Dissertations / Theses on the topic 'DNA model'

The increasing volume of biological data requires finding new ways to save these data in genetic banks. The target of this work is design and implementation of a novel algorithm for compression of DNA sequences. The algorithm is based on aligning DNA sequences agains a reference sequence and storing only diferencies between sequence and reference model. The work contains basic prerequisities from molecular biology which are needed for understanding of algorithm details. Next aligment algorithms and common compress schemes suitable for storing of diferencies agains reference sequence are described. The work continues with a description of implementation, which is follewed by derivation of time and space complexity and comparison with common compression algorithms. Further continuation of this thesis is discussed in conclusion.

A model for DNA recombination uses 4-valent rigid vertex graphs, called assembly graphs. An assembly graph, similarly to the projection of knots, can be associated with an unsigned Gauss code, or double occurrence word. We define biologically motivated reductions that act on double occurrence words and, in turn, on their associated assembly graphs. For every double occurrence word w there is a sequence of reduction operations that may be applied to w so that what remains is the empty word, [epsilon]. Then the nesting index of a word w, denoted by NI(w), is defined to to be the least number of reduction operations necessary to reduce w to [epsilon]. The nesting index is the first property of assembly graphs that we study. We use chord diagrams as tools in our study of the nesting index. We observe two double occurrence words that correspond to the same circle graph, but that have arbitrarily large differences in nesting index values. In 2012, Buck et al. considered the cellular embeddings of assembly graphs into orientable surfaces. The genus range of an assembly graph [Gamma], denoted gr([Gamma]), was defined to be the set of integers g where g is the genus of an orientable surface F into which [Gamma] cellularly embeds. The genus range is the second property of assembly graphs that we study. We generalize the notion of the genus range to that of the genus spectrum, where for each g [isin] gr([Gamma]) we consider the number of orientable surfaces F obtained from [Gamma] by a special construction, called a ribbon graph construction, that have genus g. By considering this more general notion we gain a better understanding of the genus range property. Lastly, we show how one can obtain the genus spectrum of a double occurrence word from the genus spectrums of its irreducible parts, i.e., its double occurrence subwords. In the final chapter we consider constructions of double occurrence words that recognize certain values for nesting index and genus range. In general, we find that for arbitrary values of nesting index [ge] 2 and genus range, there is a double occurrence word that recognizes those values.

Global threats to treatment of bacterial infections due to antibiotic resistance (AR) have been on the rise in recent years. Current diagnostic tests identify bacteria by using blood culture, which takes more than 24 hours. This study focuses on the fluorescent labeling of DNA derived from bacterial AR genes (KPC & VIM) and other model DNAs using oligreen dye (OG) and molecular beacons (MB). A NanoDrop 3300 fluorospectrometer was used to take fluorescence measurements. Linear dynamic range and labeling efficiency were dependent on the following optimized conditions: dilution factor of OG (200 fold), buffer (20 mM Tris HCl, pH 8), and heat treatment of 95 °C for 15 min.Fluorescence analysis of a target DNA with a designed MB showed signal-to-background of 10 with our buffer only and 20 with our buffer and 25% ethanol. I also demonstrated a simple microfluidic device capable of detecting AR genes using model DNAs, magnetic beads, and designed MBs for assays of µ50 L volume. This study provides a first step towards detecting MB-DNA complexes by a simple, low cost, and fast non-amplified method, which may be used to detect AR genes in clinical samples in the future.

We address the problem of the DNA sequences developing a "dynamical" method based on the assumption that the statistical properties of DNA paths are determined by the joint action of two processes, one deterministic, with long-range correlations, and the other random and delta correlated. The generator of the deterministic evolution is a nonlinear map, belonging to a class of maps recently tailored to mimic the processes of weak chaos responsible for the birth of anomalous diffusion. It is assumed that the deterministic process corresponds to unknown biological rules which determine the DNA path, whereas the noise mimics the influence of an infinite-dimensional environment on the biological process under study. We prove that the resulting diffusion process, if the effect of the random process is neglected, is an a-stable Levy process with 1 < a < 2. We also show that, if the diffusion process is determined by the joint action of the deterministic and the random process, the correlation effects of the "deterministic dynamics" are cancelled on the short-range scale, but show up in the long-range one. We denote our prescription to generate statistical sequences as the Copying Mistake Map (CMM). We carry out our analysis of several DNA sequences, and of their CMM realizations, with a variety of techniques, and we especially focus on a method of regression to equilibrium, which we call the Onsager Analysis. With these techniques we establish the statistical equivalence of the real DNA sequences with their CMM realizations. We show that long-range correlations are present in exons as well as in introns, but are difficult to detect, since the exon "dynamics" is shown to be determined by theentaglement of three distinct and independent CMM's. Finally we study the validity of the stationary assumption in DNA sequences and we discuss a biological model for the short-range random process based on a folding mechanism of the nucleic acid in the cell nucleus.

