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Dempsey, Nunez Laura. "Spectrum of mutations in MMAA identified by high resolution melting analysis." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=110535.
Full textAbstract:
The gene product of MMAA is required for the intracellular metabolism of cobalamin (Cbl). Mutations in this gene lead to the cblA class of disorders, characterized by isolated methylmalonic aciduria. We have been concerned that somatic cell methods of diagnosis may miss patients with mild cellular phenotypes. A high resolution melting (HRM) analysis assay was developed to rapidly scan the coding exons and flanking intronic regions of the MMAA genes for variants. DNA from 96 unaffected reference individuals, 72 patients with complementation confirmed cblA, and 181 patients with elevated isolated methylmalonic acid, who could not be diagnosed using complementation analysis, were scanned by HRM. Suspected variants were confirmed using Sanger sequencing. In the cblA cohort, HRM correctly identified all previously known mutations as well as an additional 22 variants, 10 of which had not been previously reported. Novel variants included one duplication (C.551dupG, p.C187LfsX3), one deletion (c.387delC, p.Y129YfsX13), one splice site mutation (c.440-2A>G, splice site), 4 missense mutations (c.748G>A, p.E520K; c.820G>A, p.G274S; c.627G>T, p.R209S; c.826A>G, p.K276E), and 3 nonsense mutations (c.960G>A, p.W320X; c.1075C>T, p.E359X; c.1084C>T, p.Q362X). All novel missense variants, listed above, affect highly conserved residues and are predicted to be damaging. Scanning of MMAA in the 181 undiagnosed samples revealed a single novel heterozygous missense change (c.821G>A, p.G274D).Le produit génique du MMAA est nécessaire pour le métabolisme de la cobalamine intracellulaire (Cbl). Des mutations dans ce gène conduisent à la classe de maladies cblA, caractérisé par l'acidurie méthylmalonique isolée. Nous avons été concernés que les méthodes de diagnostic de cellules somatiques peuvent manquer les patients atteints phénotypes cellulaires moins sévère. Une teste de fusion à haute résolution a été développé pour balayer rapidement les exons codantes et les régions introniques adjacentes du gène MMAA pour des variantes. Nous avons testé l'ADN à partir de 96 personnes de référence qui ne sont pas touchés, 72 patients atteints de cblA confirmé par complémentation et 181 patients présentant une élévation de l'acide méthylmalonique isolée, qui ne pouvaient pas être diagnostiquée à l'aide d'analyse de complémentation. Les variantes suspectes ont été confirmées à l'aide de séquençage Sanger. Dans la cohorte cblA, l'analyse de fusion à haute résolution a correctement identifié toutes les mutations connues antérieurement, ainsi que 22 autres variantes, dont 10 n'avaient pas été signalés précédemment. Nouveaux variantes inclus une duplication (C.551dupG, p.C187LfsX3), une délétion (c.387delC, p.Y129YfsX13), une mutation du site d'épissage (c.440-2A> G, site d'épissage), 4 mutations faux-sens (c. 748G> A, p.E520K; c.820G> A, p.G274S; c.627G> T, p.R209S; c.826A> G, p.K276E), et 3 mutations non-sens (c.960G> A, p.W320X; c.1075C> T, p.E359X; c.1084C> T, p.Q362X). Toutes les variantes faux-sens nouveaux, énumérés ci-dessus, affectent des résidus hautement conservés et sont prévus pour être endommageant. L'analyse de MMAA dans les 181 échantillons non diagnostiqués a révélé un seul changement faux-sens hétérozygote (c.821G> A, p.G274D).
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Illson, Margaret. "Spectrum of mutations in MMAB identified by high resolution melting analysis." Thesis, McGill University, 2012. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=110564.
Full textAbstract:
Pathogenic variants in the MMAB gene (OMIM 607958) are responsible for the cblB class of cobalamin-responsive methylmalonic aciduria (MMA) (OMIM 251110). MMAB encodes cobalamin adenosyltransferase, a mitochondrial enzyme responsible for the formation of adenosylcobalamin (AdoCbl). AdoCbl subsequently functions as a cofactor for methylmalonyl-CoA mutase (MCM) during the isomerization of L-methylmalonyl-CoA to succinyl-CoA. Somatic cells studies have been used to evaluate patient samples for cobalamin related disorders. Due to high basal levels of propionate incorporation, some patients with mild MMA biochemical phenotypes cannot be diagnosed by complementation analysis. A high resolution melting analysis (HRMA) assay was developed to rapidly scan the coding exons and flanking intronic regions for variants in the MMAB gene.Three cohorts of samples were scanned by HRMA: an unaffected reference population, 42 samples assigned to the cblB by complementation analysis and 181 patients with unresolved isolated MMA. HRMA correctly identified all of the previously reported mutations in the cblB cohort as well as seven additional variants including a novel nonsense variant (c.12C>A, p.C4X). Scanning of the unresolved MMA cohort identified six samples containing MMAB variants. Two samples, WG3948 and WG4034, contained compound heterozygous variants. They shared a c.572 G>A (p.R191Q) mutation. WG3948, the index case for this study, was found to have c.398 C>T (p.S133F) as the second mutation, and WG4034, the second patient, contained a novel variant c.394 C>T (p.C132R). Samples from four other affected patients contained a single variant. The c.572 G>A (p. R191Q) was found in both WG3546 and WG4090. WG3759 contained a c.521C>T ( p.S174L) substitution, and WG4029 contained a novel c.185 C>T (p.T62M) substitution. The identification of two patients with compound heterozygous variants in the MMAB gene suggests the existence of an infrequent but distinct atypical cblB phenotype. This subclass is characterized by levels of propionate incorporation and of AdoCbl synthesis within reference ranges, preventing diagnosis by somatic cell studies.Des variantes pathogéniques dans le gène MMAB (OMIM 607958) sont responsables de la classe cblB d'acidurie méthylmalonique (AMM) respondant à la cobalamine (OMIM 251110). MMAB encode cobalamine adénosyltranférase, une enzyme mitochondriale responsable de la formation de l'adénosylcobalamine (AdoCbl). AdoCbl fonctionne par la suite en tant que cofacteur pour méthylmalonyl-CoA mutase (MCM) durant l'isomérisation de L-méthylmalonyl-CoA vers succinyl-CoA. Des analyses sur des cellules somatiques ont été utilisées pour évaluer des échantillons de patients pour des troubles reliés à la cobalamine. En raison de niveaux de base élevés d'incorporation de propionate, certains patients présentant des phénotypes biochimiques bénins d'AMM ne peuvent être diagnostiqués par analyse de complémentation. Une analyse de fusion à haute résolution (AFHR) a été développée pour balayer rapidement les exons codants et les régions introniques avoisinnantes pour des variantes dans le gène MMAB.Trois cohortes d'échantillons ont été balayées par AFHR : une population de référence non-affectée, 42 échantillons assignés au groupe cblB par analyse de complémentation et 181 patients avec une AMM isolée sans diagnostique. L'AFHR a correctement identifié toutes les mutations précédemment rapportées dans la cohorte cblB ainsi que sept variantes additionelles, incluant une nouvelle variante non-sens (c.12C>A, p.C4X). Le balayage de la cohorte avec de l'AMM isolée a identifié six échantillons contenant des variantes dans MMAB. Deux échantillons, WG3948 et WG4034, étaient des porteurs de variantes hétérozygotes composés. Ils partageaient la mutation c.572G>A (p.R191Q). WG3948, le cas index pour cette étude, était porteur du c.398C>T (p.S133F) pour la deuxième mutation et WG4034, le deuxième patient, contenait une nouvel variante c.394C>T (p.C132R). Les échantillons provenant de quatre autres patients atteints contenait une seule variante. Le c.572G>A (p.R191Q) a été trouvé dans WG3546 et WG4090. WG3759 contenait une substitution c.52C>T (p.S174L), et WG4029 contenait une nouvelle substitution c.185C>T (p.T62M).L'identification de deux patients avec des variantes hétérozygotes composées dans le gène MMAB suggère l'existence d'un phénotype rare mais distinct de cblB. Cette sous-classe est charactérisée par des niveaux d'incorporation de propionate et de synthèse d'AdoCbl dans les valeurs normales, empêchant le diagnostique par analyse des cellules somatiques.
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Souza, Roberto Antonio de. "Genotipagem de linhagens de Yersinia spp. por high-resolution melting analysis." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/60/60135/tde-27062013-151724/.
