Dissertations / Theses on the topic 'Displacement amplification'

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

Next-generation sequencing technologies are revolutionising our ability to characterise and investigate infectious diseases. Utilising the power of high throughput sequencing, this study reports, the development of a sensitive, non-PCR based, unbiased amplification method, which allows the rapid and accurate sequencing of multiple microbial pathogens directly from clinical samples. The method employs Φ29 DNA polymerase, a highly efficient enzyme able to produce strand displacement during the polymerisation process with high fidelity. Problems with DNA secondary structure were overcome and the method optimised to produce sufficient DNA to sequence from a single bacterial cell in two hours. Evidence was also found that the enzyme requires at least six bases of single stranded DNA to initiate replication, and is not capable of amplification from nicks. Φ29 multiple displacement amplification was shown to be suitable for a range of GC contents and bacterial cell wall types as well as for viral pathogens. The method was shown to be able to provide relative quantification of mixed cells, and a method for quantification of viruses using a known standard was developed. To complement the novel molecular biology workflow, a data analysis pipeline was developed to allow pathogen identification and characterisation without prior knowledge of input. The use of de novo assemblies for annotation was shown to be equivalent to the use of polished reference genomes. Single cell Φ29 MDA samples had better assembly and annotation than non-amplification controls, a novel finding which, when combined with the very long DNA fragments produced, has interesting implications for a variety of analytical procedures. A sampling process was developed to allow isolation and amplification of pathogens directly from clinical samples, with good concordance shown between this method and traditional testing. The process was tested on a variety of modelled and real clinical samples showing good application to sterile site infections, particularly bacteraemia models. Within these samples multiple bacterial, viral and parasitic pathogens were identified, showing good application across multiple infection types. Emerging pathogens were identified including Onchocerca volvulus within a CSF sample, and Sneathia sanguinegens within an STI sample. Use of Φ29 MDA allows rapid and accurate amplification of whole pathogen genomes. When this is coupled with the sample processing developed here it is possible to detect the presence of pathogens in sterile sites with a sensitivity of a single genome copy.

The suitability of electromagnetic actuating devices for application to machines with ever more demanding response time specifications is discussed, with the proposal that piezoelectric actuator technology can produce practical devices with faster response times than solenoids, for example. This thesis discusses and validates the proposition that the performance of piezoelectric ceramic actuators makes them viable devices for inclusion in high-speed machine applications, where rapid clutching using two-state actuation is required. Further, techniques are devised and explored for the design and application of these devices using displacement amplifying structures, which lead to the utilisation of engineering methods of relatively low precision. This is highly advantageous as to date, the piezoelectric multilayer actuator has usually been associated with high precision engineering. Applications of piezoelectric ceramic technology are reviewed, and the mechanical and electrical properties of these materials are discussed. Literature covering applications of piezoelectric actuators in relation to clutches, motors and positioners is also reviewed. This data search revealed many devices or systems where the displacement amplification of piezoelectric actuators was exploited in some way, but failed to show any devices where the high efficiency of these amplifying structures was either primary or even necessarily achieved. Indeed, it was concluded that in the absence of such applications or methodologies, a fruitful area of research might be to explore these methodologies. This work is a core element of this thesis. Using two basic topologies, devices producing efficient transformation of high-force, small movement two-state actuation, to larger movement with lower force, have been designed (using flexural hinge methods), manufactured, tested and analyzed. Hydraulic transformers have been briefly investigated and ultimately rejected on the grounds of comparative complexity. For any displacement amplifying or transforming device, applications for these systems are widely varying, but criteria for advantageous employment of the piezoelectric approach, as opposed to electromagnetic, are established. Design techniques which are partially analytical and partially experiential are proposed, which in practice exhibit adeptness for producing well-optimised designs. These methods are incorporated into special purpose structure-designer computer programs. Several design examples are detailed, and their performance analyzed in comparison with the modelling techniques and design program predictions. The application of these displacement amplifiers is discussed by example, to two discrete motion machines, both of which have been designed specifically to demonstrate the possibilities of using piezoelectric technology to regulate discrete motion drives. It is shown that the speed of response of the devices is such, that the concept of zero-velocity clutching with the intention of minimising wear, is feasible.

The availability of DNA samples that are of adequate quality and quantity is essential for any genetic analysis. The fields of forensic biology and clinical diagnostic pathology testing often suffer from limited samples that yield insufficient DNA material to allow extensive analysis. This study examined the utility of a recently introduced whole genome amplification method termed Multiple Displacement Amplification (MDA) for amplifying a variety of limited sample types that are commonly encountered in the fields of forensic biology and clinical diagnostics. The MDA reaction, which employs the highly processive bacteriophage φ29 DNA polymerase, was found to generate high molecular weight template DNA suitable for a variety of downstream applications from low copy number DNA samples down to the single genome level. MDA of single cells yielded sufficient DNA for up to 20,000,000 PCR assays, allowing further confirmatory testing on samples of limited quantities or the archiving of precious DNA material for future work. The amplification of degraded DNA material using MDA identified a requirement for samples of sufficient quality to allow successful synthesis of product DNA templates. Furthermore, the utility of MDA products in comparative genomic hybridisation (CGH) assays identified the presence of amplification bias. However, this bias was overcome by introducing a novel modification to the MDA protocol. Future directions for this work include investigations into the utility of MDA products in short tandem repeat (STR) assays for human identifications and application of the modified MDA protocol for testing of single cell samples for genetic abnormalities.

One of the great challenges of microbial analysis in natural environments derives from the fact that a large proportion of microorganisms present is not culturable in standard conditions. To overcome this, various molecular approaches are widely used. Multiple Displacement Amplification (MDA) is especially useful as it can be applied in conjunction with other techniques to identify genes derived from individual microbial cells. The aim of this project was to study freshwater samples obtained from various environments in Ireland in order to identify dominant bacterial species and key genes associated with them that were likely to be involved in biodegradation of contaminating compounds. In order to do this, various molecular approaches were applied, with the most important being MDA assisted PCR. The tlree fresh water environments studied were: commercial bottled water, agriculturally contaminated ground water obtained from Co. Kilkenny, Ireland and ground water samples obtained from a diesel-contaminated site in Northern Ireland. In these environments, dominant bacterial strains were identified using MDA assisted PCR. Strains related to Rhodoferax ferrireducens were identified in commercial bottled water; Pseudomonas fluorescens and Polaromonas sp. in agriculturally-contaminated ground water samples; Dechloromonas aromatica and Pseudomonas putida in diesel-contaminated ground water samples. Functional genes (nitrate reductase and naphthalene dioxygenase) which are known to be involved in biodegradation were shown to be present in some of the strains. It was shown that the narG gene (nitrate reductase) was associated with strains related to Pseudomonas fluorescens, Alicycliphilus denitrificans and Polaromonas naphthalenivorans detected in agricultural-contaminated ground water. Naphthalene dioxygenase gene (nahAc) was associated with strains related to Pseudomonas fluorescens, Pseudomonas putida and nagAc (another naphthalene dioxygenase gene) with Ralstonia pickettii detected III ground water samples obtained from a diesel-contaminated site in Northern Ireland. To achieve a more comprehensive characterization of the studied environments corresponding 16S rRNA gene libraries were obtained and analysed. Sequences found in these libraries were affiliated with Proteobacteria, Bacteroidetes, Firmicutes, Chloroflexi, Actinobacteria and Cyanobacteria

Many of our closest relatives, the primates, are endangered and could be extinct in a near future. To increase the knowledge of non-human primate genomes, and at the same time acquire information on our own genomic evolution, studies using high-throughput technologies are applied, which raises the demand for large amounts of high quality DNA.

