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DNA damage response in in vitro matured oocytesAtamian, Elisa Karine 17 June 2016 (has links)
The reproductive lifetime of a woman is limited primarily by her age. The state of an oocyte represents the central determinant of the fate of an ovarian follicle as well as embryo development throughout maturation. Oocyte reserve and oocyte quality are two major determinants of the likelihood of achieving pregnancy for a woman. Assisted Reproductive Technology (ART) has provided a valuable alternative for women attempting to conceive at an older age, however even with ART the likelihood of a live birth also decreases with increased age.
Mammalian oocytes undergo meiotic maturation in preparation for ovulation and fertilization. Throughout most of its lifetime the oocyte remains arrested in the dictyate stage of prophase of meiosis I (MI), also called the germinal vesicle (GV) stage, until the follicle receives a hormonal signal to progress through meiosis. Only a small fraction of the follicles present in the ovaries receive this signal, while the rest remain unresponsive.
The DNA damage response (DDR) is activated in the presence of double stranded breaks (DSBs) in DNA and can induce various cellular responses including senescence or cell cycle arrest, and/or apoptosis, also known as programmed cell death. Telomeres mediate senescence in most cells. Telomeres consist of tandem DNA repeats and associated proteins, which cap and protect chromosome ends. Telomeres and their associated proteins form a loop at the ends of chromosomes, which buries them. This telomere complex is called shelterin. Shelterin prevents the ends of chromosomes from triggering a DNA damage response. However, with each round of DNA replication chromosomes lose small segments of their telomeres. Telomere attrition also can arise in non-dividing cells via the action of oxygen radicals.
We hypothesize that germinal vesicle arrest, which occurs in some oocytes retrieved for ART that fail to progress through meiosis, is associated with telomere attrition and the associated cellular senescence pathway induced by DNA damage. Previous studies have identified higher levels of DNA damage foci in isolated GV arrested oocytes compared to those that progress through the meiotic cell cycle. Our studies confirm the presence of the DNA damage response (DDR) regulator, ATM, at higher levels in GV stage oocytes versus those that have matured to later stages. Immunostaining shows a near 50% increase in presence of ATM in arrested oocytes.
Confirming the role of the DDR in cell cycle arrest during oocyte maturation could highlight a new target for strategies to improve ART technology and increase the likelihood of achieving pregnancy later in life.
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Chaînes de Markov régulées et approximation de Poisson pour l'analyse de séquences biologiques / Drifting Markov models and Poisson approximation for analysis of biological sequencesVergne, Nicolas 11 July 2008 (has links)
Cette thèse présente le développement, en vue de l'analyse statistique des séquences d'ADN, de nouveaux modèles permettant de prendre en compte l'hétérogénéité de ces séquences : les chaînes de Markov régulées (DMM pour drifting Markov model). Afin d'éviter l'homogénéité supposé par les modèles de Markov et de Markov cachés, nous permettons à la matrice de transition de varier du début à la fin de la séquence. A chaque position, nous avons une matrice de transition différente. Ces modèles peuvent être vus comme une alternative mais aussi comme un outil complémentaire aux modèles de Markov cachés. Nous avons considéré des dérives polynomiales ainsi que des dérives par splines polynomiales. Nous avons estimé nos modèles de multiples manières puis évalué la qualité de ces estimateurs avant de les utiliser en vue d'applications telle la recherche de mots exceptionnels. Nous avons mis en oeuvre le software DRIMM, dédié à l'estimation de nos modèles. / This document propose the conception, in the way of statistical analysis of DNA sequences, of new models which permit to take into account the heterogeneity of these sequences : the drifting Markov models (DMM). In order to avoid homogeneity of Markov models or hidden Markov models, we allow the transition matrix to vary from the beginning to the end of the sequence. At each position, we obtain a different transition matrix. DMM can be seen as a competitive model to the HMM one but it over all can be understood as a complementary tool: the hidden models of an HMM, usually fixed Markov chains can be replaced by DMM. Along this work, we consider polynomial drift or drift by polynomial splines. We estimate our models by different ways, evaluate their qualities and used them in biological applications such as the search of rare words. We develop the software DRIMM, dedicated to estimation of DMM.
