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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
81

Arylhydrazine carcinogenesis and the synthesis of C8-arylpurine oligonucleotides a study of DNA adduct affects on DNA conformation and stability /

Daft, Jonathan R., January 1900 (has links)
Thesis (Ph. D.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains xi, 323 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 191-211).
82

Etude expérimentale de la stabilité, sélectivité d'appariement et dynamique d'oligonucléotides DNA-DNA et LNA-DNA

Boccongelli, Marina 20 March 2008 (has links)
Le traitement et le diagnostic de maladies d'origine génétique suscite un grand intérêt à l'heure actuelle. De par leur spécificité d'appariement avec les acides nucléiques, les oligonucléotides possèdent un grand potentiel dans ce domaine. Ils se heurtent toutefois à des limitations majeures, dont leur faible stabilité en milieu physiologique et la difficulté qu'ils ont à franchir les membranes biologiques. De nombreuses équipes de recherche s'intéressent, afin de pallier ces limitations, à la conception et à la synthèse d'oligonucléotides chimiquement modifiés. Parmi ceux-ci, les Locked Nucleic Acids (LNA), présentant une modification qui consiste en l'insertion d'un pont −O−CH2− entre l'atome C2' et l'atome C4' du sucre, constituent une famille qui semble posséder les propriétés requises. Ils sont considérés comme des candidats très prometteurs en tant qu’agents thérapeutiques et qu’outils de diagnostic du génome. La caractérisation de la stabilité et de la sélectivité d'appariement entre les LNA et les acides nucléiques naturels est, dans ce contexte, important.<p><p>Dans ce travail, nous avons étudié la stabilité, la sélectivité d'appariement ainsi que la dynamique de la structure double brin d'un oligonucléotide hybride LNA-DNA, et nous avons comparé ces propriétés à celles d'un oligonucléotide DNA-DNA de même séquence. Ce dernier est constitué de 11 paires de bases formées par l'appariement du brin 5'-GCGTGTGTGCG-3' avec le brin 3'-CGCACACACGC-5'. Dans le cas de l'hybride, les nucléotides du second brin sont tous remplacés par des LNA.<p><p>La stabilité a été étudiée expérimentalement par différentes techniques :spectroscopie d'absorption UV, calorimétrie différentielle à balayage, résonance magnétique nucléaire et calorimétrie à titrage isotherme. Ces études montrent que la stabilité du duplexe hybride est plus importante que celle du naturel, et que ce phénomène s'explique par un terme entropique plus favorable pour la formation du duplexe LNA-DNA que pour la formation du duplexe DNA-DNA.<p><p>La sélectivité d'appariement a été étudiée en comparant la stabilité des deux oligonucléotides étudiés avec celle d'oligonucléotides présentant un mésappariement dans la séquence. Nos résultats montrent que la sélectivité d'appariement du brin LNA n'est pas significativement différente de celle du brin DNA. Ce résultat ne doit cependant pas être généralisé car nous n'avons testé qu'une position centrale pour le mésappariement.<p><p>L'étude de la dynamique de la structure des oligonucléotides a été effectuée par RMN et porte sur la caractérisation de la cinétique de l'ouverture individuelle des paires de bases. Nous observons que la durée de vie de l'état fermé des paires de bases G-C est supérieure dans l'oligonucléotide LNA-DNA, tandis que l'état fermé des paires A-T semble posséder une durée de vie supérieure dans l'oligonucléotide DNA-DNA.<p><p>Au cours de ce travail de thèse nous avons pu caractériser les facteurs énergétiques à la base de la stabilité accrue des oligonucléotides chimiquement modifiés de type LNA. Nous avons montré que leur sélectivité d’appariement n’est pas toujours supérieure à celle des oligonucléotides naturels et dépend des séquences impliquées. Enfin, nous avons mis en évidence les différences entre la dynamique de la structure d’un oligonucléotide possédant des LNA et celle d’un duplexe DNA. / Doctorat en Sciences de l'ingénieur / info:eu-repo/semantics/nonPublished
83

Étude expérimentale de la stabilité, sélectivité d'appariement et dynamique d'oligonucléotides DNA-DNA et LNA-DNA

