Maternal binge alcohol consumption has been linked to congenital birth defects in the fetus. Said defects include abnormalities in heart development, a category of disease referred to as Congenital Heart Disease. Given the prevalence of Congenital Heart Disease, with a study showing around 49.9% of women having at least participated in binge alcohol consumption at least once during the early stages of their pregnancy and Congenital Heart Disease being linked to various complications in adulthood, this is a topic relevant to the clinical setting. Alcohol consumption has been linked to decreases in DNA methylation, which generally increases transcriptional expression of nearby genes. This thesis will focus on how alcohol affects the genomic-wide epigenetics of the embryonic heart with the aim of identifying specific genes and sites within those genes that are affected by alcohol exposure in utero. We hypothesize that embryonic mouse hearts exposed to ethanol will show a differential methylation pattern characteristic of hypomethylation versus control hearts not exposed to ethanol. To test this hypothesis, we used oral gavage to administer ethanol to pregnant mice at embryonic age E9.5 (a time associated with heart chamber formation). Maternal mice were sacrificed at E11.5, embryonic hearts were removed, and DNA was extracted for further experimentation with whole genome bisulfite sequencing. Analysis of whole genome bisulfite sequencing data showed a slight trend towards hypomethylation but suggested no significant changes in the overall methylation pattern in embryonic mouse hearts at the genomic level, but we have independently identified several genes whose expression is depressed in the embryonic mouse following a single maternal binge ethanol dose at E9.5, and thus we are investigating potential alcohol-induced DNA methylation alterations in specific target genes of interest. Future investigations into gene and site-specific DNA methylation profiles as well as other epigenetic modifications should prove useful in our quest to learn how maternal alcohol consumption causes cardiac malformations leading to congenital heart disease.

No description available.

Epitranscriptomics, the study of RNA modifications, has become a hotspot of research over the last decade. Over 170 unique modifications have been discovered with a widespread occurrence in a diverse range of RNAs. 5-methylcytosine, m5C, is an evolutionarily conserved and reversable modification that regulates the stability and export of tRNAs, rRNAs, and mRNAs. m5C has recently been implicated in many biological phenomena including tumorigenesis, embryonic cell expansion and differentiation, brain development, and neuronal functions. While we are just beginning to understand the functions of m5C, a gold standard of m5C detection has yet to be established due to the low signal-to-noise presence of m5C. In this work, we utilize RNA bisulfite sequencing as a transcriptome-wide approach to understand the computational and chemical parameters needed to optimize m5C discovery in the mitochondria and the developing brain. In Chapter 1, we systematically evaluate four preparation conditions of bisulfite sequencing to identify potential presence of m5C-mRNAs localized to the mitochondria in neuronal stem cells. In tandem, we utilize unique molecular identifiers and a consortium of control template transcripts to evaluate sources of false positive m5C sites that may emerge from sequencing errors, PCR amplification, and the inadequate bisulfite conversion of transcripts. While improvements to mitochondrial transcript bisulfite conversion and false positive filtering were observed, no mitochondrial mRNAs were identified to be methylated, indicating no or very few methylated cytosines in mitochondrial mRNAs and the need for improved chemical methods to detect mitochondrial m5C-mRNAs if any. In Chapter 2, we employ the computational approaches established in Chapter 1 to survey the m5C landscape of the developing mammalian brain. We discover a general increase in unique m5C sites in mouse whole brain tissue when compared to neuronal cell cultures. Of these sites, we found the post-natal day 0 and 17 brain time points to undergo significant methylation level changes in comparison to the 6-week-old brain. These differentially methylated sites were significantly enriched for brain development, synaptic development, and transcriptional control gene network pathways. In Chapter 3, we expand on our findings in Chapter 2 to understand the impact of m5C reader FMRP and m5C eraser TET1 loss in the mouse post-natal day 17 brain. Among a set of m5C sites identified in wildtype or knockout samples, few were differentially methylated after protein ablation, suggesting m5C may rely on compensatory enzymes. Using FMRP-RNA pulldown assays to validate FMRP binding positions, we identified Ralbp1 to be hypermethylated and overexpressed in Fmr1-KO brain tissues. RalBP1 is a binding protein responsible for the endocytosis of AMPA receptors, a process critical for neuronal long term depression and brain development. / Doctor of Philosophy / Ribonucleic acid (RNA) is the product of deoxyribonucleic acid (DNA) transcription and the precursor to protein translation. Chemical modifications can be made to the bases of DNA, known as epigenetic modifications, to elicit new functions and responses to the environment. Epitranscriptomics refers to the study of RNA modifications that also serve unique roles and functions depending on the type of modification made. Here, we study the presence of 5-methylcytosine, a methyl group added to the cytosine (C) base of RNA. This modification is found throughout all branches of life and is known to promote the stability and export of many RNA types. Recently, studies have utilized many techniques including RNA bisulfite sequencing to find links between the presence of m5C-RNAs and cancer progression, stem cell development, and brain development. RNA bisulfite sequencing