Um die Wirkung herpesviral-kodierter FcgammaRezeptoren auf wirtskodierte zelluläre FcgammaRezeptoren und IgG-vermittelten Effektorfunktionen untersuchen zu können, einen methodisch neuen Ansatz wurde entwickelt, der die Detektion FcgammaR-aktivierender Antikörper ermöglicht. Dieses neuartige Assay beinhaltet die Kokultivierung virusinfizierter Zellen, die mit virusspezifischen IgG-Antikörpern opsoniert sind, mit FcgammaR-zeta BW5147-Transfektanten als Reporterzellen. Diese stabilen Transfektanten exprimieren chimäre Rezeptoren, die aus der extrazellulären Domäne der zellulären FcgammaRezeptoren bestehen, welche mit der TM und intrazellulären Domäne der murinen CD3zeta-Kette fusioniert wurden. Die Aktivierung der CD3zeta-Kette führt zu einer IgG-dosisabhängigen mIL-2 Sekretion, die im ELISA gemessen werden kann. Die FcgammaR-spezifische immune IgG könnte eine wichtige biologische Rolle in der antiviralen Immunabwehr spielen. Herpesviren exprimieren auf der Oberfläche infizierter Zellen viral-kodierte Fc-bindende Glykoproteine. Um zu bestimmen, ob virale FcgammaRezeptoren die IgG-abhängige Aktivierung von wirtskodierten FcgammaRezeptoren beeinflussen können, wurde das oben beschriebene Assay angewandt. Es wurde festgestellt, dass der HCMV-kodierte FcgammaR gp68 die Aktivierung und die nachfolgende Signalkaskade von CD16>CD32=CD64 inhibiert, während der HCMV-kodierte FcgammaR gp34 die Aktivierung von CD16>CD64>CD32 inhibiert. In klarem Kontrast dazu wirkt der HSV-kodierte FcgammaR gE, der CD16 Aktivierung vermindert, CD32 hingegen nur sehr schwach und CD64 gar nicht beeinflußt. Der MCMV-kodierte FcgammaR m138/fcr-1 vermindert die Aktivierung des murinenCD16. Zusammenfassend betrachtet zeigen die ermittelten Daten, dass es sich bei den herpesviral-kodierten FcgammaRezeptoren um hierarchische und redundante Antagonisten der wirtskodierten zellulären FcgammaRezeptoren handelt. Herpesviral-kodierte FcgammaRezeptoren wirken somit der Aktivierung des Immunsystems entgegen. / To study the possible interference of the herpesviral vFcgammaRs with the host FcgammaRs and IgG-mediated effector functions, a new methodological approach to detect FcgammaR activating antibodies was developed. The novel assay comprises the co-cultivation of virus infected cells upon opsonization with immune IgG antibodies and the stably transfected FcgammaR-zeta BW5147 transfectants as responder cells. The transfectants express chimeric receptors bearing the extracellular domain of the host FcgammaRs fused to the transmembrane and tail domains of the murine CD3zeta chain. Triggering the CD3zeta chain is sufficient to elicit IL-2 secretion in a dose dependent manner which is measured in an ELISA. The setup of the new assay provides a defined effector cell population bearing one Fcgamma receptor on the surface, which becomes activated in the presence of immune IgG antibodies bound to the native viral antigens displayed on the surface of infected cells. The assay system allows us to detect and quantify Fc gamma receptor-activating immune IgG in an FcgammaR-specific way, which is thought to have an important biological function in antiviral defense. Several alpha- and beta- herpesviruses express on the surface of infected cells virally encoded Fc binding glycoproteins. The assay described above was applied to determine if the viral FcgammaRs are able to impair IgG-mediated activation of host FcgammaRs. In a systematic approach, the effect on each host FcgammaR by each of the herpesviral FcgammaR was investigated. It was found that HCMV FcgammaR gp68 affects activation and downstream signaling of CD16 > CD32 = CD64, while gp34 attenuates CD16 > CD64 > CD32. In clear contrast, HSV gE impairs CD16 activation and weakly CD32, but has no effect on CD64. Furthemore, MCMV m138/fcr-1 diminishes activation of mouse CD16. Taken together, this data uncover herpesviral FcgammaRs as hierarchical and redundant antagonists precluding host FcgammaRs from triggering immune responses.