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O gênero Yersinia pertence à família Enterobacteriaceae e compreende 17 espécies. Y. pestis, Y. pseudotuberculosis e Y. enterocolitica são reconhecidamente patógenos de humanos e animais. Y. pestis cause a peste. Y. pseudotuberculosis e Y. enterocolitica são agentes causadores, sobretudo, de gastroenterites transmitidas por água e alimentos. As demais 14 espécies são, usualmente, consideradas não-patogênicas, com exceção de Y. ruckeri sorogrupo O:1 que causa infecções em peixes. Nas últimas décadas, a tipagem molecular tornou-se uma importante ferramenta nos estudos filogenéticos de numerosos micro-organismos e o desenvolvimento de sistemas de tipagem rápidos e baratos pode facilitar os estudos epidemiológicos de infecções bacterianas. No presente estudo objetivou-se desenvolver um método de genotipagem de Yersinia spp. baseado em high-resolution melting analysis (HRMA) para diferenciar os single-nucleotide polymorphisms (SNPs) presentes nas sequências dos genes 16S rRNA, glnA, gyrB, hsp60 e recA e aplicá-lo na tipagem de 40 linhagens de Y. pseudotuberculosis e 50 linhagens de Y. enterocolitica, bem como separar por HRMA as espécies Y. pseudotuberculosis e Y. enterocolitica. Os SNPs foram determinados nas sequências dos loci acima citados a partir de um conjunto de 119 linhagens de Yersinia spp. depositadas no GenBank/EMBL/DDBJ. Foram encontrados nas sequências dos genes analisados de Y. pseudotuberculosis, Y. enterocolitica, Y. bercovieri, Y. rohdei, Y. intermedia, Y. mollaretii e Y. ruckeri 10, 10, 9, 6, 4, 1 e 1 SNPs, respectivamente. Nenhum SNP foi encontrado nas sequências analisadas de Y. pestis e um grande número de SNPs foi encontrado nas sequências analisadas de Y. frederiksenii, Y. kristensenii e Y. massiliensis, o que impossibilitou a genotipagem dessas espécies por HRMA. As demais espécies não foram analisadas. Foram desenhados pares de primers para flanquear os SNPs encontrados em cada espécie de Yersinia testada. Usando um conjunto de primers espécie-específicos, a diversidade genética de cada espécie de Yersinia foi determinada por HRMA e a análise filogenética foi baseada na sequência concatenada composta pelos nucleotídeos identificados em cada fragmento analisado. O agrupamento foi realizado com o software BioNumerics usando o método UPGMA com 1.000 replicatas de bootstrap. A árvore filogenética ii construída para Y. pseudotuberculosis agrupou as linhagens em clusters bio-sorogrupo específicos. As linhagens do bio-sorogrupo 1/O:1 foram agrupadas em um cluster e as linhagens do bio-sorogrupo 2/O:3 em outro. A árvore filogenética construída para Y. enterocolitica agrupou as linhagens em três grupos. As linhagens altamente patogênicas, do biotipo 1B, foram agrupadas em um cluster, as linhagens de média patogenicidade, dos biotipos 2, 3, 4 e 5, foram agrupadas em um segundo cluster e as linhagens consideradas nãopatogênicas, do biotipo 1A, foram agrupadas em um terceiro cluster. O agrupamento encontrado em Y. pseudotuberculosis e Y. enterocolitica foi consistente com o perfil patogênico característico dessas duas espécies. Nenhuma correlação epidemiológica significativa foi encontrada no agrupamento de Y. bercovieri, Y. rohdei, Y. intermedia, Y. mollaretii e Y. ruckeri de acordo com os resultados de HRMA. Ademais, o método de HRMA aqui desenvolvido foi capaz de separar as espécies Y. pseudotuberculosis e Y. enterocolitica. O método de HRMA desenvolvido nesse estudo pode ser usado como uma alternativa para a genotipagem e para a diferenciação de Y. pseudotuberculosis de Y. enterocolitica. Esse método também pode complementar os métodos baseados em sequências e facilitar os estudos epidemiológicos dessas duas espécies de Yersinia.The genus Yersinia belongs to the family Enterobacteriaceae and comprises 17 species. Y. pestis, Y. pseudotuberculosis and Y. enterocolitica are well recognized human and animal pathogens. Y. pestis causes plague. Y. pseudotuberculosis and Y. enterocolitica are, usually, causative agents of food-waterborne gastroenteritis. The other 14 Yersinia species are considered to be non-pathogenic, with the exception of Y. ruckeri serogroup O:1 which causes infections in fishes. In the last few decades, molecular typing has become an important tool in phylogenetic studies of several microorganisms and the development of fast and inexpensive typing systems can facilitate epidemiological studies of bacterial infections. The present study aimed to develop a method of Yersinia spp. genotyping based on high-resolution melting analysis (HRMA) in order to differentiate the single-nucleotide polymorphisms (SNPs) present in the 16S rRNA, glnA, gyrB, hsp60 and recA sequences and apply it in the typing of 40 Y. pseudotuberculosis strains and 50 Y. enterocolitica strains, as well as, to separate by HRMA the Y. pseudotuberculosis and Y. enterocolitica species. The SNPs were determined in the sequences of the aforementioned loci using a set of 119 Yersinia strains deposited in the GenBank/EMBL/DDBJ database. It were found in the gene sequences analyzed of Y. pseudotuberculosis, Y. enterocolitica, Y. bercovieri, Y. rohdei, Y. intermedia, Y. mollaretii and Y. ruckeri 10, 10, 9, 6, 4, 1 and 1 SNPs, respectively. No SNPs was found in the analyzed sequences of Y. pestis and a large number of SNPs were found in the analyzed sequences of Y. frederiksenii, Y. kristensenii and Y. massiliensis what prevented their genotyping by HRMA. The remaining Yersinia species were not analyzed. It was designed primer pairs to flank the SNPs found in each Yersinia species tested. Using a specie-specific set of primers, the genetic diversity of each Yersinia species used was determined by HRMA and the phylogenetic analysis was based on the concatenated sequence composed by the nucleotides identified in each fragment analyzed. Clustering was performed with the software package BioNumerics using UPGMA method and 1,000 bootstrap replicates. The phylogenetic tree constructed for Y. pseudotuberculosis grouped the strains into bio-serogroups specific clusters. The strains of 1/O:1 bio-serogroup were grouped into one cluster and the strains of 2/O:3 bio-serogroup into iv other cluster. The phylogenetic tree constructed for Y. enterocolitica grouped the strains in three clusters. The highly pathogenic strains, of biotype 1B, were grouped into one cluster, the moderate pathogenic strains, of biotypes 2, 3, 4 and 5, were grouped into a second cluster and, the non-pathogenic strains, of biotype 1A, were grouped into a third cluster. The clusterization of Y. pseudotuberculosis and Y. enterocolitica were consistent with the pathogenic profile characteristic of these two Yersinia species. No significant epidemiological correlation was found in the grouping of Y. bercovieri, Y. rohdei, Y. intermedia Y. mollaretii and Y. ruckeri according to HRMA results. Moreover, the HRMA-based method develop here was able to separate the Y. pseudotuberculosis and Y. enterocolitica species. The HRMA assay developed in this study can be used as an alternative for the genotyping and the differentiation of Y. pseudotuberculosis and Y. enterocolitica. This method can also complement sequence-based methods and facilitate epidemiological studies of these two Yersinia species.
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Darbandy, Ashna. "Optimization of High Resolution Melting Analysis for Detection of KRAS Gene Mutations." Thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-130751.
Full textAbstract:
Background: Mutations of the KRAS oncogene occur in a variety types of human tumors. By assessing the mutation status of KRAS, clinicians can predict patient response to anti-EGFR therapy such as cetuximab (Erbitux®) or panitumumab (Vectibix®) in patients with metastatic colorectal cancer. The aim of this study was to optimize a real-time PCR method followed by high resolution melting analysis (HRM) in a single step for detection of most common mutations within the KRAS gene. Methods: Seven DNA samples with predefined KRAS mutations and 19 tumor samples from patients with metastatic colorectal cancer were used. KRAS mutation detection was performed by direct sequencing as well as HRM. Optimization was performed using touchdown PCR and co-amplification at lower denaturation-temperature PCR. Results: All DNA samples were successfully analyzed with direct sequencing and HRM. Moreover, the improved amplification efficiency and sensitivity was achieved using optimized PCR run protocol. Conclusion: HRM is a simple, inexpensive and reliable method for mutation detection within KRAS. By applying HRM as prescreening method would help reduce labour, time and costs.
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Burrows, Adria Michelle. "A comparative ancestry analysis of Y-chromosome DNA haplogroups using high resolution melting." University of the Western Cape, 2018. http://hdl.handle.net/11394/6536.
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>Magister Scientiae - MScThe objective of this study is to deduce paternal ancestry using ancestry informative single nucleotide polymorphisms (SNPs) by means of High Resolution Melting (HRM). This was completed by producing a multiplex system that was designed in a hierarchical manner according to the YSNP tree. This project mainly focused on African ancestry and was used to infer paternal ancestral lineages on the Johannesburg Coloured population. South Africa has a diverse population that has ancestral history from across the globe. The South African Coloured population is the most admixed population as it is derived from at least five different population groups: these being Khoisan, Bantu, Europeans, Indians and Southeast Asians. There have been studies done on the Western Cape/ Cape Town Coloured populations before but this study focused on the Johannesburg Coloured population.
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Michelle, Burrows Adria. "A comparative ancestry analysis of Y-chromosome DNA haplogroups using high resolution melting." University of the Western Cape, 2018. http://hdl.handle.net/11394/6489.
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Magister Scientiae - MSc (Biotechnology)The objective of this study is to deduce paternal ancestry using ancestry informative single nucleotide polymorphisms (SNPs) by means of High Resolution Melting (HRM). This was completed by producing a multiplex system that was designed in a hierarchical manner according to the YSNP tree. This project mainly focused on African ancestry and was used to infer paternal ancestral lineages on the Johannesburg Coloured population. South Africa has a diverse population that has ancestral history from across the globe. The South African Coloured population is the most admixed population as it is derived from at least five different population groups: these being Khoisan, Bantu, Europeans, Indians and Southeast Asians. There have been studies done on the Western Cape/ Cape Town Coloured populations before but this study focused on the Johannesburg Coloured population. The first step was to design the multiplex system. This was done by using inhouse SNPs. A total of seven multiplexes were designed and optimised, each consisting of two, three or four different SNPs respectively. A total of 143 saliva and buccal samples were collected from male Johannesburg Coloureds. DNA was extracted from the saliva samples using an optimised organic method. DNA was extracted from the buccal samples using an optimised salting out method. DNA was successfully extracted from 77 of the male samples. A total of 69 samples were screened using Multiplex 1; of the 69 samples 56 samples were successfully screened to infer the paternal lineage of the samples. The results show that the most frequent haplogroup of the Johannesburg male samples was haplogroup CF (39%). The second most frequent haplogroup was haplogroup DE (38%). Under further analysis of haplogroup DE it was seen that 37% of those samples were derived for the haplogroup E1b1b.
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PIMENTEL, ROMERO CESAR HUGO. ""INNOVATIVE SIGNAL PROCESSING TECHNIQUES IN BIOENGINEERING: COMPRESSED SENSING AND HIGH RESOLUTION DNA MELTING ANALYSIS"." Doctoral thesis, Università degli studi di Ferrara, 2021. http://hdl.handle.net/11392/2487913.
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The first part starts with an introduction of the Compressed Sensing (CS) theory. After that, an overview of the state-of-the art of some CS adaptations by using additional priors in the different stages is presented in order to explain the new CS adaptation proposed in this work. Some of the CS adaptations reviewed are confronted using synthetic signals, synthetic electrocardiograph (ECG) signals and electroencephalographic (EEG) signals. The second part provides general concepts of biology and current developments in bioinformatics to read and and analyze the DNA. Questions like what a sequencer does?, What kind of data it produces? and How can be analyzed this data? are intended to be answered. Another reliable technique used in the analysis of the DNA without sequencing is the High Resolution Melting (HRM) curves analysis, this technique is used to find differences between two strands of DNA. HRM is also discussed to finally design a HRM analysis software.La prima parte inizia con un'introduzione alla teoria del Compressed Sensing (CS). Successivamente, viene presentata una panoramica dello stato dell'arte di alcuni adattamenti CS utilizzando nelle diverse fasi, questo per spiegare il nuovo adattamento CS proposto in questo lavoro. Alcuni degli adattamenti CS esaminati vengono confrontati utilizzando segnali sintetici, segnali di elettrocardiografo sintetico (ECG) e segnali elettroencefalografici (EEG). La seconda parte fornisce concetti generali di biologia e sviluppi attuali della bioinformatica per leggere e analizzare il DNA. Domande come Cosa fa un sequencer? Che tipo di dati produce? e Come possono essere analizzati questi dati? sono destinati a ricevere una risposta. Un'altra tecnica affidabile utilizzata nell'analisi del DNA senza sequenziamento è l'analisi High Resolution Melting (HRM) curves, questa tecnica viene utilizzata per trovare differenze tra due filamenti di DNA. Si studia anche le HRM curves per progettare finalmente un software di analisi.