uses chemical applications to convert non-methylated "C"s to the RNA base "U", while retaining a "C" signature on methylated "C"s. However, due to the extremely low presence of RNA-m5C in comparison to DNA-m5C, sources of noise make it difficult to identify a true m5C signal. Because of this discrepancy, established analytical methods based on DNA biology may not be suitable for RNA analysis. To address shortcomings in current detection methods of RNA-m5C, we performed systematic analysis of 1) different preparation methods for improved m5C detection methods and 2) computational approaches for the filtering of false positive m5C sites, as described in Chapter 1. To achieve these goals, we expanded the breadth of analytical methods by including unique molecular identifiers and expanding the set of control RNA sequences to better grasp how false positive sites might be introduced into non-methylated sequences. While noticeable improvements were made to control RNA sequence false positive detection, we found that most mitochondrial RNAs did not carry the same m5C signatures as RNAs from other sources. Because of this difference, we could not conclude that mitochondrial mRNAs were methylated. Therefore, we suggest that future studies may need to develop better or alternative methods for the detection of mitochondrial RNA-m5Cs. In Chapters 2 and 3, we utilize the computational methods developed in Chapter 1 to understand how m5C levels change throughout the development of a mouse's brain. By investigating the m5C profiles of mouse newborn, young child, and juvenile brains, we found significant changes in m5C levels specific to certain RNAs. These RNAs are associated with neuronal growth, development, and maturation, which may have implications for m5C's role in cognitive development, intellectual disabilities, and neurodegenerative disorders. To discover if these RNAs could be affected by the absence of m5C-specific proteins, we created mice deficient in a protein m5C reader, FMRP, and an m5C eraser protein, TET1. Interestingly, we did not find a significant difference in mice deficient in the proteins, indicating m5C may rely on multiple proteins to serve redundant functions. However, one RNA, Ralbp1, was found to be significantly methylated in FMRP deficient models. This RNA is essential for developmental changes in the brain as well as neuronal growth and could be an interesting target for future research.

RNA epigenetics or Epitranscriptomics has emerged as a new field for understanding the post-transcriptional regulation of gene expression by RNA modifications. Among numerous types of RNA modifications, RNA cytosine-5 methylation (5-mrC) is recognized as an important epitranscriptomic mark that modulates mRNA transportation, stability and translation. In chapter 1, we summarize the currently available approaches to detect 5-mrC modification at global, transcriptome-wide and locus-specific levels, and compare the corresponding advantages and disadvantages of the techniques. We further focus on the bioinformatics data analysis of RNA bisulfite sequencing datasets by comparing existing packages with respect to key parameters for alignment and methylation calling and filtering of potentially false positive 5-mrC sites. To investigate the dynamic regulation of 5-mrC modification, as described in chapter 2, we adopt a widely used neuronal activity model, and perform RNA sequencing (RNA-seq) and RNA bisulfite sequencing (RNA BS-seq) to profile gene expression as well as transcriptome-wide 5-mrC modification. We have identified distinct gene expression profiles and differentially methylated 5-mrC sites (DMS) in neurons upon activation, and the genes with DMS sites are enriched with mitochondrial and synaptic functions. Moreover, it reveals a negative correlation between RNA methylation and mRNA expression in mouse cortical neurons during neuronal activity. Thus, these findings identify the dynamic regulation of 5-mrC modification during neuronal activity and reveal a potential link between RNA methylation and mRNA expression. In chapter 3, we investigate the effect of folate, a methyl-donor, on RNA cytosine-5 methylation (5-mrC) modification in adult mouse neural stem cells (NSCs). Compared to the control, NSCs cultured in folate deficiency or supplementation condition have shown no changes in mRNA expression, but significant changes in mRNA translation efficiency. RNA bisulfite sequencing of both total and polysome poly(A) RNA samples shows distinct 5-mrC profiles in NSCs treated with different concentrations of folic acid. It also shows consistent hypermethylation in polysome mRNAs than that in total mRNAs. This study presents the comprehensive influence of folate deficiency and supplementation on RNA cytosine-5 methylation and mRNA translation. / Doctor of Philosophy / RNA epigenetics, a collection of RNA modifications, has recently emerged as an exciting, new field for understanding post-transcriptional regulation of gene expression. RNA cytosine-5 methylation (5-mrC) is one of the most well-known RNA modifications that modulates mRNA export, stability and translation. In the first chapter, we summarize the currently available methods for the measurement of 5-mrC modification. We highlight one of the techniques, RNA bisulfite sequencing (RNA BS-seq) and focus on the bioinformatics data analysis of RNA BS-seq datasets. We have compared several existing tools in regard of the key parameters in data analysis. In the second chapter, we adopt a widely used neuronal activity model to study the dynamic regulation of RNA cytosine-5 methylation (5-mrC). We perform RNA-seq and RNA BS-seq in neurons in response to stimulation. We have identified numerous differentially expressed genes and differentially methylated 5-mrC sites in activated neurons and find that these DMS-related genes are associated with mitochondrial and synaptic functions. Furthermore, we identify a negative correlation between RNA methylation and mRNA expression, indicating a potential role of 5-mrC modification in the regulation of mRNA expression. In the third chapter, we investigate the influence of a nutrient supplement, folic acid, on 5-mrC modification in adult mouse neural stem cells. Compared to the control, NSCs cultured in folate deficiency or supplementation condition have shown no changes in mRNA expression, but significant changes in mRNA translation efficiency. We perform RNA bisulfite sequencing of both total poly(A) RNA samples and polysome poly(A) RNA samples. We identify distinct 5-mrC profiles in NSCs treated with different concentrations of folic acid. It shows consistent hypermethylation in polysome mRNAs than that in total mRNAs. This study presents the comprehensive influence of folate deficiency and supplementation on RNA cytosine-5 methylation and mRNA translation.