Orientador: Mario Antonio Bernal Rodriguez
Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin
Made available in DSpace on 2018-08-30T22:42:12Z (GMT). No. of bitstreams: 1 TelloCajiao_JohnJames_M.pdf: 2614936 bytes, checksum: e5bdfc91b42434b003cad0b5fa850afb (MD5) Previous issue date: 2016
Resumo: O objetivo deste trabalho é estudar a influência da conformação do DNA na probabilidade de dano direto produzido por partículas ionizantes. Além disso, os fundamentos mecanicísticos do modelo Linear-Quadrático são investigadas através de um modelo biofísico desenvolvido neste trabalho, baseado na TADR (Teoria da Ação Dual da Radiação). Para este fim, três modelos geométricos do material genético foram construídos. Os modelos têm resolução atomística e levam em conta ? 10^9 pares de base (bps) nas configurações A,B e Z do DNA. A partir de um único bp, os diferentes níveis organizacionais no interior do núcleo da célula foram criados por meio de transformações lineares. Em seguida, o código Monte Carlo (MC) GEANT4-DNA foi usado para simular o transporte de prótons de 0.5, 1, 5, 7 e 10 MeV assim como de partículas alfa de 2, 5, 7 e 10M eV . O número de partículas em cada caso é de tal modo que as doses absorvidas estão entre 0.5 ? 16Gy. Os três modelos foram consistentes com as dimensões das estruturas reais. Em particular, os modelos foram compatíveis com a exigência de que o diâmetro da cromatina seja de 30 nm, bem como com os volumes bp reportados em outros trabalhos. Os rendimentos tanto das quebras totais quanto das quebras duplas (TSBY e DSBY) foram obtidos para cada qualidade de radiação. Além disso, a probabilidade de impacto (SHP) definida como a razão entre o volume do DNA e o volume núcleo, foi calculada teoricamente e a partir das simulações. O modelo biofísico em conjunto com as simulações MC forneceu o número de lesões letais (N_LL) em função da dose, para prótons de 0,5 e 10 MeV, e para partículas alfa de 2 e 10 MeV . Os N_LL puderam ser divididos em aqueles criados por uma única trajetória e aqueles originados pela interacção de duas trajetórias. Concluiu-se que o TSBY é praticamente determinada pela SHP e depende fracamente da qualidade de radiação incidente. No entanto, o DSBY mostrou forte dependência tanto da conformação do DNA quanto da qualidade de radiação. Isto é devido à relação entre a capacidade de agrupamento das deposições de energia para uma radiação dada e o empacotamento do DNA. Por outro lado, a análise dos mecanismos de produção de dano, baseada na TADR e testada com o modelo biofísico desenvolvido, mostraram que os efeitos de uma única trajetória (de primeira ordem) dependem linearmente com a dose. Além disso, os efeitos inter-trajetórias seguem um comportamento quadrático com a dose, com um termo linear que influencia o mecanismo de primeira ordem. Isto significa que o comportamento linear-quadrático do N_LL com a dose, tem fundamentos mecanicistas, pelo menos, na primeira fase do dano
Abstract: The aim of this work is to study the influence of the DNA conformation on the probability of direct damage induction by ionizing particles. Also, the mechanistic grounds of the Linear-Quadratic radiobiological model are investigated through the eyes of a home-made biophysical model based on the DRAT (Dual Radiation Action Theory). To this end, three geometrical models of the genetic material were constructed. The models have atomistic resolution and account for ? 10^9 base pairs (bps) in the A-, B- and Z-DNA configurations. Starting from a single bp, the different organizational levels inside the cell nucleus were created by means of linear transformations. Next, the Monte Carlo (MC) code GEANT4-DNA was used to simulate the transport of protons of 0.5, 1, 5, 7 and 10 MeV , and alpha particles of 2, 5, 7 and 10 MeV. The number of particles in each case is such that the absorbed doses range between 0.5 Gy and 16 Gy. The three models proved to be consistent with the dimensions of the real structures. In particular, the models were compatible with the 30 nm chromatin fiber diameter requirement as well as with the bp volumes reported in other works. The Total and Double Strand Break Yields (TSBY and DSBY) were obtained for every radiation quality. Also, the Site-Hit Probability (SHP) defined as the total target to the nucleus volume ratio, was computed theoretically and from the simulations. The biophysical model in conjunction with the MC simulations furnished the number of lethal lesions (N_LL) as a function of dose, for protons of 0.5 and 10 MeV , and for alpha particles of 2 and 10 MeV . The N_LL could be split into those created by a single track and those originated by interaction of two tracks. It is concluded that the TSBY is practically determined by the SHP and depends weakly on the incident radiation quality. Nevertheless, the DSBY showed strong dependence on both the DNA conformation and the radiation quality. This is due to the interplay between the energy deposition clustering capacity of a given radiation and the DNA spatial packing. On the other hand, the analysis of the mechanisms of damage production based on the DRAT and tested with the biophysical model developed, showed that single-track (first order) effects depend linearly on the dose. Moreover, inter-track effects follows a quadratic behavior with the dose, having a linear term that influences the first order mechanism. This means that the Linear-Quadratic behavior of the N_LL with the dose, has mechanistic groundings at least at the first stage of the damage
Mestrado
Física
Mestre em Física
1370449/2014
CAPES

Selection of signi?cant genes via expression patterns is important in a microarray problem. Owing to small sample size and large number of variables (genes), the selection process can be unstable. This research proposes a hierarchical Bayesian model for gene (variable) selection. We employ latent variables in a regression setting and use a Bayesian mixture prior to perform the variable selection. Due to the binary nature of the data, the posterior distributions of the parameters are not in explicit form, and we need to use a combination of truncated sampling and Markov Chain Monte Carlo (MCMC) based computation techniques to simulate the posterior distributions. The Bayesian model is ?exible enough to identify the signi?cant genes as well as to perform future predictions. The method is applied to cancer classi?cation via cDNA microarrays. In particular, the genes BRCA1 and BRCA2 are associated with a hereditary disposition to breast cancer, and the method is used to identify the set of signi?cant genes to classify BRCA1 and others. Microarray data can also be applied to survival models. We address the issue of how to reduce the dimension in building model by selecting signi?cant genes as well as assessing the estimated survival curves. Additionally, we consider the wellknown Weibull regression and semiparametric proportional hazards (PH) models for survival analysis. With microarray data, we need to consider the case where the number of covariates p exceeds the number of samples n. Speci?cally, for a given vector of response values, which are times to event (death or censored times) and p gene expressions (covariates), we address the issue of how to reduce the dimension by selecting the responsible genes, which are controlling the survival time. This approach enables us to estimate the survival curve when n << p. In our approach, rather than ?xing the number of selected genes, we will assign a prior distribution to this number. The approach creates additional ?exibility by allowing the imposition of constraints, such as bounding the dimension via a prior, which in e?ect works as a penalty. To implement our methodology, we use a Markov Chain Monte Carlo (MCMC) method. We demonstrate the use of the methodology with (a) di?use large B??cell lymphoma (DLBCL) complementary DNA (cDNA) data and (b) Breast Carcinoma data. Lastly, we propose a mixture of Dirichlet process models using discrete wavelet transform for a curve clustering. In order to characterize these time??course gene expresssions, we consider them as trajectory functions of time and gene??speci?c parameters and obtain their wavelet coe?cients by a discrete wavelet transform. We then build cluster curves using a mixture of Dirichlet process priors.

Thesis (Ph. D.)--University of Florida, 2001.
Title from first page of PDF file. Document formatted into pages; contains xviii, 294 p.; also contains graphics. Vita. Includes bibliographical references (p. 284-293).

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 2002.
Includes bibliographical references (leaves 129-135).
Sanger's method of chain termination is the method of choice in DNA sequencing, where electrophoresis is used to separate the different sized DNA. In the past decade, microfabricated capillary devices have been developed and are increasingly used to perform DNA electrophoresis. While tremendous progress has been made in the process, sample injection has not been well understood. In an earlier study, images of sample injection obtained using video microscopy showed sharp sample stacking peak at the trailing edge of the sample plug. This thesis examines the underlying physics that explain the behavior of DNA in microcapillary electrophoresis. A developed model captures the dynamics of the major electrolytes in the system. The applied voltage and the conductivity profile determine the local electric field. The electric field drives the analyte transport. The analyte consists of DNA molecules of various fragment sizes. Since the DNA concentration is smaller than the electrolyte concentration by a few orders of magnitude, its concentration does not affect the conductivity. The major components of the sample are identified, and role during injection is investigated. Analytical studies of the electrolyte boundary dynamics and evolution and the transport of DNA are presented. The effect of the buffer, applied voltage during injection, and sample mobility on stacking are shown.
(cont.) A numerical model is implemented to quantitatively predict the stacking of DNA in microcapillary electrophoresis. The numerical model has been developed for the 1-dimensional case. The model is verified using analytical results. Results of numerical models that predict the behavior of DNA under experimental conditions are presented. The numerical model is compared with real experimental data to evaluate its predictive power. Preliminary numerical simulations have also been done for 2-dimensional geometries. A procedure has been developed for design of injector lengths to obtain a given resolution of separation in a microcapillary channel of specified length. Strategies for optimization are presented for improving the performance of the devices.
by Alok Srivastava.
Ph.D.

In this thesis we investigate the self-assembly and structural properties of DNA origamis, which are large-scale DNA nanostructures comprising thousands of DNA base pairs, using the coarse-grained model of DNA called oxDNA. We use simulations of oxDNA, with both a "brute-force" approach and with a rareevent method, to probe how the origamis self-assemble from single strands of DNA in solution. With the brute-force approach we are able to simulate the complete assembly of a small 384-base-pair origami at a high strand concentration, and identify kinetic traps under certain conditions, as well as cooperativity between staple strands and optimal assembly windows. These findings help to rationalise certain successful design strategies. Using the rare-event method we are able to use the lower strand concentrations typical of experimental conditions and make more quantitative measurements, identifying sequential staple binding for our origami design at these conditions, and probing how a staple binds each of its domains. We introduce oxDNA2, an enhanced version of oxDNA, which has improved predictive power for origami structure, as well as other improvements such as salt dependence. We verify that the model reproduces well the structural properties of Holliday junctions, which are a vital feature of DNA origamis. We then use the model to investigate the basic structural properties of 2D and 3D origamis, characterising their generic structural features such as, for 2D structures, the "weave pattern," where adjacent double helices push apart away from the junctions, and "corrugation," a systematic, out-of-plane bending of the double helices. We find a good agreement with experiment where data is available.

