériorité par rapport au protocole standard. Enfin, la mise en place d'un système de détection des micro-colonies de M. tuberculosis sur MOD9 par imagerie en temps réel Advencis-Biosystem a permis de réduire le temps de détection de M. tuberculosis à 3,2 jours avec le protocole chlorhexidine/MOD9/Advencis, avec un record mondial de détection en 25h
Isolation of Mycobacterium tuberculosis by culture is the gold standard for the diagnosis of tuberculosis. The aim of my thesis work was to simplify and improve the culture diagnosis of tuberculosis. At first we started with a bibliographic study, comparing step by step the different techniques and protocols that have been used for the diagnosis of tuberculosis. This work has allowed us to update our tuberculosis diagnosis protocol, starting with the implementation of a "Tuberculosis-kit" consisting of chlorhexidine containing containers for the recovery and decontamination of non-invasive specimens, followed by culture on an egg-based medium, a micro- colonies detection using an inverted microscope or an automated real-time imaging incubator system and finally an identification using mass spectrometry. We established a new chlorhexidine- based decontamination method that we showed to be more efficient for the recovery and isolation of M. tuberculosis than the standard NALC-NaOH method. Than we developed a new serum-based culture medium, the MOD9 that we showed in a comparative study to be superior to the reference LJ medium for the recovery of M. tuberculosis. In a second study we proved that our chlorhexidine/MOD9 protocol was superior to the standard NALC-NaOH/Bactec 960 MGIT protocol for the isolation of M. tuberculosis. And finally the implementation of a real time imaging system for the detection of M. tuberculosis micro-colonies on MOD9 permits us to dramatically reduce the detection time from 15 days with the standard NALC-NaOH/Bactec 960 MGIT protocol to 3.2 days with our 0.7%-chlorhexidine/MOD9/Advencis-Biosystem protocol with a world record detection time of 25h

Les travaux de recherche dans ce mémoire s’inscrivent dans l'étude et la commande d'un nouveau concept du groupe électrogène hybride permettant de réduire la puissance du moteur diesel (downsizing) d’économiser du carburant et d’améliorer le comportement de la génératrice synchrone durant les régimes transitoires. La solution adoptée consiste à mettre en parallèle à la génératrice synchrone un système de stockage d'énergie. Ce système est composé d'un onduleur avec un supercondensateur du côté du bus continu. L’objectif de la thèse a été de dimensionner l’ensemble supercondensateur /convertisseur statique et de développer une commande ayant des performances optimales pour obtenir le meilleur compromis entre l’énergie échangée dans le supercondensateur et l’efficacité sur la vitesse du groupe et sur l’amplitude des tensions de l’alternateur. Une commande par retour d’état avec intégration de l’écart en utilisant l’approche LMIs a été mise en place pour la synthèse des régulateurs des boucles de courants de l'onduleur. Une deuxième commande a été développée pour réguler la tension variable aux bornes du supercondensateur. Un simulateur regroupant l’alternateur et le système de stockage a été développé pour la mise au point de ces commandes. Toutes les validations ont été faites sur une maquette expérimentale spécifiquement élaborée pour cette thèse. Les essais ont été menés avec un moteur d’entraînement électrique et sur un diesel. En conclusion, les essais expérimentaux ont mis en évidence l’apport important de cette hybridation sur les variations de vitesse du diesel et sur la tension aux bornes de l’alternateur lors d’important impact ou de délestage de la charge
The research in this thesis are part of study and control of a new concept of hybrid generator to reduce the power of the diesel engine (downsizing) in order to save fuel and improve the behavior of the synchronous generator during transients. The adopted solution is to place in parallel with the synchronous generator an energy storage system. The latter consists of an inverter with a super capacitor on the DC bus. The aim of the thesis was to scale the entire supercapacitor / static converter and to develop a control law having the best performance with the best compromise between the energy exchanged in the supercapacitor, efficiency, the group speed and voltage amplitude of the generator. A feedback control condition with integration of the deviation using LMI's approach has been established for the synthesis of loop current regulators from the inverter.A second control law was developed to regulate the variable voltage across the supercapacitor. A simulator combining generator and storage system has been developed to test these commands.All validations were made on an experimental test rig specifically developed for this thesis. The tests were conducted with an electric drive motor in the test platform of the LIAS and with a diesel in that of the Leroy Somer Motors company.Finally, experimental tests have highlighted the significant contribution of this hybridization on the diesel speed variationsand on the terminal voltage of the alternator during impact or load shedding