Boccongelli, Marina 20 March 2008 (has links)
Le traitement et le diagnostic de maladies d'origine génétique suscite un grand intérêt à l'heure actuelle. De par leur spécificité d'appariement avec les acides nucléiques, les oligonucléotides possèdent un grand potentiel dans ce domaine. Ils se heurtent toutefois à des limitations majeures, dont leur faible stabilité en milieu physiologique et la difficulté qu'ils ont à franchir les membranes biologiques. De nombreuses équipes de recherche s'intéressent, afin de pallier ces limitations, à la conception et à la synthèse d'oligonucléotides chimiquement modifiés. Parmi ceux-ci, les Locked Nucleic Acids (LNA), présentant une modification qui consiste en l'insertion d'un pont −O−CH2− entre l'atome C2' et l'atome C4' du sucre, constituent une famille qui semble posséder les propriétés requises. Ils sont considérés comme des candidats très prometteurs en tant qu’agents thérapeutiques et qu’outils de diagnostic du génome. La caractérisation de la stabilité et de la sélectivité d'appariement entre les LNA et les acides nucléiques naturels est, dans ce contexte, important. Dans ce travail, nous avons étudié la stabilité, la sélectivité d'appariement ainsi que la dynamique de la structure double brin d'un oligonucléotide hybride LNA-DNA, et nous avons comparé ces propriétés à celles d'un oligonucléotide DNA-DNA de même séquence. Ce dernier est constitué de 11 paires de bases formées par l'appariement du brin 5'-GCGTGTGTGCG-3' avec le brin 3'-CGCACACACGC-5'. Dans le cas de l'hybride, les nucléotides du second brin sont tous remplacés par des LNA. La stabilité a été étudiée expérimentalement par différentes techniques : spectroscopie d'absorption UV, calorimétrie différentielle à balayage, résonance magnétique nucléaire et calorimétrie à titrage isotherme. Ces études montrent que la stabilité du duplexe hybride est plus importante que celle du naturel, et que ce phénomène s'explique par un terme entropique plus favorable pour la formation du duplexe LNA-DNA que pour la formation du duplexe DNA-DNA. La sélectivité d'appariement a été étudiée en comparant la stabilité des deux oligonucléotides étudiés avec celle d'oligonucléotides présentant un mésappariement dans la séquence. Nos résultats montrent que la sélectivité d'appariement du brin LNA n'est pas significativement différente de celle du brin DNA. Ce résultat ne doit cependant pas être généralisé car nous n'avons testé qu'une position centrale pour le mésappariement. L'étude de la dynamique de la structure des oligonucléotides a été effectuée par RMN et porte sur la caractérisation de la cinétique de l'ouverture individuelle des paires de bases. Nous observons que la durée de vie de l'état fermé des paires de bases G-C est supérieure dans l'oligonucléotide LNA-DNA, tandis que l'état fermé des paires A-T semble posséder une durée de vie supérieure dans l'oligonucléotide DNA-DNA. Au cours de ce travail de thèse nous avons pu caractériser les facteurs énergétiques à la base de la stabilité accrue des oligonucléotides chimiquement modifiés de type LNA. Nous avons montré que leur sélectivité d’appariement n’est pas toujours supérieure à celle des oligonucléotides naturels et dépend des séquences impliquées. Enfin, nous avons mis en évidence les différences entre la dynamique de la structure d’un oligonucléotide possédant des LNA et celle d’un duplexe DNA.
84