La ségrégation des chromosomes chez les cellules eucaryotes et des plasmides chez les bactéries ont en commun une phase initiale de "recherche et capture" pendant laquelle des fibres explorent le volume de la cellule à partir d'une position spécifique. Schématiquement des polymères croissent à partir d'un point et essayent d'atteindre des cibles réparties dans un espace confiné. Etonnement, les fibres impliquées présentent un même type de dynamique. Nous présentons d'abord un modèle d'instabilité dynamique basée sur l'idée populaire que la croissance du polymère est permise par une coiffe à l'extrémité de la fibre. Le modèle inclut des interactions longitudinales entre les unités de monomère aux extrémités de chaque protofilament, et un 'sauvetage par effet cliquet'. Nous examinons ensuite le mécanisme de ségrégation des plasmides RI chez la bactérie. Des simulations basées sur les observations expérimentales existantes sont utilisées pour analyser les différentes étapes du processus et tester les différentes contributions et de la fasciculation des fibres. Nous montrons que le processus actif de ségrégation peut atteindre un niveau élevé de fidélité à un coût énergétique modéré pour la bactérie hôte. Enfin, nous étudions la phase de "recherche et capture" chez la levure bourgeonnante. Nous avons calculé la probabilité de capture d'un chromosome par microtubule et avons trouvé que des effets de confinement peuvent améliorer grandement. Des observations expérimentales réalisées supportent l'hypothèse selon laquelle les microtubules possèdent un certain degré de liberté de rotation à leur point d'ancrage avec le corps du pôle du fuseau
Eukaryotic chromosome segregation and plasmid partitioning in bacteria share an initial phase of 'search and capture' during \ growing from a localized position probe the cell to find the DNA. In higher eukaryotes, the fibers are nucleated from the mien organizing center (MTOC), in yeast from the spindle pole body and in type II plasmid prokaryotic segregation they are bound ParC complex. Schematically, polymers grow from one point and try to reach targets distributed in a confined space. Remarkably the involved fibers exhibit the same kind of dynamic. We first study a model of dynamic instability based on the idea that a cap at the tip of the fiber maintains growth. The model longitudinal interactions between the terminal tubulins of each protofilament and 'gating rescues' between neighbouring protofilaments We also build a simulation to analyze the three-component segregation system of the RI plasmids in bacteria. We test the cont filament annealing or bundling during the segregation process. The effective cost of the RI plasmids segregation process for t calculated and we concluded that this active process is a cheap way to ensure DNA segregation compare to a passive mechanic. Finally we study the search and capture phase in budding yeast. We calculate the probability of capture per microtubule and find that confinement effects such as microtubule sweeping along the nuclear membrane can lead to an efficient mechanism. By live light microscopy experiments, we show that microtubules are able to pivot at their anchorage point within the spindle pole body, which dramatically reduced the search time

This paper considers the statistical mechanics occupation of the edge of a single helix of DNA by simple polymers. Using Fisher's exact closed form solution for dimers on a two-dimensional lattice, a one-dimensional lattice is created mathematically that is occupied by dimers, monomers, and holes. The free energy, entropy, average occupation, and total charge on the lattice are found through the usual statistical methods. The results demonstrate the charge inversion required for a DNA helix to undergo DNA condensation.

This thesis discusses about possibilities of construction colour DNA spectrograms and about patterns which can be detected there. Spectrograms as tools of spectral analysis give us a simultaneous view of the local frequency throughout the nucleotide sequence. They are suitable for gene identification or gene regions identification, determination of global character about whole chromosomes and also give us a chance for the discovery of yet unknown regions of potential significance. For purpose of this kind of DNA analysis is possible to use digital signal processing methods. We can apply them on only after conversion of DNA sequence to numerical representation. Selection of correct numerical representation affects how well will be reflected biological features in numerical record which we need for another use in digital signal analysis.

Japanese encephalitis virus (JEV), a member of the family flaviviridae, is one of the most important pathogens of the developing countries, causing high mortality and morbidity amongst children. The present study is aimed at the development of a DNA vaccine for Japanese Encephalitis (JE). As a first step towards developing a DNA vaccine for JE, an eukaryotic expression plasmid encoding the envelope (E) glycoprotein of Japanese Encephalitis Virus (pCMXENV) was constructed. This plasmid expresses the E protein intracellularly, when transfected into Vero cells in culture. Several independent immunization and intracerebral (i.e.) JEV challenge experiments were carried out and the results indicate that 51% and 59% of the mice are protected from lethal i.e. JEV challenge, when immunized with pCMXENV via intramuscular (i.m.) and intranasal (i.n.) routes respectively. JEV-specific antibodies were not detected in pCMXENV-immunized mice either before or after challenge. JEV-specific T cells were observed in mice immunized with pCMXENV, which increased significantly after JEV challenge indicating the presence of vaccination-induced memory T cells. Enhanced production of interferon-y (EFN-y) and complete absence of interleukin-4 (IL-4) in splenocytes of pCMXENV-immunized mice on restimulation with JEV antigens in vitro indicated that the protection is likely to be mediated by T helper (Th) lymphocytes of the Thl sub type. These results demonstrated that immunization with a plasmid DNA expressing intracellular form of JEV E protein confers significant protection against i.e. JEV challenge even in the absence of detectable antiviral antibodies. We then examined the potency of JEV DNA vaccines as well as that of the inactivated mouse brain derived BIKEN vaccine in the i.e. challenge model. The results indicate that all the mice immunized with BIKEN JE vaccine were protected against i.e. JEV challenge while 50% protection was observed in case of mice immunized with pJME or pJNSl and 38% protection was observed in pCMXENV-immunized mice. Immunization with both pJME and pJNSl resulted in 66% protection. These results indicate that the BIKEN JE vaccine confers better protection against i.e. JEV challenge than DNA vaccines. The fact that the BIKEN vaccine conferred better protection against i.e. JEV challenge than DNA vaccines indicated that the i.e. JEV challenge model can be exploited further to examine the potency of different DNA vaccine constructs. Towards this goal, we constructed plasmids that encode secretory or nonsecretory forms of JEV E protein and examined their potency in the i.e. JEV challenge model. Our results indicate that i.m. immunization of mice with plasmid encoding secretory form of JEV E protein confers higher level (75%-80%) protection than those encoding nonsecretory forms. Cytokine analysis of splenocytes isolated from DNA immunized mice after stimulation in vitro with JEV revealed that immunization with plasmid encoding secretory form of JEV E protein induces both Thl and Th2 responses while those encoding nonsecretory forms induce only Thl type of response. Thus, synthesis of secretory form of JEV E protein results in an altered immune response leading better protection against i.e. JEV challenge. Based on our studies, we propose that both cellular and humoral immune responses play a key role in protective immunity against i.e. JEV challenge and DNA vaccines that can induce higher levels of neutralizing antibodies will be as efficient as the BIKEN vaccine in conferring protection against i.e. JEV challenge.