碩士
國立臺灣大學
海洋研究所
103
Billfish is an important fishery resource all over the world, and is usually marketed as sashimi, filleted, fish floss and sauce. Most billfish products in Taiwan were only labeled with “billfish” in their ingredients. Therefore, species identification of the billfish products is necessary and will benefit the conservation and regulation of these fishery resourcs. In this study, we developed a real-time PCR approach to identify the six billfish species：Swordfish (Xiphias gladius), Sailfish (Istiophorus platypterus), Black marlin (Istiompax indica), Striped marlin (Kajikia audax), Blue marlin (Makaira nigricans) and Shortbill spearfish (Tetrapturus angustirostris). The whole system contained 14 primers, including 12 species-specific primers and two universal primers designed for discriminating the six billfish from non-billfish species. The 12 species-specific primers showed clear discrimination among the six billfish species, with ΔCt values of the target species ranging between -2.23 ~ 3.93 and the limit of detection is 0.4 ng/μL. In order to minimize the cost and time of whole identification system, we also tested the applicability of the multiplex qPCR by combining several species-specific primers in a single reaction to identify the species. The result showed that the six species can be identified according to the melting curve pattern and Tm values for X. gladius： Tm1 = 75.0 ± 0.0 °C, Tm2 = 83.5 ± 0.0 °C, I. platypterus：Tm1 = 75.2 ± 0.3 °C, Tm2 = 80.5 ± 0.2 °C, I. indica：Tm1 = 77.5 ± 0.0 °C, Tm2 = 80.5 ± 0.0 °C, K. audax：Tm = 80.5 ± 0.0 °C, M. nigricans：Tm1 = 76.0 ± 0.0 °C, Tm2 = 80.5 ± 0.0 °C, T. angustirostris：Tm = 77.0 ± 0.0 °C. An identification protocol for the six billfish species was established in this study. The methods is efficient, sensitive, and cheaper than other conventional methods, which will be helpful for fishery management and protecting the consumer’s rights.

碩士
大葉大學
環境工程學系碩士班
96
This study was conducted with the application of denaturing gradient gel electrophoresis (DGGE), and real-time quantitative polymerase chain reaction (real-time PCR) molecular biotechnology for monitoring the permeable reactive barrier (PRB) in the relation with BTEX decomposition efficiency and the distribution of microbial community. Various amounts of nitrogen nutrients and BTEX were added to examine the treatment efficiencies. It was shown that the high sodium nitrate amount had improved the BTEX removal, which was an evidence of effects on BTEX treatment. The results of benzene and toluene removal efficiencies revealed that it was completely degraded for both two compounds at the concentrations of 20, 40 and 80 ppm. Increasing the concentrations of these two compounds to 120, 160, 240 and 320 ppm resulted in the decreasing in treatment efficiencies, and the concentrations remained 40, 60, 65, 90 and 100 % for benzene, and 10, 40, 55, 90 % for Toluene, respectively. Furthermore, the microbial variations at various concentrations were consistent via optical density (OD), DGGE analysis and real-time PCR results. Each column test was conducted for 20 days to investigate the effectiveness of oxygen releasing compound (ORC). It was indicated that the highest dissolved oxygen was achieved, which was 5.08 mg/L (equal to 0.25 mg O2/day/g-ORC) at 40 % of CaO2. The results of long-term stability tests of oxygen releasing from PRB system showed that: (1) Oxygen released from ORC was sufficient for the demand of bacteria. (2) In shock-loading of BTEX tests, the removal efficiencies were reduced by 21%, 19%, 17% and 10 % for benzene, toluene, ethylbenzene and xylene, respectively. (3) Removal efficiencies were then recovered in the ascending order of as follow: xylene> ethylbenzene> benzene> toluene. (4) ORC can be used for 40 days. (5) DGGE analysis showed the changing in the microbial community structure before (13 groups) and after shock-loading (reduced to 9 groups), that implied the shock-loading was harmful to bacteria. (6) The results from real-time PCR in the study of catechol 2,3-dioxygenase gene revealed that the quantification of this gene has been declined after shock-loading, but it was latter well again at the 79th day.

