The genomes of all eukaryotic organisms are packaged into chromatin, the fundamental unit of which is the nucleosome. Since the proposal of the nucleosome as the primary repeating unit of chromatin structure in 1974, it has become clear that the biological roles of nucleosomes extend far beyond simple DNA packaging and include virtually all processes involving the genome. Despite the integral roles of nucleosomes in many fundamental biological processes, the relative contributions of the cellular cues and sequence features that directly govern their arrangement on genomic DNA remain unclear. In this Thesis, I characterise the sequence preferences of nucleosomes using data sets derived from genome-wide studies. I describe the analysis of data derived from the first genome-wide map of in vivo nucleosome occupancy across a eukaryotic genome (in this case, the budding yeast, Saccharomyces cerevisiae). Using these data, I construct a sequence-based linear model of nucleosome occupancy that takes into account structural features of DNA (which correlate with simple base composition) as well as transcription factor (TF) binding site information, which has significant ability to predict nucleosome occupancy in vivo. I go on to test particular aspects of this model and show that genetic perturbation of TFs that the in vivo model deems important (Abf1, Reb1, and Rsc3) have the expected effects, an increase in nucleosome occupancy over their cognate binding sites as well as a reduction in transcription from the corresponding genes, suggesting that these factors are required for promoter function and definition. I also confirm that in vitro nucleosome occupancy correlates highly with sequence features important for nucleosome occupancy in vivo and go on to develop a simple model for nucleosome occupancy based solely on histone-DNA interactions. This model suggests that base composition (G+C content) is a dominant feature in determining intrinsic nucleosome occupancy. Finally, I apply a model of intrinsic nucleosome occupancy to the human genome and show that there is a fundamental difference in intrinsic nucleosome occupancy at regulatory regions across species. This finding illustrates a potential functional consequence of variation in base composition in eukaryotic genomes.

The genomes of all eukaryotic organisms are packaged into chromatin, the fundamental unit of which is the nucleosome. Since the proposal of the nucleosome as the primary repeating unit of chromatin structure in 1974, it has become clear that the biological roles of nucleosomes extend far beyond simple DNA packaging and include virtually all processes involving the genome. Despite the integral roles of nucleosomes in many fundamental biological processes, the relative contributions of the cellular cues and sequence features that directly govern their arrangement on genomic DNA remain unclear. In this Thesis, I characterise the sequence preferences of nucleosomes using data sets derived from genome-wide studies. I describe the analysis of data derived from the first genome-wide map of in vivo nucleosome occupancy across a eukaryotic genome (in this case, the budding yeast, Saccharomyces cerevisiae). Using these data, I construct a sequence-based linear model of nucleosome occupancy that takes into account structural features of DNA (which correlate with simple base composition) as well as transcription factor (TF) binding site information, which has significant ability to predict nucleosome occupancy in vivo. I go on to test particular aspects of this model and show that genetic perturbation of TFs that the in vivo model deems important (Abf1, Reb1, and Rsc3) have the expected effects, an increase in nucleosome occupancy over their cognate binding sites as well as a reduction in transcription from the corresponding genes, suggesting that these factors are required for promoter function and definition. I also confirm that in vitro nucleosome occupancy correlates highly with sequence features important for nucleosome occupancy in vivo and go on to develop a simple model for nucleosome occupancy based solely on histone-DNA interactions. This model suggests that base composition (G+C content) is a dominant feature in determining intrinsic nucleosome occupancy. Finally, I apply a model of intrinsic nucleosome occupancy to the human genome and show that there is a fundamental difference in intrinsic nucleosome occupancy at regulatory regions across species. This finding illustrates a potential functional consequence of variation in base composition in eukaryotic genomes.

