Characterization of the CRISPR-Cas subtype I-B proteins Cas6b and Cas8b of Methanococcus maripaludis C5

The CRISPR-Cas system is an adaptive immune system found in archaea and bacteria to defend themselves against mobile genetic elements (e.g. phages). The system employs base complementarity of small RNA species (crRNAs) to target the foreign nucleic acids for degradation. The hallmark of the system is the CRISPR array or locus, which is composed of repetitive DNA sequences (repeats) that are interspersed by unique sequences (spacers). Spacer sequences can be derived from earlier encounters with viruses and, as part of the crRNAs, confer the base complementarity during a reoccurring attack. During the ongoing battle between prokaryotes and viruses diverse CRISPR-Cas systems evolved into three main types that are further subdivided. This thesis shows the first characterization of a subtype I-B CRISPR-Cas system. RNA-Seq data proved the in vivo activity of this CRISPR-Cas system in Methanococcus maripaludis C5. The data further revealed that the crRNAs are always composed of a complete spacer sequence flanked by an 8 nt 5' repeat tag and a 2 nt 3' repeat tag. Eigth cas genes were identified for M. maripaludis. Two Cas proteins, Cas8b and an annotated hypothetical protein were characterized in more detail. The hypothetical protein was shown to be the endoribonuclease responsible for the single-turnover catalysis of precursor crRNA into mature crRNA and was termed Cas6b. The reaction performed by Cas6b yields the 8 nt 5' terminal tag of the mature crRNAs. Despite sharing only low sequence identity of 11 %, the two Cas6 proteins of M. maripaludis and Pyrococcus furiosus could be well aligned using a structural model of Cas6b and the crystal structure of P. furiosus Cas6. Cas6b mutant analysis was used to determine four amino acid residues (lysine 30, histidine 38, histidine 40 and tyrosine 47) that comprise the catalytic site of Cas6b. The RNA binding properties of Cas6b were determined and showed a dimerization upon binding to a non-cleavable substrate. Further analyses including RNA crosslinking experiments followed by mass spectrometry identified a methionine residue (M185) that tightly coordinated to a uridine (U15) of the repeat sequence. Cas6b activity assays employing differently structured repeat variants of M. maripaludis and a 37 nt repeat sequence of Clostridium thermocellum could show, that the processing reaction performed by Cas6b does not recognize a secondary structure of the substrate. In addition to the verification to the in vivo activity of the CRISPR-Cas system, the RNA-Seq data also revealed a varying abundance pattern of crRNAs. To assess the crRNA abundance a experimental procedure was designed, which was aimed to analyse the influence of spacer sequences on a) the processing by Cas6 and b) the stability of crRNAs. With the help of this global approach influences of the spacer length and spacer sequence on the crRNA maturation and in vitro stability were recognized. In this context, future experiments will also determine further possible influences on crRNA abundance including i) crRNA loading into the Cas protein interference complex (Cascade) and ii) possible regulatory effects in terms of crRNA utilization dependent regulation. The characterization of the subtype-specific protein Cas8b revealed a splitting of the recombinant protein into two defined fragments. The exact point of cleavage was determined by Edman sequencing and provides evidence for a proteolytic cleavage of the full-length protein (either autocatalytically or by a protease). Other CRISPR-Cas subtypes were reported to contain two proteins serving as small and big subunit of the interference complex Cascade. For subtype I-B on the other hand Cas8b was found to be the only equivalent to these two proteins and it was proposed that the identified cleavage generates the large and small Cascade subunit. A biochemical analysis of Cas8b with respect to its putative roles during CRISPR-Cas immunity showed an unspecific binding to nucleic acids while no nucleolytic cleavage was observed. Possible functions of Cas8b are discussed and future studies will focus on the analysis of the protein functions in the context of a complete Cascade

Spatial hole burning in thin-disk lasers and twisted-mode operation

Spatial hole burning prevents single-frequency operation of thin-disk lasers when the thin disk is used as a folding mirror. We present an evaluation of the saturation effects in the disk for disks acting as end-mirrors and as folding-mirrors explaining one of the main obstacles towards single-frequency operation. It is shown that a twisted-mode scheme based on a multi-order quarter-wave plate combined with a polarizer provides an almost complete suppression of spatial hole burning and creates an additional wavelength selectivity that enforces efficient single-frequency operation.Comment: 14 pages, 16 figure