Towards DNA-Bodies : A Novel Polymer Structure for Biological Recognition

Lövdahl, Paul January 2010 (has links)
<p><p>There are different kinds of recognition molecules that specifically can detect and bind target molecules. Antibodies, with their two light and two heavy chains can detect and bind any kind of antigens. Molecular imprinting is a technology to prepare specific polymers that selectively bind target molecules. The technology has received wide attention in recent years because it provides a viable method for creating a polymer that is complementary in shape and binding sites to a template. The synthesized polymer is called a molecularly imprinted polymer (MIP) or a plastibody. Molecular imprinting shows promise in diverse areas as chromatography, antibody mimics, solid phase extraction and more. An alternative to molecular imprinting and other types of recognition has recently been postulated where DNA polymers based on functionalized polynucleotides build up a polymer network that are able to specifically recognize a target. This approach is characterized by binding of oligonucleotides carrying functional organic groups, to the target molecule followed by connecting the functionalized oligonucleotides forming a DNA polymer that specifically recognizes the target. The polymer is called a DNAbody. Thus, a DNAbody is a polymer structure based on DNA conjugated with functional organic groups which specifically can detect and bind to a template. The DNA-bodies can be copied and produced in larger amounts by PCR. This study showed that at least one functional oligonucleotide was able to interact with the target antibody. It was also seen that some interaction occurred between the DNA and protein. The results also indicated that it is possible to perfom DNA polymerase reactions in presence of an antibody.</p></p>
85

Impact of Ligand Shell Architecture on Structure and Reactivity of DNA Aptamer-Linked Gold Nanoparticle Assemblies

Baldock, Brandi 27 October 2016 (has links)
DNA-functionalized gold nanoparticles (DNA-NPs) have enormous potential as building blocks for materials due to their ability to specifically recognize and respond to target molecules and surfaces. The ability of DNA aptamers to adopt different conformations and bind either complementary DNA sequences or analyte molecules allows them to mediate nanoparticle assembly or disassembly, generating selective colorimetric responses. Aptamer-mediated nanoparticle assembly and disassembly is sensitive to the nanoparticle ligand shell composition and structure, yet these topics have not been extensively explored. In this dissertation, a method for determining the ligand shell composition of DNA-NPs is described and a framework for understanding the impact of the DNA assembly arrangement and recognition strand density upon aptamer-mediated nanoparticle assembly and disassembly is developed. Design rules for creating sensors with desired properties are elucidated, leading to creation of sensors with improved detection limits and quantification ranges. A technique was needed to determine the number of DNA strands of any base composition attached to gold nanoparticles (AuNPs) of any core size. A rapid, convenient and inexpensive method to quantify the number of label-free DNA strands attached to AuNPs was therefore developed. This technique was extended to determine two different DNA sequences bound to AuNPs using UV-visible and fluorescence spectroscopy. Based on the results of quantifying the ligand shells of DNA-NPs functionalized with two sequences, disulfide-terminated DNA non-specifically adsorbs and then rearranges to specifically bind the gold surface. The position of the AuNPs and DNA strands within DNA-NP assemblies had a profound influence on their ability to assemble and sense adenosine. Assemblies designed for large inter-AuNP spacing were stable but unable to sense adenosine. Assemblies designed for short inter-AuNP spacing were unstable until the DNA ligand shell was diluted. AuNPs functionalized with the fewest number of aptamers produced assemblies with the lowest detection limit and apparent disassociation constant and the largest analyte quantification range. Increasing the number of aptamer strands per AuNP increased the cooperativity of the AuNP disassembly response to adenosine. This dissertation includes previously unpublished co-authored material.
86

Desenvolvimento de aptâmeros específicos para aplicação como radiofármacos na identificação de bactérias / Development of aptamers for use as radiopharmaceuticals in the bacterial infection