In study I and II, we evaluated the multiple displacement amplification (MDA) technique, a whole genome amplification method, on a wide range of DNA sources, such as blood, hair and semen, by comparing MDA products to genomic DNA as templates for several commonly used genotyping methods. In general, the genotyping success rate from the MDA products was in concordance with the genomic DNA. The quality of sequences of the mitochondrial control region obtained from MDA products from blood and non-invasively collected semen samples was maintained. However, the readable sequence length was shorter for MDA products.

Few studies have focused on the genetic variation in the nuclear genes of non-human primates. In study III, we discovered 23 new single nucleotide polymorphisms (SNPs) in the Y-chromosome of the chimpanzee. We designed a tag-microarray minisequencing assay for genotyping the SNPs together with 19 SNPs from the literature and 45 SNPs in the mitochondrial DNA. Using the microarray, we were able to analyze the population structure of wild-living chimpanzees.

In study IV, we established 111 diagnostic nucleotide positions for primate genera determination. We used sequence alignments of the nuclear epsilon globin gene and apolipoprotein B gene to identify positions for determination on the infraorder and Catarrhini subfamily level, respectively, and sequence alignments of the mitochondrial 12S rRNA (MT-RNR1) to identify positions to distinguish between genera. We designed a microarray assay for immobilized minisequencing primers for genotyping these positions to aid in the forensic determination of an unknown sample.

Multiple Displacement Amplification (MDA) has been used to uniformly amplify bacterial genomes present in small samples, providing abundant targets for molecular analysis. The purpose of this investigation was to combine MDA and checkerboard DNA-DNA hybridization to examine the microbiota of endodontic infections. 66 samples were collected from teeth with endodontic infections. Non-amplified and MDA amplified samples were analyzed by checkerboard DNA-DNA hybridization for levels and proportions of 77 bacterial taxa. Counts, % DNA probe counts and % of teeth colonized for each species in amplified and non-amplified samples were computed. Significance of differences for each species between amplified and non- amplified samples was determined using the Wilcoxon signed ranks test and adjusted for multiple comparisons. The average amount of DNA present in clinical samples ranged from 6.80 (± 5.2) ng before to 6.26 (± 1.73) ìg after MDA. 70 of the 77 DNA probes hybridized with one or more of the non-amplified samples, while all probes hybridized with at least one sample after amplification. Most commonly detected species at levels > 104 in both amplified and non-amplified samples were Prevotella tannerae and Acinetobacter baumannii at frequencies ranging from 89-100% of samples. The mean number (± SEM) of species at counts >104 in amplified samples was 51.2 ± 2.2 and in non-amplified samples was 14.5 ± 1.7. The combination of MDA and checkerboard DNA-DNA hybridization demonstrated the presence of wide range of bacterial species in endodontic samples and could facilitate studies evaluating the microbiota of endodontic infections.
A técnica do Multiple Displacement Amplification (MDA) vem sendo utilizado para amplificar uniformemente o genoma bacteriano presente em pequenas amostras, fornecendo grandes melhorias nas análises moleculares. O propósito desta pesquisa foi associar o MDA e a hibridização DNA-DNA (checkerboard) para examinar a microbiota de infecções endodônticas. Sessenta e seis amostras foram coletadas de infecções endodônticas. As amostras não amplificadas e aquelas amplificadas pelo MDA foram analisadas pelo checkerboard para a determinação dos níveis e proporções de 77 taxas bacterianas. Computaram-se a contagem, percentagem do total de DNA e percentagem de dentes colonizados para cada espécie em amostras amplificadas e não amplificadas. As diferenças significantes para cada espécie entre as amostras amplificadas e não amplificadas foram determinadas utilizando-se o teste de Wilcoxon e ajustado para comparações múltiplas. A quantidade média de DNA presente nas amostras clínicas variou de 6,80 (± 5,2) ng sem amplificação a 6,26 (± 1,73) ìg após a utilização do MDA. Setenta das 77 sondas de DNA hibridizaram com uma ou mais das amostras não amplificadas, enquanto todas as sondas hibridizaram com no mínimo uma amostra após a amplificação. As espécies mais comumente detectadas no nível > 104 células bacterianas, nas amostras amplificadas e não amplificadas, foram Prevotella tannerae e Acinetobacter baumannii numa freqüência que variou de 89-100% das amostras. O número médio (± SEM) de espécies nas contagens >104 células bacterianas, nas amostras amplificadas, foi de 51,2 ± 2,2 e, nas não amplificadas, foi de 14,5 ± 1,7. A combinação do MDA e da hibridização DNA-DNA (checkerboard) demonstrou a presença de uma grande variedade de espécies bacterianas nas amostras endodônticas demonstrando sua utilidade naqueles estudos que avaliam a microbiota presente nas infecções endodônticas.

Vid klinisk enbaspolymorfi (SNP) analys inkluderas DNA-kontroller med kända genotyper i varje analysomgång för att säkerställa riktigheten vad gäller analysresultatet. DNA-kontrollerna har en central roll för resultatens trovärdighet vid genotypningen. Vissa kontrollprover som används är av sällsynt genotyp och kan vara mycket svåra att få tag på. Detta arbete har utförts för att undersöka om det går att erhålla DNA-material från sällsynta genotyper med hjälp av helgenomamplifiering och på så sätt säkerställa en tillgång till dessa. I arbetet testades helgenomamplifiering med hjälp av två olika kit. De helgenomamplifierade produkternas kvantitet och kvalitet analyserades och jämfördes med det ursprungliga DNA:t, med avsikt att redogöra för det mest fördelaktiga kitet för SNP-analys i kliniskt syfte. Båda helgenomamplifierings-kiten påvisade god förmåga att amplifiera genomiskt DNA med hög kvalité. Helgenomamplifierat DNA från det bästa kitet sekvenserades och här var skillnader mellan ursprungligt och helgenomamplifierat DNA marginella. Vid sekvensanalys av ett 464 baspar långt fragment av faktor II genen och 585 baspar långt fragment av ApoE genen på fem helgenomamplifierade DNA-prover påvisades endast en eventuell diskrepans.
Clinical single nucleotide polymorphisms (SNP) analysis includes DNA controls with known genotypes in each run to ensure the accuracy of the analysis results. DNA controls have a central role for the credibility of the results in the genotyping process. Some of the used control samples are rare and can be very difficult to obtain. This work was carried out to investigate whether it is possible to obtain DNA from samples with a rare genotype using whole genome amplification and as a result ensure access to these samples. In this work the whole genome amplification method was tested by two different kits. The quantity and quality of the whole genome amplification products were analyzed and compared with the original DNA, with the intention to describe the most advantageous kit for clinical SNP analysis. Both tested kits demonstrated a good ability to amplify genomic DNA with high quality. Whole genome amplified DNA from the best kit was sequenced and the difference between the original DNA and whole genome amplified DNA was negligible. Sequence analysis of 464 base pairs of the factor II gene and 585 base pairs of the ApoE gene in five whole genome amplified DNA samples indicated only one possible discrepancy.