Im Rahmen dieser Arbeit wurde ein neuartiges 3D Fluoreszenz Mikroskop, OMX gennant, entworfen und gebaut. Ein umfassender Design-Neuansatz erlaubt es den neuen Anforderungen der aktuellen Biologie bezüglich erhöhter Auflösung in Zeit und Raum Rechnung zu tragen. Mit Ausnahme vom Auflegen des Objektträgers sind alle Aspekte des Mikroskops Computer-gesteuert. Einen Großteil der Software floß in ein neues, eigenständiges Open-Source Projekt ein. Es erlaubt die Verarbeitung sehr großer, mehrdimensionaler Bilddaten, und die Entwicklung neuartiger Algorithmen in einer flexiblen Oberfläche. OMX hat zwei Betriebsarten: Im ersten Modus können bis zu 100 Bilder pro Sekunde mit optischer Auflösung in mehreren Farbkanälen aufgenommen werden. Dies entspricht etwa 10 3D Bildern pro Sekunde. Im zweiten Modus können mit der Structured Illumination Mikroskopie fixierte Präparate mit eine Auflösung unterhalb des Abbe-Limits untersucht werden. Im zweiten Teil dieser Arbeit, stelle ich erste Forschungsergebnisse von OMX vor. Drosophila X-chromosomen markiert mit GFP-MSL3 wurden in situ im sub-Sekunden Bereich beobachtet. Mit Hilfe neuentwickelter Algorithmen konnte ich die Chromosomendynamik analysieren. Das Falten und Entfalten von Bereichen eines Chromosoms wurde als Funktion der Zeit darstellen. Chromosomenstrukturen wurden mit Hilfe der SIM an fixierten primären embryonalen Kulturen untersucht. Unterstrukturen von 100-200nm sind erkennenbar. Viele Bilder zeigen eine DNA-reiche Hülle die einen DNA-armen Chromosomenkern umgibt. Ausserdem habe ich polytäne Chromosomen mit SIM aufgenommen. Bandstrukturen zeigen sich mit deutlich erhöhter Detailklarheit, und Längsfasern sind sichtbar, die ansonsten nur vom Elektronenmikroskop her bekannt sind. Als weiteres Beispiel der verbesserten Auflösungsfähigkeit habe ich Kernporen untersucht. In mit DAPI gefärbten Mauszellen zeigen diese sich als dunkle Punkte mit einer Größe von etwa 120nm. / A novel fluorescence 3D wide-field light microscope called OMX, was designed and implemented. The novel design addresses improved speed and resolution requirements of current biology research. After designing and building the microscope body I designed and implemented the needed computer software for the eight computers required to operate OMX. Over the course of the project I also designed and implemented a new Open-Source software platform for algorithm development and image analysis. It focuses on very large multi-dimensional image data handling and visualization in general. OMX can operate in two modes: In the first mode a live specimen can be observed at optical resolution (approx. 250nm) at speeds up to 100 sections per second simultaneously in multiple wavelength channels. This equals about 10 3D images per second. The second mode is for observing fixed preparations at resolutions below the Abbe diffraction limit using Structured Illumination Microscopy (SIM). This produces 3D volumetric image data with lateral resolution near 100nm and axial resolution of about 200nm. In the second part of this thesis I show first results achieved using the OMX microscope. Chromosome dynamics was analyzed using various newly developed image analysis algorithms. Sub-second motion was observed for in situ Drosophila X-chromosomes tagged with GFP-MSL3. Parts of the chromosome can be traced within the nucleus and time-series data shows its folding and unfolding as a function of time. Chromosome structure was imaged using SIM on formaldehyde fixed primary embryonic cultures stained with DAPI. Features of the sub-structure with sizes around 100-200nm were apparent. Many chromosomes show an outer layer along the chromatin axis appearing persistently denser in DNA than the central core. Polytene chromosomes were imaged using SIM. Band patterns are visible in much more detail than in conventional deconvolution microscopy and longitudinal fibers known only from electron microscopy were visible.