碩士
國立臺灣大學
植物病理與微生物學研究所
94
Papaya ringspot is one of the most destructive diseases of papaya and it is a limiting factor for papaya industry. This disease first occurred in Taiwan in 1975, and it has spreading widely throughout all of papaya-cultivated areas in Taiwan. It is caused by Papaya ringspot virus (PRSV), which belongs to Potyvirus, Potyviridae. According to the different symptoms in papaya hosts, PRSV was divided into three major strains including SM (severe mottling), DF (severe mottling with leaf-deformation) and SMN (severe mottling with necrosis and quick decline) strains. SM was a predominant strain of PRSV in the field of Taiwan several years ago. However, DF and SMN have recently become newly rising dominant strains. Almost full-length of genomic sequences of three PRSV strains were determined in the previous study. Continuous sequencing was conducted in this study. The complete nucleotide sequences (10326 bases) of three strains were determined by application of 5’ RACE and 3’ RACE systems. They encode a polyprotein of 3344 amino acids with a 5’ untranslated region of 85 nucleotides and a 3’ untranslated region of 209 nucleotides. The results of alignment of full genomic nucleotide sequences demonstrated that SM is 97.3% identical to DF, and SMN is 97.0% and 96.8% identical to SM and DF. Most genes in the PRSV genome among the three strains are 95~100% homologous, but the P1 gene and 5’ UTR region are about 95% and 92% homologous among three different strains, respectively. Phylogenic analysis of various international PRSV isolates revealed that SM, DF and SMN are close to the Yung-Kang isolate (another PRSV isolate from Taiwan), but far away from the Thailand and Hawaii isolates. The rapid assay based on reverse transcription-polymerase chain reaction (RT-PCR) with the primer pair PRSV-857 was developed for the detection of PRSV in previous study. Another primer pair PRSV-829 was devised in our study, and it has been proven to achieve better specificity and sensitivity in PRSV detections. In addition, the real-time RT-PCR technology was applied for quantitative monitoring of different PRSV strains in papaya hosts. Both “SYBR Green” and “TaqMan primer/probe” methods were adopted to develop the quantitative detection of PRSV with real-time RT-PCR, and “TaqMan primer/probe” method obtained more satisfactory results. A TaqMan primer/probe combination (named TP-PRSV), selected from the conserved regions of coat protein gene, was designed for the common detections of PRSV. Three primer/probe kits (named TP-DF, TP-SM, and TP-SMN), selected from the variable regions of P1 gene, were also developed for the strain-specific quantitative detection of the DF, SM and SMN strain, respectively. Our devised real-time RT-PCR assays were further applied to monitor the virus multiplicative dynamics in papaya hosts in the inoculation tests with different PRSV strains. When the papaya hosts (cultivar FR) were inoculated with the DF strain, PRSV could be detected 10 days after inoculation. The replication curve (DF) was increasing linearly from the 10th to 16th day, stationary from the 16th to 22th day, and reaching to the peak 24 days after inoculation. When the papayas were inoculated with the SMN strain, PRSV could be detected 10 days after inoculation. The replication curve (SMN) was increasing linearly from the 10th to 18th day, and reaching to the peak 18 days after inoculation. When the papayas were inoculated with both DF and SMN strains, DF could be detected 16 days after inoculation. The replication curve (DF) was increasing linearly from the 16th to 22th day, and reaching to the peak 22 days after inoculation. On the other hand, SMN could be detected 18 days after inoculation. The replication curve (SMN) was increasing linearly from the 18th to 22th day, and reaching to the peak 22 days after inoculation. The development of real-time RT-PCR assays of PRSV is helpful to the ecological studies of papaya ringspot disease.

Lui Yuk Sun.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2001.
Includes bibliographical references (leaves 138-148).
Abstracts in English and Chinese.