Establishment of relationships for predicting the lateral drift demands of near-fault ground motions is one of the major challenges in earthquake engineering. Excessive lateral drifts caused by earthquake ground motions are the major causes of structural damage observed in structures. In this study, some of the fundamental characteristics of near-fault ground motions are examined. Response characteristics of elastic frame structures to near-fault ground motions are investigated. An approximate method for estimating the elastic ground story and interstory drifts for regular frame type structures is presented. Inelastic displacement demands imposed on elasto-plastic single degree of freedom (SDOF) systems subjected to near-fault ground are examined. Three equations for estimating the maximum lateral inelastic displacement demand from the maximum elastic displacement demand are established. Two of these equations relate the inelastic and elastic displacement demands through natural period and strength reduction factor. The third equation relates the inelastic and elastic displacement demands through the ratio of natural period to pulse period and the strength reduction factor. Efficiency of the natural period to pulse period ratio for estimating the inelastic displacement ratio is shown. Error statistics of the proposed equations are presented and compared with similar studies in the literature. According to the results, these equations can be used for quick and rough estimates of displacement demands imposed on regular elastic moment resisting frames and elasto-plastic single degree of systems.

Multiple Displacement Amplification (MDA) has been used to uniformly amplify the genome from bacterial species in different types of oral samples. MDA is particularly useful in small samples, since it generates abundant target for microbial analysis. The aim of the present study was to combine MDA and Checkerboard DNA-DNA hybridization to evaluate the microbiota of endodontic infections in deciduous teeth A total of 35 children, 4 to 10 years old, having teeth with intact roots or less than 2/3 of physiological root resorption were involved in this study. Forty root canal samples were collected.and amplified. Amplified samples were analyzed by checkerboard DNA-DNA hybridization for levels of 83 bacterial taxa. . Percentages of teeth colonized by each species at different thresholds in amplified samples were computed. Levels of bacterial species present in different clinical conditions were analyzed. Significance of differences between mean proportions of each species were determined for root canal samples taken from teeth with (open tooth) or without (closed tooth) pulp chamber exposure to oral cavity, sinus tract, swelling, and pain. Significance of differences for each species in these clinical scenarios was sought with Kruskall-Wallis test. The mean amount of DNA (± SD) in the samples prior to amplification was 5.2 (± 4.7) ng. After MDA, samples contained, on average, 6.05 (± 2.3) ìg of DNA. Eighty of 83 DNA probes hybridized with one or more samples. Most prevalent bacterial species at levels > 104 bacterial cells were Actinomyces naeslundii 1 and Prevotella intermedia, both present in 93.8%.of sampled teeth. The mean number of species (± SEM) detected per tooth at the > 104 level was 20.19 (± 3.27).The most commonly detected species at this level were Actinomyces naeslundii 1 and Prevotella intermedia When mean DNA probe counts x 105 (± SEM) were analyzed, the most abundant species were A. naeslundii 1 (17.07±3.17), Prevotella nigrescens (1.12 ± 0.55) and P. intermedia (1.01 ± 0.30). Eikenella corrodens, Haemophilus aphrophilus, and Helicobacter pylori were not detected in any of the samples. Upon the analysis of the microbiota associated with the different clinical signs and symptoms investigated, statistically significant differences could be detected in a few of them. Twenty seven species were statistically significantly increased in the open tooth group. A. naeslundii 1, Veillonella parvula, Gemella morbillorum. Streptococcus oralis, Aggregatibacter actinomycetemcomitans and Neisseria mucosa were statistically significant increased in teeth with pulp chamber exposure to oral cavity. P. intermedia, Neisseria. mucosa, Streptococcus anginosus, Selenomonas noxia and Streptococcus sanguinis were detected in higher mean counts in teeth without sinus tract. There were no statistically significant differences in the microbiota associated with presence or absence of swelling. Painful teeth presented increased counts of Prevotella nigrescens and Prevotella oris. The microbiota associated with root canals from deciduous teeth seems to be more complex than previously anticipated In conclusion, results suggest that selected species are associated with the clinical signs and symptoms detected in primary root canal infections.
O Mulltiple Displacement Amplification (MDA) tem sido utilizado para amplificação uniforme do genoma de espécies bacterianas em diferentes amostras da cavidade oral. O MDA é particularmente útil em pequenas amostras, visto que o mesmo gera uma quantidade de amostra de DNA abundante para a análise microbiana. O objetivo do presente estudo foi avaliar a microbiota de infecções endodônticas de dentes decíduos. Um total de 35 crianças, de 4 a 10 anos de idade, apresentando dentes com raízes intactas ou menos que 2/3 de rizólise foram envolvidas no estudo. Quarenta amostras foram coletadas e amplificadas pela técnica do MDA. As amostras amplificadas foram analisadas pela hibridização DNA-DNA (Checkerboard) para taxas de 83 espécies bacterianas. Foram computadas as porcentagens de dentes colonizados por cada uma das espécies em diferentes limiares nas amostras amplificadas. Os níveis das espécies bacterianas encontradas em diferentes condições clínicas foram analisados. A significância das diferenças entre as proporções de cada espécie foram determinadas para amostras de canais radiculares de dentes com ou sem câmara pulpar exposta à cavidade oral, fístula, edema, e dor. A significância das diferenças para cada espécie nos diferentes cenários clínicos foi analisada pelo teste Kruskall-Wallis. A quantidade de DNA (± DP) nas amostras antes da amplificação era 5.2 (± 4.7) ng. Após o MDA, as amostras continham, em média, 6.05 (± 2.3) ìg de DNA. Oitenta das 83 sondas de DNA hibridizaram com uma ou mais amostras. As espécies bacterianas mais prevalentes em níveis > 104 células bacterianas foram Actinomyces naeslundii 1 e Prevotella intermedia, ambas presentes em 93.8% dos dentes analisados. O número médio de espécies (± DPM) detectadas por dente no nível de > 104 foi 20.19 (± 3.27). As espécies mais comumente encontradas neste nível foram Actinomyces naeslundii 1 e Prevotella intermédia. Quando a média de sondas de DNA x 105 (± DPM) foi analisada, as espécies mais abundantes foram A. naeslundii 1 (17.07±3.17), Prevotella nigrescens (1.12 ± 0.55) e P. intermedia (1.01 ± 0.30). Eikenella corrodens, Haemophilus aphrophilus, e Helicobacter pylori não foram detectados em nenhuma das amostras. Em relação à análise da microbiota associada a diferentes sinais e sintomas clínicos, diferenças estatisticamente significantes foram detectadas em algumas situações. Vinte e sete amostras foram estatisticamente significantes ao serem encontradas em maiores contagens em dentes abertos. A. naeslundii 1, Veillonella parvula, Gemella morbillorum. Streptococcus oralis, Aggregatibacter actinomycetemcomitans e Neisseria mucosa foram estatisticamente significantemente encontradas em maior número em dentes com exposição da câmara pulpar à cavidade oral. P. intermedia, Neisseria mucosa, Streptococcus anginosus, Selenomonas noxia e Streptococcus sanguinis foram detectados em contagens médias mais altas em dentes sem fístula. Não houve diferenças estatisticamente significantes na microbiota associada à presença ou ausência de edema. Dentes com dor apresentaram contagens elevadas de Prevotella nigrescens e Prevotella oris. A microbiota associada a canais radiculares de dentes decíduos demonstra ser mais complexa do que antes imaginado. Em conclusão, os resultados sugerem que espécies selecionadas estão associadas com os sinais e sintomas clínicos detectados em infecções endodônticas de dentes decíduos.