Microfluidics has revolutionized how molecular biology studies are conducted. It permits profiling of genomic and epigenomic features for a wide range of applications. Microfluidics has been proven to be highly complementary to NGS technology with its unique capabilities for handling small volumes of samples and providing platforms for automation, integration, and multiplexing. In this thesis, we focus on three projects (diffusion-based PCR, MID-RRBS, and SurfaceChIP-seq), which improved the sensitivities of conventional assays by coupling with microfluidic technology. MID-RRBS and SurfaceChIP-seq projects were designed to profiling genome-wide DNA methylation and histone modifications, respectively. These assays dramatically improved the sensitivities of conventional approaches over 1000 times without compromising genomic coverages. We applied these assays to examine the neuronal/glial nuclei isolated from mouse brain tissues. We successfully identified the distinctive epigenomic signatures from neurons and glia. Another focus of this thesis is applying electrical field to investigate the intracellular contents. We report two projects, drug delivery to encapsulated bacteria and mRNA extraction under ultra-high electrical field intensity. We envision rapid growth in these directions, driven by the needs for testing scarce primary cells samples from patients in the context of precision medicine. / Ph. D.

Large-scale bisulfite treatment and short reads sequencing technology allows comprehensive estimation of methylation states of Cs in the genomes of different tissues, cell types, and developmental stages. Accurate characterization of DNA methylation is essential for understanding genotype phenotype association, gene and environment interaction, diseases, and cancer. The thesis work first evaluates the performance of several commonly used bisulfite short read mappers and investigates how pre-processing data might affect the performance. Aligning bisulfite short reads to a reference genome remains a challenging task. In practice, only a limited proportion of bisulfite treated DNA reads can be mapped uniquely (around 50-70%) while a significant proportion of reads (called multireads) are aligned to multiple genomic locations. The thesis outlines a strategy to improve the mapping efficiencies of the existing bisulfite short reads software by finding unique locations for multireads. Analyses of both simulated data and real hairpin bisulfite sequencing data show that our strategy can effectively assign approximately 70% of the multireads to their best locations with up to 90% accuracy, leading to a significant increase in the overall mapping efficiency. The most common and essential downstream task in DNA methylation analysis is to detect differential methylated cytosines (DMCs). Although many statistical methods have been applied to detect DMCs, inconsistency in detecting differential methylated sites among statistical tools remains. We adapt the wavelet-based functional mixed models (WFMM) to detect DMCs. Analyses of simulated Arabidopsis data show that WFMM has higher sensitivities and specificities in detecting DMCs compared to existing methods especially when methylation differences are small. Analyses of monozygotic twin data who have different pain sensitivity also show that WFMM can find more relevant DMCs related to pain sensitivity compared to methylKit. In addition, we provide a strategy to modify the default settings in both WFMM and methylKit to be more tailored to a given methylation profile, thus improving the accuracy of detecting DMCs. Population growth and climate change leave billions of people around the world living in water scarcity conditions. Therefore, utility of reclaimed water (treated wastewater) is pivotal for water sustainability. Recently, researchers discovered microbial regrowth problems in reclaimed water distribution systems (RWDs). The third part of the thesis involves: 1) identifying fundamental conditions that affect proliferation of antibiotic resistance genes (ARGs), 2) identifying the effect of water chemistry and water age on microbial regrowth, and 3) characterizing co-occurrence of ARGs and/or mobile genetics elements (MGEs), i.e., plasmids in simulated RWDs. Analyses of preliminary results from simulated RWDs show that biofilms, bulk water environment, temperature, and disinfectant types have significant influence on shaping antibiotic resistant bacteria (ARB) communities. In particular, biofilms create a favorable environment for ARGs to diversify but with lower total ARG populations. ARGs are the least diverse at 300C and the most diverse at 220C. Disinfectants reduce ARG populations as well as ARG diversity. Chloramines keep ARG populations and diversity at the lowest rate. Disinfectants work better in bulk water environment than in biofilms in terms of shaping resistome. Network analysis on assembly data is done to determine which ARG pairs are the most co-occurred. Bayesian network is more consistent with the co-occurrence network constructed from assembly data than the network based on Spearman's correlation network of ARG abundance profiles. / Ph. D.