Die humorale Immunantwort eines Organismus auf ein Pathogen äußert sich in einer Veränderung des Antikörperrepertoires. Eine quantitative Untersuchung dieses Prozesses erfordert aufgrund der enormen Komplexität des Immunsystems, die Verwendung von Hochdurchsatztechniken, wie Peptid-Mikroarrays. Bisher gibt es nur wenige Studien, die die Verlässlichkeit von Mikroarray-Bindungsmessungen untersuchen. In dieser Arbeit werden Bewertungskriterien für die Qualität von Antikörper-Peptid-Bindungsstudien unter Verwendung der Peptid-Mikroarraytechnologie herausgearbeitet, mit dem Ziel, diese Hochdurchsatzmethode für qualitative und quantitative Antikörper-Peptid-Bindungsmessungen zu optimieren. Anhand eines Modellsystems, das aus dem monoklonalen anti-p24 (HIV-1) Antikörper CB4-1 und 26 verschiedenen Peptiden, die mit unterschiedlicher Affinität an CB4-1 binden, besteht, werden systematisch die Bindungsdissoziationskonstanten der jeweiligen Antikörper-Peptid-Komplexe mit den durch Peptid-Mikroarray-Bindungsmessungen erhaltenen Signalintensitäten verglichen. Darüber hinaus wird in dieser Arbeit die Messung von Serumantikörperbindungsprofilen gegenüber Zufallspeptidbibliotheken als Methode der serologischen Diagnostik verwendet. Anhand dreier Beispieldatensätze wird die serologische Diagnose von Infektionskrankheiten, Autoimmunkrankheiten und von Krebs mittels Zufallspeptid-Mikroarrays demonstriert. Mithilfe von Merkmalsselektion werden Peptide selektiert, die besonders geeignet sind, um zwischen gesunden und kranken Individuen zu unterscheiden. Besondere Bedeutung wird der Untersuchung der Robustheit der Methode gegenüber schwankenden experimentellen Bedingungen beigemessen. Die vorliegende Arbeit gibt Aufschluss über vorhandene Probleme der Mikroarray-Technologie, stellt Lösungsansätze vor und arbeitet bedeutende Weiterentwicklungen auf dem Weg hin zu einer minimal-invasiven serologischen Diagnostik heraus, die kein a priori Wissen über Antigene voraussetzt. / The humoral immune response to a pathogen is associated with specific changes in the antibody repertoire. Because of the enormous complexity of the immune system, a quantitative determination of this process requires high-throughput measuring tools, such as the peptide microarray technology. There are only few reports that determine the technological reliability of peptide microarray studies. By using a model system, composed of the anti-p24 (HIV-1) monoclonal antibody CB4-1 and an array of 26 different peptides for which the CB4-1 binding affinity has independently been measured, the dissociation constants of antibody-peptide complexes are systematically compared with obtained signal intensities. The assignment of serum-antibody binding profiles using random peptide microarrays for the purpose of serological diagnostics constitutes a major part of this work. By means of three sample data sets, the ability of random peptide microarrays to serve as method of serological diagnosing infectious diseases, autoimmune diseases and cancer is demonstrated. By means of feature selection, the peptides that are exceptionally appropriate to discriminate between the investigated groups are identified. This study attaches special importance to the reliability and robustness of extracted microarray data. This thesis indicates present problems of the peptide microarray technology and presents further developments on the way to minimal-invasive serological diagnostics that do not require any a priori knowledge about antigens, and thus about the investigated diseases.

Fragestellung: Das von Dixon 1991 beschriebene tierexperimentelle Modell der 'controlled cortical impact injury'(CCII) wurde zur Untersuchung pathophysiologischer und pathomorphologischer Veränderungen nach traumatischer Hirnkontusion angewandt. Magnetresonanztomographische Techniken (MRT) einschließlich der diffusionswichtenden Bildgebung (DWI) wurden genutzt, um den Zeitverlauf der Hirnödementwicklung zu erfassen, eine Differenzierung unterschiedlicher Ödemformen zu ermöglichen und einen Blut-Hirn-Schrankenschaden zu detektieren. Desweiteren wurde die MRT genutzt, um den neuroprotektiven Effekt des NO-Synthase-Pathway-Modulators Lubeluzol, der bereits im Modell der zerebralen Ischämie nachgewiesen werden konnte, zu untersuchen. Material und Methoden: An 46 Sprague Dawley Ratten wurde eine links parieto-temporale Kontusion appliziert. Die Tiere wurden bis zu 7 Tage nach Trauma magnetresonanztomographisch untersucht. 36 Tiere erhielten Lubeluzol resp. Plazebo. Ergebnisse: Die T2-gewichtete Bildgebung zeigte eine maximale Ödemausbreitung 24 - 48 Stunden nach Trauma. Es ließ sich mithilfe der DWI ein Kontusionskern von einem Kontusionsrand differenzieren. Der Kontusionskern zeichnete sich bis 48 Stunden nach Trauma durch eine Abfall des apparenten Diffusionskoeffizienten (ADC) aus, einem zytotoxischem Ödem entsprechend, der Kontusionsrand wies während des gesamten Untersuchungszeitraums einen ADC-Anstieg auf, als Ausdruck eines vasogenen Ödems. Die T1-gewichtete Bildgebung konnte nach Kontrastmittel (KM)-Applikation durch die KM-Extravasation eine über 7 Tage anhaltende Störung der Blut-Hirnschranke detektieren. In der Lubeluzol-Studie ließ sich anhand der ADC-Veränderungen, des Ödemausmasses oder physiologischer Parameter wie Blutdruck, intrakranieller Druck oder Hirnschwellung kein signifikanter Unterschied zwischen den Tieren der Substanz- bzw. Plazebo-Gruppe aufzeigen. Schlußfolgerung: Die in dem Modell der CCII induzierte traumatische Hirnkontusion zeichnet sich bis 48 Stunden nach Trauma durch einen zytotoxischen Kontusionskern und einen diesen umgebenen vasogenen Kontusionrand aus. Desweiteren konnte ein anhaltender Blut-Hirnschrankendefekt nachgewiesen werden. Ein neuroprotektiver Effekt des Lubeluzols konnte in diesem Traumamodell