Design recommendations for steel plate shear wall (SPSW) systems have recently been introduced into seismic provisions for steel buildings. Response modification, overstrength, and displacement amplification factors for SPSW systems presented in the design codes were based on professional experience and judgment. A numerical study has been undertaken to evaluate these factors for SPSW systems. Forty four unstiffened SPSWs possessing different geometrical characteristics were designed based on the recommendations given in the AISC Seismic Provisions. Bay width, number of stories, story mass, and steel plate thickness were considered as the prime variables that influence the response. Twenty records were selected to include the variability in ground motion characteristics. In order to provide a detailed analysis of the post-buckling response, three-dimensional finite element analyses were conducted for the 44 structures subjected to the selected suite of earthquake records. For each structure and earthquake record two analyses were conducted in which the first one includes geometrical nonlinearities and the other one includes both geometrical and material nonlinearities, resulting in a total of 1760 time history analysis. In this thesis, the details of the design and analysis methodology are given. Based on the analysis results response modification, overstrength and displacement amplification factors for SPSW systems are evaluated.

Protoplast fusion of Lolium perenne and Lotus corniculatus for gene introgression by Sanjeev V. Raikar Lolium perenne is one of the most important forage crops globally and in New Zealand. Lotus corniculatus is a dicotyledonous forage that contains valuable traits such as high levels of condensed tannins, increased digestibility, and high nitrogen fixing abilities. However, conventional breeding between these two forage crops is impossible due to their markedly different taxonomic origin. Protoplast fusion (somatic hybridisation) provides an opportunity for gene introgression between these two species. This thesis describes the somatic hybridisation, the regeneration and the molecular analysis of the putative somatic hybrid plants obtained between L. perenne and L. corniculatus. Callus and cell suspensions of different cultivars of L. perenne were established from immature embryos and plants were regenerated from the callus. Of the 10 cultivars screened, cultivars Bronsyn and Canon had the highest percentage of callus induction at 36% each on 5 mg/L 2,4-D. Removal of the palea and lemma which form the seed coat was found to increase callus induction ability of the embryos. Plant regeneration from the callus was achieved when the callus was plated on LS medium supplemented with plant growth regulators at different concentrations. Variable responses to shoot regeneration was observed between the different cultivars with the cv Kingston having the lowest frequency of shoot formation (12%). Different factors affecting the protoplast isolation of L. perenne were investigated. The highest protoplast yield of 10×106 g-1FW was obtained when cell suspensions were used as the tissue source, with enzyme combination ‘A’ (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. Development of microcolonies was only achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. All the shoots regenerated from the protoplast-derived calli were albino shoots. The highest protoplast yield (7×106 g-1FW) of L. corniculatus was achieved from cotyledons also with enzyme combination ‘A’ (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. The highest plating efficiency for L. corniculatus of 1.57 % was achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. The highest frequency of shoot regeneration (46%) was achieved when calli were plated on LS medium with NAA (0.1 mg/L) and BA (0.1 mg/L). Protoplast fusion between L. perenne and L. corniculatus was performed using the asymmetric somatic hybridisation technique using PEG as the fusogen. L. perenne protoplasts were treated with 0.1 mM IOA for 15 min and L. corniculatus protoplasts were treated with UV at 0.15 J/cm2 for 10 min. Various parameters affecting the fusion percentage were investigated. Successful fusions were obtained when the fusions were conducted on a plastic surface with 35% PEG (3350 MW) for 25 min duration, followed by 100 mM calcium chloride treatment for 25 min. A total of 14 putative fusion colonies were recovered. Shoots were regenerated from 8 fusion colonies. Unexpectedly, the regenerated putative hybrid plants resembled L. corniculatus plants. The flow cytometric profile of the putative somatic hybrids resembled that of L. corniculatus. Molecular analysis using SD-AFLP, SCARs and Lolium specific chloroplast microsatellite markers suggest that the putative somatic hybrids could be L. corniculatus escapes from the asymmetric protoplast fusion process. This thesis details a novel Whole Genome Amplification technique for plants using Strand Displacement Amplification technique.