In this thesis, we show how to create an approximate Markov model for the DNA. This model is constructed by encoding the DNA nucleotides into finite length symbolic sequences, referred to as words, and creating a 2D symbolic space for the DNA, where points plotted in that space represent words. From the construction of our model, we are able to specify words for the DNA, their lengths and how they can be organised together in groups of symbolic similarities. The model also allows the construction of a network of the DNA, where the nodes represent group of words and the edges connecting two nodes a measure of the likelihood that words in a group are mapped to another strongly correlated group of words after 1 shift in the nucleotide sequence. The model is then applied to reduce the complexity of the DNA, by considering the most relevant group of words that carry most of the information of the DNA. We were able to show that in the E. coli's 2/5th of the information is lost by neglecting only 3 groups of words. The model was then applied to construct measures of similarity between genes and predictability of genes in different organisms. We then study the long-term behaviour of group of words in our Markov model by analysing their recurrence properties. For some group of words, the statistics of returns was theoretically estimated from statistical properties of our model. The groups of words that contribute more to the DNA's random nature provide a simple way to analytically estimate the statistics of returns of words belonging to these groups. As an application of the recurrence analysis, we were able to show that the coding regions of the DNA contribute more to its random character.

Immune modulation is one treatment modality which is being explored in the context of human diseases and disorders. Methods of such manipulation which use gene therapy have an advantage in the treatment of chronic diseases such as autoimmunity because they are less invasive and more persistent. Furthermore, naked plasmid has advantages as a vector over other methods: it is more persistent and less immunogenic and cytotoxic than viral vectors, and simpler than DNA-conjugate vectors. Thus, naked plasmid is a viable alternative treatment to study in the context of an autoimmune disease such as diabetes mellitus, despite it's disadvantages of low transfection and expression rates. Here, we demonstrate that treatment of an autoimmune model, the non-obese diabetic (NOD) mouse, with an autoantigen to which a signal sequence had been added was protective, even in the apparent absence of secretion of that gene product. In contrast, treatment with the native cDNA of the same antigen was not protective. Furthermore, we show by immunohistochemistry that gene expression is still detectable in the muscle 22 weeks after injection. Other experiments demonstrate that multiple vaccinations with the altered form of the antigen were essentially as protective as a single vaccination following by multiple injections of blank plasmid, suggesting an important role for immunostimulatory sequences in bacterial DNA in causing surveying dendritic cells to migrate out of the tissue and present antigen in draining lymph nodes. Attempts to study the results of DNA vaccination by comparing immunization via different routes were inconclusive.
We have demonstrated that DNA vaccination of an autoimmune model with a autoantigen can delay disease. The simplicity and economy of such vectors and the benefits they have for the treatment of chronic disease in contrast to more inflammatory viral vectors, support future research into their use in the treatment of autoimmune diseases.

DNA methylation is an important epigenetic mark spanning all of life's kingdoms. In humans, DNA methylation has been associated with a wide range of age-related pathologies, including type II diabetes and cancer. More recently, in humans, changes in DNA methylation at specific positions in the genome have been found to be predictive of chronological age. Interestingly, DNA methylation age is also predictive of health status and time-to-death. A better understanding of what these DNA methylation changes represent and whether they might be causative in the ageing process will be important to ascertain. However, at present there is no animal model system with which this process can be studied at a mechanistic level. Furthermore, it is becoming increasingly apparent that many disease states that increase in prevalence with age are not caused by all cells within the individual, but are often the result of changes to a subset of cells. This underscores the importance of studying these processes at the single cell level. The recent advances in single cell sequencing approaches now mean that we can study multiple layers of biology within the same single cell, such as the epigenome and the transcriptome (scM&T-Seq). Unfortunately, we are still only able to probe these important aspects of single cell biology in a static sense. This is a major limitation in the study of ageing because ageing and age-related disease processes are inherently dynamic. As such, it is incumbent upon us to develop approaches to assay single cell biology in a dynamic manner. 
In this thesis, I describe an epigenetic age predictor in the mouse. This predictor is tissue-independent and can accurately predict age (with an error of 3.33 weeks) and can record deviations in biological age upon interventions including ovariectomy and high fat diet both of which are known to reduce lifespan. Next, I describe the analysis of a homogeneous population of muscle satellite cells (MuSCs) that I have interrogated at the single cell level, using single cell combined transcriptome and methylome sequencing (scM&T-seq). I found that with age there was increased global transcriptional variability and increased feature-specific methylome variability. These findings explain the loss of functionality of these cells with age. Lastly, I describe two imaging approaches to study DNA methylation dynamically in single cells. Using these methods, I demonstrate that it is possible to accurately determine methylation status across a wide spectrum of global methylation levels and that by using such approaches novel information about dynamic methylation processes can be obtained. These methods represent the first to study DNA methylation dynamically.

One of the most important controversies in the field of cancer epigenetics is the question of whether aberrant CpG island methylation of tumour suppressor genes can cause cancer or whether such abnormalities of DNA methylation are secondary to the malignant process. We address this question by prospectively causing abnormal methylation events in vivo. We have produced transgenic mice which over-express the de novo methyltransferase Dnmt3b under the widely expressed CAG promoter. Tg(Dnmt3b)+mice develop normally and are fertile, but die at 4-5 months, developing dilated cardiomyopathy. CpG island methylation is globally increased, and abnormal methylation of specific genes, including the tumour suppressor genes Cdkn1a, Cdkna2a and Hic1 is detectable. However, there is no spontaneous cancer incidence. Crossing the mice with the intestinal tumour prone Apc^+/-min mice leads to a modest increase in polyp number and the frequency of dysplastic tumours, although methylation of the Apc gene is not detectable in normal mucosa from Dnmt3b overexpressing mice, and methylation of the Apc promoter occurs with equal frequency in microdissected tumours from Apc^+/- mice regardless of Tg(Dnmt3b)+ genotype. The results show that increases of methylation can be well tolerated, suggesting that although methylation can be increased, active silencing by methylation is more unusual. In particular, the active silencing of tumour suppressor genes by de novo methylation is unlikely to be a primary event in the formation of tumours, although it may play a role in modulating tumour progression.

DNA copy number alterations (CNAs) are genetic changes that can produce adverse effects in numerous human diseases, including cancer. CNAs are segments of DNA that have been deleted or amplified and can range in size from one kilobases to whole chromosome arms. Development of array comparative genomic hybridization (aCGH) technology enables CNAs to be measured at sub-megabase resolution using tens of thousands of probes. However, aCGH data are noisy and result in continuous valued measurements of the discrete CNAs. Consequently, the data must be processed through algorithmic and statistical techniques in order to derive meaningful biological insights. We introduce model-based approaches to analysis of aCGH data and develop state-of-the-art solutions to three distinct analytical problems. In the simplest scenario, the task is to infer CNAs from a single aCGH experiment. We apply a hidden Markov model (HMM) to accurately identify CNAs from aCGH data. We show that borrowing statistical strength across chromosomes and explicitly modeling outliers in the data, improves on baseline models. In the second scenario, we wish to identify recurrent CNAs in a set of aCGH data derived from a patient cohort. These are locations in the genome altered in many patients, providing evidence for CNAs that may be playing important molecular roles in the disease. We develop a novel hierarchical HMM profiling method that explicitly models both statistical and biological noise in the data and is capable of producing a representative profile for a set of aCGH experiments. We demonstrate that our method is more accurate than simpler baselines on synthetic data, and show our model produces output that is more interpretable than other methods. Finally, we develop a model based clustering framework to stratify a patient cohort, expected to be composed of a fixed set of molecular subtypes. We introduce a model that jointly infers CNAs, assigns patients to subgroups and infers the profiles that represent each subgroup. We show our model to be more accurate on synthetic data, and show in two patient cohorts how the model discovers putative novel subtypes and clinically relevant subgroups.