in der hier applizierten Dosierung nicht nachgewiesen werden. / Objective: The controlled cortical impact injury (CCII) device, as described by Dixon 1991, was used to investigate the brain tissue damage in an animal model of severe traumatic brain injury. Magnetic resonance imaging (MRI) techniques including diffusion weighted imaging (DWI) have been applied to analyse the time course and the characteristics of edema formation and to detect blood-brain-barrier disruption. Furthermore MRI has been used to investigate a neuroprotective effect of the NO-synthase pathway modulator lubeluzole, which has proved markedly beneficial in a model of cerebral ischemia in rats. Material and Methods: a left parieto-temporal cortical contusion was inflicted upon 46 Sprague Dawley rats. Animals have been examined up to 7 days following trauma by MRI. 36 animals have been administered lubeluzole resp. placebo. Results: The most pronounced edema formation has been shown in T2-weighed imaging at 24 - 48 hours post trauma. DWI was able to distinguish between a contusion core and a contusion rim. The contusion core was marked by a decrease in the apparent diffusion coefficient (ADC) up to 48 hours post trauma, indicating cytotoxic edema, whereas the contusion rim has been characterised by vasogenic edema, as indicated by ADC-increase over the entire investigation period. In T1-weighted imaging contrast agent extravasation indicated a sustained blood brain barrier disruption up to 7 days after trauma. Compared to placebo administered rats in lubeluzole-treated animals no significant differences in ADC-changes, edema-extension or physiological parameters as blood pressure, intracranial pressure or brain swelling could be demonstrated. Conclusion: CCII induced traumatic brain injury is characterised by a cytotoxic edema up to 48 hours encircled by a vasogenic contusion rim accompanied by a sustained blood brain barrier disruption. In the model of CCII lubeluzole did not reveal a neuroprotective effect in the applied dosage.

Die Beschreibung genregulatorischer Ereignisse ist entscheidend um Zelldifferenzierung und -entwicklung zu verstehen. Dynamische Vernderungen der Chromatinstruktur, Histonmodifikationen und das Binden von Transkriptionsfaktoren an Enhancer und Promotoren, koennen mit Hilfe von genomweiten Hochdurchsatz-Sequenziertechniken wie ChIP-Seq, DNase-Seq, ATACSeqund RNA-Seq untersucht werden. In dieser Arbeit entwickele ich mehrere probabilistische Modelle fuer die Analyse von genomweiten Sequenzierungsdaten. Diese umfassen 1. einen Peak-Finder fuer ChIP-/DNase-/ATAC-Seq-Daten, der sich Replikate zunutze macht und praezise Peak-Weiten berechnet, 2. eine Pipeline um das Genom in hoher Aufloesung in eindeutige Klassen von Kombinationen von Histonmodifikationen zu segmentieren, 3. ein Bayes-Netzwerk-Modell welches multiple zeitlich aufgelste Histonmodifikations-ChIP-seq-Daten kombinatorisch clustert Klassen von regulatorischen Elementen zu identifizieren. Mit Hilfe dieser Modelle untersuchen wir die Promotorumgeben und zeigen einen Zusammenhang zwischen Chromatinstruktur und Promotordirektionalitaet. Darueber hinaus verwenden wir ein Modell zur direkten Reprogrammierung von Stammzellen in Motorneuronen durch die gezielte Expression von Transkriptionsfaktoren und analysieren die dadurch induzierten zeitlichen Vernderungen der Chromatinstruktur und Transkriptionsfaktorbindedynamik. Wir beobachten, dass Promotoren verschiedenen Chromatin-Dynamiken zur Aktivierung und Repression folgen, die mit den Chromatin-Dynamiken von Enhancer-Elementen korrelieren. Enhancer hingegen werden durch kooperatives Verhalten direkt induzierter Transkriptionsfaktoren und anderen Faktoren, die in den Stammzellen zu Beginn vorhanden waren oder im Verlaufe der Differenzierung aktiviert wurden, kontrolliert. Diese Arbeit zeigt wie wichtig Chromatin-Dynamik und ihre Beziehung zur Logik von Transkriptionsfaktoren ist, um die Veraenderungen der Genexpression zu verstehen. / Delineating transcription regulatory events is crucial to understand cell differentiation and development. Dynamic changes of chromatin structure, histone modifications and transcription factor binding to enhancers and promotors can be investigated with the aid of genome-wide high-throughput sequencing technologies such as ChIP-Seq, DNase-Seq, ATAC Seq and RNA Seq. In this thesis, I develop several probabilistic models for the analysis of genome-wide sequencing data. These include: 1. a peak finder for ChIP-Seq, DNase-Seq and ATAC Seq data, which exploits biological replicates and accurately demarcates peak widths, 2. a pipeline for high-resolution genome segmentation into unique classes of combinations of histone modifications and 3. a Bayesian network model that can co-cluster multiple time-course histone modification ChIP-Seq data sets into distinct classes of regulatory elements. With the aid of these models we investigate the promoter chromatin environment and show a link between chromatin state and transcription initiation directionality. In addition, we use a system for direct reprogramming of stem cells in motor neurons by the targeted expression of transcription factors to analyse changes in chromatin state and transcription factor dynamics during differentiation. We observe that promoters follow different chromatin dynamics for activation and repression that correlate with the chromatin dynamics of enhancer elements. Enhancers are controlled by cooperative behavior of directly induced transcription factors and other factors present in the stem cells initially, or activated in the course of differentiation. Overall, this work demonstrates the importance of understanding chromatin dynamics and their relationship to transcription factors logic in order to better explain changes in gene expression.