La plupart des bâtiments de moyenne et grande hauteur en béton armé sont munis de murs de refend ductiles afin résister aux charges latérales dues au vent et aux séismes. Les murs de refend ductiles sont conçus selon des règles conception stricts. Ces murs sont généralement conçus de façon à forcer la formation d’une rotule plastique à leur base dans l’éventualité d’un séismemajeur. Lors de la conception d’un mur, l’enveloppe des moments fléchissants ainsi que l’enveloppe des efforts tranchants dans la portion du mur situé au-dessus de la rotule plastique sont basés sur la résistance probable en flexion du mur dans la région de la rotule plastique. Plusieurs études sur les murs de refend conçus selon cette philosophie de conception on fait le constat que l’effort tranchant maximum dans un mur peut être sous-estimé lors d’un séisme, et que des rotules plastiques peuvent également se former à d’autres endroits qu’à la base du mur, ce qui constitue un mécanisme de ruine indésirable. Ces effets sont principalement attribuables à la contribution des modes supérieures à la réponse dynamique globale des bâtiments lors d’un séisme. L’effet des modes supérieurs est particulièrement important dans les bâtiments élancés de grande hauteur ayant une période propre de vibration longue. L’essai pseudo-dynamique avec sous-structure est uneméthode efficace et économique d’évaluer expérimentalement l’effet des modes supérieurs sur le comportement sismique des murs de refend dans les bâtiments. Lors de tels essais, comme la masse du bâtiment est modélisée numériquement, ceci permet de tester des structures à de relativement grandes échelles sans avoir à combattremécaniquement les forces d’inerties générées lors d’un séisme. Dans le cadre de la présente étude, la portion constituant la base d’un mur de refend correspondant à la zone de rotule plastique faisant partie d’un bâtiment de huit étages à l’échelle 1/2,75 a été testé. Les dimensions générales de la portion de mur testée étaient de 1800 mm de longueur, par 2200 mm de hauteur par 160 mm d’épaisseur. Le mur étudié a été conçu selon l’édition 2015 du Code National du Bâtiment du Canada (CNBC 2015) ainsi que selon la norme CSA A23.3-14 (Calcul des ouvrages en béton), où le facteur d’amplification de l’effort tranchant causé par l’effet des modes supérieurs n’a pas été pris en compte. Lors des essais pseudo-dynamiques avec sous-structure, une nouvelle méthode de contrôle à trois degrés de liberté convenant à des spécimens d’essai très rigides axialement a été développée et validée. Une procédure novatrice de redémarrage d’un essai interrompu en cours de route a également été développée et validée. Lors des essais, le bâtiment de huit étages incluant la portion de mur dans le laboratoire a été soumis à trois séismes. Le premier séisme était de très faible intensité, l’intensité du deuxième séisme correspondait au séisme de conception, et le troisième séisme correspondait au séisme de conception dont l’intensité a été doublé. Durant les deux séismes de forte intensité, le mur testé s’est comporté de manière ductile et des fissures de cisaillement et de flexion importantes ont été observées. Même si l’effort tranchant maximum mesuré durant le séisme de conception a atteint 2,16 fois la valeur de conception du mur, et 3,01 fois la valeur de conception du mur dans le cas du séisme amplifié, aucun mécanisme de ruine n’a été observé. Suite aux essais pseudo-dynamiques avec sous-structure, un essai par poussée progressive a également été effectué. Les résultats des essais pseudo-dynamiques avec sous-structure portent à croire que la valeur de l’effort tranchant de conception d’un mur selon la norme CSA A23.3-14 est sous-estimé. De plus, l’essai poussée progressive a permis de démontrer que lemur était beaucoup plus résistant qu’anticipé, puisque l’effort tranchant avait été sous-estimé lors de la conception. L’essai par poussée progressive a également permis de démontrer que le mur peut atteindre des niveaux de ductilité en déplacement supérieur à celui prévu par la norme CSA A23.3-14.
Abstract: Most mid- and high-rise reinforced concrete (RC) buildings rely on RC structural walls as their seismic force resisting system. Ductile RC structural walls (commonly called shear walls) designed according to modern building codes are typically detailed to undergo plastic hinging at their base. Both the design moment envelope for the remaining portion of the wall and the design shear forces are evaluated based on the probable flexural resistance of the wall in the plastic hinge region. Several analytical studies have shown that so-designed structural walls can be subjected to shear forces in excess of the design values. Plastic hinging can also develop in the upper portion of the walls. These effects are mainly attributed to higher mode response and, hence, are more severe in taller or slender walls with long fundamental periods. Considering the literature, there is a significant uncertainty regarding the behavior of the structural walls under the higher mode of vibrations excited under earthquake excitations. Hybrid testing is an effective experimentalmethod to study the natural behaviour of structures such as shear walls. The hybrid testing method enables the simulation of the seismic response of large structural elements like RC shear walls without the need to include large masses typically encountered in multi-storey buildings. In this study a barbell shaped RC shear wall specimen of 1800mm in length including a 300mm × 300mm boundary element at each end that is 2200mm in height, and 160mm thick was investigated. A test specimen corresponding to the base plastic hinge zone of an 8-storey shear wall was tested in a laboratory evolvement whilst the reminder of the building structure was modeled numerically. The reference wall was scaled down by a factor of 1/2.75 to obtain dimensions of the test specimen. The RC wall was designed in accordance with the 2015 edition of the National Building Code of Canada (NBCC 2015) and the Canadian Standard Association A23.3-14 code. The amplification of the base design shear force accounting for the inelastic effects of higher modes specified by the CSAA23.3-14 standard was not taken into account in order to evaluate the amplification experimentally. In order to investigate the response of ductile RC walls under earthquake ground motions and track the effect of the higher vibration modes on the shear force demand, three earthquakes with different intensities were applied on the hybrid model successively. The RC wall exhibited a ductile behaviour under the ground motions and flexural and shear cracks developed all over the height of the wall. In spite of amplifying the shear force demand by a factor of 2.16 under the design level earthquake and 3.01 under a high intensity earthquake, no shear failure was observed. The test results indicated that the amplification of the design shear forces at the base of ductile RC shear walls are underestimated by the CSAA23.3-14 standard. A new method for controlling three degrees of freedomin hybrid simulation of the earthquake response of stiff specimens was developed and verified in this study. Also, an innovative procedure to restore an interrupted hybrid test was programmed and verified. The hybrid tests were followed by a push-over test under a lateral force distribution equal to the square root of sum of the squares of the first five modes in order to evaluate the displacement ductility of the RC wall. Findings of the final push-over test showed that the tested ductile RC wall can withstand higher displacement ductilities than the presented levels in the NBCC 2015.

This study deals with the organisation of collective movements in ants in presence of environmental heterogeneities, particularly in situations involving crowding. We chose two species of ants, characterised by different degrees of polymorphism, as well as by their mode of food transport. Our aim is to identify the link between the behavioural rules observed at the scale of the individual and the spatio-temporal organisation observed at the scale of the group. Ndependently of the species, we found that the regulation of traffic in crowding situations depends both on interattraction processes, via the communication through the chemical trail, and on dispersion phenomena. These latter vary as a function of the size of the individuals and of the task they achieve, but give rise to comparable organisations in the two species studied. The originality of this work lies in the fact that it shows that the mechanisms of dispersion allowing the regulation of the traffic and the prevention of crowding are a by-product of the interattraction processes.