Thesis: S.M., Massachusetts Institute of Technology, Department of Biology, 2014.
Cataloged from PDF version of thesis.
Includes bibliographical references (pages 47-49).
Complex DNA nanostructures have proven difficult to assemble from starting materials. Inefficient nanostructure assembly constitutes a barrier to the widespread use of DNA nanotechnology and is difficult to investigate experimentally due to the complicated nature of the assembly. This work introduces a type of tile assembly model, the isothermal tile assembly model (iTAM). The iTAM seeks to capture the behavior of assembling DNA tile nanostructures to identify design factors and reaction conditions which improve assembly yields. Simulations using the iTAM model explain the experimental observation that only a narrow range of temperatures permit optimal isothermal assembly of tile-based DNA nanostructures. This narrow temperature range reflects a balance between the stabilization of non-designed interactions at low temperatures and the destabilization of the overall designed structure at high temperature. Simulations based on the iTAM are effective at estimating the temperature of optimal assembly unique to 25 two-dimensional tile designs, with an mean error of estimation of 4.6 degrees C. Results from the iTAM indicate that optimal assembly temperatures are determined largely by the strength of tile-tile domain interactions. For a given tile design, tile concentration and the length of time represent convenient axes of control over tile assembly. Kinetic trapping that blocks complete assembly of a tile design is likely to be overcome by increasing the both temperature and tile concentration in the assembly reaction. Such a change also substantially decreases the computationally predicted time required for complete assembly.
by Benjamin Steele.
S.M.

Background. DNA methylation (DNAm) is a removable chemical modification to the DNA sequence intimately associated with genomic stability, cellular identity, and gene expression. DNAm patterning reflects joint contributions from genetic, environmental, and behavioral factors. As such, differences in DNAm patterns may explain interindividual variability in risk liability for complex traits like major depression (MD). Hundreds of significant DNAm loci have been identified using cross-sectional association studies. This dissertation builds on that foundational work to explore novel statistical approaches for longitudinal DNAm analyses. Methods. Repeated measures of genome-wide DNAm and social and environmental determinants of health were collected up to six times across pregnancy and the first year postpartum as part of the Pregnancy, Race, Environment, Genes (PREG) Study. Statistical analyses were completed using a combination of the R statistical environment, Bioconductor packages, MplusAutomate, and Mplus software. Prenatal maternal DNAm was measured using the Infinium HumanMethylation450 Beadchip. Latent growth curve models were used to analyze repeated measures of maternal DNAm and to quantify site-level DNAm latent trajectories over the course of pregnancy. The purpose was to characterize the location and nature of prenatal DNAm changes and to test the influence of clinical and demographic factors on prenatal DNAm remodeling. Results. Over 1300 sites had DNAm trajectories significantly associated with either maternal age or lifetime MD. Many of the genomic regions overlapping significant results replicated previous age and MD-related genetic and DNAm findings. Discussion. Future work should capitalize on the progress made here integrating structural equation modeling (SEM) with longitudinal omics-level measures.

Le modèle de Poland-Scheraga (PS) est le modèle standard pour étudier la transition de dénaturation de deux brins d’ADN complémentaires et de même longueur. Ce modèle a fait l’objet d’une attention remarquable car il est exactement résoluble dans sa version homogène. Le caractère résoluble est lié au fait que le modèle PS homogène peut être mis en correspondance avec un processus de renouvellement discret. Dans la littérature biophysique une généralisation du modèle, obtenue en considérant des brins non complé- mentaires et de longueurs différentes, a été considérée et le caractère résoluble s’étend à cette généralisation substantielle. Dans cette thèse, nous présentons une analyse mathématique du modèle de Poland- Scheraga généralisé. Nous considérons d’abord le modèle homogène et nous exploitons que les deux brins de la chaîne peuvent être modélisés par un processus de renouvellement en deux dimensions. La distribution K(⋅) de l’emplacement (bidimensionnel) du premier contact entre les deux brins est supposée de la forme K(n+m) = (n+m)−α−2L(n+m) avec α ≥ 0 et L(⋅) à variation lente et correspond à une boucle avec n bases dans le premier brin et m dans le deuxième. Nous étudions la transition de localisation-délocalisation et nous montrons l’existence des transitions à l’intérieur de la phase localisée. Nous présentons ensuite des estimations précises sur les propriétés de chemin du modèle. Ensuite, nous étudions la version désordonnée du modèle en incluant une séquence de variables aléatoires indépendantes identiquement distribuées à deux indices. Nous nous concentrons sur l’influence du désordre sur la transition de dénaturation: nous voulons déterminer si la présence des inhomogénéités modifie les propriétés critiques du système par rapport au cas homogène. Nous prouvons que le désordre est non pertinent si α < 1 et nous montrons que pour α > 1, les points critiques gelés et recuits diffèrent (basant sur les techniques de coarse graining et la méthode des moments fractionnaires), ce qui prouve la présence d’un régime de désordre pertinent
The Poland-Scheraga (PS) model is the standard basic model to study the denaturation transition of two complementary and equally long strands of DNA. This model has enjoyed a remarkable attention because it is exactly solvable in its homogeneous version. The solvable character is related to the fact that the homogeneous PS model can be mapped to a discrete renewal process. In the bio-physical literature a generalization of the model, allowing different length and non complementarity of the strands, has been considered and the solvable character extends to this substantial generalization. In this thesis we present a generalized version of the PS model that allows mismatches and non complementary strands (in particular, the two strands may be of different lengths). We consider first the homogeneous model and we exploit that this model can be mapped to a bivariate renewal process. The distribution K(⋅) of the location (in two dimensions) of the first contact between the two strands is assumed to be of the form K(n + m) = (n + m)−α−2L(n + m) with α ≥ 0 and L(⋅) slowly varying and corresponds to a loop with n bases in the first strand and m in the second. We study the localization-delocalization transition and we prove the existence of transitions inside the localized regime. We then present precise estimates on the path properties of the model. We then study the disordered version of the model by including a sequence of inde- pendent and identically distributed random variables with two indices. We focus on the influence of disorder on the denaturation transition: we want to determine whether the presence of randomness modifies the critical properties of the system with respect to the homogeneous case. We prove that the disorder is irrelevant if α < 1. We show also that for α > 1, the quenched and annealed critical points differ (basing on coarse graining techniques and fractional moment method), proving the presence of a relevant disorder regime

DNA nanotechnology is a rapidly developing field that creates nanoscale devices from DNA, which enables novel interfaces with biological material. Their therapeutic use is envisioned and applications in other areas of basic science have already been found. These devices function at physiological conditions and, owing to their molecular scale, are subject to thermal fluctuations during both preparation and operation of the device. Troubleshooting a failed device is often difficult and we develop models to characterise two separate devices: DNA walkers and DNA origami. Our framework is that of continuous-time Markov chains, abstracting away much of the underlying physics. The resulting models are coarse but enable analysis of system-level performance, such as ‘the molecular computation eventually returns the correct answer with high probability’. We examine the applicability of probabilistic model checking to provide guarantees on the behaviour of nanoscale devices, and to this end we develop novel model checking methodology. We model a DNA walker that autonomously navigates a series of junctions, and we derive design principles that increase the probability of correct computational output. We also develop a novel parameter synthesis method for continuous-time Markov chains, for which the synthesised models guarantee a predetermined level of performance. Finally, we develop a novel discrete stochastic assembly model of DNA origami from first principles. DNA origami is a widespread method for creating nanoscale structures from DNA. Our model qualitatively reproduces experimentally observed behaviour and using the model we are able to rationally steer the folding pathway of a novel polymorphic DNA origami tile, controlling the eventual shape.