Iêda Mendes Ferreira 26 February 2013 (has links)
A dificuldade na detecção precoce de focos infecciosos específicos causados por bactérias tem aumentado a necessidade de pesquisar novas técnicas para este fim, uma vez que estes focos necessitam de tratamento prolongado com antibióticos e, em alguns casos, até mesmo drenagem ou, se for o caso, a remoção de próteses ou enxertos. A detecção de infecções bacterianas por cintilografia teria a vantagem de uma imagem de corpo inteiro, desde que traçadores específicos estejam disponíveis. Esse estudo visa a obtenção de aptâmeros específicos para identificação de bactérias para uso futuro como radiofármaco. A metodologia SELEX (do inglês Systematic Evolution of Ligands by Exponential Enrichment) pode gerar oligonucleotídeos (aptâmeros) que são capazes de se ligar com alta afinidade e especificidade a alvos específicos, desde pequenas moléculas até proteínas complexas, usando ciclos de enriquecimento e amplificação. Aptâmeros podem ser marcados com diferentes radionuclídeos tais como 99mTc, 18F e 32P. Aptâmeros anti-peptideoglicano, o principal componente da parede celular externa bacteriana, foram obtidos através da SELEX. Células inteiras de Staphylococcus aureus também foram utilizadas para a realização da SELEX para células (cell-SELEX). A seleção dos aptâmeros foi realizada por meio de dois procedimentos distintos. Esses foram o processo A em que foram realizados 15 ciclos da SELEX nos quais a separação dos oligonucleotídeos ligados ao peptideoglicano dos não ligados foi efetuada por filtração e o processo B em que foram realizados 15 ciclos com a separação realizada por centrifugação, seguidos de 5 ciclos de cell-SELEX. A SELEX teve início com um pool de ssDNA (DNA de fita simples). Para o processo A, inicialmente a biblioteca de ssDNA foi incubada com o peptideoglicano e a amplificação dos oligonucleotídeos que foram capazes de se ligar ao peptideoglicano foi realizada por PCR (Polymerase Chain Reaction). Os oligonucleotídeos amplificados foram novamente incubados com o peptideoglicano, amplificados e purificados. Ao fim de 15 ciclos de seleção, os oligonucleotídeos selecionados foram clonados. O produto de recombinação foi utilizado para transformar a bactéria Escherichia coli Top10F. O DNA plasmidial de 40 colônias selecionadas foram extraídos e quantificados. Os plasmídeos foram sequenciados, duas sequências diferentes (Antibac1 e Antibac2) foram obtidas e as estruturas secundárias determinadas. Os aptâmeros obtidos foram sintetizados e marcados com 32P. Os aptâmeros marcados foram incubados com células de S. aureus e a quantidade de ssDNA ligado às bactérias foi determinado por espectrometria de cintilação líquida. A biblioteca de oligonucleotídeos marcada com 32P foi usada como controle. Para o aptâmero Antibac1 a radiação foi 28 vezes maior do que a obtida com o controle e para o aptâmero Antibac2 22 vezes. Um ensaio de especificidade foi conduzido com os aptâmeros marcados utilizando-se células de S. aureus, E.coli, Candida albicans e fibroblastos humanos. Para os dois aptâmeros (Antibac1 e Antibac2) a ligação às células bacterianas foi significativamente superior à verificada para C. albicans e fibroblastos, demonstrando a especificidade dos mesmos para identificação de bactérias. Para o processo B após os 15 ciclos da SELEX foi realizada a cell-SELEX que começou com o produto do 15o ciclo sendo incubado com células de S.aureus. Ao fim de 5 ciclos de seleção, os oligonucleotídeos selecionados foram clonados e sequenciados como no processo A. Onze diferentes sequências foram obtidas de 21 clones e as estruturas secundárias foram determinadas. Os aptâmeros obtidos pelo processo A apresentaram alta afinidade e especificidade para bactérias. Os aptâmeros obtidos pelo processo B serão avaliados quanto a estes parâmetros em trabalhos futuros. / The difficulty in early detection of specific foci caused by bacteria in the bacterial infection has raised the need to search for new techniques for this purpose, since these foci require prolonged treatment with antibiotics and in some cases even drainage or, if applicable, removal of prostheses or grafts. Detection of bacterial infections by scintigraphy had the advantage that a whole body image could be obtained, since specific tracers were available. This study aims to obtain aptamers specific for bacteria identification for future use as radiopharmaceutical. The SELEX (Systematic Evolution of Ligands by Exponential Enrichment) methodology can generate oligonucleotides (aptamers) that are able to bind with high affinity and specificity to a specific target, from small molecules to complex proteins, by using rounds of enrichment and amplification. Aptamers can be labeled with different radionucleotides such as 99mTc, 18F and 32P. In this study, aptamers anti-peptidoglycan, the main component of the bacterial outer cell wall, were obtained through SELEX. Whole cells of Staphylococcus aureus were also used to perform the SELEX to cells (cell-SELEX). The selection of aptamers was performed by two different procedures (A and B). The A process has been accomplished by 15 SELEX rounds in which the separation of the oligonucleotides bound to the peptidoglycan of unbound ones was performed by filtration. In the B process 15 SELEX rounds were performed using the centrifugation for this separation, followed by 5 rounds cell-SELEX. The SELEX started with a pool of ssDNA (single stranded DNA). For A process, initially a library of ssDNA was incubated with peptidoglycan and the amplification of oligonucelotides that were able to bind to peptidoglycan was performed by PCR (Polymerase Chain Reation). The amplified oligonucleotides were again incubated with peptidoglycan, amplified and purified. At the end of 15 selection rounds the selected oligonucleotides were cloned. The product of recombination was used to transform Escherichia coli Top10F. The plasmid DNA from 40 selected colonies were extracted and quantified. The plasmids were sequenced, two different sequences (Antibac1 and Antibac2) were obtained and their secondary structures determined. The aptamers obtained were synthesized and labeled with 32P. The labeled aptamers were incubated with S. aureus cells and the amount of radiolabeled ssDNA was determined by liquid scintillation spectrometry.
87