Lolium perenne is one of the most important forage crops globally and in New Zealand. Lotus corniculatus is a dicotyledonous forage that contains valuable traits such as high levels of condensed tannins, increased digestibility, and high nitrogen fixing abilities. However, conventional breeding between these two forage crops is impossible due to their markedly different taxonomic origin. Protoplast fusion (somatic hybridisation) provides an opportunity for gene introgression between these two species. This thesis describes the somatic hybridisation, the regeneration and the molecular analysis of the putative somatic hybrid plants obtained between L. perenne and L. corniculatus. Callus and cell suspensions of different cultivars of L. perenne were established from immature embryos and plants were regenerated from the callus. Of the 10 cultivars screened, cultivars Bronsyn and Canon had the highest percentage of callus induction at 36% each on 5 mg/L 2,4-D. Removal of the palea and lemma which form the seed coat was found to increase callus induction ability of the embryos. Plant regeneration from the callus was achieved when the callus was plated on LS medium supplemented with plant growth regulators at different concentrations. Variable responses to shoot regeneration was observed between the different cultivars with the cv Kingston having the lowest frequency of shoot formation (12%). Different factors affecting the protoplast isolation of L. perenne were investigated. The highest protoplast yield of 10×10⁶ g⁻¹FW was obtained when cell suspensions were used as the tissue source, with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. Development of microcolonies was only achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. All the shoots regenerated from the protoplast-derived calli were albino shoots. The highest protoplast yield (7×10⁶ g⁻¹FW) of L. corniculatus was achieved from cotyledons also with enzyme combination 'A' (Cellulase Onozuka RS 2%, Macerozyme R-10 1%, Driselase 0.5%, Pectolyase 0.2%), for 6 h incubation period in 0.6 M mannitol. The highest plating efficiency for L. corniculatus of 1.57 % was achieved when protoplasts were plated on nitrocellulose membrane with a L. perenne feeder layer on PEL medium. The highest frequency of shoot regeneration (46%) was achieved when calli were plated on LS medium with NAA (0.1 mg/L) and BA (0.1 mg/L). Protoplast fusion between L. perenne and L. corniculatus was performed using the asymmetric somatic hybridisation technique using PEG as the fusogen. L. perenne protoplasts were treated with 0.1 mM IOA for 15 min and L. corniculatus protoplasts were treated with UV at 0.15 J/cm² for 10 min. Various parameters affecting the fusion percentage were investigated. Successful fusions were obtained when the fusions were conducted on a plastic surface with 35% PEG (3350 MW) for 25 min duration, followed by 100 mM calcium chloride treatment for 25 min. A total of 14 putative fusion colonies were recovered. Shoots were regenerated from 8 fusion colonies. Unexpectedly, the regenerated putative hybrid plants resembled L. corniculatus plants. The flow cytometric profile of the putative somatic hybrids resembled that of L. corniculatus. Molecular analysis using SD-AFLP, SCARs and Lolium specific chloroplast microsatellite markers suggest that the putative somatic hybrids could be L. corniculatus escapes from the asymmetric protoplast fusion process. This thesis details a novel Whole Genome Amplification technique for plants using Strand Displacement Amplification technique.

Le travail proposé est le résultat d’une collaboration entre le laboratoire Physique de la matière condensée et nanosciences de Monastir, le laboratoire Laplace de Toulouse et le laboratoire Hubert Curien de Saint-Étienne. Il s’inscrit dans un projet visant à étudier théoriquement la propagation des ondes électromagnétiques dans des dispositifs passifs à base du matériau magnétique anisotrope. La présence d’une couche magnétique dans telle structure a de nombreux avantages : non réciprocité́, performances intéressantes, isolation importante, …Plus précisément, c’est la fabrication des dispositifs non-réciproques tels que les isolateurs et les circulateurs. L’étude théorique d’une telle structure avec plusieurs couches de substrats anisotropes nécessite l’utilisation des logiciels commerciaux. Ces logiciels ont l’avantage de permettre l’étude de structures très complexes, mais ont pour principaux inconvénients leur coût d’achat et capacité́ mémoire nécessaire. C’est pourquoi il est indispensable d’une part de comprendre le comportement des matériaux magnétiques utilisés dans la structure : il s’agit de modéliser le matériau magnétique par un tenseur de perméabilité dépendant de son état d’aimantation. L’objectif de cette thèse est la modélisation électromagnétique par la méthode SDA numérique des structures complexes avec des milieux anisotropes dispersifs. Cette approche, est basé sur le calcul numérique de la fonction de Green spectrale par la méthode de l’opérateur transverse (MOT), sera en mesure d’atteindre un niveau élevé de précision dans la description de l’interaction du signal haute fréquence avec une structure constituée d’un milieu magnétique anisotrope saturé. Les résultats de simulation ont été validés avec d’autres résultats numériques publiés dans la littérature et le logiciel commercial HFSS dans le cas d’une ligne microruban imprimée sur une couche isotrope (diélectrique). Ils ont été numériquement validés avec la littérature pour la même structure mais présence d’un substrat magnétique anisotrope pour différentes directions de polarisation. Ensuite, Ils ont été validés aussi par confrontation avec les résultats expérimentaux pour une ligne coplanaire fabriquée sur un substrat de ferrite-LTCC transversalement aimanté par un champ magnétique statique
The work proposed here took place in Hubert Curien Laboratory in collaboration with Laplace Laboratory and Laboratory of Monastir. It is part of a project aiming for the theoretical study of the propagation of the electromagnetic waves inside passive devices fabricated with anisotropic magnetic material. Using a magnetic substrate in such structure has many advantages: non reciprocity, high isolation...More precisely, the fabrication of nonreciprocal de- vice such as isolator and circulator. The numerical study of such structure containing many anisotropic magnetic layers needs to use commercial software. As a principal inconvenience is their purchase cost and their necessary memory capacity. In this case, it is necessary to understand the behavior of the magnetic material used in the structure: it is a question of modeling the magnetic material by a permeability tensor depending on its state of magnetization. The aim of this thesis is the electromagnetic modeling of multilayers structures with complex media using the numerical spectral domain approach. The numerical SDA is based on the numerical computation of the Green’s function with the transverse operator formalism. The numerical results are validated with published numerical data for a microstrip line printed on isotropic or anisotropic magnetic material. They are then compared with an excellent agreement with numerical data provided from software based on the finite elements method (HFSS). For more credibility of our numerical approach, the results of simulation about the complex propagation constant are compared with a good much with measured data for coplanar waveguide fabricated on a low temperature co-fired ceramic (LTCC-ferrite). The validation was done in a large band of frequency from 1 to 12 GHz