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Modélisation du chromosome bactérien en vue de la conception de réseaux biologiques de régulation dans l'espace cellulaire / Modeling of the bacterial chromosome to design biological regulatory network in the celular spaceLepage, Thibaut 12 January 2015 (has links)
La structure spatiale de l'ADN est fortement affectée par le surenroulement. Lorsqu'il est surenroulé, l'ADN circulaire (ou linéaire et contraint topologiquement aux extrémités) se replie et forme des boucles appelées plectonèmes, rapprochant dans l'espace des régions éloignées du chromosome, perturbant ainsi de l réseau de régulation de la transcription de la cellule. Les chromosomes bactériens sont surenroulés négativement et ce surenroulement est connu pour jouer un rôle important dans la régulation de la transcription. Cependant, à cause de la nature globale de la contrainte topologique associée à l'ADN, les méthodes actuelles ne sont capables de simuler que de petites molécules (jusqu'à quelques milliers de paires de bases, KB). Cette thèse présente un nouvel algorithme utilisé pour réaliser des simulations de Monte-Carlo d'ADN surenroulé, basé sur une molécule. Cet algorithme a été utilisé pour réaliser des simulations de longues molécules (plusieurs dizaines de KB) et apporter un nouvel éclairage sur des questions encore débattues à propos de la structure de l'ADN surenroulé à cette échelle. Cette méthode permet d'étudier l'effet de la position des gènes le long de l'ADN sur leur co-localisation et leur co-régulation, et laisse entrevoir la possibilité de simuler le repliement d'un chromosome bactérien entier. / Superhelicity strongly affects the 3D structure of DNA. When supercoiled, circular DNA (or linear DNA with topolocically constrained ends) folds and forms loops called plectonemes, bringing some distant parts of the chromosme close to one another in space, thus perturbing the transcription regulation network of the cell. Bacterial chromosomes are negatively supercoiled and superhelicity is known to play a important role in the regulation of the transcription. However, due to the global nature of the topological constraint imposed to the DNA, current methods have only been able to simulate small moelcules (up to a few kilobasepairs, KB). This thesis presents a novel algorithm used to performed Monte-Carlo simulations of supercoiled DNA, featuring a local approach to the topological constraint via the computation of the twist of the molecule. Using this efficient algorithm, stimulations of long molecules (tens of KB) were performed and shed a new light on debated questions about the structure of supercoiled DNA at this scale. This method allows to study the effect of the position of genes along the DNA on their co-localisation and co-regulation, and to envision the possibility of simulating the folding of a whole bacterial chromosome.
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Development of impedimetric DNA sensor for diagnosis of Human Papillomavirus type 18 infection / Desenvolvimento de um sensor de DNA impedimétrico para o diagnóstico de infecção por Papilomavirus Humano tipo 18Correr, Wagner Rafael 17 December 2014 (has links)
Currently, the most common strategy employed to detect DNA sequences is PCR (Polymerase Chain Reaction). Nevertheless, in the last few years research on DNA biosensors has increased significantly. Such sensors represent an alternative to PCR in the detection of specific DNA sequences, once they exhibit fast response, low limits of detection, and require simpler sample preparation. The development of a biosensor for detection of DNA from Human Papillomavirus type 18 is reported. To immobilise DNA probe onto indium-tin oxide (ITO) electrodes, a silanisation was carried out using 3-Aminopropyltryethoxysilane (APTES). Silanisation was studied and optimised using ultra-violet absorption spectroscopy, atomic force microscopy, fluorescence microscopy, and cyclic voltammetry. After immobilisation, the hybridisation with target sequence is detected by changes in surface properties of ITO electrode by Cyclic Voltammetry and Electrochemical Impedance Spectroscopy, using the Ferri-Ferrocyante redox couple. The detection of synthetic target sequence was performed in the range of 12.5 to 100 nM, and 300nM for PCR products. The sensor did not show significative response for non-complementary sequence at 50 nM. This sensor can be applied for fast and low cost detection of HPV genetic material at nanomolar levels. / A estratégia mais empregada atualmente na detecção de sequência de DNA é a PCR (Reação em Cadeira da Polimerase). Contudo, nos últimos anos, a pesquisa em biossensores de DNA tem aumentado significativamente. Estes sensores representam uma alternativa a PCR na detecção de sequências específicas de DNA, uma vez que exibem resposta rápida, baixos limites de detecção e requerem preparação simples da amostra. Nesta dissertação descrito o desenvolvimento de um biossensor para a detecção do DNA do Papilomavirus Humano tipo 18. A fim de imobilizar a sequência de captura de DNA em eletrodos de óxido de estanho e índio (ITO), realizou-se uma silanização usando 3-Aminopropiltrietoxisilano (APTES). A reação de silanização foi estudada e otimizada através das técnicas de Espectroscopia de Absorção Ultravioleta, Microscopia de Força Atômica, Microscopia de Fluorescência e Voltametria Cíclica. Após a imobilização, a hibridização com a sequência alvo é detectada através de alterações nas propriedades de superfície do eletrodo através de Voltametria Cíclica e Espectroscopia de Impedância Eletroquímica, usando o par redox Ferri-ferrocianeto. A detecção da sequência alvo sintética foi realizada no intervalo de 12.5 a 100 nM, e para o produto de PCR, 300 nM. O sensor não demonstrou resposta significativa para sequência não complementar a 50 nM. Este sensor pode ser aplicado na detecção rápida e de baixo custo de material genético do HPV a níveis nanomolares.