DNA methylation is an epigenetic mechanism critical for tissue development, cell specification and cellular function. Mammalian brains consist of millions to billions of neurons and glial cells that can be subdivided into many distinct types of cells. We hypothesize that brain methylomes are heterogeneously methylated across different types of cells and the transcription factors play key roles in brain methylome programming. To dissect brain methylome heterogeneity, in Chapter 2, we first focused on the identification of cell-subset specific methylated (CSM) loci which demonstrate bipolar DNA methylation pattern, i.e., hypermethylated in one cell subset but hypomethylated in others. With the genome-scale hairpin bisulfite sequencing approach, we demonstrated that the majority of CSM loci predicted likely resulted from the methylation differences among brain cells rather than from asymmetric DNA methylation between DNA double strands. Importantly, we found that putative CSM loci increased dramatically during early stages of brain development and were enriched for GWAS variants associated with neurological disorder-related diseases/traits. It suggests the important role of putative CSM loci during brain development, implying that dramatic changes in functions and complexities of the brain may be companied by a rapid change in epigenetic heterogeneity. To explore epigenetic regulatory mechanisms during brain development, as described in Chapter 3, we adopted unbiased data-driven approaches to re-analyze methylomes for human and mouse frontal cortices at different developmental stages. We predicted Egr1, a transcriptional factor with important roles in neuron maturation, synaptic plasticity, long-term memory formation and learning, plays an essential role in brain epigenetic programming. We performed EGR1 ChIP-seq and validated that thousands of EGR1 binding sites are with cell-type specific methylation patterns established during postnatal frontal cortex development. More specifically, the CpG dinucleotides within these EGR1 binding sites become hypomethylated in mature neurons but remain heavily methylated in glia. We further demonstrated that EGR1 recruits a DNA demethylase TET1 to remove the methylation marks at EGR1 binding sites and activate downstream genes. Also, we found that the frontal cortices from the knockout mice lacking Egr1 or Tet1 share strikingly similar profiles in both gene expression and DNA methylation. Collectively, the study in this dissertation reveals EGR1 programs the brain methylome together with TET1 during postnatal development. This study also provides new insights into how life experience and neuronal activity may shape the brain methylome. / Ph. D. / DNA methylation is a widespread epigenetic mark on DNA, serving as a “switch” to turn on or off gene expression. It plays essential roles in cellular functions, tissue development. Mammalian brains contain millions to billions of neurons and glial cells, which can be further divided into many different types of cells. We hypothesize that brain cells have different methylation profiles across the genome, and transcriptional factors play important roles in programming methylation in the mammalian brain genome. To study the diversity of methylation profiles across the genomes of different brain cells, in Chapter 2, we first focused on the identification of cell-subset specific methylated (CSM) genomic regions which show bipolar DNA methylation pattern, i.e., hypermethylated in one type of cell but hypomethylated in others. By applying a technique called the genome-scale hairpin bisulfite sequencing to mouse frontal cortices, we demonstrated that the majority of CSM genomic regions predicted likely resulted from the methylation differences among brain cells, rather than from methylation differences between DNA double strands. Surprisingly, we found that these predicted CSM genomic regions increased dramatically during early stages of brain development and were enriched for GWAS variants associated with neurological disorder-related diseases/traits. It suggests the importance of predicted CSM genomic regions, implying that dramatic changes in brain function and structure may be companied by a rapid change in DNA methylation diversity during brain development. To explore underlying epigenetic mechanisms during brain development, as described in Chapter 3, we re-analyzed methylomes for human and mouse frontal cortices at different developmental stages, and predicted Egr1, a transcriptional factor with important roles in neuron maturation, synaptic plasticity, long-term memory formation and learning, plays an essential role in brain methylome programming. We found thousands of EGR1 binding sites showed cell-type specific methylation patterns, and were established during postnatal frontal cortex development. More specifically, the methylation level of these EGR1 binding sites was low in mature neurons but pretty high in glial cells. We further demonstrated that EGR1 recruits a DNA demethylase TET1 to remove the methylation marks at EGR1 binding sites and activate downstream genes. Also, we found that the frontal cortices from the Egr1 knockout or Tet1 knockout mice show strikingly similar profiles in both gene expression and DNA methylation. Collectively, the study in this dissertation reveals EGR1 works together with TET1 to program the brain methylome during postnatal development. This study also provides new insights into how life experience and neuronal activity may shape the brain methylome.