Homologous genetic recombination occurs during the life cycle of virtually every organism Genetic studies especially in prokaryotes and fungi have defined the rules of recombination, led to the characterization of alternate pathways and to the development of molecular models The biochemistry of homologous genetic recombination has advanced most productively in bacteria and fungi due to the extensive genetic understanding of these organisms The identification of mutants defective in homologous recombination, purification and characterization of the gene products that participate in recombination has brought the ultimate goal of reconstituting a cell-k free system for Eschenchia coli, at least with naked DNA substrates, closer to reality.

In the eukaryotic nucleus, an immense length of DNA is compactly packaged to generate an ordered three-dimensional hierarchical structure called chromatin (van Holde, 1988; Wolffe, A.P, 1998). This organization forms a template for various DNA transaction processes like replication, transcription, recombination etc. The different stages of organization of the chromatin finally results in the 10,000-fold compaction observed in the metaphase chromosome. The problem of how the fibres of chromatin are folded has interested biologists and biochemists for decades. It has long been recognized that the Histones play a major part in this folding. However, the distinctly different roles of the Histones H2A, H2B, H3 and H4 on one hand and the lysine rich Histones such as Histone H1 and its cognates on the other, were not understood until after the discovery of the nucleosomes in the early 1970s. Some of the early insights into the structure of chromatin came through the digestion of nuclear chromatin with calcium-dependent endonucleases like micrococcal nuclease. A repeating kinetic intermediate of about 200 bp of DNA with Histones was obtained (Simpson, 1978). Based on repeating pattern of micrococcal nuclease digested chromatin and structural studies, Kornberg (1974) proposed that chromatin is composed of a flexible chain of repeating units of 100 A0 diameter. These units were termed as "nucleosomes" (Oudet et al, 1975). It then became clear that the Histones H2A, H2B, H3 and H4 were constituents of the nucleosome core particle whereas the lysine rich Histone H1 was somehow associated with the linker DNA between core particles. Hence, the formers are called core Histones and the latter as linker Histones. On further digestion of nucleosome, a nucleosome core was obtained in which wrapping of 146 bp of DNA about the Histone octamer to form the core particle provided the first level of folding. Electron microscopy and X-ray diffraction techniques suggested that this particle is a disk, 57 A0 thick and 110 A0 in diameter, and that the DNA is wound around the Histone core (Finch et al, 1977), But this cannot account for the many thousand-fold condensation of the DNA in the eukaryotic nucleus. The "string of beads" structure observed obviously could not satisfy the compaction requirement. It soon became evident that there exists some level of higher order folding of the chromatin fiber. In a classical paper, Finch and Klug (1976), showed that the extended nucleosomal filaments condense into irregular fibers of about 30 nm diameter in the presence of low concentrations of Mg 2+. Based on the data from earlier structural studies, these authors proposed a solenoid model in which nucleosomes were wrapped into a regular helix with a pitch of about 11nm. Later, it was observed that the formation of well defined fibers requires the presence of lysine rich Histones such as Histone H1.

The rise of high-throughput scientific experimentation and data collection has introduced new classes of statistical and computational challenges. The technologies driving this data explosion are subject to complex new forms of measurement error, requiring sophisticated statistical approaches. Simultaneously, statistical computing must adapt to larger volumes of data and new computational environments, particularly parallel and distributed settings. This dissertation presents several computational and theoretical contributions to these challenges. In chapter 1, we consider the problem of estimating the genome-wide distribution of nucleosome positions from paired-end sequencing data. We develop a modeling approach based on nonparametric templates that controls for variability due to enzymatic digestion. We use this to construct a calibrated Bayesian method to detect local concentrations of nucleosome positions. Inference is carried out via a distributed HMC algorithm that scales linearly in complexity with the length of the genome being analyzed. We provide MPI-based implementations of the proposed methods, stand-alone and on Amazon EC2, which can provide inferences on an entire S. cerevisiae genome in less than 1 hour on EC2. We then present a method for absolute quantitation from LC-MS/MS proteomics experiments in chapter 2. We present a Bayesian model for the non-ignorable missing data mechanism induced by this technology, which includes an unusual combination of censoring and truncation. We provide a scalable MCMC sampler for inference in this setting, enabling full-proteome analyses using cluster computing environments. A set of simulation studies and actual experiments demonstrate this approach's validity and utility. We close in chapter 3 by proposing a theoretical framework for the analysis of preprocessing under the banner of multiphase inference. Preprocessing forms an oft-neglected foundation for a wide range of statistical and scientific analyses. We provide some initial theoretical foundations for this area, including distributed preprocessing, building upon previous work in multiple imputation. We demonstrate that multiphase inferences can, in some cases, even surpass standard single-phase estimators in efficiency and robustness. Our work suggests several paths for further research into the statistical principles underlying preprocessing. / Statistics