Analysis of oligonucleotides by matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOF-MS). / CUHK electronic theses & dissertations collection

January 2001 (has links)
Li Yiu-Ching. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 123-132). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.
88

Cellular uptake and effect of phosphorothioated antisense oligodeoxynucleotides against glucose transporter 1 and glucose transporter 5 on breast tumor MCF-7 cells.

January 1999 (has links)
by Tsui Hong Teng. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 174-181). / Abstracts in English and Chinese. / A CKNO WLED GMENTS --- p.7 / ABSTRACT --- p.8-10 / Chapter Chapter 1: --- Introduction: --- p.11-44 / Chapter 1.1) --- Glucose transporters / Chapter 1.2) --- Glucose transporters and cancers / Chapter 1.3) --- Antisense strategies / Chapter 1.4) --- Cellular uptake of oligonucleotides / Chapter 1.5) --- Hyperthermia and combined treatments / Chapter Chapter 2: --- Materials and methods --- p.45-60 / Chapter 2.1) --- Materials: / Chapter 2.1a) --- Cell lines and culture media / Chapter 2.1b) --- Oligonucleotides synthesis / Chapter 2.1c) --- Chemicals / Chapter 2.2) --- Methods: / Chapter 2.2a) --- Oligonucleotide design / Chapter 2.2b) --- Oligonucleotide treatment / Chapter 2.2c) --- Flow cytometry / Chapter 2.2d) --- Confocal microscopy / Chapter 2.2e) --- MTT assay for cytotoxicity or cell proliferation / Chapter Chapter 3: --- Cellular uptake of oligonucleotide spontaneously and Lipofectin-aided: --- p.61-85 / Chapter 3.1) --- Introduction / Chapter 3.2) --- Flow cytometric studies / Chapter 3.3) --- Confocal microscopic studies / Chapter 3.4) --- Cytotoxic effect of Lipofectin alone on MCF-7 cells / Chapter 3.5) --- Discussion / Chapter Chapter 4: --- Hyperthermia can enhance oligonucleotide uptake: --- p.86-118 / Chapter 4.1) --- Introduction / Chapter 4.2) --- Flow cytometric studies / Chapter 4.3) --- Confocal microscopic studies / Chapter 4.4) --- Cytotoxic effect of hyperthermia on MCF-7 cells / Chapter 4.5) --- FITC-ODN uptake in survival cells by propidium iodide (PI) exclusion method for hyperthermia / Chapter 4.6) --- Discussion / Chapter Chapter 5: --- The antiproliferative effects of antisense molecules against Glut-1 and 5 on MCF- 7 cells transfected by Lipofectin: --- p.119-146 / Chapter 5.1) --- Introduction / Chapter 5.2) --- The growth curve of MCF-7 cells / Chapter 5.3) --- The calibration of MTT assay / Chapter 5.4) --- The effect of antisense Glut-1 concentration without Lipofectin on MCF-7 cells / Chapter 5.5) --- The effect of antisense Glut-1 concentration with Lipofectin on MCF-7 cells / Chapter 5.6) --- The effect of antisense Glut-5 concentration without Lipofectin on MCF-7 cells / Chapter 5.7) --- The effect of antisense Glut-5 concentration with Lipofectin on MCF-7cells / Chapter 5.8) --- The effect of transfection time of antisense Glut-1 on MCF-7 cells / Chapter 5.9) --- The effect of transfection time of antisense Glut-5 on MCF-7 cells / Chapter 5.10) --- The effect of transfection time of antisense Glut-5 for higher concentration on MCF-7 cells / Chapter 5.11) --- The effect of antisense Glut-1 to Lipofectin (w/w) ratio on MCF-7 cells / Chapter 5.12) --- The effect of antisense Glut-1 to Lipofection (w/w) ratio for higher transfection time on MCF-7 cells / Chapter 5.13) --- The effect of antisense Glut-5 to Lipofectin (w/w) ratio on MCF-7 cells / Chapter 5.14) --- Discussion / Chapter Chapter 6: --- Combined treatments: --- p.147-162 / Chapter 6.1) --- Introduction / Chapter 6.2) --- The effect of combined treatment of antisense Glut-1 combined with antisense Glut-5 on MCF-7 cells / Chapter 6.3) --- The chronic effect of hyperthermia for 5 hours on MCF-7 cells / Chapter 6.4) --- The effect of combined treatment between antisense Glut-1 and hyperthermia on MCF-7 cells / Chapter 6.5) --- The net effect of antisense Glut-1 in combined treatment between hyperthermia and antisense Glut-1 on MCF-7 cells / Chapter 6.6) --- The effect of combined treatment between antisense Glut-5 and hyperthermia on MCF-7 cells / Chapter 6.7) --- The net effect of antisense Glut-5 in combined treatment between hyperthermia and antisense Glut-5 on MCF-7 cells / Chapter 6.8) --- Discussion / Chapter Chapter 7: --- Discussion: --- p.163-173 / Chapter Chapter 8: --- References: --- p.174-181
89