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Tsang, Ho-yin, and 曾皓言. "Detection of clinically silent beta-globin gene mutations in Chinese using high resolution melting analysis." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B48334182.
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Mutations in the beta-globin (β-globin) gene cause beta-thalassaemia (β-thalassaemia).The screening strategy for β-thalassaemiais based on the value of mean corpuscular volume (MCV) from the complete blood count (CBC) data. Current laboratory practice considers blood samples with MCV higher than 80fL as normal. No further assessment will be done on these samples. However, there are clinically silent β-globin gene mutations with MCV higher than 80fL, for example, heterozygous haemoglobin E (HbE). The importance of finding out this kind of mutations is due to the serious outcome when they occur together with classic β thalassaemia mutations in compound heterozygous states, which may produce a condition mimicking β thalassaemia major.
The method used to recognize the presence of clinically silent β-globin gene mutations should be robust and with high sensitivity. High resolution melting (HRM) is a suitable technique to screen gene mutations. It is fast and convenient. The process is completed in a closed system without any post PCR manipulation. The sensitivity is up to a single nucleotide change. Using HRM for mutations screening followed by confirmation with sequencing can reduce time and cost of testing clinically silent β-globin gene mutations on a large scale.
This study first shows the ability of HRM in detecting various types of β-globin gene mutations. The technique is then applied to detect clinically silent β-globin gene mutations in a group of high school students with normal CBC data. Mutations with different clinically significance were found. The frequency of mutation found in the samples of the study suggests that screening for β-globin gene mutation may be worthwhile in subjects with MCV higher than 80fL.published_or_final_version
Pathology
Master
Master of Medical Sciences
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Ho, Sophia KW, and 何廣慧. "Detection of clinically silent alpha-globin gene mutations in Chinese using high resolution melting analysis." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/206558.
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α-thalassemia is an inherited globin gene disorder commonly found among the Chinese population. It is composed of both non-deletional and deletional α-globin gene mutations. Classical α-thalassemia presents with red cell microcytosis but silent cases with a normal mean corpuscular volume (MCV) are also seen. Routine laboratory testing methods for large-scale detection of silent α-thalassemia mutations are onerous and time-consuming. Furthermore, methods such as denaturing high performance liquid chromatography (HPLC) or denaturing gradient gel electrophoresis (DGGE) for scanning of point mutations are costly and they require post-PCR separation. High resolution melting (HRM) analysis is an economical, sensitive, and fast method for large scale point mutation scanning. Contamination is significantly reduced with HRM because the process is performed in a closed-tube environment and does not require post-PCR manipulation. We used HRM and multiplex gap-PCR analysis to determine the prevalence of silent α-thalassemia carriers in Hong Kong.
Of the 223 hematologically normal blood samples scanned by Roche LightCycler 480®, HRM did not show any sample with a non-deletional α-globin gene mutation of clinical significance. α-multiplex gap-PCR analysis revealed 36 samples (16.1%) with single α-globin gene deletions. The detection of single α-globin gene deletions in samples with a MCV greater than 80 fL indicates that the previously reported prevalence of α-thalassemia mutations in our Chinese population based on MCV screening is under-estimated. The data also suggest that non-deletional α-thalassemia mutations presenting with a normal MCV are very rare, and they most likely present with microcytosis.
The fact that most silent α-thalassemia mutations are due to large deletions supports the use of traditional molecular techniques such as gap-PCR for their detection. HRM can be used as an adjunct tool for large-scale population screening of non-deletional mutations. This study provides more accurate data on the prevalence of silent α-thalassemia carriers in the Hong Kong Chinese population. The information will facilitate genetic counseling and risk assessment in families carrying α-thalassemia mutations.published_or_final_version
Pathology
Master
Master of Medical Sciences
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Ozbak, Hani. "The application of High Resolution Melting Analysis (HRMA) for rapid detection of bacteria responsible for bloodstream infections." Thesis, University of Manchester, 2013. https://www.research.manchester.ac.uk/portal/en/theses/the-application-of-high-resolution-melting-analysis-hrma-for-rapid-detection-of-bacteria-responsible-for-bloodstream-infections(b3d5c15b-9541-44c2-873c-f7a32fc60282).html.
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Background: The diagnosis of bloodstream infection is a significant challenge for healthcare providers and is often associated with severe illness (sepsis) and poor outcomes. Rapid detection and identification of pathogens followed by characterisation of antibiotic resistance could help direct early treatment and improve patient care. Standard blood culture methods, which usually take 2-5 days to complete, can confirm if there is a bacteraemia or not in suspected patients. However, molecular approaches have been developed and are being increasingly investigated to overcome disadvantages of culture. One of the main potentials of molecular techniques is that they should be able to identify pathogens within a short time which could help clinicians treat patients earlier with rational antimicrobial therapy and limit overuse of antibiotic exposure. Objectives: To present the development and optimisation of a simple, rapid and cost-effective Real Time PCR methods combined with a High Resolution Melting Analysis (HRMA) approach, to detect and identify common bacteria associated with bloodstream infections. Approach: 16S rRNA and Gram classification primers were used on a broad range real-time PCR for molecular Gram typing and HRMA in a single run. Differentiation of bacterial species was achieved using a multi-parameter, decision-tree approach based on Gram typing, grouping according to melting temperature (Tm) and sequential comparisons of melting profiles (Curve shapes) against reference organisms. Findings: A preliminary validation was undertaken by blinded analysis of 53 consecutive bloodstream isolates from a clinical microbiology laboratory. 50 isolates contained organisms present on the panel and 96% of these were identified correctly at genus or species level. A correct Gram classification was reported for all 53 isolates. The strategy of amplification of the bacterial signal to an appropriate level using a short term pre-culture system (STPCS) for up to 12 hours prior to HRMA analysis significantly improved the overall sensitivity of the assay in spiked blood. Conclusion: This study suggests that a PCR-HRMA approach could be used as an alternative cheap approach to other molecular approaches for rapid detection and identification of bacteria responsible for >95% of bloodstream infections especially when combined with a Short Term Pre-Culture System (STPCS). Such development together with the current standard culture-based methods could allow clinicians to establish more effective management and treatment of patients with suspected bloodstream infection at an earlier stage than is possible with only current culture-based approaches.
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Loiacono, M. "MOLECULAR CHARACTERIZATION BY RT-REAL TIME PCR AND HIGH RESOLUTION MELTING ANALYSIS FOR FOOD SAFETY AND VETERINARY DIAGNOSTICS." Doctoral thesis, Università degli Studi di Milano, 2015. http://hdl.handle.net/2434/338720.
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This PhD thesis is the outcome of a range of activities and experimental results aimed to a better characterization of the risk that Escherichia coli and other microrganisms and parasites may pone to the health of animals and finally humans. One of the main activity of the present project was based on the test hypothesis that the virulence profile of E. coli strains toward bovine mammary gland can be modulated by the interaction with the host cells. These hypothesis were tested through a gene expression study of some virulence factors of six E. coli strains when co-cultured with a bovine mammary cell line, since the in vitro models represent both an essential tool to investigate the biological mechanistic of mastitis, and an efficient alternative to animal experiments. Preliminarly, a meta-analysis of existing literature studies on the available bovine mammary cell lines was performed, resulting in the selection of MAC-T as the most responsive cell line to bacteria causing mastitis. The E. coli strains used for the coculture experiments with MAC-T cells were isolated from different types of bovine mastitis (acute, chronic and undetermined) and from a VTEC food-borne strain associated to human clinical disease (O157). An upregulation of the virulence factor eae (intimin) in all but one the analyzed mastitis strains following co-culture with MAC-T cell line was detected through RT-reat time PCR, and also the adherence virulence factor ycd and the b12 gene were upregulated in some strains, overall suggesting the possibility that mastitic E. coli strains can acquire a more risky molecular profile when exposed to the bovine mammary cells. This finding may have clear implications on the risk assessment related to the E. coli strains in bovine mammary tissue and milk. In addition, with the aim to improve the current methodologies for foodborne risk analysis linked to E. coli, the project activity provided a preliminary research for the setup and validation of new protocols based on real time PCR-High Resolution Melting Analysis, a widely used technique to target sequence polymorphisms of the same gene in different species without the need to perform DNA sequencing or to use species-specific probes, to help the identification of putative verocytotoxic status in E. coli strains of O26 serogroup, and other serotypes, isolated from bovine milk.
Since the applications of HRMA for the characterization of microorganisms can not be limited to food safety, but can be developed for a large number of issues linked to general veterinary diagnostics, among the objectives of this PhD project some new real-time PCR-HRMA coupled methods were also developed, providing a contribution to the advancement of the existing molecular tools for sensitive and effective species identification, or variant/mutation screening, applied to different foodborne and veterinary pathogens. Thus, new HRMA-based protocols were designed and tested for the identification of Pseudomonas spp responsible for chromatic alterations in mozzarella cheese, for the detection and differentiation of Dirofilaria repens and D. immitis in canine blood samples, for the detection of the mutation site associated to FQ resistance in Staphylococcus pseudintermedius isolated from canine diagnostic samples, and for discrimination of the two most common microsporidial parasites in honeybees, Nosema apis and N. ceranae. Overall, these new HRMA-based assays could represent additional tools for epidemiological studies, routine disease assessment and therapeutical decisions.
The possibility to identify the presence of risk-predictive SNPs in E. coli isolates using these newly established HRMA-based protocols is a novel, and simpler, opportunity with respect to the current, and more complex, surveillance strategies that are based on the amplification of stx genes together with other virulence factors for the evaluation of VTEC status. In the future, a possible way forward of this research is represented, on one side, by the deeper assessment of the reciprocal modulation between E. coli mastitis-derived strains and immortalized MAC-T cells using high-throughput RNA sequencing, and on the other side by a large scale validation of the HRMA-based evaluation of risk-predictive SNPs in order to improve the current approaches. And overall, the established HRMA-based protocols when extensively validated would be highly suitable for routine veterinary diagnostics applied to field investigation, as quick and sensitive single step protocols allowing specific and sensitive detection of the targets with shorter analysis time and reduced cost, in parallel or in alternative to the classical approaches.
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Brecht, Gadean. "Genetic analysis of mitochondrial DNA within Southern African populations." UWC, 2020. http://hdl.handle.net/11394/7365.