The aim of this thesis is the development and benchmarking of computational methods for the analysis of high-throughput data from tiling arrays and next-generation sequencing. Tiling arrays have been a mainstay of genome-wide transcriptomics, e.g., in the identification of functional elements in the human genome. Due to limitations of existing methods for the data analysis of this data, a novel statistical approach is presented that identifies expressed segments as significant differences from the background distribution and thus avoids dataset-specific parameters. This method detects differentially expressed segments in biological data with significantly lower false discovery rates and equivalent sensitivities compared to commonly used methods. In addition, it is also clearly superior in the recovery of exon-intron structures. Moreover, the search for local accumulations of expressed segments in tiling array data has led to the identification of very large expressed regions that may constitute a new class of macroRNAs. This thesis proceeds with next-generation sequencing for which various protocols have been devised to study genomic, transcriptomic, and epigenomic features. One of the first crucial steps in most NGS data analyses is the mapping of sequencing reads to a reference genome. This work introduces algorithmic methods to solve the mapping tasks for three major NGS protocols: DNA-seq, RNA-seq, and MethylC-seq. All methods have been thoroughly benchmarked and integrated into the segemehl mapping suite. First, mapping of DNA-seq data is facilitated by the core mapping algorithm of segemehl. Since the initial publication, it has been continuously updated and expanded. Here, extensive and reproducible benchmarks are presented that compare segemehl to state-of-the-art read aligners on various data sets. The results indicate that it is not only more sensitive in finding the optimal alignment with respect to the unit edit distance but also very specific compared to most commonly used alternative read mappers. These advantages are observable for both real and simulated reads, are largely independent of the read length and sequencing technology, but come at the cost of higher running time and memory consumption. Second, the split-read extension of segemehl, presented by Hoffmann, enables the mapping of RNA-seq data, a computationally more difficult form of the mapping task due to the occurrence of splicing. Here, the novel tool lack is presented, which aims to recover missed RNA-seq read alignments using de novo splice junction information. It performs very well in benchmarks and may thus be a beneficial extension to RNA-seq analysis pipelines. Third, a novel method is introduced that facilitates the mapping of bisulfite-treated sequencing data. This protocol is considered the gold standard in genome-wide studies of DNA methylation, one of the major epigenetic modifications in animals and plants. The treatment of DNA with sodium bisulfite selectively converts unmethylated cytosines to uracils, while methylated ones remain unchanged. The bisulfite extension developed here performs seed searches on a collapsed alphabet followed by bisulfite-sensitive dynamic programming alignments. Thus, it is insensitive to bisulfite-related mismatches and does not rely on post-processing, in contrast to other methods. In comparison to state-of-the-art tools, this method achieves significantly higher sensitivities and performs time-competitive in mapping millions of sequencing reads to vertebrate genomes. Remarkably, the increase in sensitivity does not come at the cost of decreased specificity and thus may finally result in a better performance in calling the methylation rate. Lastly, the potential of mapping strategies for de novo genome assemblies is demonstrated with the introduction of a new guided assembly procedure. It incorporates mapping as major component and uses the additional information (e.g., annotation) as guide. With this method, the complete mitochondrial genome of Eulimnogammarus verrucosus has been successfully assembled even though the sequencing library has been heavily dominated by nuclear DNA. In summary, this thesis introduces algorithmic methods that significantly improve the analysis of tiling array, DNA-seq, RNA-seq, and MethylC-seq data, and proposes standards for benchmarking NGS read aligners. Moreover, it presents a new guided assembly procedure that has been successfully applied in the de novo assembly of a crustacean mitogenome. / Diese Arbeit