The spectral analysis of nonstationary categorical time series using local spectral envelope

Most classical methods for the spectral analysis are based on the assumption that the time series is stationary. However, many time series in practical problems shows nonstationary behaviors. The data from some fields are huge and have variance and spectrum which changes over time. Sometimes,we are interested in the cyclic behavior of the categorical-valued time series such as EEG sleep state data or DNA sequence, the general method is to scale the data, that is, assign numerical values to the categories and then use the periodogram to find the cyclic behavior. But there exists numerous possible scaling. If we arbitrarily assign the numerical values to the categories and proceed with a spectral analysis, then the results will depend on the particular assignment. We would like to find the all possible scaling that bring out all of the interesting features in the data. To overcome these problems, there have been many approaches in the spectral analysis. Our goal is to develop a statistical methodology for analyzing nonstationary categorical time series in the frequency domain. In this dissertation, the spectral envelope methodology is introduced for spectral analysis of categorical time series. This provides the general framework for the spectral analysis of the categorical time series and summarizes information from the spectrum matrix. To apply this method to nonstationary process, I used the TBAS(Tree-Based Adaptive Segmentation) and local spectral envelope based on the piecewise stationary process. In this dissertation,the TBAS(Tree-Based Adpative Segmentation) using distance function based on the Kullback-Leibler divergence was proposed to find the best segmentation

Doctor of Philosophy

dissertationAdenosine deaminases that act on RNA (ADARs) deaminate adenosines in doublestranded RNA (dsRNA) to produce inosines. The extent an adenosine is edited depends on the sequence context of the target adenosine. Human ADAR2 (hADAR2) has a 5' nearest neighbor preference of U>A>C>G and a 3' preference of G>C>U«A, but it is not known which amino acids mediate these preferences. Previous studies show that preferences are derived mainly from the catalytic domain. Thus, we adapted a previously reported screen in yeast to identify mutations in the hADAR2 catalytic domain that allow editing of an adenosine in context of a disfavored triplet, GAC. A favored triplet, UAG, was used as the positive control. Hairpin substrates containing disfavored GAC and favored UAG were based on the R/G editing site of GRIA2 (glutamate receptor, ionotropic, AMPA 2) pre-mRNA, a well-studied endogenous substrate for hADAR2. Four mutants that edited GAC more than WT hADAR2 (E488Q, V493T, N597K and N613K) and one mutant that did not edit GAC (T490A) were further characterized by determining their binding affinity, catalytic rate, base-flipping and preferences to understand the effect of these mutations on ADAR reactivity. Gel-shift assays showed two mutants, N597K and N613K, had ~2-fold higher binding affinity compared to WT hADAR2, suggesting these mutants may have been selected in the screen due to tighter binding. Other mutants E488Q, T490A and V493T, which are on a highly conserved loop close to the active site, showed similar binding affinity as WT hADAR2 for both UAG and GAC, indicating discrimination was not derived from differences in binding affinity. We also determined catalytic rates, and probed base-flipping by substituting the target adenosine with the fluorescent base analog 2-aminopurine (2-AP). Remarkably, with both UAG and GAC substrates, mutants with similar binding affinity showed a correlation between catalytic rate and base-flipping, as indicated by a change in 2-AP fluorescence intensity (FI). Our data provide the first information on the residues important for preferences, and point to a conserved loop as key. Unexpectedly, our data suggest that hADAR2's preferences are derived from differences in base-flipping, rather than direct recognition of the neighboring base