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>Magister Scientiae - MScAs human beings we are curious about our origin and ancestry. A curiosity has led to an investigation of human evolution and expansion across the world by means of population genetics and phylo-genetics by evaluating a region in Southern Africa that is largely unknown. The objective of this study was to develop a quick, inexpensive and accurate hierarchical diagnostic screening system of the MtDNA phylogenetic tree, AI-SNPs in the mtDNA genome by using High Resolution Melting analysis to evaluate the population composition and ancestral haplogroups of Southern African populations in Limpopo. The admixture between the ‘Khoesan’ hunter-gatherers, herders and the Bantu speaking populations led to population growth and expansion in Limpopo. This has contributed to populations settling in Limpopo and has thus shaped the ancestral contemporary populations. No research on these individuals residing in Limpopo has been done before, thus an investigation of their ancestral origin was necessary. A total of 760 saliva samples were collected from individuals residing in Limpopo. Only 500 saliva samples were extracted by means of an optimized salting out technique. Five hundred extracted genomic samples were genotyped by means of a quick, inexpensive High-resolution melting analysis. Of the 500 samples, the genotyping results showed 95 individuals derived for the L3 haplogroup which gives a 19% ratio of individuals screened with Multiplex 1. Only 56 individuals were derived for the L1 haplogroup, which gives a percentage of 11%. A total of 249 individuals were derived for the L0 haplogroup, making up a 50% of the total individuals genotyped. Only 100 samples were derived for L0a, making up 20% of individuals screened with Multiplex 1. Of the 95 samples derived for the L3 haplogroup, the results showed 87 individuals to be ancestral for both M and N, making up 91.57% of individuals screened with Multiplex 2. http://etd.uwc.ac.za/. In population genetics using SNPs to infer population history and ancestral origin has become significant, this study allowed researchers to evaluate population groups by investigating their genetic markers and the application of the results allowed for downstream analyses. Finally, this study provides a quick and simple screening method for the selection of lineages that are of interest for further studies.
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Raphela, Mashikoane Pinky Jane. "Molecular characterization of Mycoplasma synoviae in chickens in South Africa using single-stranded conformation polymorphism and high-resolution melting curve analysis of the vlhA gene." Diss., University of Pretoria, 2012. http://hdl.handle.net/2263/26196.
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Mycoplasma synoviae (Ms) causes respiratory infection and synovitis in chickens and turkeys and is an economically important pathogen of poultry worldwide. It is critically important to characterize Ms strains, especially in countries in which poultry flocks are vaccinated with the live attenuated Ms strain MS-H. Vaccination with this vaccine may cause sero-conversion and persistence of the vaccine strain in the respiratory tract and will frequently result in positive Ms cultures and PCR results. Vaccination of flocks therefore complicates the diagnosis of Ms by the presence of detectable antibodies in the blood. Many diagnostic techniques cannot distinguish between the vaccine strain and wild type strain. Single stranded conformation polymorphism (SSCP) and real-time PCR with high melting curve (HRM) analysis can discriminate between the different Ms strains obtained from the field and also distinguish them from the live vaccinestrains. These techniques provide effective tools for the further study of the epidemiology and spread of Ms strains in chickens in South Africa. This project was undertaken to establish whether SSCP and HRM analyses can be used effectively to discriminate between Ms field isolates and the vaccine strain. Mycoplasma synoviae DNA was extracted from samples and conventional PCR was performed using oligonucleotide primers complementary to the single-copy conserved 5’ end of the variable lipoprotein and haemagglutinin encoding gene (vlhA). Twenty six samples were separated by agarose gel electrophoresis and prepared for SSCP and real-time PCR and HRM curve analysis. Results obtained from SSCP were compared to real-time PCR/HRM. Differences obtained by SSCP and melting curve analysis between different isolates were confirmed by sequencing. Results obtained from the different techniques differentiated the strains from the vaccine strain (isolate Ms10), which had a different melting temperature to the others and a different band pattern on the SSCP gel. These results confirmed that HRM and SSCP methods can be used to detect and discriminate between Mycoplasma synoviae field isolates and the vaccine strain.Dissertation (MSc)--University of Pretoria, 2012.
Veterinary Tropical Diseases
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Al_griw, Huda Hm. "Molecular detection of bloodstream pathogens in critical illness." Thesis, University of Manchester, 2012. https://www.research.manchester.ac.uk/portal/en/theses/molecular-detection-of-bloodstream-pathogens-in-critical-illness(5f143a31-3694-454c-8940-5ae434f1eb31).html.
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Background: Critically ill patients are at particular risk of developing bloodstream infection. Such infections are associated with the development of sepsis, leading to a marked increase in mortality rate. Early detection of the causative organism and appropriate antibiotic treatment are therefore critical for optimum outcome of patients with nosocomial infection. Current infection diagnosis is based on standard blood culture techniques. However, microbiological culture has a number of limitations, not least that it takes several days to confirm infection and is therefore not useful in directing the early treatment with antibiotics. New techniques based on the detection of pathogen DNA using real-time polymerase chain reaction (PCR) technology have the potential to address these limitations but their clinical utility is still to be proved. Objectives: Develop and evaluate novel PCR-based approaches to bloodstream infection diagnosis in critical illness based on detection and identification of bacterial and fungal DNA in blood. Methods: A range of commercial and 'in-house' PCR-based assays for detection of bacterial and fungal DNA were developed and/or optimised for use in clinical blood samples. These included LightCycler SeptiFast, a CE-marked multi-pathogen assay for common bloodstream pathogens, BactScreen and GramScreen, broad spectrum bacterial assays based on 16S rRNA gene and real-time PCR assays developed to detect a range of clinically important fungal pathogens. Novel approaches to speciation of pathogen DNA using melting temperature (Tm) profiling and high resolution melting analysis (HRMA) were developed. Clinical evaluation of assays was either on blinded clinical isolates or blood samples from critically ill patients with clinical suspicion of bloodstream infection against conventional microbiological culture. Several techniques aimed at improving extraction of pathogen DNA from blood were also investigated. Results: The CE-marked commercial assay SeptiFast showed analytical sensitivity and specificity of 79% and 83% respectively. Concordance with positive culture results was good but high levels of 'false positives' were detected possibly attributed to detection of free pathogen DNA not associated with viable pathogens. The predictive value of a negative SeptiFast test was 98% suggesting that absence of pathogen DNA is a strong indicator of absence of infection. Further studies were aimed at detailed optimisation and validation of 16S rRNA gene real-time PCR assays for bacterial DNA. BactScreen and GramScreen were able to detect a broad range of clinically important bacteria down to <50 CFU/ml blood. A preliminary comparative evaluation against SeptiFast showed BactScreen gave excellent concordance with blood culture results with minimal false positive results compared to SeptiFast. Efficient extraction of pathogen DNA was shown to be a key factor in determining analytical sensitivity and several protocols were evaluated. Low cost approaches to speciation of bacterial DNA were developed by combining broad range real-time PCR with HRMA. A novel HRMA method based on Tm profiling was shown to identify 89% and 96% of blinded clinical isolates at species or genus level respectively. Real-time PCR/HRMA approaches were also successfully developed for detection and identification of fungal pathogens including a range of Candida and Aspergillus species associated with bloodstream fungal infection. Conclusions: These studies have highlighted some of the key factors that need to be considered when developing and validating PCR based assays for pathogen DNA detection in blood. A set of novel tools have been developed for rapid detection and identification of bacterial and fungal pathogens that could address the challenges of infection diagnosis based on pathogen DNA detection. Further work is required, not least in development of more efficient pathogen DNA extraction and detailed clinical validation but the tools described here have the potential to provide cost effective solutions to aid infection diagnosis that would be complementary to current culture-based methods. The provision of time critical information could have a positive impact on clinical decision-making leading to more effective management and treatment of patients with suspected bloodstream infection.
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Sedláková, Lucie. "Analýza DNA izolované z různých typů probiotických výrobků s využitím PCR v reálném čase a HRM analýzy." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2016. http://www.nusl.cz/ntk/nusl-240562.
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The aim of this diploma thesis was to introduce real-time PCR with high-resolution melting analysis for Bifidobacterium species. Currently a small number of publication, dealing with identification of Bifidobacterium species using high-resolution melting analysis, is available. According to publications dealing with identification of lactic acid bacteria were selected primers P1V1 and P2V1, LAC1 and LAC2, LsppUPF and LsppUPR, V3F and V3R, V6F and V6R. Using this primers bacterial DNA was amplified by real-time PCR with high-resolution melting analysis. After evaluation of the measured results efficiency of selected primers was verified on DNA izolated from complex sample of probiotic product. After further optimisation real-time PCR with high-resolution melting analysis could be suitable using selected primers for Bifidobacterium species.
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Pires, Elisabete Sofia Videira. "Real-Time PCR, High Resolution Melting - aplicações forenses." Master's thesis, Universidade de Aveiro, 2012. http://hdl.handle.net/10773/10747.
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Mestrado em Biologia Molecular e CelularApontada como a maior revolução científica na área forense desde a descoberta das impressões digitais, a identificação humana por meio da análise do DNA tornou-se uma poderosa ferramenta de investigação, auxiliando na elucidação de casos forenses, baseando-se cientificamente na existência de polimorfismos genéticos ao longo do genoma em indivíduos diferentes, que faz com que cada pessoa possua um código genético único. Com a introdução da real-time PCR nas investigações forenses, tornou-se possível uma análise sensível e específica de regiões polimórficas tanto no genoma nuclear como no mitocondrial, a partir de quantidades ínfimas de DNA obtidas de amostras altamente degradadas ou com baixo número de cópias. A quantificação do DNA é um procedimento importante na análise forense e deve ser efetuado, previamente, a qualquer análise de DNA. A união entre a bioinformática e a genética forense propiciou a criação de métodos de análise específicos, como a HRM, muito útil na genotipagem de SNPs, de extrema importância na investigação forense. Foi elaborada uma revisão bibliográfica com o objetivo de conhecer as aplicações forenses da real-time PCR e os respetivos métodos, tendo se confirmado então a aplicabilidade deste método na área forense.
Listed as the greatest revolution in forensic science since the discovery of fingerprints, identification by analyzing human DNA has become a powerful research tool, helping to elucidate forensic cases, scientifically based on the existence of genetic polymorphisms throughout the genome at different individuals, which causes that each person has a unique genetic code. With the introduction of real-time PCR in forensic investigations, it became possible a sensitive and specific analysis of polymorphic regions both in the mitochondrial and nuclear genome, from minute quantities of DNA obtained from samples highly degraded or low copy number. The quantification of DNA is an important procedure in forensic analysis and must be made in advance to any DNA analysis. The union between forensic genetics and bioinformatics led to the creation of specific analysis methods, such as HRM, very useful in scanning and genotyping of SNPs, of utmost importance in forensic investigation. A literature review has been prepared in order to meet the forensic applications of real-time PCR and related methods, and so been confirmed the applicability of this method in the forensic field.