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Empirical Bayes, Bayes factors and deoxyribonucleic acid fingerprintingBasu, Ruma January 2017 (has links)
The central theme in this thesis is Empirical Bayes. It starts off with application of Bayes and Empirical Bayes methods to deoxyribonucleic acid fingerprinting. Different Bayes factors are obtained and an alternative Bayes factor using the method of Savage is studied both for normal and non- normal priors. It then moves on to deeper methodological aspects of Empirical Bayes theory. A 1983 conjecture by Carl Morris on the parametric empirical Bayes prediction intervals for the normal regression model is studied and an improvement suggested. Carlin and Louis’ (1996) parametric empirical Bayes prediction interval for the same model is also dealt with analytically while their approach had been primarily numerical. It is seen that both of these intervals have the same coverage probability up to a certain order of approximation and they have the same expected length up to the same order of approximation. Both the intervals are equal tailed up to the same order of approximation. Then the corrected proof of an important published result by Datta, Ghosh and Mukerjee (2000) is provided using first principles of probability matching. This result is relevant to our work on parametric empirical Bayes prediction intervals.
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Revisão taxonômica e biogeografia de Atlantirivulus santensis Köhler, 1906 (Rivulidae, Cyprinodontiformes)Ywamoto, Eric Venturini. January 2019 (has links)
Orientador: Claudio de Oliveira / Resumo: Atlantirivulus santensis é amplamente distribuída ao longo das drenagens costeiras de São Paulo, Brasil. Algumas evidências de variação morfológica dentro dessa espécie sugerem que possam existir espécies novas que ainda não tenham sido descritas. Nesse sentido, o objetivo do presente estudo é verificar se existem diferentes linhagens de A. santesis utilizando a ferramenta do DNA Barcoding, realizar a revisão taxonômica de A. santesis através de caracteres morfológicos e estabelecer os limites de distribuição desta espécie com o intuito de contribuir para futuros estudos de sistemática dos rivulídeos. Foram analisadas 68 sequências parciais da região Citocromo c Oxidase I do DNA mitocondrial (com aproximadamente 550 pares de bases) de espécimes de A. santensis (Ubatuba, Maresias, Bertioga, São Paulo, Santos, Mongaguá, Pedro de Toledo, Itanhaém e Peruíbe), além de Atlantirivulus simplicis encontrada no limite norte (Paraty-RJ) da distribuição de A. santensis e Atlantirivulus ribeirensis, limite sul, para fins comparativos, como grupos externos. Os dados obtidos foram analisados com os softwares Geneious v.4.8.5, BioEdit v5.0.9 e Mega 7.0. Os resultados foram graficamente representados em dendrogramas de Neighbour-Joining (NJ), Automatic Barcode Gap Definition (ABGD) e PTP. As análises geraram três clusters (mais os dois grupos externos) dentro de A. santensis. O primeiro grupo é composto por espécimes de Peruíbe, Pedro de Toledo e Itanhaém, o segundo por Santos, Mongaguá e São... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Atlantirivulus santensis is widely distributed along the coastal drains of São Paulo, Brazil. Some evidence of morphological variation within this species suggests that there may be new species that have not yet been described. In this sense, the objective of the present study is to verify if there are different strains of A. santesis using the tool of the DNA Barcoding, to carry out the taxonomic revision of A. santesis through morphological characters and to establish the distribution limits of this species with the intention to contribute for future studies of rivulide systematics. We analyzed 68 partial sequences of the cytochrome c Oxidase I region of the mitochondrial DNA (approximately 550 base pairs) of A. santensis specimens (Ubatuba, Maresias, Bertioga, São Paulo, Santos, Mongaguá, Pedro de Toledo, Itanhaém and Peruíbe), in addition to Atlantirivulus simplicis found in the northern boundary (Paraty-RJ) of the distribution of A. santensis and Atlantirivulus ribeirensis(Juquiá-SP), southern limit, for comparative purposes, as external groups. The data obtained were analyzed with the software Geneious v.4.8.5, BioEdit v5.0.9 and Mega 7.0. The results were graphically represented in NeighborJoining (NJ), Automatic Barcode Gap Definition (ABGD) and PTP dendrograms. The analyzes generated three clusters (plus the two external groups) within A. santensis. The first group consists of specimens from Peruíbe, Pedro de Toledo and Itanhaém, the second from Santos, Mongaguá and ... (Complete abstract click electronic access below) / Mestre
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Theoretical investigation of cisplatin-deoxyribonucleic acid crosslink products.January 2004 (has links)
Fu Annie Yuen Yee. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 97-108). / Abstracts in English and Chinese. / ABSTRACT (ENGLISH) --- p.iii / ABSTRACT (CHINESE) --- p.iv / ACKNOWLEDGMENTS --- p.v / TABLE OF CONTENTS --- p.vi / LIST OF FIGURES --- p.ix / LIST OF TABLES --- p.xi / Chapter CHAPTER ONE: --- BACKGROUND INFORMATION --- p.1 / Chapter 1.1 --- Introduction --- p.1 / Chapter 1.2 --- Deoxyribonucleic Acid --- p.1 / Chapter 1.2.1 --- Nomenclature and Symbols --- p.1 / Chapter 1.2.2 --- Torsion Angles --- p.4 / Chapter 1.2.3 --- Conformation --- p.5 / Chapter 1.3 --- DNA Studies --- p.8 / Chapter 1.3.1 --- Base --- p.8 / Chapter 1.3.2 --- Base-Pair --- p.10 / Chapter 1.3.3 --- Summary --- p.11 / Chapter 1.4 --- Cisplatin Studies --- p.11 / Chapter 1.4.1 --- Reaction --- p.12 / Chapter 1.4.2 --- Cisplatin-DNA Products --- p.14 / Chapter 1.4.3 --- Summary --- p.15 / Chapter 1.5 --- Scope of This Thesis --- p.15 / Chapter CHAPTER TWO: --- COMPUTATION AND METHODOLOY --- p.17 / Chapter 2.1 --- Introduction --- p.17 / Chapter 2.2 --- Hartree-Fock Approximation --- p.17 / Chapter 2.3 --- Geometry Optimization --- p.18 / Chapter 2.4 --- Molecular Orbital (MO) Calculation --- p.20 / Chapter 2.5 --- Verification of Methodology --- p.20 / Chapter 2.5.1 --- Backbone Torsion Angles --- p.20 / Chapter 2.5.2 --- N7-N7 Distance --- p.26 / Chapter 2.5.3 --- Location of HOMO --- p.30 / Chapter 2.6 --- Summary --- p.31 / Chapter CHAPTER THREE: --- UNDERSTANDING OF THE CISPLATIN-DNA CROSSLINKS --- p.33 / Chapter 3.1 --- Introduction --- p.33 / Chapter 3.2 --- MO Analysis --- p.33 / Chapter 3.3 --- Potential Binding Sites of DNA --- p.34 / Chapter 3.3.1 --- "1,2-d(GpG) Intrastrand Crosslink" --- p.40 / Chapter 3.3.2 --- "l,2-d(ApG) Intrastrand Crosslink" --- p.40 / Chapter 3.3.3 --- "l,3-d(GpXpG) Intrastrand Crosslink" --- p.41 / Chapter 3.3.4 --- d(GpC)d(GpC) Interstrand Crosslink --- p.41 / Chapter 3.3.5 --- d(GpXpC)d(GpXpC) Interstrand Crosslink --- p.41 / Chapter 3.3.6 --- Summary --- p.42 / Chapter 3.4 --- Empirical Selection Rule --- p.44 / Chapter 3.4.1 --- Convention --- p.44 / Chapter 3.4.2 --- Selection of Potential HOMO Location (or Active Site) --- p.45 / Chapter 3.4.3 --- Selection of Potential HOMO-Nearby