The Advantages Of Paramagnetic NMR

In der Kernspinresonanzspektroskopie (NMR) treten drei Effekte auf, die paramagnetische und diamagnetische Moleküle in isotroper Lösung unterscheiden: residuale dipolare Kopplung (RDC), Pseudokontaktverschiebung (PCS) und paramagnetische Relaxationsverstärkung (PRE). Alle drei Effekte sind abhängig von intermolekularen Winkeln und Abständen und können daher Informationen über die Struktur und Dynamik des Moleküls liefern. Um diese Informationen zu erhalten, muss das Molekül paramagnetische Eigenschaften aufweisen. Eine der heutzutage gebräuchlichen Methoden verwendet kleine molekulare Tags, die paramagnetische Metallionen koordinieren. Die meisten dieser Tags binden über eine Disulfidbrücke an Cysteine an der Proteinoberfläche. Um diese Methode für DNA anzuwenden werden daher neue Taggingstrategien benötigt. Im Rahmen dieser Arbeit wurde eine modifizierte Nukleobase synthetisiert, mit der ein Schwefelatom in die DNA eingebracht werden kann. Diese Methode erlaubt es, jeden Tag an die DNA zu binden, der als Verbindungsmethode eine Disulfidbrücke nutzt. Mit der Nukleobase wird eine Kohlenstoff-Dreifachbindung in die DNA eingefügt und mit Hilfe einer dipolaren Cycloaddition wird die freie Thiolgruppe eingebracht. Die modifizierte Nukleobase wurde erfolgreich an einem selbstkomplementären DNA-Strang (24 Nukleobasen) getestet. Die Nukleobase wurde während der Synthese der DNA eingefügt und der mit Lutetium, Terbium oder Thulium vorbeladene Cys-Ph-TAHA Tag wurde über eine Disulfidbrücke an die DNA gebunden. Die Beladung des Tags und die Taggingreaktion verliefen hierbei quantitativ. Nach diesem Erfolg war es ein Hauptaspekt dieser Arbeit, eine verlässliche und reproduzierbare Aufreinigungs- und Probenvorbereitungsmethode zu entwickeln. Diesem Punkt kommt besondere Bedeutung zu, da das Phosphatrückgrat der DNA, im Gegensatz zu Proteinen, Metallionen koordinieren kann. Im Theorieteil dieser Arbeit ist eine komplette Herleitung der drei Hauptmerkmale paramagnetischer NMR gegeben. Diese Herleitung beginnt bei Grundbegriffen des Magnetismus und neben den Gleichungen für RDCs, PCSs und PREs werden Ausdrücke für den dipolaren Hamiltonoperator, Kreuzrelaxationsraten, kreuzkorrelierte Relaxationsraten, durch Alignment induzierte RDCs, Korrelationsfunktionen und spektrale Dichten gegeben. Das zweite Thema dieser Arbeit basiert auf einem weiteren paramagnetischen Effekt. Um der reduzierten Empfindlichkeit der Kernspinresonanzspektroskopie verglichen mit anderen Spektroskopiemethoden entgegenzuwirken, wurden viele Methoden entwickelt, die auf eine Erhöhung der Polarisierung der Atomkerne zielen, d.h. um sogenannte hyperpolarisierte Kerne zu erzeugen. Eine dieser Methoden, die photochemisch erzeugte dynamische Kernpolarisierung (photo CIDNP), basiert auf kurzlebigen Radikalen, die durch direkte Laserbestrahlung der Probe im Magneten erzeugt werden. Im Rahmen dieser Arbeit wurde ein photo CIDNP Aufbau entworfen, gebaut und getestet. Die ersten Experimente und Resultate mit Triethylendiamin, L-Tyrosin und 3-Fluor-L-tyrosin zeigen die Vorteile und Grenzen dieser Methode auf. Für 3-Fluor-L-tyrosin wurde eine komplette Analyse des Relaxationsverhaltens, einschließlich der Kreuzrelaxation und der kreuzkorrelierten Relaxation, durchgeführt

Topological sorting and self-assembly of knotted molecules: models and simulations

