Heterochromatin und epigenetische Kontrolle der Centromere in Dictyostelium discoideum

Heterochromatin Protein 1 (HP1) is an evolutionarily conserved protein required for formation of a higher-order chromatin structures and epigenetic gene silencing. The objective of the present work was to functionally characterise HP1-like proteins in Dictyostelium discoideum, and to investigate their function in heterochromatin formation and transcriptional gene silencing. The Dictyostelium genome encodes three HP1-like proteins (hcpA, hcpB, hcpC), from which only two, hcpA and hcpB, but not hcpC were found to be expressed during vegetative growth and under developmental conditions. Therefore, hcpC, albeit no obvious pseudogene, was excluded from this study. Both HcpA and HcpB show the characteristic conserved domain structure of HP1 proteins, consisting of an N-terminal chromo domain and a C-terminal chromo shadow domain, which are separated by a hinge. Both proteins show all biochemical activities characteristic for HP1 proteins, such as homo- and heterodimerisation in vitro and in vivo, and DNA binding activtity. HcpA furthermore seems to bind to K9-methylated histone H3 in vitro. The proteins thus appear to be structurally and functionally conserved in Dictyostelium. The proteins display largely identical subnuclear distribution in several minor foci and concentration in one major cluster at the nuclear periphery. The localisation of this cluster adjacent to the nucleus-associated centrosome and its mitotic behaviour strongly suggest that it represents centromeric heterochromatin. Furthermore, it is characterised by histone H3 lysine-9 dimethylation (H3K9me2), which is another hallmark of Dictyostelium heterochromatin. Therefore, one important aspect of the work was to characterise the so-far largely unknown structural organisation of centromeric heterochromatin. The Dictyostelium homologue of inner centromere protein INCENP (DdINCENP), co-localized with both HcpA and H3K9me2 during metaphase, providing further evidence that H3K9me2 and HcpA/B localisation represent centromeric heterochromatin. Chromatin immunoprecipitation (ChIP) showed that two types of high-copy number retrotransposons (DIRS-1 and skipper), which form large irregular arrays at the chromosome ends, which are thought to contain the Dictyostelium centromeres, are characterised by H3K9me2. Neither overexpression of full-length HcpA or HcpB, nor deletion of single Hcp isoforms resulted in changes in retrotransposon transcript levels. However, overexpression of a C-terminally truncated HcpA protein, assumed to display a dominant negative effect, lead to an increase in skipper retrotransposon transcript levels. Furthermore, overexpression of this protein lead to severe growth defects in axenic suspension culture and reduced cell viability. In order to elucidate the proteins functions in centromeric heterochromatin formation, gene knock-outs for both hcpA and hcpB were generated. Both genes could be successfully targeted and disrupted by homologous recombination. Surprisingly, the degree of functional redundancy of the two isoforms was, although not unexpected, very high. Both single knock-out mutants did not show any obvious phenotypes under standard laboratory conditions and only deletion of hcpA resulted in subtle growth phenotypes when grown at low temperature. All attempts to generate a double null mutant failed. However, both endogenous genes could be disrupted in cells in which a rescue construct that ectopically expressed one of the isoforms either with N-terminal 6xHis- or GFP-tag had been introduced. The data imply that the presence of at least one Hcp isoform is essential in Dictyostelium. The lethality of the hcpA/hcpB double mutant thus greatly hampered functional analysis of the two genes. However, the experiment provided genetic evidence that the GFP-HcpA fusion protein, because of its ability to compensate the loss of the endogenous HcpA protein, was a functional protein. The proteins displayed quantitative differences in dimerisation behaviour, which are conferred by the slightly different hinge and chromo shadow domains at the C-termini. Dimerisation preferences in increasing order were HcpA-HcpA << HcpA-HcpB << HcpB-HcpB. Overexpression of GFP-HcpA or a chimeric protein containing the HcpA C-terminus (GFP-HcpBNAC), but not overexpression of GFP-HcpB or GFP-HcpANBC, lead to increased frequencies of anaphase bridges in late mitotic cells, which are thought to be caused by telomere-telomere fusions. Chromatin targeting of the two proteins is achieved by at least two distinct mechanisms. The N-terminal chromo domain and hinge of the proteins are required for targeting to centromeric heterochromatin, while the C-terminal portion encoding the CSD is required for targeting to several other chromatin regions at the nuclear periphery that are characterised by H3K9me2. Targeting to centromeric heterochromatin likely involves direct binding to DNA. The Dictyostelium genome encodes for all subunits of the origin recognition complex (ORC), which is a possible upstream component of HP1 targeting to chromatin. Overexpression of GFP-tagged OrcB, the Dictyostelium Orc2 homologue, showed a distinct nuclear localisation that partially overlapped with the HcpA distribution. Furthermore, GFP-OrcB localized to the centrosome during the entire cell cycle, indicating an involvement in centrosome function. DnmA is the sole DNA methyltransferase in Dictyostelium required for all DNA(cytosine-)methylation. To test for its in vivo activity, two different cell lines were established that ectopically expressed DnmA-myc or DnmA-GFP. It was assumed that overexpression of these proteins might cause an increase in the 5-methyl-cytosine(5-mC)-levels in the genomic DNA due to genomic hypermethylation. Although DnmA-GFP showed preferential localisation in the nucleus, no changes in the 5-mC-levels in the genomic DNA could be detected by capillary electrophoresis.