befasst sich mit der Entwicklung und dem Benchmarken von Verfahren zur Analyse von Daten aus Hochdurchsatz-Technologien, wie Tiling Arrays oder Hochdurchsatz-Sequenzierung. Tiling Arrays bildeten lange Zeit die Grundlage für die genomweite Untersuchung des Transkriptoms und kamen beispielsweise bei der Identifizierung funktioneller Elemente im menschlichen Genom zum Einsatz. In dieser Arbeit wird ein neues statistisches Verfahren zur Auswertung von Tiling Array-Daten vorgestellt. Darin werden Segmente als exprimiert klassifiziert, wenn sich deren Signale signifikant von der Hintergrundverteilung unterscheiden. Dadurch werden keine auf den Datensatz abgestimmten Parameterwerte benötigt. Die hier vorgestellte Methode erkennt differentiell exprimierte Segmente in biologischen Daten bei gleicher Sensitivität mit geringerer Falsch-Positiv-Rate im Vergleich zu den derzeit hauptsächlich eingesetzten Verfahren. Zudem ist die Methode bei der Erkennung von Exon-Intron Grenzen präziser. Die Suche nach Anhäufungen exprimierter Segmente hat darüber hinaus zur Entdeckung von sehr langen Regionen geführt, welche möglicherweise eine neue Klasse von macroRNAs darstellen. Nach dem Exkurs zu Tiling Arrays konzentriert sich diese Arbeit nun auf die Hochdurchsatz-Sequenzierung, für die bereits verschiedene Sequenzierungsprotokolle zur Untersuchungen des Genoms, Transkriptoms und Epigenoms etabliert sind. Einer der ersten und entscheidenden Schritte in der Analyse von Sequenzierungsdaten stellt in den meisten Fällen das Mappen dar, bei dem kurze Sequenzen (Reads) auf ein großes Referenzgenom aligniert werden. Die vorliegende Arbeit stellt algorithmische Methoden vor, welche das Mapping-Problem für drei wichtige Sequenzierungsprotokolle (DNA-Seq, RNA-Seq und MethylC-Seq) lösen. Alle Methoden wurden ausführlichen Benchmarks unterzogen und sind in der segemehl-Suite integriert. Als Erstes wird hier der Kern-Algorithmus von segemehl vorgestellt, welcher das Mappen von DNA-Sequenzierungsdaten ermöglicht. Seit der ersten Veröffentlichung wurde dieser kontinuierlich optimiert und erweitert. In dieser Arbeit werden umfangreiche und auf Reproduzierbarkeit bedachte Benchmarks präsentiert, in denen segemehl auf zahlreichen Datensätzen mit bekannten Mapping-Programmen verglichen wird. Die Ergebnisse zeigen, dass segemehl nicht nur sensitiver im Auffinden von optimalen Alignments bezüglich der Editierdistanz sondern auch sehr spezifisch im Vergleich zu anderen Methoden ist. Diese Vorteile sind in realen und simulierten Daten unabhängig von der Sequenzierungstechnologie oder der Länge der Reads erkennbar, gehen aber zu Lasten einer längeren Laufzeit und eines höheren Speicherverbrauchs. Als Zweites wird das Mappen von RNA-Sequenzierungsdaten untersucht, welches bereits von der Split-Read-Erweiterung von segemehl unterstützt wird. Aufgrund von Spleißen ist diese Form des Mapping-Problems rechnerisch aufwendiger. In dieser Arbeit wird das neue Programm lack vorgestellt, welches darauf abzielt, fehlende Read-Alignments mit Hilfe von de novo Spleiß-Information zu finden. Es erzielt hervorragende Ergebnisse und stellt somit eine sinnvolle Ergänzung zu Analyse-Pipelines für RNA-Sequenzierungsdaten dar. Als Drittes wird eine neue Methode zum Mappen von Bisulfit-behandelte Sequenzierungsdaten vorgestellt. Dieses Protokoll gilt als Goldstandard in der genomweiten Untersuchung der DNA-Methylierung, einer der wichtigsten epigenetischen Modifikationen in Tieren und Pflanzen. Dabei wird die DNA vor der Sequenzierung mit Natriumbisulfit behandelt, welches selektiv nicht methylierte Cytosine zu Uracilen konvertiert, während Methylcytosine davon unberührt bleiben. Die hier vorgestellte Bisulfit-Erweiterung führt die Seed-Suche auf einem reduziertem Alphabet durch und verifiziert die erhaltenen Treffer mit einem auf dynamischer Programmierung basierenden Bisulfit-sensitiven Alignment-Algorithmus. Das verwendete Verfahren ist somit unempfindlich gegenüber Bisulfit-Konvertierungen und erfordert im Gegensatz zu anderen Verfahren keine weitere Nachverarbeitung. Im Vergleich zu aktuell eingesetzten Programmen ist die Methode sensitiver und benötigt eine vergleichbare Laufzeit beim Mappen von Millionen von Reads auf große Genome. Bemerkenswerterweise wird die erhöhte Sensitivität bei gleichbleibend guter Spezifizität erreicht. Dadurch könnte diese Methode somit auch bessere Ergebnisse bei der präzisen Bestimmung der Methylierungsraten erreichen. Schließlich wird noch das Potential von Mapping-Strategien für