Objetivo. Ao mesmo tempo em que a obesidade aumenta no mundo todo e se torna cada vez mais um problema de Saúde Pública, o stress aumenta no cotidiano das pessoas e na busca pela sobrevivência. Verificar a possível associação entre prevalências de obesidade centralizada e indicadores de stress é o objetivo desse trabalho. Métodos. Utilizam-se dados de um estudo transversal, com informações de 298 mulheres de 20 a 59 anos, moradoras de um município da Grande São Paulo, as quais responderam questionários especialmente elaborados para avaliar o stress psicológico. O diagnóstico de obesidade centralizada foi feito através da medida da circunferência da cintura (CC) e da razão cinturaquadril (RCQ). O stress psicológico foi medido em escores atribuídos às respostas dos questionários e classificado em 3 categorias: isento, resistência e exaustão. A análise estatística foi realizada mediante dois modelos de regressão linear generalizada múltipla entre a variável resposta obesidade centralizada em duas categorias (sim, não) e o stress psicológico em três fases (isento, resistência e exaustão), controlando-se as variáveis demográficas: idade e escolaridade. Resultados. As prevalências de obesidade centralizada foram semelhantes nos dois modelos, respectivamente 40,6 % e 42% para CC e RCQ. As prevalências de stress psicológico foram 61,7% e 8,4% para as fases resistência e exaustão. As associações entre a categoria sim foram positivas e significantes, respectivamente para CC e RCQ (RP 1,51, P 0,028 e RP 1,52, P 0,022) com o stress na fase de exaustão, com o aumento da idade (RP 1,02, P 0,001 e RP 1,01, P 0,002) e com baixa escolaridade (RP 0,67, P 0,030 e RP 0,59, P 0,005). O teste de tendência foi positivo (P 0,029) para a categoria sim do RCQ e aumento das categorias de stress. Conclusões. A fase de exaustão do stress mostrou associação positiva e significante com a obesidade centralizada nos dois modelos estudados, CC e RCQ. O teste significante de tendência com a RCQ sugere efeito gradativo das fases do stress sobre a obesidade centralizada. São necessários, no entanto, outros estudos que comprovem a associação da obesidade centralizada com o stress subdividido em categorias. / Objective. When observing modern life nowadays we find out that, at the same time that obesity increases all around the world and becomes a real concern to public health authorities, we also see stress proliferating in peoples everyday life, specially in the fight for survival. The purpose of this work is to verify the association between prevalence of abdominal obesity and stress indicators. Methods. This work uses given data of a transversal study, containing information of 298 women aged between 20 and 59, inhabitants of the Great São Paulo, who had been submitted to questionnaires especially formulated to evaluate psychological stress. The diagnosis of abdominal obesity was made using two models: measuring Waist Circumference (WC) and Waist - Hip ratio (WHR). Psychological stress was measured in scores attributed to answers of the questionnaires and classified in 3 categories: Exempt, Resistance and Exhaustion. The statistics analysis were carried through two models of multiple generalized linear regression between the variable which is the answer- abdominal obesity focused in two categories (Yes, No) and psychological stress focused in three categories (Exempt, Resistance, Exhaustion) maintaining under control the demographic variables such as age and scholarship. Results. The results referring to the prevalence of abdominal obesity were similar in the two models showing respectively 40.6% and 42% for WC and WHR. The results on the prevalence of psychological stress were 61.7% and 8.4% respectively for the phase of Resistance and the phase of Exhaustion. The associations in the Yes category were classified as being positive and significant, for WC and WHR respectively, Prevalence Ratio PR 1,51, significancy P 0,028 and PR 1,52, P 0,022 for the stress in the phase of Exhaustion, when considered also an increase in age (PR 1,02, P 0,001 and PR 1,01, P 0,002) and a decrease in the level of education (PR 0,67, P 0,030 and PR 0,59, P 0,005) The trend analysis was positive (P 0,029) for the increase of the WHR and the categories of stress. Conclusions. The phase of Exhaustion of Stress showed positive and significant association with the Abdominal Obesity in the two models, WC and WHR. The positive results in the trend tests with the WHR suggest that abdominal obesity may be gradually affected by the phases of stress. Nevertheless, there is the need of further investigation to confirm the association between abdominal obesity and the various categories of stress.