The continuation of the periodic table up to Z = 172.

The chemical elements up to Z = 172 are calculated with a relativistic Hartree-Fock-Slater program taking into account the effect of the extended nucleus. Predictions of the binding energies, the X-ray spectra and the number of electrons inside the nuclei are given for the inner electron shells. The predicted chemical behaviour will be discussed for a11 elements between Z = 104-120 and compared with previous known extrapolations. For the elements Z = 121-172 predictions of their chemistry and a proposal for the continuation of the Periodic Table are given. The eighth chemical period ends with Z = 164 located below Mercury. The ninth period starts with an alkaline and alkaline earth metal and ends immediately similarly to the second and third period with a noble gas at Z = 172. Mit einem relativistischen Hartree-Fock-Slater Rechenprogramm werden die chemischen Elemente bis zur Ordnungszahl 172 berechnet, wobei der Einfluß des ausgedehnten Kernes berücksichtigt wurde. Für die innersten Elektronenschalen werden Voraussagen über deren Bindungsenergie, das Röntgenspektrum und die Zahl der Elektronen im Kern gemacht. Die voraussichtliche Chemie der Elemente zwischen Z = 104 und 120 wird diskutiert und mit bereits vorhandenen Extrapolationen verglichen. Für die Elemente Z = 121-172 wird eine Voraussage über das chemische Verhalten gegeben, sowie ein Vorschlag für die Fortsetzung des Periodensystems gemacht. Die achte chemische Periode endet mit dem Element 164 im Periodensystem unter Quecksilber gelegen. Die neunte Periode beginnt mit einem Alkali- und Erdalkalimetall und endet sofort wieder wie in der zweiten und dritten Periode mit einem Edelgas bei Z = 172.

CD-Systems of Restarting Automata

Die vorliegende Arbeit behandelt Restartautomaten und Erweiterungen von Restartautomaten. Restartautomaten sind ein Werkzeug zum Erkennen formaler Sprachen. Sie sind motiviert durch die linguistische Methode der Analyse durch Reduktion und wurden 1995 von Jancar, Mráz, Plátek und Vogel eingeführt. Restartautomaten bestehen aus einer endlichen Kontrolle, einem Lese/Schreibfenster fester Größe und einem flexiblen Band. Anfänglich enthält dieses sowohl die Eingabe als auch Bandbegrenzungssymbole. Die Berechnung eines Restartautomaten läuft in so genannten Zyklen ab. Diese beginnen am linken Rand im Startzustand, in ihnen wird eine lokale Ersetzung auf dem Band durchgeführt und sie enden mit einem Neustart, bei dem das Lese/Schreibfenster wieder an den linken Rand bewegt wird und der Startzustand wieder eingenommen wird. Die vorliegende Arbeit beschäftigt sich hauptsächlich mit zwei Erweiterungen der Restartautomaten: CD-Systeme von Restartautomaten und nichtvergessende Restartautomaten. Nichtvergessende Restartautomaten können einen Zyklus in einem beliebigen Zustand beenden und CD-Systeme von Restartautomaten bestehen aus einer Menge von Restartautomaten, die zusammen die Eingabe verarbeiten. Dabei wird ihre Zusammenarbeit durch einen Operationsmodus, ähnlich wie bei CD-Grammatik Systemen, geregelt. Für beide Erweiterungen zeigt sich, dass die deterministischen Modelle mächtiger sind als deterministische Standardrestartautomaten. Es wird gezeigt, dass CD-Systeme von Restartautomaten in vielen Fällen durch nichtvergessende Restartautomaten simuliert werden können und andererseits lassen sich auch nichtvergessende Restartautomaten durch CD-Systeme von Restartautomaten simulieren. Des Weiteren werden Restartautomaten und nichtvergessende Restartautomaten untersucht, die nichtdeterministisch sind, aber keine Fehler machen. Es zeigt sich, dass diese Automaten durch deterministische (nichtvergessende) Restartautomaten simuliert werden können, wenn sie direkt nach der Ersetzung einen neuen Zyklus beginnen, oder ihr Fenster nach links und rechts bewegen können. Außerdem gilt, dass alle (nichtvergessenden) Restartautomaten, die zwar Fehler machen dürfen, diese aber nach endlich vielen Zyklen erkennen, durch (nichtvergessende) Restartautomaten simuliert werden können, die keine Fehler machen. Ein weiteres wichtiges Resultat besagt, dass die deterministischen monotonen nichtvergessenden Restartautomaten mit Hilfssymbolen, die direkt nach dem Ersetzungsschritt den Zyklus beenden, genau die deterministischen kontextfreien Sprachen erkennen, wohingegen die deterministischen monotonen nichtvergessenden Restartautomaten mit Hilfssymbolen ohne diese Einschränkung echt mehr, nämlich die links-rechts regulären Sprachen, erkennen. Damit werden zum ersten Mal Restartautomaten mit Hilfssymbolen, die direkt nach dem Ersetzungsschritt ihren Zyklus beenden, von Restartautomaten desselben Typs ohne diese Einschränkung getrennt. Besonders erwähnenswert ist hierbei, dass beide Automatentypen wohlbekannte Sprachklassen beschreiben.