Active Site --- p.47 / Chapter 3.4.4 --- Applications --- p.48 / Chapter 3.5 --- Cisplatin --- p.51 / Chapter 3.6 --- Cisplatin-DNA Crosslinks --- p.52 / Chapter 3.6.1 --- "l,2-d(GpG) and l,2-d(ApG) Intrastrand Crosslinks" --- p.52 / Chapter 3.6.2 --- "l,2-d(ApG) versus l,2-d(GpA) Intrastrand Crosslinks" --- p.53 / Chapter 3.6.3 --- "l,3-d(GpXpG) Intrastrand and d(GpXpC)d(GpXpC) Interstrand Crosslinks" --- p.54 / Chapter 3.6.4 --- Platination at Terminal Positions --- p.55 / Chapter 3.7 --- Structural Parameters --- p.59 / Chapter 3.7.1 --- Optimized Geometries --- p.59 / Chapter 3.7.2 --- DNA Sequences from PDB --- p.67 / Chapter 3.7.3 --- Backbone Torsion Angles --- p.70 / Chapter 3.8 --- Summary --- p.70 / Chapter CHAPTER FOUR: --- CONCLUDING REMARKS --- p.72 / APPENDIX I BACKBONE TORSION ANGLES AND SUGAR RING CONFORMATION OF THE OPTIMIZED GEOMETRIES --- p.74 / APPENDIX II BACKBONE TORSION ANGLES OF THE EXPERIMENTAL SEQUENCES FROM NUCLEIC ACID DATABASE (NDB) --- p.82 / REFERENCES --- p.97
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DNA-based logicBader, Antoine January 2018 (has links)
DNA nanotechnology has been developed in order to construct nanostructures and nanomachines by virtue of the programmable self-assembly properties of DNA molecules. Although DNA nanotechnology initially focused on spatial arrangement of DNA strands, new horizons have been explored owing to the development of the toehold-mediated strand-displacement reaction, conferring new dynamic properties to previously static and rigid structures. A large variety of DNA reconfigurable nanostructures, stepped and autonomous nanomachines and circuits have been operated using the strand-displacement reaction. Biological systems rely on information processing to guide their behaviour and functions. Molecular computation is a branch of DNA nanotechnology that aims to construct and operate programmable computing devices made out of DNA that could interact in a biological context. Similar to conventional computers, the computational processes involved are based on Boolean logic, a propositional language that describes statements as being true or false while connecting them with logic operators. Numerous logic gates and circuits have been built with DNA that demonstrate information processing at the molecular level. However, development of new systems is called for in order to perform new tasks of higher computational complexity and enhanced reliability. The contribution of secondary structure to the vulnerability of a toehold-sequestered device to undesired triggering of inputs was examined, giving new approaches for minimizing leakage of DNA devices. This device was then integrated as a logic component in a DNA-based computer with a retrievable memory, thus implementing two essential biological functions in one synthetic device. Additionally, G-quadruplex logic gates were developed that can be switched between two topological states in a logic fashion. Their individual responses were detected simultaneously, establishing a new approach for parallel biological computing. A new AND-NOT logic circuit based on the seesaw mechanism was constructed that, in combination with the already existing AND and OR gates, form a now complete basis set that could perform any Boolean computation. This work introduces a new mode of kinetic control over the operation of such DNA circuits. Finally, the first example of a transmembrane logic gate being operated at the single-molecule level is described. This could be used as a potential platform for biosensing.
