Refine
Departments, institutes and facilities
Document Type
- Doctoral Thesis (5) (remove)
Year of publication
- 2015 (5) (remove)
Has Fulltext
- no (5)
Keywords
- Analytik (1)
- Biomass (1)
- Biomasse (1)
- Chemische Analyse (1)
- Enhancer (1)
- Genregulation (1)
- Krebsforschung (1)
- Lignin (1)
- Nachwachsender Rohstoff (1)
- Phenol-Formaldehyd-Harze (1)
The generation and maintenance of intricate spatiotemporal patterns of gene expression in multicellular organisms requires the establishment of complex mechanisms of transcriptional regulation. Estimations that up to one million enhancers exist in the human genome accentuates the utmost importance of this type of cis-regulatory element for gene regulation. However, surprisingly little is known about the mechanisms used to temporarily or permanently activate or inactivate enhancers during cellular differentiation. The current work addresses the question how enhancer regulation can be achieved.
Using the chemokine (C-C motif) ligand gene Ccl22 as a model, the first example is based on the question how the activation of an enhancer can be prevented in a physiological context. Ccl22 is expressed by myeloid cells, such as dendritic cells, upon exposure to inflammatory stimuli. The expression in other cell types, such as fibroblasts, is prevented by the strong accumulation of H3K9me3 at the enhancer's proximal region. This accumulation is attenuated in myeloid cells through activity of the stimulus-induced demethylase Jmjd2d. To tease out which genomic fragment or fragments in the Ccl22 locus could be responsible for the maintenance of enhancer inactivity, potentially through the recruitment of H3K9 methyltransferases, the enhancer repressing capacity of 1 kb fragments of the gene locus was analysed in retroviral reporter assays. It was found that a fragment adjacent to the Ccl22 enhancer that overlaps with a member of a subfamily of long interspersed nuclear elements (LINEs) showed strong repressive potential on a model enhancer. Subsequent retroviral reporter assays with LINEs from loci of other stimulus-dependent genes identified additional LINE fragments that exhibit strong enhancer repressive capacity. These findings suggest a mechanism for enhancer silencing involving LINEs.
The second example concentrates on the inactivation of an enhancer during colorectal cancer (CRC) progression. The adenoma to carcinoma transition during CRC progression often is accompanied by a downregulation of the tumour suppressor gene EPHB2. The EMT inducing factor SNAIL1 strongly downregulated EPHB2 expression in a CRC cell model. To gain insights into the transcriptional regulation of EPHB2, potential cis-regulatory elements in the EPHB2 upstream region were analysed using reporter assays. A cell-type-specific enhancer was identified and subsequent chromatin analyses revealed a correlation between enhancer chromatin conformation and EPHB2 expression in different CRC cell lines. Additionally, the overexpression of the murine Snail1 induced chromatin changes at the EPHB2 enhancer towards a poised, transcriptionally silent chromatin conformation. Mutational analyses of the minimal enhancer region pinpointed three transcription factor binding motifs to be essential for full enhancer activity. Different binding patterns between CRC cell lines at the TCF/LEF motif were subsequently identified. Furthermore, a switch from TCF7L2 to LEF1 occupancy was found upon overexpression of Snail1 in vitro and in vivo. The generation of LS174T CRC cells overexpressing LEF1 confirmed the involvement of LEF1 in the downregulation of EPHB2 and the competitive displacement of TCF7L2. This part of the work demonstrated that the SNAIL1 induced downregulation of EPHB2 is dependent on the decommissioning of a transcriptional enhancer and led to a hypothetical model involving LEF1 and ZEB1.
In summary, this work highlighted two distinct mechanisms for enhancer regulation. One mechanism is based on enhancer repressive LINE fragments that might prevent stimulus-dependent enhancer activation. In the second, enhancer silencing was shown to be based on a competitive transcription factor binding mechanism.
Over the last 50 years, the controlled motion of robots has become a very mature domain of expertise. It can deal with all sorts of topologies and types of joints and actuators, with kinematic as well as dynamic models of devices, and with one or several tools or sensors attached to the mechanical structure. Nevertheless, the domain has not succeeded in standardizing the modelling of robot devices (including such fundamental entities as “reference frames”!), let alone the semantics of their motion specification and control. This thesis aims to solve this long-standing problem, from three different sides: semantic models for robot kinematics and dynamics, semantic models of all possible motion specification and control problems, and software that can support the latter while being configured by a systematic use of the former.
Lignin ist bereits ein intensives Gebiet der Forschung, allerdings werden Verknüpfungen zwischen Quelle, Aufschlussmethode und Einsatz in der Literatur kaum beschrieben. In der vorliegenden Arbeit werden Lignine von verschiedenen Quellen (Weizenstroh, Buche, Nadelholz) und Aufschlussmethoden (AFEX, Wasserdampfaufschluss, Organosolv, Saure Hydrolyse) analytisch erfasst und hinsichtlich ihres Einsatzes in polymeren Materialien charakterisiert. Eine breite Auswahl an Methoden wurden eingesetzt, FT-IR- Spektroskopie, UV-Vis, 31P-NMR, GPC, Pyrolyse-GC/MS, sowie HPLC zur Bestimmung der Reinheit gemäß des NREL-Standard-Protokolls. Thermische Analysen, wie TGA und DSC zeigten Glasübergangstemperaturen um 120°C, sowie Zersetzungstemperaturen zwischen 340°C und 380°C. Die Ergebnisse weisen für das Organosolv-Buchenholz-Lignin hochreine Fraktionen auf, die bis dato noch nicht erreicht wurden. Die Ergebnisse dieser Arbeit identifizien die Organosolv-Buchenholz-Lignine als ein verwertbares Produkt im Hinblick auf die Anwendung in Polyurethanen sowie Phenol-Formaldehydharzen.
In this doctoral thesis the curing process of visible light-curing (VLC) dental composites and 3D printing rapid prototyping (RP) materials are investigated with the focus on dielectric analysis (DEA). This method is able to monitor the curing of resins in an alternating electric fringe field with adjustable frequencies and is often used for cure control of composites manufacturing in the aviation and automotive industry but hardly established in dental science or RP method development. It is capable of investigating very fast initiation and primary curing processes using high frequencies in the kHz-range. The aim of the Thesis is a better understanding of the curing processes with respect to curing parameters such as resin composition, viscosity, temperature, and for light-curing composites also light intensity and irradiation depth. Due to the nature of both dental and RP systems an application of specific experimental set-up had to be designed allowing for the generation of reproducible and valid results. Subsequently, different evaluation methods were developed to characterize the curing behavior of both material types. A special focus was paid to the determination of kinetic parameters from DEA measurements. Reaction rates of the curing of the corresponding thermosets were calculated and applied to the ion viscosity curves measured by DEA to evaluate reaction kinetic parameters. For the dental composites it could be clearly shown that the initial curing rate is directly proportional to light intensity and not to its square root as proposed by many others authors. A good description of the curing behaviour of 3DP RP materials was also achieved assuming a reaction order smaller than one. This data provides the base for the kinetic modeling of polymerization and curing processes proposed within the Thesis.