Statistical Uncertainty in Quantitative Neutron Radiography

We demonstrate a novel procedure to calibrate neutron detection systems commonly used in standard neutron radiography. This calibration allows determining the uncertainties due to Poisson-like neutron counting statistics for each individual pixel of a radiographic image. The obtained statistical errors are necessary in order to perform a correct quantitative analysis. This fast and convenient method is applied to data measured at the cold neutron radiography facility ICON at the Paul Scherrer Institute. Moreover, from the results the effective neutron flux at the beam line is determined

NEUROCIÊNCIAS E O CONCEITO DE CULPABILIDADE NO DIREITO PENAL

The author analyzes what are the consequences that the discoveries from the neuroscience bring to criminal law, especially to its concept of culpability and, ultimately, to the very conception of Law, that we have today. In this sense, it addresses the criminal concept of culpability, as it is understood from the perspective of free will and from the normative conceptions. The author also studies the options that criminal scholars can adopt to face neuroscientists theses.O autor analisa quais consequências as descobertas das neurociências trazem para o Direito penal, especialmente para o seu conceito de culpabilidade e, também, em última análise, para a própria concepção de Direito, que temos atualmente. Nesse sentido, aborda o conceito penal de culpabilidade, como ele é compreendido a partir da perspectiva de livre arbítrio e das concepções normativas, examinando também as opções que os penalistas podem adotar para enfrentar as teses neurocientíficos.&nbsp

CAPTURE AND ANALYSIS OF SENSOR DATA FOR ASTHMA PATIENTS

Worldwide more than 230 million people suffer from asthma. Reliable and timely guidance for indi-viduals to minimize their risk for asthma attacks is not available. This is largely due to the fact that asthma symptoms are often caused by multiple environmental and personal factors. Many of them are neither captured nor systematically analysed. This is addressed by the project ActOnAir. It aims at a comprehensive capture of health factors and the environmental exposure of individuals, as well as a subsequent analysis in real-time. For this purpose the ActOnAir system provides a mobile sensor box for data collection, a sensor data integration and processing platform, a data mining component and a smartphone application for patients. This contribution outlines the design objectives of the ActOnAir system and discusses corresponding key requirements. The related system architecture is introduced and first results from a prototype implementation are sketched

Auswirkungen der Krise auf Schwellenländer: Welches Entwicklungsmodell hat sich bewährt?

Die Jahre vor der Finanzkrise waren durch ein rasches Produktionswachstum in der Weltwirtschaft insgesamt, vor allem aber durch ein beeindruckendes Wachstum der BRIC-Staaten und weiterer Schwellenländer gekennzeichnet. Nach Ansicht von Thomas Mirow, Präsident der European Bank for Reconstruction and Development, London, war der Entwicklungsweg der Länder Mittel- und Osteuropas bis 2008 erfolgreich. Das Entwicklungsmodell der Transformationsländer sei jedoch vielschichtig zu beurteilen und erfordere für die Zukunft Handlungsbedarf. Nach der Analyse von Gunther Schnabl, Universität Leipzig, ist der Aufholprozess, der durch inländische Kapitalakkumulation im Sektor der handelbaren Güter getrieben ist, überholt. In Mittel- und Osteuropa, Ostasien, Lateinamerika und den rohstoffexportierenden Ländern werden Wachstumsprozesse durch den Zustrom von Kapital getrieben. Diese Entwicklungsstrategie habe aber auch Schattenseiten. Die Frequenz und Dimension von Krisen sei gestiegen. Dies könne mit der Flucht volatilen Kapitals in die entwickelten Kapitalmärkte enden. Michael Knogler, Osteuropa-Institut, Regensburg, wirft einen Blick auf das osteuropäische Aufholmodell eines exportorientierten Wachstums, mit einem kreditfinanzierten Technologieimport als Entwicklungsstrategie. Dies führte dazu, dass das Wachstum in den Jahren ab 2002 von Investitionen und Krediten aus den westlichen Ländern abhängig war. Als Folge der Finanzkrise könnten westliche Kreditgeber ihre Auslandsengagements zurückfahren, so dass eine Verlangsamung des Konvergenzprozesses wahrscheinlich sei. Klaus-Jürgen Gern, Institut für Weltwirtschaft, Kiel, ist der Meinung, dass die Schwellenländer, zumindest eine bestimmte Gruppe, den vor der Krise erreichten Wachstumspfad weiter verfolgen können. Für eine stärkere Abkoppelung der Schwellenländer vom Wachstum in den Industrieländern sei jedoch eine Stärkung der Binnennachfrage vor allem in den Schwellenländern, deren Wachstum bislang exportgetrieben war, notwendig.Finanzmarktkrise, Weltwirtschaft, Wirtschaftswachstum, Osteuropa, Industriestaaten, Schwellenländer, EU-Staaten