Targeting DNA has the advantage over proteins for cancer remediation because of the fewer copies of the ligands required for the desired therapeutic effect. Traditionally, covalent DNA binders like alkylating agents have been used to induce genetic instability through the formation of DNA lesions and strand breaks, leading to cellular apoptosis. The primary drawback of this treatment is the non-specific binding that affects both cancerous and non-cancerous cells. G-quadruplexes are the DNA secondary structures that are present in abundance near the promoter regions of the oncogenes and are involved in the regulation of their activities. A ligand-mediated stabilization of G-quadruplexes in the promoter regions and down-regulation of the associated oncogenes have been validated. In contrast to alkylating agents, G-quadruplex ligands induce genetic stabilization through non-covalent interactions. They can be designed to interact specifically with G-quadruplex DNA over duplex DNA, which reduce side effects arising from the off-targeting. G-quadruplex ligands invariably have the large planar aromatic moiety to interact with G-quadruplexes through π- π stacking interactions. For determining the size effect of the aromatic moiety on stabilization of G-quadruplexes, a series of ligands were synthesized by conjugating nucleobases or 1,10-phenanthroline with an aminoglycoside, neomycin. The resulting conjugates increased the binding affinity synergistically and enabled us to study the effect of the stacking moiety required for G-quadruplex stabilization. Nucleobase-neomycin conjugates did not show stabilization stabilize of human telomeric G-quadruplex. 1,10-Phenanthroline-neomycin conjugate (7b) on the other hand binds to human telomeric G-quadruplex with a Ka of (8.92.4)×108 M-1 and inhibits telomerase activity at 1.56 µM probably through G-quadruplex stabilization. Moving forward, we further enlarged the aromatic moiety by tethering two 1,10-phenantholine molecules together through a five-atom linker. The resulting molecule (2-Clip-phen) was conjugated with various amino-containing side chains. 2-Clip-phen derivatives showed at least 30 times weaker binding to duplex DNA over G-quadruplex DNA. In addition, compounds showed a preference for the antiparallel G-quadruplex conformation over parallel and hybrid G-quadruplex conformations, as shown in the CD spectroscopy studies. Ligands 11 and 13 induced the formation of an antiparallel G-quadruplex from random coils and stabilize it to 60 oC (Tm) in a salt-free condition. Mass spectrometry study showed the formation of a two-tetrad G-quadruplex with the 2-Clip-phen ligand. Docking study showed that the ligand interacts most favorably with antiparallel G-quadruplex conformation, which is supported further by the larger thermal stabilization effect on antiparallel G-quadruplex compared with other G-quadruplex conformations. Our study suggests that 2-Clip-phen can be used as a scaffold for designing G-quadruplex binding ligands that preferentially bind to antiparallel G-quadruplexes, which has never been reported before.

碩士
國立高雄第一科技大學
機械與自動化工程研究所
99
In this study, a micro feeding tool holder with a displacement magnification mechanism is provided for improving the feeding resolution and providing tool-servo function of an existing ultraprecision machine. The feeding stroke of the tool holder is amplified by a bellows-type hydraulic device with two different cross-sectional areas. Due to the nature of incompressible liquid in the closed chamber of the hydraulic device, the tiny displacement of the PZT actuator can be amplified based on the area ratio of the two bellows. In this paper, detail design of the tool holder is described and fundamental characteristics are presented. According to the experimental results, a displacement of 54.35 um was obtained with a displacement amplification ratio of 4.4 when the PZT actuator was excited by an applied voltage of 100 V. The stroke and feeding resolution of the tool holder is 100 um and 10 nm, respectively. In addition, the stiffness, natural frequency, and bandwidth were obtained as 1.42 N/um, 150 Hz and 130 Hz, respectively. In order to perform real-time control of the tool holder, the program was configured by FPGA (Field Programmable Gate Array) module; and the cutting experiments were base on an embedded Real-time controller. Through cutting experiment for a flat surface of oxygen-free copper (OFC) workpiece, a measured roughness of Ra 27 nm was obtained. In addition, to perform cutting experiments for an aspheric surface, a peak-to-valley (PV) form error was obtained as 1.03 um under open-loop control and a PV form error was reduced to 0.6 um under proportional-integral (PI) closed-loop control. The effectiveness of proposed micro tool holder was successfully demonstrated.

碩士
國立臺灣大學
土木工程學研究所
100
In this study, 838 ground motion records from the 1999 Chi-Chi earthquake in Taiwan are used to compute the amplification factors between average spectral acceleration (velocity, displacement) in acceleration (velocity, displacement) sensitive region and peak ground acceleration (velocity, displacement). The results are compared with those presented in literatures. This study also examines the impact of site condition (VS30), closest site-to-source distance and hanging/foot-wall effect on amplification factors. The study shows that (1) among the amplification factors for spectral acceleration, velocity and displacement, those for spectral velocity have highest dependency on VS30 and distance; (2) amplification factors for spectral acceleration and velocity are similar to those proposed by Newmark and Hall in 1982s, and those for displacement are higher than the factors developed in other studies.

碩士
國立高雄應用科技大學
機械工程系
106
In this thesis, Taguchi quality design is applied and the gray relational method is used to explore the optimal design of the hinge of flexible mechanism applied in the displacement magnification mechanism. In the study, three models of flexible hinges and displacement amplifying mechanisms with different geometric shapes were established by using SolidWorks drawing software, and an appropriate force was applied as a force to simulate the finite element analysis software ANSYS Workbench and find out through Taguchi quality design. The best combination of the parameters of the displacement amplification mechanism and the influencing factors on the quality characteristics are used as the best solution to predict the combination of the various factors. Then the analysis of variance and the interaction check are carried out to ensure the correctness of the prediction. The gray correlation method was used to integrate multiple optimal target to maximize efficiency, so, in the multi-quality design features to determine the best combination of conflict problems encountered will be resolved in order to get the best solution. The results show that the optimization method of this study can obviously integrate the overall quality of the displacement and look at the characteristics of low-stress and low-quality. The best combination of parameters for the aluminum alloy material, the hinge length of 5 mm opening, the geometry S-type, the hinge thickness of 0.3 mm, the material thickness of 5 mm, and the hinge opening 10 mm, is conformed with the verification results. Also meet the expected goals and can successfully solve the conflict between multi-objective quality characteristics to achieve the best combination of both displacement and contingency.