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A história evolutiva da família de fatores transcricionais E2F e seu envolvimento na resposta ao dano de DNARauber, Rafael January 2016 (has links)
As proteínas E2 promoter binding factor (E2F) estão presentes em quase todos os organismos eucarióticos e são essenciais para o controle de muitos processos celulares, como a progressão do ciclo celular, divisão celular, replicação do DNA, e apoptose. A família E2F compreende dois tipos diferentes de proteínas: os E2F típicos e os E2F atípicos, que diferem estruturalmente e possuem funções específicas. Desde a descoberta desta família gênica muitos estudos focaram nos seus aspectos funcionais, porém a história evolutiva desta família gênica estava fracamente entendida em plantas. Nossas descobertas sugerem que os E2F típicos são mais similares a proteína ancestral que os E2F atípicos (DEL). As proteínas E2F surgiram logo após a emergência das espécies eucarióticas, enquanto as proteínas DEL parecem ter surgido antes da separação entre fungos, metazoários e plantas. As proteínas DEL provavelmente emergiram por uma duplicação parcial do gene que codificava a proteína E2F ancestral. Nossos dados também sugerem que a divergência entre E2Fs ativadores e repressores emergiu duas vezes durante a evolução, uma na linhagem embriófita de plantas e outra na linhagem metazoária. Os E2Fs típicos possuem membros que são reguladores positivos para os seus genes alvo e dados recentes estabeleceram um link entre a resposta a danos ao DNA e genes E2F específicos em células de mamíferos, porém o envolvimento da família genica E2F na sinalização ao reparo de DNA em plantas ainda permanece desconhecido. Nós estudamos o envolvimento dos genes E2F em resposta a dano genotóxico em plantas de arroz e Arabidopsis thaliana. Foram conduzidos experimentos com plântulas de arroz e arabidopsis usando UV-B e MMS (Methyl Methanesulfonate) como fonte de danos genotóxicos. Em resposta a estes estresses E2F ativadores específicos aumentaram sua expressão relativa quando comparados com plantas em condições controle. Os resultados obtidos nos experimentos de estresse genotóxicos nos levaram a sugerir que membros específicos da família E2F de arroz e arabidopsis estão envolvidos com as respostas ao dano de DNA. / The E2 promoter binding factor (E2F) proteins are present in almost all eukaryotic organisms and are essential to control several cellular processes, such as the cell cycle progression, cell division, DNA replication, and apoptosis. The E2F family comprises two different types of proteins: the typical E2Fs and atypical E2Fs, which differ structurally and have specific functions. Since the discovery of this gene family several studies have focused on the functional aspects, but the evolutionary history of this gene family was poorly understood in plants. Our findings suggest that typical E2F is more similar to the ancestral protein than atypical E2F (DEL). The E2F proteins arose early after the emergence of the eukaryotic species, while DEL proteins appear to have arisen after the separation of fungi, metazoan and plants. The DEL proteins probably emerged through a partial duplication from the gene that encodes the ancient E2F protein. Our data also suggest that the divergence of the E2Fs between activators and repressors emerged twice during evolution, once in the embryophyte lineage and another in the metazoan lineage. The typical E2Fs has members that are positive regulators for their target genes and recent data have established a link between response to DNA damage and specific E2F genes in mammalian cells, but the involvement of the E2F gene family in the signaling of DNA repair in plants still unknown. We studied the involvement of E2F genes in response to genotoxic damage in rice and Arabidopsis thaliana plants. Experiments were conducted with rice and A. thaliana plantlets using UV-B and MMS (Methyl Methanesulfonate) as genotoxic damage souce. In response to these stresses specific activator E2F genes increased their relative expression compared to plants in control condition. The results obtained in the genotoxic stress experiments lead us to suggest that specific members of the E2F family of rice and arabidopsis are involved with responses to DNA damage.