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 110-128).

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Sepsis is defined as a life-threatening organ dysfunction that results in systemic activation of coagulation and inflammation in response to microbial infection. Neutrophil extracellular traps (NETs) have shown to be an important interface between innate immunity and coagulation in sepsis. The major structural components of NETs are nucleosomes (DNA-histone complexes). Although nucleosomes do not modulate coagulation, there are conditions where DNA and histones dissociate from each other in the circulation (e.g. in the presence of heparan sulfate or therapeutic heparin binding histones, or DNase digestion of DNA). In vitro, purified DNA was reported to activate coagulation, but this procoagulant activity has been questioned due to isolation methods that yield DNA that is contaminated with other procoagulant molecules. On the other hand, histones have been shown to not only activate coagulation but are cytotoxic to endothelial cells. However, their contribution to the pathogenesis of sepsis has yet to be determined in an in vivo model. Understanding the contribution of DNA, histones, and nucleosomes to the pathogenesis of sepsis may allow us to develop novel therapies that may prove targeting multiple components of NETs (i.e. DNA and histones) may be beneficial. Consequently, in this thesis, we (1) identified methods of DNA purification that produce DNA that is free of contamination and confirmed the procoagulant properties of the isolated DNA, (2) determined the harmful effects of DNA, histones, and nucleosomes cytotoxicity, coagulation, and inflammation in vitro and in vivo, (3) and then we explored the possibility of targeting both DNA and histones using a combination approach of DNase I and heparin in a mouse model of sepsis. Since heparin is administered to patients as a thromboprophylaxis and DNase I is a potential therapy in sepsis, it is important to understand any potential drug-drug interactions. / Thesis / Doctor of Philosophy (PhD) / Sepsis is a type of blood poisoning that occurs when the body has an over reactive response to an infection. This can lead to tissue damage, organ failure, and death. Sepsis is recognized as a global health priority. The death rate from sepsis is high between 15% to 30%, suggesting that an improved understanding of how sepsis leads to death may develop into new therapies. Recently, it was discovered that high levels of free-floating DNA and histones in the blood can predict death in sepsis. The DNA and histones are likely released by white blood cells in response to trying to fight off the infection. In test tubes, free-floating DNA can trigger clotting of blood. DNA often exists in blood together with histones. In test tubes, histones can kill blood vessels and make blood thicker. However, no one has confirmed that DNA and/or histones are harmful to mammals and contributes to death in sepsis. Some new studies show that getting rid of DNA with injections of DNase I minimally increases survival in mice. Other studies show that removing histones with a treatment called heparin shows a small increase in survival in mice. Heparin is also a blood thinner and decreases inflammation. No one knows if these drugs used together can improve sepsis survival. Because both drugs on their own show some survival improvement in sepsis, perhaps using them together will cure sepsis. This thesis has three objectives: (1) to confirm the clotting properties of free-floating DNA, (2) to find out if DNA and/or histones contributes to death in sepsis, and (3) if using a combination of DNase I and heparin can cure sepsis in a mouse model. Finding new therapies for sepsis can save millions of people's lives and decrease the financial burden on society and healthcare systems.

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