Die Untersuchung der posttranskriptionellen Genregulation erfordert eine eingehende Kenntnis vieler molekularer Prozesse, die auf RNA wirken, von der Prozessierung im Nukleus bis zur Translation und der Degradation im Zytoplasma. Mit dem Aufkommen von RNA-seq-Technologien können wir nun jeden dieser Schritte mit hohem Durchsatz und Auflösung verfolgen. Ribosome Profiling (Ribo-seq) ist eine RNA-seq-Technik, die darauf abzielt, die präzise Position von Millionen translatierender Ribosomen zu detektieren, was sich als ein wesentliches Instrument für die Untersuchung der Genregulation erweist. Allerdings ist die Interpretation von Ribo-seq-Profilen über das Transkriptom aufgrund der verrauschten Daten und unserer unvollständigen Kenntnis des translatierten Transkriptoms eine Herausforderung. In dieser Arbeit präsentiere ich eine Methode, um translatierte Regionen in Ribo-seq-Daten zu erkennen, wobei ein Spektralanalyse verwendet wird, die darauf abzielt, die ribosomale Translokation über die übersetzten Regionen zu erkennen. Die hohe Sensibilität und Spezifität unseres Ansatzes ermöglichten es uns, eine umfassende Darstellung der Translation über das menschlichen und pflanzlichen (Arabidopsis thaliana) Transkriptom zu zeichnen und die Anwesenheit bekannter und neu-identifizierter translatierter Regionen aufzudecken. Evolutionäre Konservierungsanalysen zusammen mit Hinweisen auf Proteinebene lieferten Einblicke in ihre Funktionen, von der Synthese von bisher unbekannter Proteinen einerseits, zu möglichen regulatorischen Rollen andererseits. Darüber hinaus zeigte die Quantifizierung des Ribo-seq-Signals über annotierte Genemodelle die Translation mehrerer Transkripte pro Gen, was die Verbindung zwischen Translations- und RNA-Überwachungsmechanismen offenbarte. Zusammen mit einem Vergleich verschiedener Ribo-seq-Datensätze in menschlichen und planzlichen Zellen umfasst diese Arbeit eine Reihe von Analysestrategien für Ribo-seq-Daten als Fenster in die vielfältigen Funktionen des exprimierten Transkriptoms. / The study of post-transcriptional gene regulation requires in-depth knowledge of multiple molecular processes acting on RNA, from its nuclear processing to translation and decay in the cytoplasm. With the advent of RNA-seq technologies we can now follow each of these steps with high throughput and resolution. Ribosome profiling (Ribo-seq) is a popular RNA-seq technique, which aims at monitoring the precise positions of millions of translating ribosomes, proving to be an essential tool in studying gene regulation. However, the interpretation of Ribo-seq profiles over the transcriptome is challenging, due to noisy data and to our incomplete knowledge of the translated transcriptome. In this Thesis, I present a strategy to detect translated regions from Ribo-seq data, using a spectral analysis approach aimed at detecting ribosomal translocation over the translated regions. The high sensitivity and specificity of our approach enabled us to draw a comprehensive map of translation over the human and Arabidopsis thaliana transcriptomes, uncovering the presence of known and novel translated regions. Evolutionary conservation analysis, together with large-scale proteomics evidence, provided insights on their functions, between the synthesis of previously unknown proteins to other possible regulatory roles. Moreover, quantification of Ribo-seq signal over annotated transcript structures exposed translation of multiple transcripts per gene, revealing the link between translation and RNA-surveillance mechanisms. Together with a comparison of different Ribo-seq datasets in human cells and in Arabidopsis thaliana, this work comprises a set of analysis strategies for Ribo-seq data, as a window into the manifold functions of the expressed transcriptome.