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Konečná, Jana. "PROBIOTICKÉ GENY POTRAVINÁŘSKY VÝZNAMNÝCH BAKTERIÍ MLÉČNÉHO KVAŠENÍ." Doctoral thesis, Vysoké učení technické v Brně. Fakulta chemická, 2019. http://www.nusl.cz/ntk/nusl-402108.
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Isolation of deoxyribonucleic acid (DNA) is an important step in the molecular diagnostics of microorganisms. A high quality of isolated DNA is necessary for DNA amplification by the polymerase chain reaction (PCR). The conventional DNA isolation using phenol chloroform extraction and DNA precipitation in ethanol is time consuming and requires the use of toxic phenol. Magnetic separation techniques using magnetic solid particles are one of modern methods to speed up the nucleic acids isolation. The aim of this work was to use two different types of magnetic particles for solidphase DNA extraction. The amounts of DNA in separation mixtures were measured using ultraviolet spectrophotometry (UV). The first experimental conditions were tested on chicken erythrocytes DNA. Phosphate buffer (pH 7, 7.6 and 8) was used for adsorption of DNA on magnetic particles. It was shown that approximately almost one half of DNA was adsorbed to the particles. The elution conditions of DNA were also optimized. Secondly, bacterial DNA was tested. This DNA eluted from the particles was in PCR ready quality. High resolution melting (HRM) curve analysis is a simple, low-cost method for amplicon discrimination and easy connection with real-time polymerase chain reaction (PCR). In this contribution, we report rapid species identification of strains belonging to the Lactobacillus group using HRM-PCR. Three different DNA isolation methods were used in this work: phenol extraction, separation using magnetic particles and commercial kit. Ten sets of targeted gene fragments primers (LAC1 – LAC2, LAC2 – LAC4, P1V1 – P2V1, Gro F – Gro R, 3BA-338f – Primer 1, V1F – V1R, CHAU - V3F – CHAU - V3R, CHAU - V6F – CHAU - V6R, poxcDNAFw – poxPromRVC, poxcDNAFw – poxPromRVT) were tested for amplification of the 16S rRNA gene. Use of GroF/R and LAC2/4 primers pairs successfully identify strains belong to the Lactobacillus group. The variance between used extraction methods for evidence of HRM curves was found.
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Konečná, Jana. "Využití molekulárně biologických technik pro identifikaci a analýzu probiotických bakterií." Doctoral thesis, Vysoké učení technické v Brně. Fakulta chemická, 2019. http://www.nusl.cz/ntk/nusl-402114.
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Isolation of deoxyribonucleic acid (DNA) is an important step in the molecular diagnostics of microorganisms. A high quality of isolated DNA is necessary for DNA amplification by the polymerase chain reaction (PCR). The conventional DNA isolation using phenol-chloroform extraction and DNA precipitation in ethanol is time-consuming and requires the use of toxic phenol. Alternative method of DNA isolation is use of commercially available kits which, however, are expensive and their efficiency is low. Magnetic separation techniques using magnetic solid particles are one of modern methods to speed up the nucleic acids isolation. The aim of this work was to use two different types of magnetic particles for solid-phase DNA extraction. Magnetic microparticles P(HEMA – co – GMA) containing –NH2 group and nanoparticles PLL, whitch contains polylysine. The amounts of DNA in separation mixtures were measured using ultraviolet spectrophotometry (UV). The first experimental conditions were tested on chicken erythrocytes DNA. Phosphate buffer (pH 7, 7.6 and 8) was used for adsorption of DNA on magnetic particles. It was shown that approximately almost one half of DNA was adsorbed on the particles. The elution conditions of DNA were also optimized. Secondly, bacterial DNA was tested. After optimalization, the developed method was used for DNA isolation from real food supplements. This DNA eluted from the particles was in PCR ready quality. High resolution melting (HRM) curve analysis is a simple, low-cost method for amplicon discrimination and easy connection with real-time polymerase chain reaction (PCR). In this thesis, we report rapid species identification of strains belonging to the Lactobacillus group using HRM-PCR. Three different DNA isolation methods were used in this work: phenol extraction, separation using magnetic particles and commercial kit. Ten sets of targeted gene fragments primers (LAC1 – LAC2, LAC2 – LAC4, P1V1 – P2V1, Gro F – Gro R, 3BA-338f – Primer 1, V1F – V1R, CHAU - V3F – CHAU - V3R, CHAU - V6F – CHAU - V6R, poxcDNAFw – poxPromRVC, poxcDNAFw – poxPromRVT) were tested for amplification of the 16S rRNA gene. Use of GroF/R and LAC2/4 primers pairs successfully identify strains belong to the Lactobacillus group. The variance between used extraction methods for evidence of HRM curves was found.
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Knápková, Monika. "Použití vysokorozlišovací analýzy křivek tání ke studiu baktérií mléčného kvašení." Master's thesis, Vysoké učení technické v Brně. Fakulta chemická, 2019. http://www.nusl.cz/ntk/nusl-401890.
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Currently, there is a growing interest in the use of probiotic products, and there are many of them in the market. With the growing interest, greater emphasis is placed on the identification of declared probiotic microorganisms. Precise identification of microbial composition is often a difficult task and it requires more advanced methods especially in the field of molecular diagnostics. The diploma thesis was focused on the verification of the presence od declared probiotic microorganisms in probiotic food supplements GS Laktobacily Forte 21, Biopron 9 Premium and Linex® Forte. DNA was isolated from the complex matrices by phenol extraction, commercial kit and magnetic carriers F79/L3-PLL in the quality suitable for PCR. Subsequently, the isolated DNA was amplified by real-time polymerase chain reaction using genus- and species-specific primers. The specific PCR product was subjected to agarose gel electrophoresis, whereas species identification was not always in compliance with the data declared by producers. The next part of the thesis was focused on polymerase chain reaction with high-resolution melting analysis to distinguish bacterial strains belonging to the Lactobacillus group and to identify probiotic microorganisms present in the complex matrices of the probiotic food supplements. Eight primer sets were tested (V1F HRM a V1R-HRM, CHAU-V3F a CHAU-V3R, CHAU-V6F a CHAU-V6R, LAC2 a LAC4, LAC1 a LAC2, P1V1 a P2V1, poxcDNAFw a poxPromRVC, poxcDNAFw a poxPromRVT). Three primer pairs (V1F HRM a V1R-HRM, poxcDNAFw a poxPromRVC, poxcDNAFw a poxPromRVT) were evaluated as the most suitable for distinguishing Lactobacillus bacterial strains.
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Hamano, Yuya. "Forensic age prediction by use of methylation-sensitive high resolution melting." Kyoto University, 2018. http://hdl.handle.net/2433/232076.
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Salgado, Isa Sofia Ribeiro. "Deteção de mutações nos genes JAK2 e MPL por high resolution melting." Master's thesis, Universidade de Aveiro, 2013. http://hdl.handle.net/10773/12628.
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Mestrado em Biologia AplicadaAs NMPs representam um grupo heterogéneo de doenças clonais caracterizadas pela expansão e produção excessiva de eritrócitos, granulócitos e/ou plaquetas sanguíneas na medula óssea. Em 2005, foi identificada a mutação V617F no gene JAK2 numa elevada frequência de doentes com NMP, em especial nos doentes com PV (65-97%), TE (23-57%) e MFP (35-57%). A deteção da mutação V617F no gene JAK2 e de outras mutações funcionalmente similares, isto é, mutações no exão 12 do gene JAK2 e no exão 10 do gene MPL, foram recentemente incluídas pela Organização Mundial de Saúde, nos critérios de diagnóstico para a PV, TE e MFP. Várias técnicas têm sido descritas e aplicadas à pesquisa destas mutações. A técnica de AS-PCR (PCR alelo-específico) é considerada uma técnica de diagnóstico capaz de detetar uma mutação heterozigótica presente em apenas 1-3% das células. Recentemente, o HRM foi descrito como uma técnica simples, rápida, de baixo custo e com elevada sensibilidade e especificidade na identificação e/ou deteção de mutações. Este estudo teve como principal objetivo avaliar a eficácia da técnica de HRM na deteção da mutação V617F-JAK2, das mutações no exão 12 do gene JAK2 e do exão 10 do gene MPL, numa série de 160 amostras de doentes com diagnósticos de NMP. A técnica de HRM demonstrou uma especificidade de 100% e uma sensibilidade ligeiramente inferior (98,4%) na deteção da mutação V617F, quando comparada com a técnica utilizada por rotina no GDPN para a deteção desta mutação (AS-PCR). Na pesquisa de mutações no exão 12 do gene JAK2 e exão 10 do gene MPL, a técnica de HRM permitiu detetar 100% dos casos com mutação. Os resultados deste estudo sugerem que o HRM tem uma utilização limitada na deteção da mutação V617F do gene JAK2, embora se tenha revelado uma técnica adequada ao rastreio rápido das mutações do exão 12 do gene JAK2 e do exão 10 do gene MPL. No presente estudo, foram detetadas mutações nos genes JAK2 e MPL em 80,6% dos doentes com PV, em 32,0% dos doentes com TE, em 33,3% dos doentes com MFP e em 33,3% dos doentes com NMP não classificadas.
Myeloproliferative neoplasms (MPNs) are a heterogeneous group of clonal diseases characterized by increased and excessive proliferation of erythrocytes, granulocytes and/or platelets in the bone marrow. In 2005, several groups identified the presence of the V617F mutation in the JAK2 gene in a significant proportion patients with PV (65-97%), ET (23-57%) and PMF (35-57%). Detection of the JAK2 mutation or other functionally similar mutation, such as JAK2 exon 12 mutations or MPL exon 10 mutations, have recently been included in the essential diagnostic criteria for PV, ET and PMF by the World Health Organization. Several techniques have been used to detect these mutations. AS-PCR (allele specific PCR) is considered a diagnostic tool capable of detecting a heterozygous mutation present in only 1-3% of mutated cells. Recently, HRM was described as a simple, fast and cost effective technique with high sensitivity and specificity that allows the detection and identification of mutations. The present study aimed at the evaluation of HRM as a diagnostic tool to detect JAK2-V617F, JAK2 exon 12 mutations or MPL exon 10 mutations, in 160 samples of MPNs patients. HRM revealed a 100% specificity and a slightly lower sensitivity (98,4%) in the V617F mutation detection when compared to AS-PCR. HRM detected all positive cases with JAK2 exon 12 mutations or MPL exon 10 mutations. Our results suggest that HRM is of limited use to detect the JAK2-V617F mutation. However, it is a suitable technique for mutation screening of JAK2 exon 12 mutations or MPL exon 10 mutations. In this study, JAK2 and MPL mutational frequency was 80,6% in PV, 32,0% in TE, 33,3% in PMF and in unclassifiable MPNs patients.