Assemblierungen mit der Einführung eines neuen, Kristallisation-genanntes Verfahren zur unterstützten Assemblierung aufgezeigt. Es enthält Mapping als Hauptbestandteil und nutzt Zusatzinformation (z.B. Annotationen) als Unterstützung. Dieses Verfahren ermöglichte die erfolgreiche Assemblierung des kompletten mitochondrialen Genoms von Eulimnogammarus verrucosus trotz einer vorwiegend aus nukleärer DNA bestehenden genomischen Bibliothek. Zusammenfassend stellt diese Arbeit algorithmische Methoden vor, welche die Analysen von Tiling Array, DNA-Seq, RNA-Seq und MethylC-Seq Daten signifikant verbessern. Es werden zudem Standards für den Vergleich von Programmen zum Mappen von Daten der Hochdurchsatz-Sequenzierung vorgeschlagen. Darüber hinaus wird ein neues Verfahren zur unterstützten Genom-Assemblierung vorgestellt, welches erfolgreich bei der de novo-Assemblierung eines mitochondrialen Krustentier-Genoms eingesetzt wurde.

Single stage and two-stage sodium sulfite cooking were carried out on either spruce, pine or pure pine heartwood chips to investigate the influence of several process parameters on the initial phase of such a cook down to about 60 % pulp yield. The cooking experiments were carried out in the laboratory with either a lab-prepared or a mill-prepared cooking acid and the temperature and time were varied. The influences of dissolved organic and inorganic components in the cooking liquor on the final pulp composition and on the extent of side reactions were investigated. Kinetic equations were developed and the activation energies for delignification and carbohydrate dissolution were calculated using the Arrhenius equation. A better understanding of the delignification mechanisms during bisulfite and acid sulfite cooking was obtained by analyzing the lignin carbohydrate complexes (LCC) present in the pulp when different cooking conditions were used. It was found that using a mill-prepared cooking acid beneficial effect with respect to side reactions, extractives removal and higher stability in pH during the cook were observed compared to a lab-prepared cooking acid. However, no significant difference in degrees of delignification or carbohydrate degradation was seen.  The cellulose yield was not affected in the initial phase of the cook however; temperature had an influence on the rates of both delignification and hemicellulose removal. It was also found that the  corresponding activation energies increased in the order:  xylan, glucomannan, lignin and cellulose. The cooking temperature could thus be used to control the cook to a given carbohydrate composition in the final pulp. Lignin condensation reactions were observed during acid sulfite cooking, especially at higher temperatures. The LCC studies indicated the existence of covalent bonds between lignin and hemicellulose components with respect to xylan and glucomannan. LCC in native wood showed the presence of phenyl glycosides, ϒ-esters and α-ethers; whereas the α-ethers  were affected during sulfite pulping. The existence of covalent bonds between lignin and wood polysaccharides might be the rate-limiting factor in sulfite pulping. / The sulfite pulping process is today practised in only a small number of pulp mills around the globe and the number of sulfite mills that use sodium as the base (cation) is less than five. However, due to the increasing interest in the wood based biorefinery concept, the benefits of sulfite pulping and especially the sodium based variety, has recently gained a lot of interest. It was therefore considered to be of high importance to further study the sodium based sulfite process to investigate if its benefits could be better utilized in the future in the production of dissolving pulps. Of specific interest was to investigate how the pulping conditions in the initial part of the cook (≥ 60 % pulp yield) should be performed in the best way. Thus, this thesis is focused on the initial phase of sodium based single stage bisulfite, acid sulfite and two-stage sulfite cooking of either 100 % spruce, 100 % pine or 100 % pine heartwood chips. The cooking experiments were carried out with either a lab prepared or a mill prepared cooking acid and the temperature and cooking time were varied. Activation energies for different wood components were investigated as well as side reactions concerning the formation of thiosulfate. LCC (Lignin carbohydrates complexes) studies were carried out to investigate the influence of different cooking conditions on lignin carbohydrate linkages.