Obwohl EtG seit dem Jahr 2000 intensiv als Alkoholmarker in Haaren beforscht wird, bietet die Thematik weiterhin Raum für Forschung, insbesondere im Bereich der instrumentellen Analytik. Ziel der vorliegenden Arbeit ist die Beleuchtung dieser und weiterer Aspekte. Die Extraktion erfolgte überwiegend mittels der sogenannten Mikropulverisierung. Sie ermöglichte die simultane Mahlung der Haarmatrix und Extraktion des EtGs mit einem hohen Probendurchsatz. Die Selektion und anschließende Detektion erfolgte überwiegend durch HPLC-MS/MS. Die Sicherheit bei der Bestimmung des Analyten wurde durch die erfolgreiche Teilnahme an drei Ringversuchen der Society of Hair Testing (SoHT) belegt. Wiederholbedingungen wurden durch Herstellung von eigenen Haarreferenzmaterialien und die Verwendung von homogenen Fremdhaarmaterialien sichergestellt. Zur Evaluierung der Stabilität von EtG wurden zwei Haarmaterialien unter thermischen Stressbedingungen eingelagert und mit dem Gehalt von Referenzproben verglichen. Der Analyt zeigte außergewöhnliche Stabilität unter den gewählten Bedingungen. Ebenso erfolgte eine Beurteilung des Zerstörungsgrads von EtG im Haar durch oxidierende Substanzen, einhergehend mit der Entwicklung eines zerstörungsfreien Schnelltests mittels FTIR zur Detektion von oxidierten Cysteinspezies in Haaren. Das Modellsystem Barthaar wurde für zwei Experimentreihen etabliert: die Korrelation des EtG-Gehaltes im Barthaar nach Aufnahme definierter Alkoholmengen und den Nachweis von glucuronidierten Spezies im Barthaar nach Aufnahme der korrespondierenden Muttersubstanzen. Während keine eindeutige Korrelation zwischen aufgenommener Alkoholmenge und EtG-Gehalt im Barthaar hergestellt werden konnte, war es durchaus möglich, zwei glucuronidierte Metabolite von Arzneistoffen im Barthaar nach Konsum der Ausgangssubstanzen nachzuweisen. / Although EtG is subject to extended research since the year 2000, the topic still holds headroom for further experiments, especially when it comes to the field of instrumental analysis. The goal of the present thesis was the clarification of crucial analytical and further aspects. The extraction was mostly carried out using the so-called micropulverisation. It rendered the simultaneous milling of the hair matrix and extraction of EtG possible with a high sample throughput. Selection of the analyte and following detection was mainly carried out using HPLC-MS/MS. The quality of analysis was ensured by the successful participation in three interlaboratory tests carried out by the Society of Hair Testing (SoHT). Repetitive conditions were ensured by manufacturing of own hair reference materials as well as by the usage of homogeneous external hair materials. Two hair materials were treated under thermal stress conditions and the EtG values were compared to reference samples to verify the analytes stability. EtG showed extraordinary stability under the chosen conditions. Likewise, an assessment of the degree of EtG decay after oxidative treatment as well as the development of a nondestructive assay via FTIR to detect oxidized cysteine species were established. The model system beard hair was arranged for the conduction of two experimental series: the correlation of the EtG content in beard hair after defined oral consumption of ethanol and the detection of glucuronidation of the corresponding parent substances after consumption. Whilst no distinct correlation could be observed for the ethanol experiment, it was possible to provide evidence for the existence of two glucuronized metabolites of drugs after consumption of the parent compounds.

There is a growing need for accurate distribution data for both common and rare plant species for conservation planning and ecological research purposes. A database of more than 500 observations for nine tree species with different ecological and geographical distributions and a range of frequencies of occurrence in south-eastern New South Wales (Australia) was used to compare the predictive performance of logistic regression models, generalised additive models (GAMs) and classification tree models (CTMs) using different data restriction regimes and several model-building strategies. Environmental variables (mean annual rainfall, mean summer rainfall, mean winter rainfall, mean annual temperature, mean maximum summer temperature, mean minimum winter temperature, mean daily radiation, mean daily summer radiation, mean daily June radiation, lithology and topography) were used to model the distribution of each of the plant species in the study area. Model predictive performance was measured as the area under the curve of a receiver operating characteristic (ROC) plot. The initial predictive performance of logistic regression models and generalised additive models (GAMs) using unrestricted, temperature restricted, major gradient restricted and climatic domain restricted data gave results that were contrary to current practice in species distribution modelling. Although climatic domain restriction has been used in other studies, it was found to produce models that had the lowest predictive performance. The performance of domain restricted models was significantly (p = 0.007) inferior to the performance of major gradient restricted models when the predictions of the models were confined to the climatic domain of the species. Furthermore, the effect of data restriction on model predictive performance was found to depend on the species as shown by a significant interaction between species and data restriction treatment (p = 0.013). As found in other studies however, the predictive performance of GAM was significantly (p = 0.003) better than that of logistic regression. The superiority of GAM over logistic regression was unaffected by different data restriction regimes and was not significantly different within species. The logistic regression models used in the initial performance comparisons were based on models developed using the forward selection procedure in a rigorous-fitting model-building framework that was designed to produce parsimonious models. The rigorous-fitting modelbuilding framework involved testing for the significant reduction in model deviance (p = 0.05) and significance of the parameter estimates (p = 0.05). The size of the parameter