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ALMEIDA, F. A. N. "AUTÊNTICAÇÃO de Ginseng Brasileiro Utilizando a Técnica de High Resolution Melting (hrm)." Universidade Federal do Espírito Santo, 2018. http://repositorio.ufes.br/handle/10/7113.
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tese_12002_Dissertação_Francine Alves Nogueira de Almeida.pdf: 2009835 bytes, checksum: d864a01c75bbc516074e2befb4c57c5c (MD5)
Previous issue date: 2018-02-20À medida que a indústria de plantas medicinais cresce, a autenticidade de seus produtos é uma questão de segurança do consumidor e não pode ser negligenciada. O Ginseng brasileiro, por exemplo, se refere a duas espécies distintas, Pfaffia glomerata e Hebanthe eriantha. Apesar da similaridade entre essas duas espécies, o que dificulta a identificação morfológica, elas possuem propriedades químicas e farmacológicas distintas. Foi proposto que a técnica de High Resolution Melting (HRM) como uma ferramenta para discriminar e identificar as espécies P. glomerata e H. eriantha utilizando uma região do gene matK e rbcL. Para tal, foram adquiridas seis amostras referência, três de cada uma das espécies citadas, e 60 amostras comerciais, vendidas como Ginseng brasileiro, por meio de compra física ou online. O DNA foi extraído pelos métodos CTAB e por Kit. Os primers desenhados foram testados através da amplificação por PCR convencional e confirmado em gel de poliacrilamida antes da padronização da amplificação por PCR-HRM e por fim os resultados foram comparados aos de sequenciamento. Foram desenvolvidos três primers dois para o gene matK, HRM-matKD e HRM-matK, e um para o gene rbcL, HRM-rbcL. Durante a padronização notou-se a influência da temperatura de anelamento e concentração do primer no resultado da corrida, entretanto o método de extração não alterou os resultados. A análise de HRM mostrou que o primer HRM-matKD foi o que atendeu ao objetivo proposto apresentando alta sensibilidade (92-93%) e especificidade (100%) comparado ao método de sequenciamento. Foi possível validar a técnica de HRM utilizando amostras comerciais previamente sequenciadas, o que nos permite afirmar que a técnica de HRM pode ser utilizada para discriminar H. eriantha e P. glomerata. HRM é um método rápido e de baixo custo, sendo uma ferramenta confiável para identificação de espécies de Ginseng brasileiro.
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Marques, Jefferson Luiz Brum. "High-resolution electrocardiogram analysis." Thesis, University of Sheffield, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.263558.
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Chan, Ming-yan, and 陳明恩. "Rapid diagnosis of isoniazid resistant mycobacterium tuberculosis by high resolution melting (HRM) assay." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2012. http://hub.hku.hk/bib/B48333402.
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Mycobacterium tuberculosis (MTB) is a major infective agent causing human tuberculosis (TB) in the worldwide. Although tuberculosis can be treated by a six-month course of antibiotics, the prevalence of extensively drug-resistance TB (XDR-TB) made the disease becomes a global health problem. In addition to the conventional MTB detection methods, molecular methods become significant in drug resistant MTB detection which can enhance effective drug treatment.
In this study, 200 MTB respiratory specimens were collected from patients with suspected tuberculosis in Tuen Mun Hospital in Hong Kong. Based on the culture method as a gold standard for MTB detection, the presence of MTB in clinical samples was determined by IS6110single tube nested real-time PCR. In addition, by using High Resolution Melting (HRM) analysis, the presence of mutant type KatG315 gene for detecting isoniazid resistant MTB was determined.
Among 66 MTB culture positive samples, 10 samples had positive acid fast bacilli (AFB) smears giving the diagnostic sensitivity 15.1%. IS6110 single tube nested PCR was amplified in 51 specimens giving 77.2% MTB detection sensitivity and 97.8% specificity. Among 51 samples positive for IS6110 PCR, 66.7% showed successful amplification in subsequent KatG-HRM assay. Two samples were confirmed to be isoniazid (INH) resistance in Public Health Laboratory Centre (PHLC). However, there was only one sample showing detectable KatG315 mutation in clinical specimen by using HRM while the other was only detected in the corresponding culture isolate.
From the result of this study, single tube nested real-time PCR demonstrated MTB detection in clinical samples and INH resistant strain with KatG315mutationcan be detected by HRM analysis. Early detection of mycobacteria allow earlier treatment of the patient, thus transmission of the disease can be controlled.published_or_final_version
Microbiology
Master
Master of Medical Sciences
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Park, Dongwook 1959. "High-resolution laser radar performance analysis." Thesis, Massachusetts Institute of Technology, 1988. http://hdl.handle.net/1721.1/14712.
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Cury, Nathália Moreno. "Investigação de Mutações no Gene BRCA1 em Famílias Brasileiras com Suspeita da Síndrome Hereditária do Câncer de Mama e/ou Ovário." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/17/17135/tde-14062012-134410/.
Full textAbstract:
Cerca de 10% dos casos de câncer de mama e/ou ovário são caracterizados como hereditários, onde a presença de mutações germinativas no gene de suscetibilidade BRCA1 aumenta o risco de desenvolver esses cânceres durante a vida da mulher. O BRCA1 é um gene supressor tumoral envolvido na resposta de danos ao DNA, controle do ciclo celular, na remodelação da cromatina, ubiquitinação e regulação da transcrição. O presente estudo tem como objetivo central caracterizar as mutações do gene BRCA1 associadas a Síndrome Hereditária do Câncer de Mama e/ou Ovário (HBOC) em pacientes atendidos no Serviço de Aconselhamento Genético do Câncer do Hospital das Clínicas da Faculdade de Medicina de Ribeirão Preto da Universidade de São Paulo (HCFMRP/USP). Os vinte e dois éxons codificantes do BRCA1 foram analisados utilizando o método de High Resolution Melting (HRM) para triagem de mutações pontuais, seguido pelo sequenciamento de DNA dos casos selecionados para validação. A técnica de MLPA (Multiplex Ligation-dependent Probe Amplification) também foi usada para detectar grandes deleções e duplicações. Uma vez confirmada a mutação, membros da família considerados de alto risco, serão investigados para a mutação específica, a fim de proporcionar-lhes um aconselhamento genético apropriado para a detecção precoce do câncer. No presente estudo, foram investigados 41 pacientes que preencheram os critérios para o teste genético de acordo com NCCN Clinical Practice Guidelines in Oncology v.1.2010. Um total de 21 mutações foram identificadas, duas das quais são patogênicas: a deleção dos éxons 17-18 e a deleção dos éxon 19. Ambas estão localizadas no domínio BRCT do gene BRCA1, essencial para a ligação de fosfoproteínas críticas para a ativação do complexo de reparo do DNA. Outra mutação, a S616del, foi tratada como patogênica, mas apresenta informações controversas em diferentes estudos. O trabalho também identificou uma nova mutação, Val1117Ile. Um estudo de haplótipos das mutações identificadas nos pacientes foi realizado e revelou que um dos haplótipos, denominado de 6, contendo quatro resíduos mutados (871Leu, 1038Gly, 1183Arg e 1613Gly) estava presente em 50% das pacientes. O estudo de associação com 82 indivíduos saudáveis, mostrou diferença significativa (p=0,026) nos pacientes, sugerindo assim um risco aumentado de HBOC. Adicionalmente, foi analisada a mutação germinativa R337H no gene p53 para os casos suspeitos de Síndrome de Li-Fraumeni. Em síntese, o presente estudo contribui com a identificação de uma nova mutação não-sinônina no gene BRCA1 e sugere que o haplótipo 871Leu-1038Gly-1183Arg-1613Gly possa conferir risco aumentado do câncer de mama e/ou ovário em pacientes diagnosticados com HBOC.About 10% of cases of breast and/or ovary cancer are characterized as hereditary, where the presence of germline mutations in susceptibility BRCA1 gene increases the risk of developing these cancers during womans lifetime. BRCA1 is a tumor suppressor gene involved in DNA damage response, cell cycle control, chromatin remodeling, ubiquitination and transcriptional regulation. The present study aims to characterize BRCA1 gene mutations associated with Hereditary Breast/Ovary Cancer Syndrome (HBOC) in patients from the Cancer Genetic Counseling Service of the General Hospital of the Medical School of Ribeirão Preto, University of São Paulo (HCFMRP-USP). The twenty two coding exons of BRCA1 were analyzed using High Resolution Melting (HRM) method for the screening of point mutations, followed by DNA sequencing of the cases selected to validation. MLPA (Multiplex Ligation-dependent Probe Amplification) technique was also used to detect gross deletions and duplications. Once confirmed the mutation, family members most at risk will be analyzed for the specific mutation in order to provide them with an appropriate genetic counseling for early detection of cancer. In the present study, we investigated 41 patients that fulfilled the criteria for genetic testing according to NCCN Clinical Practice Guidelines in Oncology v.1.2010. A total of 21 mutations were identified, two of them are pathogenic: a deletion of exons 17-18 and a deletion of exon 19. Both of them are located in the BRCT domain of BRCA1 gene, impairing the binding of essential phosphoproteins critical to the activation of DNA repair complex. Another mutation, S616del, shows controversial information about its pathogenesis in different studies.The present study also describes a new mutation, Val1117Ile. A study of haplotypes of the mutations identified in patients was performed and revealed that one of the haplotypes, called 6, containing four mutated residues (871Leu, 1038Gly, and 1183Arg 1613Gly) was present in 50% of patients. The association study with 82 healthy subjects showed a significant difference (p = 0.026) in patients, thus suggesting an increased risk for HBOC. Additionally, the germline mutation R337H on p53 gene was also analyzed in the present study for suspected cases of Li-Fraumeni Syndrome. In summary, this study contributes to the identification of a new missense mutation in the BRCA1 gene and suggests that the haplotype-871Leu-1038Gly 1183Arg-1613Gly may confer increased risk of breast cancer and / or ovarian cancer in patients diagnosed with HBOC.
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Hytch, Martin J. "Quantitative high resolution electron microscopy." Thesis, University of Cambridge, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.317785.
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Sarlo, Rodrigo. "High-Resolution, High-Frequency Modal Analysis for Instrumented Buildings." Diss., Virginia Tech, 2018. http://hdl.handle.net/10919/84477.
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Civil infrastructure failure is hard to predict, both in terms of occurrence and impact. This is due to combination of many factors, including highly variable environmental and operational conditions, complex construction and materials, and the sheer size of these structures. Often, the mitigation strategy is visual inspection and regular maintenance, which can be time-consuming and may not address root causes of failure. One potential solution to anticipating infrastructure failure and mitigating its consequences is the use of distributed sensors to monitor the physical state of a structure, an area of research known commonly as structural health monitoring, or SHM. This approach can be applied in a variety of contexts: safety during and after natural disasters, evaluation of building construction quality and life-cycle assessment for performance based design frameworks.