Plasma- und Serumproben waren in früheren epidemiologischen Studien häufig das einzige biologische Material, das gesammelt und untersucht wurde. Diese Studien besitzen gerade durch ihren sehr langen Untersuchungszeitraum einen riesigen Informationsgehalt und wären ein unbezahlbarer Schatz für genetische Analysen. Oft ist aufgrund damals mangelnder Akquirierung jedoch keine genomische DNA verfügbar. Um die in Plasmaproben in geringer Menge vorkommende DNA verwenden zu können, extrahierten wir die DNA mit Hilfe von magnetischen Partikeln und setzten sie in eine Whole Genome Amplification (WGA) mittels Φ29-DNA-Polymerase ein. Wir stellten 88 Probenpärchen, bestehend aus einer WGA-Plasma-DNA und der korrespondierenden Vollblut-DNA derselben Person, zusammen und genotypisierten bei diesen neun hochpolymorphe Short Tandem Repeats (STR) und 25 SNPs. Die durchschnittliche innerhalb der Probenpaare auftretende Diskordanzrate betrug 3,8% für SNPs sowie 15,9% für STRs. Basierend auf den Ergebnissen der Hälfte der Probenpaare entwickelten wir einen Ausschlussalgorithmus und validierten diesen in der anderen Hälfte der Probenpaare. Mit diesem ist es möglich, zum Einen diejenigen Proben mit einer guten DNA-Qualität herauszufiltern, um Genotypisierungsfehler zu vermeiden, und zum Anderen jene Proben mit insuffizienter DNA-Qualität auszuschließen. Nachdem Proben, die fünf oder mehr homozygote Loci in dem 9-STR-Markerset aufwiesen, ausgeschlossen wurden, resultierte dies in einer Ausschlussrate von 22,7% und senkte die durchschnittliche Diskordanzrate auf 3,92% für STRs bzw. 0,63% für SNPs. Bei SNPs entspricht dieser Wert ungefähr der Fehlerquote, wie er auch bei Genotypisierungen mit Vollblut-DNA in vielen Laboratorien auftritt. Unsere Methode und das Ausschlusskriterium bieten damit neue Möglichkeiten, um zuverlässige DNA aus archivierten Plasmaproben wiederzugewinnen. Dieser Algorithmus ist auch besser geeignet, als nur die eingesetzte DNA-Menge in die WGA-Reaktion als Kriterium zu benützen
Plasma and serum samples were often the only biological material collected for earlier epidemiological studies. These studies have a huge informative content, especially due to their long follow-up and would be an invaluable treasure for genetic investigations. However, often no banked DNA is available. To use the small amounts of DNA present in plasma, in a first step, we applied magnetic bead technology to extract this DNA, followed by a whole-genome amplification (WGA) using phi29-polymerase. We assembled 88 sample pairs, each consisting of WGA plasma DNA and the corresponding whole-blood DNA. We genotyped nine highly polymorphic short tandem repeats (STRs) and 23 SNPs in both DNA sources. The average within-pair discordance was 3.8% for SNPs and 15.9% for STR genotypes, respectively. We developed an algorithm based on one-half of the sample pairs and validated on the other one-half to identify the samples with high WGA plasma DNA quality to assure low genotyping error and to exclude plasma DNA samples with insufficient quality: excluding samples showing homozygosity at five or more of the nine STR loci yielded exclusion of 22.7% of all samples and decreased average discordance for STR and SNP markers to 3.92% and 0.63%, respectively. For SNPs, this is very close to the error observed for genomic DNA in many laboratories. Our workflow and sample selection algorithm offers new opportunities to recover reliable DNA from stored plasma material. This algorithm is superior to testing the amount of input DNA

博士
國立成功大學
資源工程學系碩博士班
96
A procedure that considers topographic effects and runout behavior is proposed for analyzing seismic landslide hazards. The theoretical topographic amplification factors and corresponding amplified ground motion are calculated. By using the amplified motion, a cumulative displacement map is generated through Newmark’s displacement method. The high displacement areas are defined as the source areas of landslides. A runout simulation that identifies sliding routes and the final deposition areas of the sliding material from these source areas is performed. Finally, the complete set of landslide zones, including source, and sliding and deposition areas, is predicted. Three landslide hazard maps of the 99 Peaks region, Mt. Yan region and Mt. Baishiya region are evaluated, and the maps of actual landslides triggered by the September 21, 1999, Chi-Chi earthquake are compared with the prediction. Relative to the conventional procedure, the results show that the proposed procedures which include the topographic effect can obtain a better result for predicting the source area of landslides. And the proposed procedures, which combines topographic effects and runout simulation, can generate more accurate predictions for predicting the complete set of landslide zones, including source, and sliding and deposition areas.

碩士
國立交通大學
土木工程系所
106
During the Chi-Chi earthquake in Taiwan, it was observed that the near-fault ground motions with pulse-like signals caused some serious damages to bridge structures. This study proposes revised engineering design parameters including strength reduction factor, R, yielding strength factor, Cy, displacement amplification factor, Rd, which could provide suitable design solutions to minimize damages to bridge structures during near-fault earthquake motions. The analysis involved collection of related seismic records, focusing on the aforementioned factors. Using the commercial software, Bispec, we were able to conduct a dynamic time history analysis. This study also provided four criteria including: (1) signal processing method, (2) orientation of the records, (3) hysteretic model, and (4) numbers of records, in order to compare with the current code. Ultimately, the study would find the final standards necessary to revise the current formula based on analysis results. Based on analysis results, this study applied to the data from the Pacific Earthquake Engineering Research Center (PEER Center) and chose the 7 sample size. It also selected the maximum velocity orientation and MC model. In addition, except for the conditions mentioned above, for typical structures, the period was within 3 sec, and  was around 3 to 5. Thus, the revised formula used these ranges as a factor for consideration. For R, this study adjusted the demarcation of the period and the segments for equivalent energy and equivalent displacement to approximate the analysis results. For Cy, this study found that the relationship between Cy and S_aM/F_uM could acquire the suitable curve to fit the analysis results. For Rd, the analysis results indicated that it could not fill the requirements of using the current formula to adjust. Therefore, this study used the nonlinear regression analysis to propose the formula. To sum up, the engineering design parameters were all revised according to the analysis results. In addition, except for Rd, this study used the original pattern of the code to revise the formula and expected to make good use of the simple curve in the application of seismic engineering. The present study definitely has its limitations. In order to obtain more reliable and objective data, research on far-field ground motions including analysis should be conducted in the future.

Soil represents the largest global reservoir of microbial diversity for the discovery of novel genes and enzymes. Both stable-isotope probing (SIP) and metagenomics have been used to access uncultured microbial diversity, but few studies have combined these two methods for accessing the biotechnological potential of soil genetic diversity and fewer yet have employed functional metagenomics for recovering novel genes and enzymes for bioenergy or bioproduct applications. In this research, I demonstrate the power of combining functional metagenomics and SIP using multiple plant-derived carbon substrates and diverse soils for characterizing active soil bacterial communities and recovering glycosyl hydrolases based on gene expression. Three disparate Canadian soils (tundra, temperate rainforest and agricultural) were incubated with five native carbon (12C) or stable-isotope labelled (13C) carbohydrates (glucose, cellobiose, xylose, arabinose and cellulose). Sampling at defined time intervals (one, three and six weeks) was followed by DNA extraction and cesium chloride density gradient ultracentrifugation. Denaturing gradient gel electrophoresis (DGGE) of all gradient fractions confirmed the recovery of labeled nucleic acids. Sequencing of original soil samples and labeled DNA fractions demonstrated unique heavy DNA patterns associated with all soils and substrates. Indicator species analysis revealed many uncultured and unclassified bacterial taxa in the heavy DNA for all soils and substrates. Among characterized taxa, Salinibacterium (Actinobacteria), Devosia (Alphaproteobacteria), Telmatospirillum (Alphaproteobacteria), Phenylobacterium (Alphaproteobacteria) and Asticcacaulis (Alphaproteobacteria) were the bacterial ???indicator species??? for the heavy substrates and soils tested. Both Actinomycetales and Caulobacterales (genus Phenylobacterium) were associated with metabolism of cellulose. Members of the Alphaproteobacteria were associated with the metabolism of arabinose and members of the order Rhizobiales were strongly associated with the metabolism of xylose. Annotated metagenomic data suggested diverse glycosyl hydrolase gene representation within the pooled heavy DNA. By screening only 2876 inserts derived from the 13C-cellulose heavy DNA, stable-isotope probing and functional screens enabled the recovery of six clones with activity against carboxymethylcellulose and methylumbelliferone-based substrates.