Estudos funcionais e estruturais para a caracterização da via de captação e assimilação de sulfato em Xanthomonas axonopodis pv. citri. / Structural and functional studies for characterization of the sulfate uptake and assimilation pathway from Xanthomonas axonopodis pv. citri.

Pereira, Cristiane Tambascia 03 July 2013 (has links)
Em Escherichia coli, a assimilação de sulfato e sua redução a sulfeto é mediada por um transportador do tipo ABC codificado pelos genes sbpcysWUA e várias enzimas codificadas por cysNCDGHIJ. Embora a importância deste sistema tenha sido largamente demonstrada em outros organismos, não existem relatos na literatura sobre a via na bactéria fitopatogênica Xanthomonas axonopodis pv. citri (X. citri). Neste trabalho, utilizando uma abordagem funcional baseada em análises de bioinformática, proteômica e gene repórter, mostramos que a via de sulfato está presente e ativa em X. citri. Ainda, a partir da expressão e produção em larga escala da proteína ligadora de sulfato Sbp, realizamos ensaios de biofísica que evidenciaram a interação da proteína com sulfato e o aumento da estabilidade térmica e estrutural, obtendo-se cristais. O trabalho apresenta as primeiras evidências da funcionalidade desta via em X. citri durante o crescimento in vitro e infecção in vivo e abre pespectivas de estudos para compreensão do papel deste íon na fisiologia do microrganismo. / In Escherichia coli, the capture and reduction of sulfate to sulfide is mediated by an ABC-type transporter encoded by genes sbpcysWUA and several enzymes encoded by cysNCDGHIJ. Although the importance of this system has been widely demonstrated in other organisms, there are no reports in the literature related to the way that sulfate is assimilated and reduced in plant pathogen Xanthomonas axonopodis pv. citri (X. citri). In this work, using a functional approach based on bioinformatics analysis, proteomics and gene reporter, we show that the way is present and active in X. citri. Indeed, the sulfate binding protein was expressed and produced in large scale for biophysical assays demonstrating the interaction of the protein with sulfate and increased thermal stability and crystals was produced. The study presents the first evidence of the functionality of this pathway in X. citri during growth in vitro and in vivo infection and opens perspectives studies to understand the role of this ion in the physiology of the microorganism.
90

Effects of ammonium salts as co-matrices for the analysis of oligonucleotides by matrix-assisted laser desorption/ionization mass spectrometry.