Tato práce se zabývá buněčným cyklem kvasinky Saccgaromyces cerevisiae. Oblastí našeho zájmu je přechod mezi G1 a S fází, kde je naším cílem identifikovat velikosti buňky v době počátku DNA replikace. Nejprve se věnujeme nedávno publikovanému matematickému modelu, který popisuje mechanismy vedoucí k S fázi. Práce poskytuje detailní popis tohoto modelu, stejně jako časový průběh některých důležitých proteinů či jejich sloučenin. Dále se zabýváme pravděpodobnostním modelem aktivace replikačních počátků DNA. Nově uvažujeme vliv šíření DNA replikace mezi sousedícími počátky a analyzujeme jeho důsledky. Poskytujeme také senzitivní analýzu kritické velikosti buňky vzhledem ke konstantám popisujícím dynamiku reakcí v modelu G1/S přechodu.

During DNA replication the DNA has to be unpacked, duplicated and repacked into chromatin, which is comprised of DNA and histone proteins (Nicholson and Muller, 2008b). The coordinated replication of DNA and histone protein synthesis is vital for the correct chromatin formation (Marzluff et al., 2008). The mechanism controlling the histone gene expression is only partially understood, especially the mechanism controlling any disturbances in the coordination of DNA replication and histone protein synthesis. Previous experiments suggested that the regulation mechanism of histone balance could involve regulation by a free histone protein pool (Takami and Nakayama, 1997b; Takami and Nakayama, 1997a; Dominski et al., 2005; Kroeger et al., 1995). A mathematical model was produced by Dr Hameister (Hameister, 2012) to describe the control of histone production during S phase. The parameters used were taken from literature. Modifications were made using experimentally measured data to replace literature values (Harris et al., 1991; Clark, 2006; Strachen and Read, 2003). The aim of the project was to both optimise the model by defining parameters to reflect what occurs naturally in the cell, as well as trying to validate the model. The DNA replication rate was measured by FACS analysis and was input into the model. Histone RNA levels during S phase were measured by Northern blots and histone RNA degradation rates were analysed. To confirm that the modified DNA replication rate was producing accurate simulations, the curve produced for the mRNA levels could be compared to the experimentally measured mRNA values (Figure 4.3.1). The over expression of an H2B gene was verified using Western and Northern analysis. The validation of the model that the histone gene balance was being regulated by a free histone pool was not absolutely confirmed. However, results seen in Figure 3.6.1, showed an increase in H2B RNA degradation in the sample with the additional gene due to the additional histone proteins. Further work is required for confirmation of the regulation mechanism.

Replication of DNA is a vital process for growth and cell division in all domains of life. The mechanisms of helicase loading as well as hand-off of nascent RNA primers from the primase to the replicative polymerases during DNA synthesis are of fundamental importance. The prototype structural/functional model provided by the widely studied Gram-negative Escherichia coli DNA replication system is not universally applicable. The current model for the Gram-positive Bacillus subtilis DNA replication suggests that PolC is the main processive replicative polymerase with DnaE playing an essential but minor role during DNA synthesis. Our results suggest that DnaE is a major polymerase important for DNA replication and DNA repair. DnaE polymerase activity is stimulated in the presence of SSB, the clamp DnaN and PolC polymerase, its error-prone synthesis is modulated by the aforementioned proteins and its errors are corrected in trans by the PolC exonuclease domain, in a template specific ternary DNA-DnaE-PolC complex. Hereby, we propose a new revised model for DNA replication in Bacillus subtilis, where DnaE is the major replicative polymerase on the lagging strand and PolC, which is the major polymerase on the leading strand, provides the crucial proof-reading activity in trans. These findings revise the current model in Bacillus subtilis and suggest a division of labour between DnaE and PolC polymerases.