Automatic detection and prediction of discontinuities in laser beam butt welding utilizing deep learning

Laser beam butt welding of thin sheets of high-alloy steel can be really challenging due to the formation of joint gaps, affecting weld seam quality. Industrial approaches rely on massive clamping systems to limit joint gap formation. However, those systems have to be adapted for each individually component geometry, making them very cost-intensive and leading to a limited flexibility. In contrast, jigless welding can be a high flexible alternative to substitute conventionally used clamping systems. Based on the collaboration of different actuators, motions systems or robots, the approach allows an almost free workpiece positioning. As a result, jigless welding gives the possibility for influencing the formation of the joint gap by realizing an active position control. However, the realization of an active position control requires an early and reliable error prediction to counteract the formation of joint gaps during laser beam welding. This paper proposes different approaches to predict the formation of joint gaps and gap induced weld discontinuities in terms of lack of fusion based on optical and tactile sensor data. Our approach achieves 97.4 % accuracy for video-based weld discontinuity detection and a mean absolute error of 0.02 mm to predict the formation of joint gaps based on tactile length measurements by using inductive probes

Whole body plastination, intra-organ heterogeneity, and tissue based diagnosis – a survey

 Background: The corpse is the final structural relict of life. Its detailed analysis, the autopsy formed the basis and contributed significantly to our understanding of location, function and interaction of organs in man. Today, autopsies are performed rarely. They have been replaced by radiological in vivo visualization techniques and the analysis of organ excisions and biopsies. Which attributes do whole body preservations possess in this context? Techniques of Whole Body Analysis: In vivo imaging transfers the appearance of body organs and cellular structures in virtual images. The patient’s exposure to X-rays, fundamental particles (electrons, positrons, etc.), strong magnetic fields (nuclear resonance), or ultra sounds release the corresponding signals. The obtained images are interpreted in search for local abnormalities such as cancer, acute and chronic infections, inborn errors, hypertrophy or atrophy. Autopsies require the removal and visual inspection of organs shortly after the victim’s death. In addition, tissue probes of suspicious lesions are fixed and microscopically analyzed. The search for gene or protein abnormalities are added dependent upon the clinical history and gross findings. The whole body plastination is performed in separated steps which include fixation, anatomical dissection, forced polymer impregnation, positioning and curing. Organs and other tissue structures can be taken out of the body and separately demonstrated, or aligned and fixed within the body. Additional tissue examinations are possible at this stage, which is followed by hardening and fixation of the still flexible body. Fixation is done with heat, light or gas.   Results and Interpretation: Tissue conservation is a prerequisite to analyze and investigate in diagnosis and forecast of disease occurrence and behaviour. In history, autopsies have opened the door to localize the position and to understand the functions of organs. Today, they have been replaced by tissue banking and in vivo examinations in a wide range, especially when local lesions of organs are under investigation. Analysis of blood and serum is the main technique to search for organ dysfunction. Whole body plastination is an appropriate technique to investigate and demonstrate healthy appearance of organs, intra-organ heterogeneity, connection to and communication with neighbouring or distant organs as well as localization and distribution of organ lesions, and the associated functional impact. Perspectives: Modern societies try to inform their citizens by numerous investigations of the public health status and to improve the health condition as well as to minimize the development of behaviour associated diseases such as smoking and lung cancer, or overweight and infarction. Well performed body conservation supports these efforts. In addition, it can be considered an innovative technique to understand, diagnose, and even treat dysfunction of intra-body communication at the physical and even mental level.