Knots are ubiquitous objects and decorative elements that have been studied since antiquity. During the centuries knots have become important not only for their mysterious and elegant aspects, but also for their practical relevance. Knots in ropes, for example, have always been useful for different practical applications, from climbing to sailing, from fishing to medicine. Chains that are sufficiently long or compactified are prone to develop knots. This is a "statistical necessity" that has been conjectured by Delbruck in 1962 and mathematically proved by Sumners and Whittington nearly 30 years later. In particular, they showed that for a self-avoiding polygon, the knotting probability tends to unity as the polygon length tends to infinity. This statistical necessity makes topological entanglement a genuine characteristic of polymeric systems. In case of linear polymer chains, knots can be untied by a suitable reptation of the polymer in space and therefore the entanglement is referred as physical knots. On the other hand, if the polymer ends are joined by a cyclisation reaction, the geometrical self-entanglement becomes trapped in the form of a proper mathematical knot, whose topology cannot be changed by any geometrical rearrangement of the polymer except by cutting it. Among polymers, double-stranded DNA (dsDNA) provides an ideal system to study the spontaneous occurrence of knots. In fact, differently from proteins and RNA, metric and topological properties of dsDNA are well captured by aspecific polymer models where only the polymer contour length, persistence length and thickness come into play. Studying knots in dsDNA is informative also to understand their biological implication. The presence of knots, in fact, severely affects several cellular processes, such as transcription and replication, with detrimental effects. Fortunately, cellular mechanisms have adopted countermeasures: there exist enzimes, namely topoisomerases, that are capable of simplifying the topological complexity of the DNA entanglement by favouring the selective cross-passage of pairs of DNA strands. The action of topoisomerases has been understood thanks to the topological profiling of DNA molecules realised with gel electrophoresis. This is the typical technique that permits to sort short DNA molecules by knot type. In particular, molecules are electrically driven through the obstacles of an agarose gel, where their mobility depends on the specific knot type. However, this technique can be used to profile only relatively short DNA molecules (10-15 kb). For longer ones, gel electrophoresis resolution would severely degrades, especially for knots with high number of crossings. This raises the problem of developing novel techniques that can be applied to characterise knot types in longer DNA molecules. Here, we will use molecular dynamics simulations and theoretical approaches to discuss the possibility to use spatially modulated nanochannels to sort ring polymer by their knot type. This approach permits, in principle, to separate polymers by their topological complexity, overcoming the aformentioned limits of gel electrophoresis. The spontaneous knotting of DNA is largely controlled by events where, for example, a loop is threaded by one termini; as a result both the complexity and size of the knots, as well as their location along the DNA contour, are stochastic. This is not the case for other types of biomolecules, particularly proteins, where the folding process towards the native state is tightly controlled by their chemical composition (primary sequence) via their intra-molecular interactions. As a result, proteins whose native state is knotted always feature the same knot type in the same sequence location. Mimicking such reproducible molecular knotting processes are, at least in part, the motivation of the ongoing quest of synthetic chemistry to create synthetic molecules tied in specific knot types. In this regard, chemists succeeded in controlling chemical reactions between small building blocks to assemble molecules with a priori desired topology. The chemists who developed this set of techniques, whose contribution opened up the way to a revolutionary chemistry, were awarded with the Chemistry Nobel Prize in 2016. Despite the high interest in the topic, up to recently, only a handful of different knot types have been synthesised. The reason is due to various challenging aspects of the synthesis process. These include the choice of the suitable building blocks, their correct spatial arrangement, and, above all, the selection of the designable target topology. Not every knot type, in fact, is necessarily expected to be equally designable in practice. In this thesis, we performed a computational and theoretical study to explore which designable molecular knots could be accessible for molecular synthesis with current experimental techniques

A-to-I RNA Editing in Human Cells

RNA editing is a means of diversifying the transcriptome and regulating innate immunity. Among the different classes of enzymes that modify RNA, adenosine deaminase acting on RNA (ADAR) is a type that catalyzes adenosine-to-inosine editing on double-stranded RNA molecules to regulate cellular responses to endogenous and exogenous RNA. Of the three ADAR homologs in humans, dysregulation of ADAR1 editing due to inherited mutations leads to disorders such as Aicardi-Goutieres syndrome, an inflammatory disease that manifests in the brain and skin, and dyschromatosis symmetrica hereditaria, a skin pigmentation disorder. ADAR1 is the primary A-to-I editor of RNA in humans, and the majority of edit sites are found in a class of repetitive elements called Alu, many of which are located in introns and 3’ untranslated regions of RNA. The functional consequences of A-to-I editing are varied, although a complete lack of functional ADAR1 is usually not tolerated, as revealed by the MDA5-mediated embryonic lethality in mice lacking functional ADAR1. In human neural progenitor cells, loss of ADAR1 causes spontaneous upregulation of interferon and cell death, although the RNA triggers remain unknown. Given the importance of ADAR1-editing in maintaining homeostasis in various contexts, there is a need to understand in more detail how ADAR1 isoforms are regulated and how they individually contribute to the A-to-I RNA editome. Two ADAR1 protein isoforms, p110 (110 kDa) and p150 (150 kDa), are expressed constitutively and in response to interferon, respectively, but the contribution of each isoform to the editing landscape remains incompletely characterized, largely because of the challenges in expressing p150 without p110. We revealed that the p110 isoform can be expressed from the canonical p150-encoding mRNA due to leaky ribosome scanning downstream of the p150 start codon. Synonymous mutations introduced in the region between the p150 and p110 start codons reduce leaky scanning and usage of the p110 start codon, and cells expressing p150 constructs with these mutations produce significantly reduced levels of p110. With the ability to express p150 with significantly reduced levels of p110, the A-to-I editome can be classified in terms of p150-selective and p110-selective sites, allowing evaluation of the relative contributions of either isoform to global editing levels. Our editing analysis revealed that the majority of ADAR1-edit sites are p150-selective, although a significant proportion of ADAR1-edit sites are also shared between p150 and p110, being not dependent on presence of either isoform for editing to occur. Of the sites that are putatively p110- selective, the majority are located in introns. Finally, the ability of p150 mRNA to give rise to p110 means that p110 is also an interferon-inducible protein alongside the canonical interferon-stimulated ADAR1 isoform: p150. During the interferon response, the transcriptome changes, and many new mRNA structures, perhaps some immunogenic ones, will enter the nucleus and cytoplasm. The distribution of ADAR1 isoforms is such that p110 is mostly present in the nucleus, and p150 mostly in the cytoplasm. We propose that optimal editing in the nucleus and cytoplasm during the interferon response is achieved by the inducibility of p110 and p150, both of which share a large number of target sites