O presente estudo teve como objetivo avaliar e comparar os processos de polpação sodaantraquinona e bissulfito base magnésio para Bambusa vulgaris Schrad. com 2 anos de idade oriundos de plantios comerciais no Maranhão Brasil visando a produção de polpa celulósica branqueável e não branqueável (números kappa 30±2 e 50±2 respectivamente). A matéria-prima foi caracterizada com relação à densidade básica, composição química e dimensões de fibras; para aos processos de polpação avaliou-se o tempo total de cozimento, o fator H, o rendimento bruto, rendimento depurado, teor de rejeitos, carga de reagentes químicos e propriedades físicomecânicas das polpas. Os resultados obtidos mostram que a matéria-prima apresentou valores típicos para a espécie em questão no que diz respeito à densidade básica, dimensões e índices morfológicos das fibras e composição química. Para o processo de polpação soda-antraquinona os níveis de deslignificação determinados (números kappa) foram obtidos com cargas alcalinas de 15 e 19% (base NaOH) respectivamente e fator H de 1218; para o processo bissulfito base magnésio as polpas celulósicas com os níveis de deslignificação estabelecidos foram obtidas com uma mesma carga de reagentes químicos 26% (base SO2) e fator H de 1218 para polpas com número kappa 30±2 e 828 para polpas com número kappa 50±2; o tempo total de cozimento para o processo soda-antraquinona necessário para obtenção das polpas celulósicas desejadas são, em média, 3,8 vezes menores aos do processo bissulfito base magnésio. Adicionalmente os rendimentos depurados foram 3,0 e 4,6 pontos porcentuais superiores para as polpas com números kappa 30±2 e 50±2 respectivamente no processo soda-antraquinona. Os ensaios físicomecânicos mostram as diferenças entre as polpas tanto no que diz respeito ao processo de polpação empregado quanto no nível de deslignificação, sendo este último um fator de diferenciação dos produtos (papel) que podem ser obtidos a partir das polpas celulósicas. Comparando-se os processos de polpação no que diz respeito às propriedades físico-mecânicas observa-se a superioridade das polpas soda-antraquinona exceto nos parâmetros de drenabilidade e densidade aparente. Os resultados obtidos neste trabalho mostram que para a produção de polpa celulósica a partir de Bambusa vulgaris o processo soda-antraquinona é superior ao processo bissulfito base magnésio tanto nos aspectos de engenharia de processo (quantitativos) como nos aspectos relacionados à qualidade da polpa obtida. / The present study had as objective evaluates and to compare the soda-antraquinone and magnesium base bisulfite pulping processes for Bambusa vulgaris Schrad. with 2 years old originating from commercial plantings in Maranhão Brazil seeking the production of bleachable and unbleachable cellulosic pulp grade\'s (kappa numbers 30±2 and 50±2 respectively). The raw material was characterized regarding the basic density, chemical composition and dimensions of fibers; for to the pulping processes was evaluated the total time of cooking, H factor, total yield, screened yield, rejects, load of chemical reagents and physicalmechanical properties of the pulps. The obtained results show that the raw material presented typical values for the species in subject in what says respect to the basic density, dimensions and morphologic indexes of the fibers and chemical composition. For the soda-antraquinone pulping process the certain levels of delignification (kappa numbers) were obtained with alkaline loads of 15 and 19% (NaOH base) respectively and a H factor of 1218; for the magnesium base bisulfite process the cellulosic pulps with the established levels of delignification were obtained with a fix load of chemical reagents 26% (SO2 base) and H factor of 1218 for pulps with kappa number 30±2 and 828 for pulps with kappa number 50±2; the necessary total time of cooking to the sodaantraquinone process to obtain the wanted cellulosic pulps are, on average, 3,82 time less when compared with the bisulfite process. Additionally the screened yields were 3 and 4,63 percent superior for the pulps with numbers kappa 30±2 and 50±2 respectively in the soda-antraquinone process. The physical-mechanical tests show the differences among the pulps in what says respect to the used pulping process as in the delignification level, being this last one a factor of differentiation of the products (paper) that can be obtained starting from the cellulosic pulps. Comparing the pulping processes as what concerns of physical-mechanical properties the superiority of the soda-antraquinone pulps is observed except in freeness and density parameters. The results obtained in this work show that for the production of cellulosic pulp from Bambusa vulgaris the soda-antraquinone process it is superior than the magnesium base bisulfite process as much in the aspects of engineering process (quantitative) as in the aspects related to the quality of the obtained pulp.