In one way or another, SHM methods always require a ``baseline,'' a set of physical features which describes the behavior of a healthy structure. Often, the baseline is defined in terms of modal parameters: natural frequencies, damping ratios, and mode shapes. Although changes in modal parameters are indicative of structural damage, they are also indicative of a slew of non-damage factors, such as signal-to-noise ratio, environmental conditions, and the characteristics of forces exciting the structure. In many cases, the degree of observed modal parameter changes due to non-damage factors can be much greater than that due to damage itself. This is especially true of low-frequency modal parameters. For example, the fundamental frequency of a building is more sensitive to global influences like temperature than local structural changes like a cracked column.
It has been proposed that extracting modal parameters at higher frequencies may be the key to improving the damage-sensitivity of SHM methods. However, for now, modal analysis of civil structures has been limited to low frequency ambient excitation and sparse sensor networks, due to practical limitations. Two key components for high-frequency modal analysis have yet to be studied: 1) Sufficient excitation at high frequencies and 2) high-resolution (high sensor density) measurements. The unifying goal of this work is to expand modal analysis in these two areas by applying novel instrumentation and experimental methods to two full-scale buildings, Goodwin Hall and Ernest Cockrell Jr. Hall. This enables realistic, practical insights into the limitations and benefits of the high-frequency SHM approach. Throughout, analyses are supported through the novel integration of uncertainty quantification techniques which so far has been under-utilized in the field.
This work is divided into three experimental areas, with approaches centering on the identification of modal parameters. The first area is the application of high spacial resolution sensor networks in combination to ambient vibration testing. The second is the implementation of a robust automation and monitoring strategy for complex dynamic structures. The third is the testing of a novel method for performing experimental modal analysis on buildings emph{in situ}. The combination of results from these experiments emphasizes key challenges in establishing reliable high-frequency, high-resolution modal parameter ``baselines'' for structural health monitoring (SHM) of civil infrastructure.
The first study presented in this work involved the identification of modal parameters from a five-story building, Goodwin Hall, using operational modal analysis (OMA) on ambient vibration data. The analysis began with a high spacial density network of 98 accelerometers, later expanding this number to 117. A second extensional study then used this data as reference to implement a novel automation method, enabling the identification of long-term patterns in the building's response behavior. Three dominant sources of ambient excitation were identified for Goodwin Hall: wind, human-induced loading, and consistent low-level vibrations from machinery, etc. It was observed that the amplitude of excitation, regardless of source, had significant effects on the estimated natural frequencies and damping ratios. Namely, increased excitation translated to lower natural frequencies and higher damping. In addition, the sources had different characteristics in terms of excitation direction and bandwidth, which contributed to significantly different results depending on the ambient excitation employed. This has significant implications for ambient-based methods that assume that all ambient vibrations are broadband random noise.
The third and final study demonstrated the viability of emph{in situ} seismic testing for controlled excitation of full-scale civil structures, also known as experimental modal analysis (EMA). The study was performed by exciting Ernest Cockrell Jr. Hall in Austin, Texas with both vertical and lateral ground waves from seismic shaker truck, T-Rex. The EMA results were compared to a standard operational modal analysis (OMA) procedure which relies on passive ambient vibrations. The study focused on a frequency bandwidth from 0 to 11 Hz, which was deemed high frequency for such a massive structure. In cases were coherence was good, the confidence comparable or better than OMA, with the added advantage that the EMA tests took only a fraction of the time. The ability to control excitation direction in EMA enabled the identification of new structural information that was not observed OMA. It is proposed that the combination of high spacial resolution instrumentation and emph{in situ} excitation have the potential to achieve reliable high-frequency characterization, which are not only more sensitive to local damage but also, in some cases, less sensitive to variations in the excitation conditions.Ph. D.
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Goode, Ashley Harford. "High resolution ultrasonic imaging system." Thesis, University of Portsmouth, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.329278.
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Cox, K. B. "High resolution analysis of dispersed seismic signals." Thesis, University of Oxford, 1986. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.375223.
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Lepidis, Polichronis. "High resolution frequency analysis in scanning probe microscopy." [S.l.] : [s.n.], 2002. http://deposit.ddb.de/cgi-bin/dokserv?idn=96834674X.
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Mundt, Laura Elena [Verfasser], and Stefan [Akademischer Betreuer] Glunz. "High-resolution analysis of perovskite absorbers in photovoltaics." Freiburg : Universität, 2018. http://d-nb.info/1181689929/34.
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Nakajima, Kaoru. "MONOLAYER ANALYSIS USING HIGH-RESOLUTION RUTHERFORD BACKSCATTERING SPECTROSCOPY." 京都大学 (Kyoto University), 2009. http://hdl.handle.net/2433/85403.
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Kyoto University (京都大学)0048
新制・論文博士
博士(工学)
乙第12399号
論工博第4030号
新制||工||1477(附属図書館)
27429
UT51-2009-M905
京都大学大学院工学研究科機械物理工学専攻
(主査)教授 木村 健二, 教授 井手 亜里, 教授 河合 潤
学位規則第4条第2項該当
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MASIELLO, IRENE. "High-resolution epigenetic analysis of the cell nucleus." Doctoral thesis, Università degli studi di Pavia, 2018. http://hdl.handle.net/11571/1214856.
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In the interphase nucleus, DNA is necessarily organized around the histone core and, then, in higher order structures. At the electron microscope, dispersed euchromatin fibers and dark heterochromatin regions can be distinguished. The latter is divided in constitutive heterochromatin, reasonably localized at the periphery nearby the nuclear envelope and far from the perichromatin region where transcription occurs, and facultative heterochromatin, containing the genes undergoing silencing and activation (regulated genes) on the surface facing the perichromatin region. Chromatin structure affects numerous and fundamental genetic processes, such as gene expression, and therefore it is strictly regulated by different mechanisms. DNA methylation prevents the binding of transcriptional factors and recruits transcriptional repressor complexes, thus forming compact and inactive chromatin, although recent studies show 5-methylcytosine to reduce nucleosome stability. 5-methylcytosine was also found in the 3’ untranslated regions to probably influence mRNA stability. Histone post-transcriptional modifications can lead both to a more compact or relaxed chromatin structure by inducing nucleosome repositioning: for instance, H3K9me3 is known as a marker of constitutive heterochromatin while H3K27me3 or H4K20me3 label facultative heterochromatin. The ionic environment is also known to profoundly affect chromatin organization: an increase of calcium or magnesium results in different types of chromatin conformations, probably with different stability.
Until now, DNA or RNA methylation, as well as histone modifications, was studied through biomolecular approaches. Here, we propose an alternative epigenetic analysis at ultrastructural level, using mouse hepatocytes to describe the distribution of 5-methylcytosine and histone modified residues on condensed chromatin regions and HeLa cells in order to detect epigenetic modifications on RNA fibrils. Treatments known to induce chromatin compaction (modification of cation concentration or heat shock) were also chosen to deeply investigate chromatin structure regulation. Therefore, cells and tissues were prepared principally for the cytochemical analysis, combining numerous immunocytochemical reactions with specific staining methods.
This process is complex to analyze and more variables have to be considered. However, our results suggest that the main function of DNA methylation and histone modifications is the gene switching-on and -off, which occurs on chromatin surface, whereas they do not seem necessarily involved in the maintaining of chromatin condensation status, probably preserved by other mechanisms to be deeply investigated. As for RNA, our data indicate that cytosine methylation is a very precocious event, probably confirming its involvement in mRNA stability, and DNMT3A is unexpectedly involved in this modification. Other studies will be carried out to understand the processes regulating chromatin structure and the role of mRNA methylation. However, the influence of the ionic environment on both chromatin structure and the epigenetic modifications should be further studied.
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Gatcombe, Christopher Peter. "Computer modelling of high resolution ultrasonic transducers." Thesis, City University London, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.236597.
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Cinar, Umut. "Road Extraction From High Resolution Satellite Images Using Adaptive Boosting With Multi-resolution Analysis." Master's thesis, METU, 2012. http://etd.lib.metu.edu.tr/upload/12614650/index.pdf.
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Road extraction from satellite or aerial imagery is a popular topic in remote sensing, and there are many road extraction algorithms suggested by various researches. However, the need of reliable remotely sensed road information still persists as there is no sufficiently robust road extraction algorithm yet. In this study, we explore the road extraction problem taking advantage of the multi-resolution analysis and adaptive boosting based classifiers. That is, we propose a new road extraction algorithm exploiting both spectral and structural features of the high resolution multi-spectral satellite images. The proposed model is composed of three major componentsfeature extraction, classification and road detection. Well-known spectral band ratios are utilized to represent reflectance properties of the data whereas a segmentation operation followed by an elongatedness scoring technique renders structural evaluation of the road parts within the multi-resolution analysis framework. The extracted features are fed into Adaptive Boosting (Adaboost) learning procedure, and the learning method iteratively combines decision trees to acquire a classifier with a high accuracy. The road network is identified from the probability map constructed by the classifier suggested by Adaboost. The algorithm is designed to be modular in the sense of its extensibility, that is
new road descriptor features can be easily integrated into the existing model. The empirical evaluation of the proposed algorithm suggests that the algorithm is capable of extracting majority of the road network, and it poses promising performance results.
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Gaslikova, Lidia. "High-resolution wave climate analysis in the Helgoland area /." Geesthacht : GKSS-Forschungszentrum, Library, 2006. http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&doc_number=015585234&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA.
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Hsu, Ching-Ming. "High resolution SIMS analysis using a chemical bevelling technique." Thesis, Imperial College London, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.243823.
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Wang, Peng. "High resolution imaging and analysis using aberration-corrected stem." Thesis, University of Liverpool, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.433775.
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Zamotaeva, Valeriya. "High-resolution FTIR spectra analysis of sulfur dioxide isotopologues." Thesis, Bourgogne Franche-Comté, 2019. http://www.theses.fr/2019UBFCK053.