Abstract (English) --- p.i
Abstract (Chinese) --- p.iii
Acknowledgements --- p.iv
Abberviations --- p.v
Table of Contents --- p.vii
List of Tables --- p.xiv
List of Figures --- p.xvi
Chapter 1. --- Introduction --- p.1
Chapter 1.1. --- Bacteriological evaluation of water --- p.1
Chapter 1.1.1. --- Indicator organisms for water quality monitoring --- p.2
Chapter 1.1.2. --- Properties defined for indicator organisms --- p.3
Chapter 1.1.3. --- Example of common indicator organisms --- p.3
Chapter 1.1.3.1. --- Total coliform group --- p.3
Chapter 1.1.3.2. --- "Fecal coliform, Escherichia coli" --- p.4
Chapter 1.1.3.3. --- Fecal Streptococcus --- p.5
Chapter 1.1.3.4. --- Klebsiella --- p.5
Chapter 1.2. --- The need for specific detection of waterborne pathogenic organisms --- p.6
Chapter 1.3. --- Common water-borne pathogenic organisms --- p.7
Chapter 1.3.1. --- Bacteria --- p.7
Chapter 1.3.1.1. --- Escherichia coli 0157:H7 --- p.7
Chapter 1.3.1.2. --- Salmonella typhimurium --- p.11
Chapter 1.3.1.3 --- Legionella pneumophila --- p.12
Chapter 1.3.2. --- Protozoa --- p.14
Chapter 1.3.3. --- Viruses --- p.15
Chapter 1.4. --- Conventional approaches for pathogens detection --- p.16
Chapter 1.4.1. --- Examples of conventional detection methods --- p.17
Chapter 1.4.2. --- Problems related to the conventional detection methods --- p.18
Chapter 1.5. --- Novel approaches for pathogens detection --- p.19
Chapter 1.5.1. --- Modifications of media --- p.19
Chapter 1.5.2. --- Antibody-based methods --- p.20
Chapter 1.5.3. --- Nucleic acid-based methods --- p.21
Chapter 1.6. --- Principles of pathogens concentration by filtration and immunomagnetic separation --- p.22
Chapter 1.7. --- Principles of pathogens detection by polymerase chain reaction --- p.24
Chapter 1.8. --- Principles of quantitative assay of water-borne pathogens using real-time PCR --- p.26
Chapter 1.9. --- Aims of this study --- p.28
Chapter 2. --- Detection of water-borne bacteria by polymerase chain reaction --- p.31
Chapter 2.1. --- Introduction --- p.31
Chapter 2.2. --- Materials and Methods --- p.35
Chapter 2.2.1 --- Bacterial strains --- p.35
Chapter 2.2.2. --- Bacterial enumeration --- p.35
Chapter 2.2.3. --- DNA extraction and purification --- p.36
Chapter 2.2.3.1. --- Boiling method --- p.36
Chapter 2.2.3.2. --- Protinesae K extraction method --- p.36
Chapter 2.2.3.3. --- Chelex extraction method --- p.37
Chapter 2.2.4. --- Targeted sequences --- p.38
Chapter 2.2.4.1. --- eaeA gene --- p.38
Chapter 2.2.4.2. --- mdh gene --- p.39
Chapter 2.2.4.3. --- flaR gene --- p.39
Chapter 2.2.5. --- PCR amplification --- p.40
Chapter 2.2.6. --- Gel electrophoresis --- p.41
Chapter 2.3. --- Results --- p.42
Chapter 2.3.1. --- Optimization of the PCR --- p.42
Chapter 2.3.2. --- Sensitivity of PCR detection --- p.42
Chapter 2.3.2.1. --- Boiling method --- p.42
Chapter 2.3.2.2. --- Proteinease K method --- p.43
Chapter 2.3.2.3. --- Chelex method --- p.43
Chapter 2.3.3. --- Specificity of PCR detection --- p.43
Chapter 2.3.3.1. --- primers targeted uidA gene --- p.44
Chapter 2.3.3.2. --- primers targeted mdh gene --- p.44
Chapter 2.3.3.3. --- primers targeted flaR gene --- p.44
Chapter 2.4. --- Discussion --- p.57
Chapter 3. --- Concentration and separation of water-borne bacteria by two-step-filtration and immunomagnetic separation --- p.61
Chapter 3.1. --- Introduction --- p.61
Chapter 3.2. --- Materials and Methods --- p.66
Chapter 3.2.1. --- Bacterial strains --- p.66
Chapter 3.2.2. --- Bacterial enumeration --- p.66
Chapter 3.2.3. --- Filtration --- p.67
Chapter 3.2.4. --- Immunomagnetic separation (IMS) --- p.68
Chapter 3.2.4.1. --- Antibodies and Magnetic beads --- p.68
Chapter 3.2.4.2. --- Binding of antibodies to magnetic beads --- p.68
Chapter 3.2.4.3. --- Immunomagnetic separation of bacteria in seeded samples --- p.70
Chapter 3.2.5. --- Determine the efficiency of filtration and immunomagnetic separation --- p.70
Chapter 3.2.6. --- DNA extraction --- p.71
Chapter 3.2.7. --- Multiplex PCR --- p.71
Chapter 3.2.8. --- PCR amplification --- p.72
Chapter 3.2.9. --- Gel electrophoresis --- p.72
Chapter 3.3. --- Results --- p.73
Chapter 3.3.1. --- Efficiency of filtration and immunomagnetic separation --- p.73
Chapter 3.3.2. --- Detection limit of PCR --- p.73
Chapter 3.3.2.1. --- Filtration and immunomagnetic separation --- p.73
Chapter 3.3.2.2. --- Influence of background flora --- p.73
Chapter 3.3.2.3 --- Shing Mun River and Lam Tsuen River --- p.77
Chapter 3.3.3. --- Multiplex PCR --- p.77
Chapter 3.4. --- Discussion --- p.91
Chapter 4. --- Quantitative assay of water-borne pathogens using real-time PCR --- p.94
Chapter 4.1. --- Introduction --- p.94
Chapter 4.2. --- Materials and Methods --- p.99