estimates and their standard errors were inspected because large estimates and/or standard errors are an indication of model degradation from overfilling or effecls such as mullicollinearily. For additional variables to be included in a model, they had to contribule significantly (p = 0.025) to the model prediclive performance. An attempt to improve the performance of species distribution models using logistic regression models in a rigorousfitting model-building framework, the backward elimination procedure was employed for model selection, bul it yielded models with reduced performance. A liberal-filling model-building framework that used significant model deviance reduction at p = 0.05 (low significance models) and 0.00001 (high significance models) levels as the major criterion for variable selection was employed for the development of logistic regression models using the forward selection and backward elimination procedures. Liberal filling yielded models that had a significantly greater predictive performance than the rigorous-fitting logistic regression models (p = 0.0006). The predictive performance of the former models was comparable to that of GAM and classification tree models (CTMs). The low significance liberal-filling models had a much larger number of variables than the high significance liberal-fitting models, but with no significant increase in predictive performance. To develop liberal-filling CTMs, the tree shrinking program in S-PLUS was used to produce a number of trees of differenl sizes (subtrees) by optimally reducing the size of a full CTM for a given species. The 10-fold cross-validated model deviance for the subtrees was plotted against the size of the subtree as a means of selecting an appropriate tree size. In contrast to liberal-fitting logistic regression, liberal-fitting CTMs had poor predictive performance. Species geographical range and species prevalence within the study area were used to categorise the tree species into different distributional forms. These were then used, to compare the effect of plant species rarity on the predictive performance of logistic regression models, GAMs and CTMs. The distributional forms included restricted and rare (RR) species (Eucalyptus paliformis and Eucalyptus kybeanensis), restricted and common (RC) species (Eucalyptus delegatensis, Eucryphia moorei and Eucalyptus fraxinoides), widespread and rare (WR) species (Eucalyptus data) and widespread and common (WC) species (Eucalyptus sieberi, Eucalyptus pauciflora and Eucalyptus fastigata). There were significant differences (p = 0.076) in predictive performance among the distributional forms for the logistic regression and GAM. The predictive performance for the WR distributional form was significantly lower than the performance for the other plant species distributional forms. The predictive performance for the RC and RR distributional forms was significantly greater than the performance for the WC distributional form. The trend in model predictive performance among plant species distributional forms was similar for CTMs except that the CTMs had poor predictive performance for the RR distributional form. This study shows the importance of data restriction to model predictive performance with major gradient data restriction being recommended for consistently high performance. Given the appropriate model selection strategy, logistic regression, GAM and CTM have similar predictive performance. Logistic regression requires a high significance liberal-fitting strategy to both maximise its predictive performance and to select a relatively small model that could be useful for framing future ecological hypotheses about the distribution of individual plant species. The results for the modelling of plant species for conservation purposes were encouraging since logistic regression and GAM performed well for the restricted and rare species, which are usually of greater conservation concern.

<p>This thesis reports on the development of low-temperature processes for transition metal carbide and MAX-phase thin film growth. Magnetron sputtering and evaporation, far from thermodynamical equilibrium, have been utilised to engineer the properties of the films by physical and chemical control. Deposition of W, W₂C and β-WC_1-x films with controlled microstructure, from nanocrystalline to epitaxial, is shown in the W-C system down to 100 ^oC. W films with upto 20 at% C exhibited an extreme solid-solution hardening effect, with a nanoindentation hardness maximum of 35 GPa. Furthermore, the design of epitaxial ternary carbide films is demonstrated in the Ti_1-xV_xC_y system in the form of controlled unit-cell parameters, strain-free films with a perfect match to the substrate, and ternary epitaxial gradient films. Moreover, phase stabilisation and pseudomorphic growth can be tuned in (Nb,Mo)C and (Ti,W)C films. The results obtained can be used for example to optimise electrical contacts in SiC high-power semiconductor devices. </p><p>A large part of this thesis focuses on the deposition of MAX-phases. These compounds constitute a family of thermally stable nanolaminates with composition M_n+1AX_n, n=1, 2 or 3, where M is an early transition metal, A is generally a group 13-14 element, and X is C or N. They show a combination of typical ceramic and metallic properties and are also machinable by virtue of the unique deformation behaviour observed only in laminates. So far, the MAX-phases have almost exclusively been prepared by high-temperature sintering and studied in bulk form. However, this thesis establishes a patented seed layer approach for successful MAX-phase thin film depositions down to 750 ^oC. For the first time, single-phase and epitaxial films of Ti₃SiC₂, Ti₃AlC₂ and Ti₂AlC have been grown. The method has also been used to synthesise a new MAX-phase, Ti₄SiC₃. In addition, two previously unreported intergrown MAX-type structures are presented, Ti₅Si₂C₃ and Ti₇Si₂C₅. Combined theoretical and experimental results show the possibility to deposit films with very low bulk resistivity and designed mechanical properties. Furthermore, the demonstration of MAX-phase and carbide multilayer films paves the way for macrostructure engineering, for example, in coatings for low-friction or wear applications.</p>