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Dans cette thèse, nous avons examiné les propriétés spectrales de l’anhydride sulfureux. Les spectres FTIR expérimentaux de nombreux isotopologues du dioxyde de soufre, 32S16O2, 34S16O2, 32S18O2 et 32S16O18O, ont été enregistrés pour la première fois dans les régions des bandes fondamentales, «chaudes», harmoniques et de combinaison. La grande variabilité des conditions expérimentales a permis d’observer et d’identifier pour la première fois des transitions appartenant aux ban¬des ro-vibrationnelles suivantes : 3v2, 3v2 - v2, 2v2 - v2 bandes de 32S16O2 ; 2v2 - v2 bande de 34S16O2 ; v1 + v2, v2 + v3, v1 + v3, 2v1, 2v3 bandes de 32S18O2 ; v1, v3, 2v1, v1 + v3, 2v3 bandes de 32S16O18O. Les problèmes spectroscopiques inverses ont été résolus pour les états étudiés avec une déviation «rms» comparable à l’incertitude expérimentale. Suite à cette analyse, environ 38 000 transitions ro-vibrationnelles appartenant à 17 états vibratoires excités ont été identifiées pour la première fois. Les données très précises obtenues sur tous les isotopologues du dioxyde de soufre ont été utilisées pour corriger les paramètres de la IPFIn this thesis we considered the spectral properties of the sulfur dioxide. The experimental FTIR spectra of numerous sulfur dioxide isotopologues, 32S16O2, 34S16O2, 32S18O2 and 32S16O18O, were first recorded in the regions of fundamental, «hot», combination and overtone bands. The wide variability of the experimental conditions gave possibility to observe and identify for the first time transitions be¬ longing to the following of ro-vibrational bands: 3v2, 3v2 - v2, 2v2 - v2 bands of 32S16O2; 2v2 - v2 band of 34S16O2; v1 + v2, v2 + v3, v1 + v3, 2v1, 2v3 bands of 32S18O2; v1, v3, 2v1, v1 + v3, 2v3 bands of 32S16O18O. The inverse spectroscopic problems were solved for the studied states with the «rms»-deviation comparable to the experimental uncertainty of the spectral line position. As a result of the analysis about 38 000 ro-vibrational transitions belonging to 17 excited vibrational states were identified for the first time. The obtained highly accurate data on all sulfur dioxide isotopologues were used to correct the parameters of the IPF
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Aboaba, Olusegun Abiola. "High-resolution multipath channel parameter estimation using wavelet analysis." Thesis, Curtin University, 2014. http://hdl.handle.net/20.500.11937/1271.
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This thesis explores the novel use of wavelet analysis as a high-resolution digital signal processing algorithm for multipath channel parameter estimation. The results obtained from this research indicate that this wavelet-based digital signal processing algorithm overcomes the resolution limitation in conventional high-resolution algorithm. This may provide a more cost-effective means of implementing channel sounding equipments for very high-resolution measurements.
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Falco, Mariachiara. "Analysis of high resolution observations of sunspots fine structures." Doctoral thesis, Università di Catania, 2016. http://hdl.handle.net/10761/3859.
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This Ph.D. thesis deals with the study of the interaction between plasma and magnetic fields on the Sun. Observations acquired during two observing campaigns, one at the Solar Swedish Tower and the other at the Richard B. Dunn Solar Telescope, provided high-resolution data which were used to study the details of some phenomena occurring in the Sun.
In particular, this work aims to extend and consolidate our knowledge on the formation and evolution of sunspots observed in the solar photosphere. The first aspect which was investigated concerns the study of the mechanism of the penumbra formation in a sunspot. In this regard, I give my contribution in explaining the behaviour of the magnetic field forming the penumbral filaments. A second aspect concerns the study of the kinematic and magnetic properties of a light bridge separating
into two parts the umbra of a sunspot. I found that there is a relationship between the upflow motions in the light bridge dark lane and the magnetic field configuration. The third aspect concerns the study of the
properties of granules in a light bridge and in the quiet Sun using a new algorithm developed in collaboration with the Department of Mathematics of the University of Catania. In particular, I compared the size, mean continuum intensity and Doppler velocity between the granules forming the light bridge and those of the quiet Sun. In the conclusion, I give my interpretation of the observed phenomena and suggest future observations to confirm these results and support theoretical models.
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Surampudi, Bala Anjani Vasudha. "High-Resolution Modeling of Steel Structures." University of Cincinnati / OhioLINK, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1504787210175847.
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Warhola, Paul J. "An analysis of alternative methods to conduct high-resolution activities in a variable-resolution simulation." Thesis, Monterey, Calif. : Springfield, Va. : Naval Postgraduate School ; Available from National Technical Information Service, 1997. http://handle.dtic.mil/100.2/ADA337495.
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Mummery, Gavin Thomas. "Developing a high-resolution bioengineering model for slope stability analysis." Thesis, University of Bristol, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.435426.
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Bagnall, Kate. "High spatial resolution analysis of metastable magnesium and titanium alloys." Thesis, University of Bristol, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.337260.
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Akilian, Mireille. "Thin optic surface analysis for high resolution X-ray telescopes." Thesis, Massachusetts Institute of Technology, 2004. http://hdl.handle.net/1721.1/34556.
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Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2004.Includes bibliographical references (p. 119-122).
The art of glass developed throughout the years has covered artifacts ranging from crude ornaments to high precision optics used in flat panel displays, hard disk drives, and x-ray telescopes. Methods for manufacturing glass sheets and further sheet shaping processes are covered. Future generation, high resolution x-ray telescopes require thin optics with large surface area to thickness ratio and a surface flatness of -500 nm. A novel method utilizing porous ceramics, which provide a thin layer of air for sheet glass to rest on during the shaping process, is investigated. The shaping process involves slumping glass on a uniform layer of air at elevated temperatures, where the viscosity of glass is low enough for it to sag under its own weight and replicate the surface it rests on. Flow in porous, rectangular air bearings is covered with both flat and grooved surfaces. The pressure distribution in the air gap between the ceramic and the glass sheet determines the surface quality of glass during slumping. The mechanical integrity of porous ceramics at elevated temperatures is investigated to predict the effect of the decrease in ceramic stiffness on the final shape of the optic.
(cont.) A metrology truss used to kinematically constrain thin optics during metrology is designed. This device mitigates the effects of external forces, such as gravity, friction, and thermal stresses, induced on the optic while being mechanically constrained, thus significantly improving the repeatability of the optic surface map measurements.
by Mireille Akilian.
S.M.
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Morgenthal, Guido. "Aerodynamic analysis of structures using high-resolution vortex particle methods." Thesis, University of Cambridge, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620717.
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Albratty, Mohammed Mofareh. "Metabonomic analysis of Drosophila mutants using high resolution mass spectrometry." Thesis, University of Strathclyde, 2013. http://oleg.lib.strath.ac.uk:80/R/?func=dbin-jump-full&object_id=18695.
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The availability of fully sequenced genomes of model organisms such as Drosophila and their subsequent annotation has afforded opportunities for reverse genetics in a complex model organism. Metabonomics used as an aid to functional genomics can be used to understand the functions of genes in living systems. Thus metabonomics has been employed to study Drosophila samples extracted from whole animals at different developmental stages or in response to external stimuli or genetic mutation. With unmatched mass resolution, accuracy, and detection sensitivity, linear ion trap - Fourier Transform Orbitrap Mass Spectrometry (LTQ-Orbitrap-MS) has the potential for high throughput metabonomic analysis. Five different but linked studies are reported in this work. A global metabolic profiling method based on electrospray ionisation mass spectrometry was developed for Drosophila melanogaster metabolites. The method involved optimizing the extraction of Drosophila metabolites followed by analysi s using liquid chromatography coupled with high-resolution mass spectrometry. The effect of extraction conditions and storage were studied, thus 750-800 metabolites were putatively identified in order to obtain the metabolite profiles of Drosophila reference strains and mutants. Metabolic studies were carried out to elucidate gene functions using established protocols. The online resource FlyAtlas.org provides detailed microarray-based expression data for the tissues and life-stages of Drosophila. Since downstream genes, such as urate oxidase are tubule-specific, an Orbitrap technology has been used to elucidate tissue specific metabolomes. Additionally, genetic interventions using designed RNA interference were also made and validated by qPCR and metabonomics. The method produced a new opportunity for metabonomics use in validating gene expressions. The xanthine oxidase inhibitor allopurinol was used to phenocopy the rosy mutation which caused the levels of xanthine and hypoxanthine to rise while the levels of uric acid fell. In addition, many unexpected metabolic changes followed this treatment with effects on the pentose phosphate pathway and tryptophan metabolism being the most marked. The yellow (y) gene was first discovered in Drosophila, but occurs in many insect species and in some bacteria. The y protein is similar to the major royal jelly proteins produced by bees. Metabolomic profiling was carried out on Oregon R (OR) and y Drosophila larvae at the third instar. There were numerous metabolic differences between the metabolic profiles of OR and y. Phenylalanine, tyrosine and DOPA were all elevated in y, as might be expected since the mutation blocks melanin biosynthesis. In addition, there were other metabolic effects including marked effects on to lysine metabolism. The white mutation of Drosophila, which affects ABC transporters, was studied with regards to its effect on pigment biosynthesis in Drosophila. In addition to the expected effects on pigme nts there were interesting male/female differences possibly related to the presence of the white gene in the X chromosome. In addition the effect of salt stess on wild type and white flies was studied. Overall, LTQ-Orbitrap-MS proved suitable for metabonomic analysis of both wild-type and mutant Drosophila and had potential in the analysis of metabolomes of single tissues. The possibility of using Orbitrap-based metabonomics in combination with Drosphila for drug testing is discussed and is a goal for the future.
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Pugh, Andrew. "Elemental analysis of glass optical fibres with high spatial resolution." Thesis, Sheffield Hallam University, 1991. http://shura.shu.ac.uk/20251/.
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The properties of glass optical fibres are very strongly dependent on the elemental concentration profiles of the fibre cores. Core dopants such as germanium define the core refractive index, which in turn defines the manner in which the light is transmitted through the fibre. Erbium in fibre cores can facilitate the operation of fibre lasers and aluminium in turn can control the erbium distribution. The aim of the project described in this thesis was to measure the elemental concentration profiles in a variety of fibre cores using X-ray microanalysis in an electron microscope. Conventional X-ray microanalysis of bulk samples has an analytical resolution in the order of a micron. With monomode optical fibre cores having cores typically three microns in diameter the resolution of the conventional technique is plainly inadequate. An experimental technique has been developed for the preparation of thin cross-sectional samples of glass optical fibres. Application of this technique has facilitated the preparation and analysis of thin film specimens with an average thickness of 400 microns. This approach has allowed analysis to be performed with an effective spatial resolution of 100-300 nm. The technique has been applied to the determination of germanium concentration in Raman fibres, to the investigation of erbium confinement in erbium doped fibres and to the investigation of inter-ionic diffusion in semiconductor doped fibres. It has been shown that the germanium, and hence refractive index, profile of germanium doped fibres is not changed by the process of fibre drawing. Evidence has been gathered supporting the theory of erbium confinement by aluminium and an important degree of elemental diffusion has been shown to take place during the drawing of semiconductor doped fibres. In addition an experimental technique has been developed for the preparation of thin cross-sectional samples of glass optical fibres.