[ES] La radiación ultravioleta está asociada a la formación de ciertas lesiones en el ADN que están en el origen del cáncer de piel. Entre las más relevantes se encuentran los daños que se producen en las bases pirimidínicas: los dímeros ciclobutánicos (CPDs) y fotoproductos (6-4) (6-4PPs). Como protección contra las fotolesiones del ADN, los organismos vivos disponen de enzimas que restauran las lesiones a su forma original, manteniendo así la integridad genética. Algunos organismos manifiestan un proceso de reparación adicional de los CPDs y los 6-4PPs, que corresponde a la foto-rreactivación y que involucra enzimas denominadas fotoliasas CPD y (6-4). En con-creto, actualmente existe una viva discusión sobre el mecanismo de reparación por la fotoliasa (6-4). El objectivo general de esta tesis doctoral ha sido estudiar la ciclorre-versión de los intermedios clave propuestos para la lesión 6-4PP para apoyar uno de los mecanismos propuestos hasta ahora. En primer lugar se ha preparado un modelo de la azetidina intermedia de la le-sión 6-4PP en secuencias TC para investigar su reparación mediante un proceso de donación de electrones a través de fotosensibilizadores con un potencial redox ade-cuado, mimetizando así el cofactor flavina de la fotoliasa (6-4). Los estudios de elec-troquímica, espectroscopía, análisis y química computacional mostraron que la posibi-lidad de inyectar un electrón al anillo de la azetidina conlleva una ciclorreversión de la azetidina bipirimidínica generando las bases timina y 6-azauracilo. También se ha evi-denciado que la transferencia de electrones sólo tiene lugar si el componente timina está presente en el modelo. En segundo lugar, se ha investigado la ciclorreversión de la azetidina mediante un proceso oxidativo en cual el anillo de azetidina dona un electrón al fotosensibilizador. La comparación con un derivado ciclobutánico mostró que la presencia del nitrógeno en el anillo de cuatro miembros disminuye el potencial redox facilitando el proceso de oxidación. En tercer lugar, el paso de ciclorreversión se ha estudiado con dos fotosensibili-zadores intrínsecos, la guanina y el daño oxidativo 8-oxoguanina (OG), unidos cova-lentemente a un CPD o a un oxetano, como modelo del intermedio formado en la reparación del daño (6-4). En conjunto, los datos de espectroscopía y análisis croma-tográfica mostraron la posibilidad de que estos fotosensibilizadores endógenos pueden actuar como dadores de electrones mimetizando, por tanto, la función del cofactor flavina en la fotoliasa. Finalmente, el anillo de azetidina ha sido incorporado en un oligonucleótido para estudiar su ciclorreversión mediante transferencia de electrones. En base a los resulta-dos de los capítulos previos, OG ha sido elegido como un fotorreductor natural. En un primer paso, una metodología ha sido desarrollada para insertar la azetidina dentro de una secuencia de oligonucleótido. Luego, la irradiación en estado estacionario del dúplex que contiene OG y la azetidina ha demostrado que la transferencia de electro-nes tiene lugar y conlleva a la ciclorreversión del heterociclo. Además, experimentos preliminares han sido llevados a cabo para evaluar la reparación del anillo de cuatro miembros, como un análogo del intermedio generado en el caso del fotoproducto (6-4), por las fotoliasas reales CPD y (6-4).
[CAT] La radiació ultravioleta està associada a la formació de certes lesions en l'ADN que podrien concluir al càncer de pell. Entre les més rellevants es troben els danys que es produïxen en les bases pirimidínicas: els dímers ciclobutánics (CPDs) i els fotopro-ductes (6-4) (6-4PPs) . Per a protegir-se de les fotolesions al l'ADN, els organismes vius disposen d'enzims que restauren les lesions a la seua forma original, mantenint així la integritat genètica. En alguns organismes els CPDs i els 6-4PPs manifesten un procés de reparació addicional, que correspon a la fotorreactivació on están involu-crats enzims denominades fotoliases CPD i (6-4) . En concret, actualment hi ha una viva discussió sobre el mecanisme de reparació per la fotoliasa (6-4) . L'objectiv gene-ral d'esta tesi doctoral ha sigut estudiar la ciclorreversió dels intermedis clau proposats per a la lesió 6-4PP com a recolzament d' un dels mecanismes proposats fins ara. En primer lloc s'ha preparat un model de l'azetidina intermèdia de la lesió 6-4PP en seqüències TC per a investigar la seua reparació per mitjà d'un procés de donació d'electrons per fotosensibilizadors amb un potencial redox adequat, mimetitzant així el cofactor flavina de la fotoliasa (6-4) . Els estudis d'electroquímica, espectroscòpia, anàlisi cromatogràfica i química computacional van mostrar que la possibilitat d'injec-tar un electró a l'anell de l'azetidina comporta una ciclorreversió de l'azetidina bipiri-midínica a les bases de timina i 6-azauracil. També s'ha evidenciat que la transferència d'electrons només té lloc si la base timina està present en el model. En segon lloc, s'ha investigat la ciclorreversión de l'azetidina mitjacant procés oxidatiu en que l'anell d'azetidina dóna un electró cap al fotosensibilizador. La compa-ració amb un derivat ciclobutánic va mostrar que la presència del nitrògen en el anell de quatre membres disminuïx el potencial redox facilitant el procés d'oxidació. En tercer lloc, el pas de ciclorreversió s'ha estudiat amb dos fotosensibilizadors intrínsecs, guanina i el dany oxidatiu 8-oxoguanina (OG) , units covalentment a un CPD o a un oxetano, com a intermedi del dany (6-4) . En conjunt, les dades d'espec-troscòpia i anàlisi cromatogràfica van mostrar la possibilitat que estos fotosensibiliza-dors endògens poden actuar com a donadors d'electrons mimetitzant, per tant, la fun-ció del cofactor flavina en la fotoliasa. Finalment, l'anell d'azetidina ha sigut incorporat en un oligonucleotid per a estu-diar la seua ciclorreversió per mitjà de una transferència electrònica. Basant-se en els resultats dels capítols previs, OG ha sigut triat com un fotorreductor natural. En un primer pas, una metodologia ha sigut desenvolupada per a inserir l'azetidina dins d'una seqüència d'oligonucleòtid. Després, la irradiació en estat estacionari del dúplex que conté OG i l'azetidina ha demostrat que la transferència d'electrons té lloc i comporta a la ciclorreversió de l'heterocicle. A més, experiments preliminars han sigut duts a terme per a avaluar la reparació de l'anell de quatre membres, com un anàleg del in-termedi en la reparació del fotoproducte (6-4) , per les fotoliases reals CPD i (6-4).
[EN] Ultraviolet radiation is associated with the formation of certain lesions in the DNA that are at the origin of skin cancer. Among the most relevant are the damages that occur at pyrimidine bases: cyclobutane dimers (CPDs) and (6-4) photoproducts (6-4) (6-4PPs). To obtain protection from DNA photolesions, living organisms have enzymes that restore the lesions to their original form, thus maintaining genetic integ-rity. In some organisms, CPDs and 6-4PPs show an additional repair process, which corresponds to photoreactivation and involves enzymes called CPD and (6-4) photol-yases. In particular, there is currently a lively discussion about the mechanism of repair by (6-4) photolyase. The general objective of this doctoral thesis has been to study the cycloreversion of the proposed intermediate of 6-4PP lesions as a key to support one of the mechanisms proposed so far. In a first place, a model of the intermediate azetidine of the 6-4PP lesion for TC sequences was prepared to investigate its repair by means of electron donation by photosensitizers with suitable redox potential, mimicking the flavin cofactor of the (6-4) photolyase. Electrochemical, spectroscopic, analytical measurements as well as computational studies showed that the injection of an electron into the azetidine ring leads to a cycloreversion of the bipyrimidine azetidine to the thymine and 6-azauracil bases. It has also been shown that electron transfer only takes place if the thymine component is present in the model. Secondly, the cycloreversion of azetidine has been investigated by means of an oxidative process in which the azetidine ring donates an electron to the photosensitiz-er. The comparison with a cyclobutane derivative showed that the presence of the nitrogen in the four-membered ring decreases the redox potential, facilitating thus the oxidation process. Third, the cycloversion step has been studied with two intrinsic photosensitizers, guanine and the oxidatively generated damage 8-oxoguanine (OG), covalently bound to a CPD or to an oxetane, as a model for the intermediate of 6-4PP repair. Altogeth-er, the spectroscopic and analytical data showed that these endogenous photosensitiz-ers can act as electron donors mimicking, thus, the function of the flavin cofactor in photolyase. Finally, azetidine ring has been incorporated in an oligonucleotide to study its cy-cloreversion by electron transfer. Based on the results of the previous chapter, OG has been chosen as a natural photoreductant. In a first step, a methodology has been developed to insert the azetidine within the oligonucleotide sequence. Then, steady-state irradiation of the duplex containing OG and the azetidine has demonstrated that the electron transfer takes place and leads to the cycloreversion of the heterocycle. In addition, preliminary experiments have been carried out to evaluate the repair of this four-membered ring, as an analog to the (6-4) photoproduct intermediate, by real CPD and (6-4) photolyases.
Fraga Timiraos, AB. (2019). Model Studies on the Photorepair of (6-4) Dimeric Lesions of DNA [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/117610
TESIS

Thesis (Ph.D.)--University of Florida, 2004.
Typescript. Title from title page of source document. Document formatted into pages; contains 117 pages. Includes Vita. Includes bibliographical references.