Patent DE10 2004 037 519 B4 “Sensor Process and Device for Determining a Physical Value“. A Proof of Concept

At present, a fraction of 0.1 - 0.2% of the patients undergoing surgery become aware during the process. The situation is referred to as anesthesia awareness and is obviously very traumatic for the person experiencing it. The reason for its occurrence is mostly an insufficient dosage of the narcotic Propofol combined with the incapability of the technology monitoring the depth of the patient’s anesthetic state to notice the patient becoming aware. A solution can be a highly sensitive and selective real time monitoring device for Propofol based on optical absorption spectroscopy. Its working principle has been postulated by Prof. Dr. habil. H. Hillmer and formulated in DE10 2004 037 519 B4, filed on Aug 30th, 2004. It consists of the exploitation of Intra Cavity Absorption effects in a two mode laser system. In this Dissertation, a two mode external cavity semiconductor laser, which has been developed previously to this work is enhanced and optimized to a functional sensor. Enhancements include the implementation of variable couplers into the system and the implementation of a collimator arrangement into which samples can be introduced. A sample holder and cells are developed and characterized with a focus on compatibility with the measurement approach. Further optimization concerns the overall performance of the system: scattering sources are reduced by re-splicing all fiber-to-fiber connections, parasitic cavities are eliminated by suppressing the Fresnel reflexes of all one fiber ends by means of optical isolators and wavelength stability of the system is improved by the implementation of thermal insulation to the Fiber Bragg Gratings (FBG). The final laser sensor is characterized in detail thermally and optically. Two separate modes are obtained at 1542.0 and 1542.5 nm, tunable in a range of 1nm each. Mode Full Width at Half Maximum (FWHM) is 0.06nm and Signal to Noise Ratio (SNR) is as high as 55 dB. Independent of tuning the two modes of the system can always be equalized in intensity, which is important as the delicacy of the intensity equilibrium is one of the main sensitivity enhancing effects formulated in DE10 2004 037 519 B4. For the proof of concept (POC) measurements the target substance Propofol is diluted in the solvents Acetone and DiChloroMethane (DCM), which have been investigated for compatibility with Propofol beforehand. Eight measurement series (two solvents, two cell lengths and two different mode spacings) are taken, which draw a uniform picture: mode intensity ratio responds linearly to an increase of Propofol in all cases. The slope of the linear response indicates the sensitivity of the system. The eight series are split up into two groups: measurements taken in long cells and measurements taken in short cells.

The Political Ecology of Salmon Aquaculture in Chile

Every German consumes per year, 15% is salmon, which is the third most popular fish in Germany after Alaska-Seelachs and Hering (Keller/Kress 2013: 9). But where does the salmon that ends up on our plates every 6th time we eat fish come from? There's no obligation for producers to declare the origin of their fish products, but if they do so, the latin name of the fish, catching method and catch area should be declared. Salmon, of which about 40% are captured in the wild and the rest brought up in aquacultures, could then be declared as follows: Salmon (salmo salar), aquaculture from Chile. Without any doubt, this makes consumption more transparent, but the standards of production – both, social and ecological ones – and the ecological impacts are still kept in the dark.