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Effect of oxidized and hyperoxidized guanine on DNA primer-template structures.January 2009 (has links)
Fenn, Dickson. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 74-81). / Abstract also in Chinese. / Title Page --- p.i / Thesis Committee --- p.ii / Acknowledgement --- p.iii / Table of Contents --- p.v / List of Tables --- p.ix / List of Figures --- p.x / List of Abbreviations and Symbols --- p.xv / Abstract --- p.xvii / Chapter 1.Chapter One: --- Introduction --- p.1 / Chapter 1.1 --- Oxidation and Hyperoxidation of Guanine --- p.1 / Chapter 1.2. --- DNA Replication --- p.2 / Chapter 1.3 --- Mutagenesis --- p.3 / Chapter 1.4 --- Literature Survey on Spiroiminodihydantoin (Sp) --- p.4 / Chapter 1.5 --- Purpose of This Work --- p.5 / Chapter 1.6 --- DNA Structure --- p.6 / Chapter 1.6.1 --- Nomenclature --- p.6 / Chapter 1.6.2 --- Torsion Angles --- p.6 / Chapter 1.6.3 --- Sugar Pucker Conformation --- p.7 / Chapter 1.6.4 --- Secondary Structures of DNA --- p.8 / Chapter 2.Chapter Two: --- Materials and Methodology --- p.10 / Chapter 2.1 --- Sample Design --- p.10 / Chapter 2.2 --- Sample Preparation --- p.11 / Chapter 2.2.1 --- DNA Synthesis and Purification --- p.11 / Chapter 2.2.2 --- HPLC Separation --- p.11 / Chapter 2.2.3 --- NMR Samples Preparation --- p.12 / Chapter 2.3 --- NMR Analysis --- p.12 / Chapter 2.3.1 --- Resonance Assignment --- p.14 / Chapter 2.3.1.1 --- Proton --- p.14 / Chapter 2.3.1.2 --- Phosphorous --- p.16 / Chapter 2.3.2 --- Sugar Pucker Conformation --- p.17 / Chapter 2.3.3 --- Backbone Conformation --- p.18 / Chapter 2.4 --- UV Melting Analysis --- p.19 / Chapter 3.Chapter Three: --- "HPLC, NMR and UV Results" --- p.21 / Chapter 3.1 --- HPLC Separation of Sp Diastereoisomers --- p.21 / Chapter 3.2 --- NMR Resonance Assignments --- p.24 / Chapter 3.2.1 --- 5'-GG Sample --- p.24 / Chapter 3.2.2 --- 5'-G(oG) Sample --- p.26 / Chapter 3.2.3 --- 5'-G(Sp) Sample --- p.29 / Chapter 3.2.4 --- 5'-T(oG) Sample --- p.31 / Chapter 3.2.5 --- 5'-T(Sp) Sample --- p.34 / Chapter 3.3 --- Sugar Pucker Conformation --- p.38 / Chapter 3.4 --- Backbone Conformation --- p.41 / Chapter 3.5 --- UV Melting --- p.43 / Chapter 4.Chapter Four: --- Effect of Spiroiminodihydantoin and 7-hydro-8-oxoguanine on Primer-Template Structures --- p.44 / Chapter 4.1 --- Overview --- p.42 / Chapter 4.2 --- NMR Investigations of the Primer-Template Models --- p.45 / Chapter 4.2.1 --- Incorporation of a dCTP Opposite a 5'-GG Template --- p.45 / Chapter 4.2.2 --- Incorporation of a dCTP Opposite a 5'-G(oG) Template --- p.46 / Chapter 4.2.3 --- Incorporation of a dCTP Opposite a 5'-G(Sp) Template --- p.48 / Chapter 4.2.4 --- Incorporation of a dATP Opposite a 5'-T(oG) Template --- p.50 / Chapter 4.2.5 --- Incorporation of a dATP Opposite a 5'-T(Sp) Template --- p.51 / Chapter 4.3 --- Effect of Sp and oG on Primer-Template Structures --- p.52 / Chapter 4.3.1 --- Misaligned Structure with a Sp-Bulge --- p.52 / Chapter 4.3.2 --- C·oG Base Pair in 5'-G(oG) --- p.54 / Chapter 4.3.3 --- Biological Implications --- p.54 / Chapter 5. --- Chapter Five: Preliminary Structural Calculations on Primer- Template Structures --- p.56 / Chapter 5.1 --- Experimental Restraints Extraction --- p.56 / Chapter 5.2 --- Experimental Restraints Distribution --- p.58 / Chapter 5.3 --- Structural Calculations --- p.60 / Chapter 5.4 --- Structural Results --- p.62 / Chapter 5.4.1 --- 5'-GG --- p.63 / Chapter 5.4.2 --- 5'-G(oG) --- p.64 / Chapter 5.4.3 --- 5'-T(oG) --- p.65 / Chapter 5.4.4 --- 5'-T(SpR) with 5'-T(Spl) Restraints --- p.66 / Chapter 5.4.5 --- 5'-T(SpR) with 5'-T(Sp2) Restraints --- p.67 / Chapter 5.4.6 --- 5'-T(SpS) with 5'-T(Spl) Restraints --- p.68 / Chapter 5.4.7 --- 5'-T(SpS) with 5'-T(Sp2) Restraints --- p.69 / Chapter 5.6 --- Structural Analysis --- p.70 / Chapter 6. --- Chapter Six: Conclusions and Future Work --- p.72 / Appendix --- p.73 / References --- p.74
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