Bevor sich eine Zelle teilt muss sie ihr gesamtes genetisches Material verdoppeln. Eukaryotische Genome werden von einer Vielzahl von Replikationsstartpunkten, den sogenannten Origins, aus repliziert, die über das gesamte Genome verteilt sind. In dieser Arbeit wird der zugrundeliegende molekulare Mechanismus quantitativ analysiert, der für die nahezu simultane Initiierung der Origins exakt ein Mal pro Zellzyklus verantwortlich ist. Basierend auf umfangreichen experimentellen Studien, wird zunächst ein molekulares regulatorisches Netzwerk rekonstruiert, welches das Binden von Molekülen an die Origins beschreibt, an denen sich schließlich komplette Replikationskomplexe (RKs) bilden. Die molekularen Reaktionen werden dann in ein Differentialgleichungssystem übersetzt. Um dieses mathematische Modell zu parametrisieren, werden gemessene Proteinkonzentrationen als Anfangswerte verwendet, während kinetische Parametersätze in einen Optimierungsverfahren erzeugt werden, in welchem die Dauer, in der sich eine Mindestanzahl von RKs gebildet hat, minimiert wird. Das Modell identifiziert einen Konflikt zwischen einer schnellen Initiierung der Origins und einer effizienten Verhinderung der DNA Rereplikation. Modellanalysen deuten darauf hin, dass eine zeitlich verzögerte Origininitiierung verursacht durch die multiple Phosphorylierung der Proteine Sic1 und Sld2 durch Cyclin-abhängige Kinasen, G1-Cdk bzw. S-Cdk, essentiell für die Lösung dieses Konfliktes ist. Insbesondere verschafft die Mehrfach-Phosphorylierung von Sld2 durch S-Cdk eine zeitliche Verzögerung, die robust gegenüber Veränderungen in der S-Cdk Aktivierungskinetik ist und außerdem eine nahezu simultane Aktivierung der Origins ermöglicht. Die berechnete Verteilung der Fertigstellungszeiten der RKs, oder die Verteilung der Originaktivierungszeiten, wird auch genutzt, um die Konsequenzen bestimmter Mutationen im Assemblierungsprozess auf das Kopieren des genetischen Materials in der S Phase des Zellzyklus zu simulieren.
Before a cell divides it has to duplicate its entire genetic material. Eukaryotic genomes are replicated from multiple replication origins across the genome. This work is focused on the quantitative analysis of the underlying molecular mechanism that allows these origins to initiate DNA replication almost simultaneously and exactly once per cell cycle. Based on a vast amount of experimental findings, a molecular regulatory network is constructed that describes the assembly of the molecules at the replication origins that finally form complete replication complexes. Using mass–action kinetics, the molecular reactions are translated into a system of differential equations. To parameterize the mathematical model, the initial protein concentrations are taken from experimental data, while kinetic parameter sets are determined using an optimization approach, in particular a minimization of the duration, in which a minimum number of replication complexes has formed. The model identifies a conflict between the rapid initiation of replication origins and the efficient inhibition of DNA rereplication. Analyses of the model suggest that a time delay before the initiation of DNA replication provided by the multiple phosphorylations of the proteins Sic1 and Sld2 by cyclin-dependent kinases in G1 and S phase, G1-Cdk and S-Cdk, respectively, may be essential to solve this conflict. In particular, multisite phosphorylation of Sld2 by S-Cdk creates a time delay that is robust to changes in the S-Cdk activation kinetics and additionally allows the near-simultaneous activation of multiple replication origins. The calculated distribution of the assembly times of replication complexes, that is also the distribution of origin activation times, is then used to simulate the consequences of certain mutations in the assembly process on the copying of the genetic material in S phase of the cell cycle.

The ICRP recommends a radiation weighting factor of one for all low-LET radiation. However, many experimental studies find inconsistencies between low-LET RBE and the ICRP's current radiation weighting factor. Generally, there is evidence that dependence exists between radiation energy and radiation RBE where lower energy radiations tend to have a greater biological effect than higher energy radiation. Specifically, the radiations of tritium and carbon K-shell x-rays have been studied in numerous experiments and the biological effects of both of these radiations are consistently greater than that of Co-60. In this work, the relationship between radiation energy and radiation effect has been investigated with the use of a newly developed double strand break (DSB) yield estimation algorithm. This algorithm makes use of a detailed solenoidal 30 nm DNA chromatin model to describe the radiation-sensitive biological target. In addition to the DNA model, NOREC, an event by event Monte Carlo code, was used in this algorithm to characterize the electron track. As an alternative to the conventional approach of computationally simulating DNA damage by spatial overlay of an electron track on DNA, this algorithm instead focuses on quantifying the distance between ionizations in an electron track and next determining the likelihood that any given ionization pair forms a DSB. The first step of the algorithm involves electron characterization while the second step relies on DNA molecule characterization. By assuming a DSB biological endpoint and determining the DSB yield as a function of electron energy, energy dependent RBE values were estimated for monoenergetic electrons from 10 eV to 1 MeV. Photon RBE values, x-ray RBE values and radionuclide RBE values were also calculated and reported in this work in addition to electron RBE values. Photon RBE values were estimated based upon the electron RBE calculation. Photon RBE values were reported from 1 eV to 10 MeV. In turn, x-ray RBE values were calculated based upon photon values for several tube voltage and filter combinations. Finally, RBE values for over 1000 radionuclides were estimated and reported.

Made available in DSpace on 2015-03-26T13:35:22Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1403023 bytes, checksum: a018f8a9517a70e3f44eed507700561d (MD5) Previous issue date: 2013-07-25
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
By performing single molecule stretching experiments with optical tweezers, we have stu- died the DNA interaction with the ligand Hoechst(33258). The mechanical properties of the complexes as a function of ligand concentration were directly determined from these measure- ments by fitting the force versus extension curve to the WormLike Chain model of semiflexible polymers. In addition, the physico-chemical parameters of the interaction were extracted from the persistence length data by using a previously developed two-sites quenched disorder sta- tistical model, allowing the determination of the binding isotherm. This model has allowed us to decouple the two different binding modes present in this system. In particular, it was found that the binding isotherm consists of two Hill-type processes, one non-cooperative and the other strongly cooperative. Finally, DNA condensation due to the interaction with the ligand was also verified and characterized here by analyzing the apparent contour length of the complexes.
Neste trabalho, fizemos experimentos de estiramentos em moléculas únicas com pinça ótica e estudamos a interação do DNA com o ligante Hoechst(33258). As propriedades mecânicas dos complexos formados como uma função da concentracao do ligante, foram diretamente determinadas a partir destas medidas, por ajuste das curvas de forca por extensão pelo modelo da cadeia vermiforme (WLC), de polímeros semiflexíveis. Além disso, os parâmetros físico-químicos da interação foram extraídos dos dados do comprimento de persistência usando um modelo estatístico de desordem de dois sítios previamente desenvolvido, permitindo a obtenção da isoterma de ligação. A aplicação do modelo nos permitiu decompor os dois modos de ligação presentes neste sistema. Em particular, encontramos que a isoterma de ligação consiste de dois processos tipo Hill, um não cooperativo e o outro fortemente cooperativo. Finalmente a condensação do DNA devido a interação com o ligante foi também verificada e caracterizada aqui por análise do comprimento de contorno aparente do complexo.

This thesis discusses the development of software architecture to support the computational investigation of random polygons in 3 space. The random polygons themselves are a simple model of long polymer chains. (A DNA molecule is one example of a polymer.) This software architecture includes "building blocks" which specify the actual manipulations and computations to be performed, and a structural framework which allows the user to specify which manipulations/computations to perform, in which order and with how many repetitions. The overall framework is designed in such a way that new building blocks can easily be added in the future. The development of three different building blocks to be used in this architecture which are entitled: Reducer, Lengthener and OutsideInLengthener are also discussed in this thesis. These building blocks manipulate the existing polygons - increasing or decreasing their size.

This research studied the Anderson localization of electrons in two-channel wires with correlated disorder and in DNA molecules. It involved an analytical calculation part where the formula for the inverse localization length for electron states in a two-channel wire is derived. It also involved a computational part where the localization length is calculated for some DNA molecules. Electron localization in two-channel wires with correlated disorder was studied using a single-electron tight-binding model. Calculations were within second-order Born-approximation to second-order in disorder parameters. An analytical expression for localization length as a functional of correlations in potentials was found. Anderson localization in DNA molecules were studied in single-channel wire and two-channel models for electron transport in DNA. In both of the models, some DNA sequences exhibited delocalized electron states in their energy spectrum. Studies with two-channel wire model for DNA yielded important link between electron localization properties and genetic information.