Chapter 4.2.1. --- Bacteria strains --- p.99
Chapter 4.2.2. --- Bacterial enumeration --- p.99
Chapter 4.2.3. --- Primers and Probes --- p.100
Chapter 4.2.3.1. --- eaeA gene --- p.101
Chapter 4.2.3.2. --- mdh gene --- p.102
Chapter 4.2.3.3. --- flaR gene --- p.102
Chapter 4.2.4. --- Targeted sequences cloning and sequencing --- p.103
Chapter 4.2.4.1. --- Amplication of targeted sequence by PCR --- p.103
Chapter 4.2.4.2. --- Purification of PCR product --- p.104
Chapter 4.2.4.3. --- Ligation with cloning vector --- p.105
Chapter 4.2.4.4. --- Transformation of E.coli DH5a cells --- p.105
Chapter 4.2.4.5. --- Plasmid DNA isolation --- p.106
Chapter 4.2.4.6. --- DNA quantitation and sequencing --- p.107
Chapter 4.2.5. --- Quantitation determination using real-time PCR --- p.108
Chapter 4.3. --- Results --- p.110
Chapter 4.3.1. --- Determination of targeted sequences --- p.110
Chapter 4.3.2. --- Reading of fluorescence intensity and data analysis --- p.110
Chapter 4.3.3. --- Sensitivity of real-time PCR --- p.114
Chapter 4.3.4. --- Specificity of real-time PCR --- p.121
Chapter 4.3.5. --- Quantitation analysis in seeded samples --- p.121
Chapter 4.4. --- Discussion --- p.131
Chapter 5. --- Conclusion and future perspectives --- p.133
Chapter 6. --- References --- p.138

碩士
國立臺灣大學
植物病理與微生物學研究所
97
Papaya ringspot , caused by the Papaya ringspot virus (PRSV), is one of the most important diseases in papaya. PRSV is divided into three major strains distinguished by the symptoms on leaves: severe mottling (SM), deformation (DF), and severe mottling with necrosis (SMN). In this study, three papaya cultivars (lines) were inoculated with PRSV (DF and SMN strain), then the Real-Time RT-PCR assay was used to perform quantitative detection to track the distribution, migration, and propagation of each PRSV in papaya hosts. Three papaya cultivars (lines) were used including Tainung No.2 (TN2), Red Lady (RL), and National Taiwan University Hybrid No.8. (NTU8). After inoculation with the DF strain of PRSV (PRSV/DF), viruses could be detected in roots, stems and leaves of NTU8 within 2 days, which is earliest among the three cultivars. Viruses were found in NTU8 leaves within 4 days post-inoculation, indicating that the viruses moved very fast in NTU8, although they did not replicate well and remained at low quantity (103 copies). PRSV/DF was monitored in roots, stems and leaves of TN2 4, 8 and 10 days after inoculation respectively; the virus quantities in leaves of TN2 reached to a peak (107~8 copies) 24 days later, which is the highest compared to RL and NTU8 . For the RL cultivar, viruses were detected in roots, stems and leaves at 7, 8, and 8 days post-inoculation respectively, and the peak quantity in leaves 24 days after inoculation reached 104 copies. In the inoculation tests with the SMN strain of PRSV (PRSV/SMN), NTU8 exhibited detectable virus levels in roots, stems and leaves 2, 2 and 7 days after inoculation respectively, and the virus quantity remained (103~4 copies) at 24 days later. PRSV/SMN in TN2 was detected in roots and stem after 8 and 4 days respectively, and reached into leaves 10 days after inoculation, where quantity could reach as high as 106 copies after 30 days. The roots, stems and leaves of RL showed detectable virus at 8, 8 and 4 days after inoculation, with quantities at about 104 copies in the leaves 24 days later. In roots usually accumulate more or the same amounts than in leaves, and stems gave rise to slightly lower quantity of viruses than in roots as a results of comparative quantitative detection among roots, stems and leaves no matter what cultivar of papaya was used. It seems that roots are very important for PRSV to propagate. The new bred papaya cultivar tolerant to PRSV, NTU8, constantly showed a good replication of PRSV/DF at 106 copies in roots, but a lower amount of 103 copies in the leaves. TN2, the susceptible cultivar to PRSV, showed more viruses in the leaves approximately 104~5 times than NTU8. This is probably one of the factors resulting in the tolerance of NTU8. When NTU8 was inoculated with PRSV/SMN, it had the virus quantity of 107~8 copies in roots, but only 103~4 copies in leaves; which is similar to the case of PRSV/DF. However, NTU8 seems to have slightly lower tolerance to PRSV/SMN. In this thesis, in addition, the Real-Time RT-PCR method performed its better sensitivity and could detect these viruses at the same time or earlier than the conventional RT-PCR method. Furthermore, this method also provides a precise relative quantification for PRSV and it is helpful to understand how the virus propagates in the host, and will be valuable for future ecological study and control of PRSV.