An investigation into the population genetics and ecology of Gracilariopsis longissima (Gracilariales, Rhodophyta) around the South West Peninsula of Britain

For the conservation and management of natural resources, a detailed knowledge of the ecology and genetics of individual species is essential. Red seaweeds are an important component of marine ecosystems but few species have been the focus of research. Gracilariopsis longissima is a poorly studied member of a family of agarophytes of commercial importance, although it is thought to occur commonly around the coast of Britain. This report provides new information about the ecology and population genetics of the species with the identification of 11 populations around the coasts of Devon and Cornwall, seven of which were in the Fal Estuary and Helford River complex. Site characteristics are described, in particular with regard to substrata which were found to be significantly different between sites. New ecological data about algal assemblages in which Gs. longissima occurs is reported for spring (four sites) and summer (five sites), with significant differences seen for all except two sites. Anecdotal reports that Gs. longissima grows mainly on smaller substrata were investigated but were unsupported by the evidence, although a non-random distribution with respect to substrate size was found, which needs further investigation. Microsatellites were newly developed for the species but were thought to be monomorphic and were not pursued. Cross-genera amplification with Gracilaria gracilis microsatellites did not provide sufficiently reliable data. A number of molecular methods were optimised and used to identify populations and investigate the genetics of three populations. These results are the first report of population genetics in the species. Intra-population genetic variation was seen to be high when estimated using RAPD primers and was accompanied by significant differentiation between the populations investigated. Biofilms occur on almost all submerged aquatic surfaces, including living organisms. Gs. longissima is no exception: epiphytes and biofilms were investigated and found to be highly diverse and well-attached, with some thalli in some populations completely obscured by overgrowth. Cleaning methods were tested, with mechanical removal proving to be the most successful. Biofilms were also found to affect RAPD profiles, confirming that cleaning of wild collected specimens was essential for reliable RAPD data to be obtained