January 1996 (has links)
by Cheng Sau Wan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves [72]-[76]). / TABLE OF CONTENTS --- p.i / ABSTRACT --- p.iv / LIST OF FIGURES --- p.vi / LIST OF TABLES --- p.x / Chapter CHAPTER ONE --- RESEARCH BACKGROUND --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Matrix-assisted laser desorption / ionization mass spectrometry (MALDI) --- p.2 / Chapter 1.2.1 --- Laser desorption methods --- p.2 / Chapter 1.2.2 --- The matrix --- p.3 / Chapter 1.2.2.1 --- Role of the matrix --- p.3 / Chapter 1.2.2.2 --- Features of the matrix --- p.4 / Chapter 1.2.3 --- Mechanisms of ion formation --- p.6 / Chapter 1.2.3.1 --- Desorption process(es) --- p.6 / Chapter 1.2.3.2 --- Ionization process(es) --- p.7 / Chapter 1.3 --- Sequencing of DNA --- p.8 / Chapter 1.3.1 --- DNA sequencing procedure --- p.10 / Chapter 1.3.1.1 --- Generation of the nested set of DNA molecules --- p.11 / Chapter 1.3.1.2 --- Sequence analysis --- p.11 / Chapter 1.3.2 --- MALDI-TOF-MS as a DNA sequencing tool --- p.12 / Chapter 1.3.3 --- MALDI analysis of oligonucleotides --- p.14 / Chapter 1.4 --- Outline of the present work --- p.16 / Chapter CHAPTER TWO --- INSTRUMENTATION AND EXPERIMENTAL --- p.18 / Chapter 2.1 --- Time-of-flight mass spectrometry (TOF-MS) --- p.18 / Chapter 2.1.1 --- Linear time-of-flight mass spectrometry --- p.18 / Chapter 2.1.2 --- Reflectron time-of-flight mass spectrometry --- p.21 / Chapter 2.1.3 --- Ion detection --- p.22 / Chapter 2.1.4 --- Vacuum system --- p.22 / Chapter 2.2 --- Instrumentation --- p.24 / Chapter 2.2.1 --- The laser system --- p.24 / Chapter 2.2.2 --- Ion source and vacuum system --- p.24 / Chapter 2.2.3 --- Flight tube and reflector --- p.27 / Chapter 2.2.4 --- The detector --- p.28 / Chapter 2.2.5 --- Data acquisition and computer control --- p.28 / Chapter 2.3 --- Experimental --- p.29 / Chapter 2.3.1 --- Sample preparation --- p.29 / Chapter 2.3.2 --- Mass spectrometric analysis --- p.30 / Chapter CHAPTER THREE --- USE OF AMMONIUM SALTS AS CO-MATRICES --- p.32 / Chapter 3.1 --- Introduction --- p.32 / Chapter 3.2 --- Experimental --- p.35 / Chapter 3.3 --- Results and Discussion --- p.36 / Chapter 3.3.1 --- Effects of counter-anions --- p.36 / Chapter 3.3.2 --- Effects of matrix materials --- p.40 / Chapter 3.4 --- Conclusions --- p.43 / Chapter CHAPTER FOUR --- USE OF POTASSIUM SALTS AS CO-MATRICES --- p.44 / Chapter 4.1 --- Introduction --- p.44 / Chapter 4.2 --- Experimental --- p.44 / Chapter 4.3 --- Results and Discussion --- p.44 / Chapter 4.3.1 --- Adduct formation --- p.49 / Chapter 4.3.2 --- Signal enhancement --- p.50 / Chapter 4.4 --- Conclusions --- p.52 / Chapter CHAPTER FIVE --- ANALYSIS OF HIGH MASS OLIGONUCLEOTIDES --- p.53 / Chapter 5.1 --- Introduction --- p.53 / Chapter 5.2 --- Experimental --- p.53 / Chapter 5.3 --- Results and Discussion --- p.54 / Chapter 5.4 --- Conclusions --- p.67 / Chapter CHAPTER SIX --- CONCLUSIONS AND FURTHER WORK --- p.68 / Chapter 6.1 --- Conclusions --- p.68 / Chapter 6.2 --- Further work --- p.70 / ACKNOWLEDGMENT --- p.A1 / REFERENCES --- p.R1 - R5

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