碩士
國立臺灣大學
植物病理與微生物學研究所
102
Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases of rice worldwide. In Taiwan, despite the attempt of developing rice blast forecasting model(s) in 1970s, nationwide disease monitoring and notification has long been relying on periodic surveys by trained plant protection personnel. The objective of this study is to first develop an approach which allows the collection and quantification of M. oryzae conidia (airborne inoculum) in the field. A modified blast disease forecasting model, using the amount of conidia along with several weather factors (including temperature, humidity, rainfall, etc.) as parameters, will then be established. We have successfully developed a cyclone-based spore trap and a standard sample processing protocol for extracting DNA from collected airspores. Using quantitative real-time PCR (qPCR) technology and a specific primer pair designed based on a Magnaporthe infection structure specific protein (mif23) gene, the amount of M. oryzae conidia can be easily quantified. While detection limit for the SYBR Green qPCR assay can be as low as 4 copy numbers of M. oryzae gDNA, the limit for reliable and accurate quantification is 10 copy numbers. For the TaqMan assay, the limit for reliable and accurate quantification is 4 copy numbers. Aiming to build a forecasting model, airspore samples, weather data, and disease severity ratings have been periodically collected from ten monitoring stations located at the paddy field and upland field blast nurseries at Chiayi Agricultural Experiment Station, a field site at Chiayi Sikou Farm, and seven field sites chosen by seven District Agricultural Research and Extension Stations in Taiwan (missing data exist for some of the monitoring stations). With our newly-developed spore trap and qPCR technique, M. oryzae spores can be detected before the appearance of leaf blast. It was observed that during the whole season, the amount of spores first increased while the field plants were commonly infected, and it then dropped after the stage of panicle development. In order to improve the handling and storage of airspore samples, we tested the effects of different treatments on the preservation of spore DNA. The optimized way would be: to avoid UV light exposure while sampling, to suspend the sample with CTAB buffer after collection, to store the sample at room temperature or 4℃, and to finish DNA extraction within two weeks. For disease modeling, we developed preliminary rice blast forecasting models for specific rice cultivars (TK9, TN11 and TC192) and multiple cultivars, on the basis of cumulative meteorological data from 1-14 or 7-14 days prior to the prediction day. It was found that the "number of spores" was not considered a significant parameter in most of the models, indicating that weather parameters such as relative humidity and hours of rainfall may be key factors favoring rice blast development. The approaches, sampling ranges and frequencies of the spore trapping and disease ratings may also have some effect on the result. Finally, to make the spore trapping technique applicable for characterization of pathogen physiological races, we developed a high resolution melt (HRM) technique which was proved to be powerful for the detection of the A, D, A+D, and C types of alleles at the pex31 (Avr-pik/kp/km) gene in M. oryzae. The differentiation limit for the HRM analysis is 25 airspores. In the future, with the use of other specific primer pairs, the spore trapping, qPCR, and HRM techniques develop in this study can be widely applied for the monitoring and detection of various airborne diseases. Since the data used for modeling in our study were from the monitoring stations at the Chiayi blast nursery and Sikou Farm, it is important to know that before the forecasting models can be widely applied, more weather data and disease severity data from multiple years, cultivars, and locations are required for model training, validation, and improvement.