SCN9A and its natural antisense transcript

Schmerz ist ein wesentlicher Bestandteil unseres Überlebens , er macht uns bewusst wo unsere körperlichen Limits liegen und schützt uns vor schädlichen Umwelteinflüssen . Schmerzen ,wie auch anderen somatosensorischen sub Modalitäten wie Sehen, Hoeren oder Geruch , eine Wahrnehmung, ein Produkt unseres Gehirns um externe Einfluesse zu verarbeiten und zu handhaben (10) (18). Abgesehen von seiner wesentlichen Rolle das eigene Überleben zu sichern , hat nicht jede Art von Schmerz und vor allem chronische Schmerzen eine funktionelle Rolle und stellt eine grosse Herausfroderung in der klinischen Behandlung da (15, 16). Derzeit leiden rund 10% der Bevölkerung unter chronischen Schmerzen aufgrund irreversibler Nervenverletzungen oder Entzündungen. Die beiden großen Klassen von Analgetika, die verwendet werden, um chronische Schmerzen (dh nicht-steroidalen Antiphlogistika und Opioide) zu behandeln haben sehr viele Nebenwirkungen und ziele nur auf die Bekaempfung der Symptome ab, anstatt die zugrunde liegenden Ursachen zu behandeln. Neue wissenschaftliche Erkenntnisse durch Knockout (31,32) , knock down und Überexpression Studien sowie Analysen von Patienten mit ungewöhnlichen Schmerzzustaenden (20) haben zeigt, dass Natriumkanäle Hauptakteure der Schmerzweiterleitung und Prozessierung darstellen. Insbesondere Nav1.7, ein TTX-empfindliche spannungsabheangiger Natrium Kanal, der hauptsächlich in sensorischen DRG-Neuronen und sympathischen Ganglien Neuronen exprimiert wird , ist von grundlegender Bedeutung in Initiation und Ausbreitung von Aktionspotentialen (9 - 21, 23) und spielt daher eine wesentliche Rolle im Schmerzen empfinden . Vererbte oder spontane Mutationen in SCN9A (Nav1.7), die zu gain of function Mutationen in Nav1.7 fuehren und in Hypersentitivteat von DRG-Neuronen resultierien, fuehren zu Krankheitsbildern wie Primary erythermalgia (PE) (14,12,21) und paroxysmale extreme Schmerzstörung (PEPD). Nonsense-Mutationen führen zum Verlust der Funktion von Nav1.7 und resultieren in Ionenkanal-assoziierten Unempfindlichkeit gegen Schmerzen (20,17). Interessanterweise hat SCN9A ein correspondierendes Antisense-Transkript auf dem gegenüberliegenden Strang, das tail to tail angeordent ist und moeglicherweise eine Rolle in der Regulation der Expression SCN9A spielt. Verschiedene Studien haben unabhängig voneinander gezeigt, dass Antisense-Transkripte Auswirkungen auf die Expression des sense Transkription des entsprechenden Gens haben, entweder durch 1) störung seine Transkription 2) RNAi, RNA-Editing-und RNA-Maskierung 3) oder durch Epigenetics und Chromatin-Remodeling . (1) Durch die Erforschung des Mechnisms durch welchen SCN9A antisense transcript sein korrespondierendes sense Gene reguliert , erhoffen wir uns einen tieferen Einblick in die molekuaren Zusammenheange die unsere Schmerempfindung beeinflussen und steuern.Desweitern erhoffen wir uns dadurch wesentlich an der Entwicklung einer neuen Klasse von Schmerzmitteln beizutragen.Pain is an essential part of our survival and keeps us in awareness of our physical limitations and protects us from harmful environmental stimuli. However pain and other somatosensory sub modalities like vision and smell are rather a perception, a product of our brain to handle and bring external sensory inputs to consciousness (10, 18). Regardless of its essential role in one’s survival, pain and in particular chronic pain which does not fulfil any physiological role, is a major clinical challenge to treat (15, 16). Currently 10 % of the population are suffering from chronic pain due to irreversible nerve injury or inflammation. Up to now the two major classes of analgesics that are used to treat chronic pain (i.e. non-steroidal anti-inflammatories and the opioids) focus more on the symptoms of pain than its underlying causes. Recent scientific discoveries obtained from mice in knockout (31,32), knock down and overexpression studies as well as analyses of patients suffering from unusual pain states (20) show that voltage gated sodium channels are key players in several pain pathways. In particular Nav1.7, a TTX sensitive voltage gated sodium channel that is mainly expressed in sensory DRG neurons and sympathetic ganglia neurons, is fundamental in initiation and propagation of action potentials (9- 21, 23) and plays an important role in pain sensation. Inherited or spontaneous mutations in SCN9A (Nav1.7) causing a gain of function result in hyperexcitability of DRG neurons and lead to Primary erythermalgia (PE) (12,14,21) and Paroxysmal extreme pain disorder (PEPD) while nonsense mutations causing loss of function of Nav1.7 cause Channelopathy-associated insensitivity to pain (20, 17). Interestingly, SCN9A has a corresponding antisense transcript encoded on the opposite strand that tail to tail overlaps with the sense transcript and might play a role in regulation of SCN9A expression. Different studies have reported independently that natural antisense transcripts have an impact on the transcription level of its corresponding sense gene either through 1) interfering with its transcription 2) RNAi, RNA editing and RNA masking 3) or through epigenetic and chromatin remodelling (1). By investigating the possible mechanism of SCN9A antisense transcripts on Nav1.7 expression, we aim to gain deeper insights into pain pathways and contribute to the development of a new specific class of analgesic drugs