Nuclear export of single native mRNA molecules observed via light sheet fluorescence microscopy and transcriptional regulation of BR2.1 during heat-shock

Eucaryotes store most of their genetic information in the nucleus. Parts of this information encode the amino acid sequence of proteins. To synthesize a protein according to the nucleotide sequence, first the corresponding DNA-sequence is transcribed by RNA-Polymerase II to mRNA. Subsequently ribosomes translate the mRNA into the correct amino acid sequence. In eucaryotes the ribosomes are localized in the cytoplasm and are separated from the nucleus by the nuclear envelope. On the one hand separation of transcription and translation enables eucaryotes to process the transcript post-transcriptionally, on the other it requires a transport of the mRNA from the nucleoplasm into the cytoplasm. The nucleoplasm is interconnected with the cytoplasm by nuclear pore complexes. Most of the nucleo-cytoplasmic trafficking is facilitated through the nuclear pore complexes. Messenger RNA is exported into the cytoplasm through the nuclear pore complexes, too. During transcription the nascent mRNA is bound by several proteins which are essential e.g. for mRNA processing and export. The complex of the mRNA and its associated proteins is called an mRNP-particle. Fully processed mRNP-particles are able to cross the permeability barrier of nuclear pore complexes. In this thesis the kinetics of the mRNA-export were measured in salivary gland cells of C. tentans at the single molecule level. Therefore, mRNA was labeled by Hrp36, which was bacterially expressed and subsequently covalently linked to a fluorescent dye. Hrp36 associates cotranscriptionally with the nascent mRNA and is part of the mRNP-particle. After microinjection, labeled Hrp36 is transported into the nucleus, via its endogenous M9-shuttle domain. As all mRNP-particles, also the labeled ones, diffuse through the nucleus after transcription is finished and can be imaged by advanced fluorescence microscopy. In this thesis it is shown that the kinetics of the mRNA-export across the nuclear prore complexes follow a broad distribution in the range of 20ms to seconds. Furthermore, only 30% of all mRNP-particles are exported after they engaged an NPC. Fitting the mRNA-export kinetics with a bimodal gamma distribution revealed average export times of t1exp = 76ms, which is governed by multiple rate limiting steps and t2exp = 158ms, which is governed by just a single rate limiting step. Therefore, the translocation of the mRNA across the nuclear pore complex is not rate limiting for protein-biosynthesis which takes on average several minutes. Trajectory analysis of export events =300ms, showed that the mRNA were localized mainly in the nuclear basket during the export process. Here proteins are localized which are crucial for the mRNP-particle quality control. These proteins bind mRNP-particles, which are only partially processed, and thereby inhibit their translocation through the nuclear pore complex until their processing is completed. Assuming that the general reaction scheme is the same for all mRNP-particles and considering the fact that these slow export events show only a single rate limiting reactions step, this export events presumably correspond to mRNP-particles, whose processing were not finished. In addition to the mRNP-particle export kinetics, the Dbp5 interaction kinetics with the nuclear pore complexes were measured. Dbp5isaRNA-helicase, which is essential form RNP- particle export. It is assumed that Dbp5 removes the transport receptors from the mRNA via its helicase activity and thereby inhibit the translocation of mRNA back into the nucleus. The interaction kinetics of Dbp5 showed two interaction times (t1Dbp5 200Hz = 26ms & t2Dbp5 200Hz = 240ms). Due to the low number of observations, the interaction times gained by fitting the data with a bimodal gamma distribution showed a high uncertainty This makes a comparison of this results with the observed mRNA-export kinetics not advisable. In the second part of the thesis a so far unknown regulation mechanism of transcription was studied. First hints to this mechanism were observed by a control experiment during the examination of the mRNA-export kinetics. Transcription can be subdivided into the four stages of initiation, early elongation, stable elongation and termination. It was previously believed that after transition into stable elongation the transcription process is either completed or terminated prematurely. The results of this thesis give evidence that the transcription process in salivary gland cells of C. tentans can be halted temporally at the stage of stable elongation by applying a heat-shock to the larvae. The halted transcription processes can be resumed after heat-shock is released. Since RNA-polymerase II is highly conserved throughout eucaryotes, it seems very likely that this regulatory mechanism is not limited to C. tentans . The transcription halt during stable elongation described here, shows that eucaryotes have a more direct and far-ranging access to transcription as believed. This direct control of transcription significantly increases the temporal dynamic of transcriptional regulation

Emission measurement and safety assessment for the production process of silicon nanoparticles in a pilot-scale facility

Emission into the workplace was measured for the production process of silicon nanoparticles in a pilot-scale facility at the Institute of Energy and Environmental Technology e.V. (IUTA). The silicon nanoparticles were produced in a hot-wall reactor and consisted of primary particles around 60nm in diameter. We employed real-time aerosol instruments to measure particle number and lung-deposited surface area concentrations and size distribution; airborne particles were also collected for off-line electron microscopic analysis. Emission of silicon nanoparticles was not detected during the processes of synthesis, collection, and bagging. This was attributed to the completely closed production system and other safety measures against particle release which will be discussed briefly. Emission of silicon nanoparticles significantly above the detection limit was only observed during the cleaning process when the production system was open and manually cleaned. The majority of the detected particles was in the size range of 100-400nm and were silicon nanoparticle agglomerates first deposited in the tubing then re-suspended during the cleaning process. Appropriate personal protection equipment is recommended for safety protection of the workers during cleanin

Caveolin-1: a critical regulator of lung fibrosis in idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive chronic disorder characterized by activation of fibroblasts and overproduction of extracellular matrix (ECM). Caveolin-1 (cav-1), a principal component of caveolae, has been implicated in the regulation of numerous signaling pathways and biological processes. We observed marked reduction of cav-1 expression in lung tissues and in primary pulmonary fibroblasts from IPF patients compared with controls. We also demonstrated that cav-1 markedly ameliorated bleomycin (BLM)-induced pulmonary fibrosis, as indicated by histological analysis, hydroxyproline content, and immunoblot analysis. Additionally, transforming growth factor β1 (TGF-β1), the well-known profibrotic cytokine, decreased cav-1 expression in human pulmonary fibroblasts. cav-1 was able to suppress TGF-β1–induced ECM production in cultured fibroblasts through the regulation of the c-Jun N-terminal kinase (JNK) pathway. Interestingly, highly activated JNK was detected in IPF- and BLM-instilled lung tissue samples, which was dramatically suppressed by ad–cav-1 infection. Moreover, JNK1-null fibroblasts showed reduced smad signaling cascades, mimicking the effects of cav-1. This study indicates a pivotal role for cav-1 in ECM regulation and suggests a novel therapeutic target for patients with pulmonary fibrosis

Ligand binding mechanism in steroid receptors; from conserved plasticity to differential evolutionary constraints

Steroid receptor drugs have been available for more than half a century, but details 24 of the ligand binding mechanism has remained elusive. We solved X-ray structures of 25 the glucocorticoid and mineralocorticoid receptors to identify a conserved plasticity at 26 helix 6-7 region that extend the ligand binding pocket towards the receptor surface. 27 Since none of the endogenous ligands exploit this region, we hypothesized that it 28 constitutes an integral part of the binding event. Extensive all atom unbiased ligand 29 exit and entrance simulations corroborate a ligand binding pathway that gives the 30 observed structural plasticity a key functional role. Kinetic measurements reveal that 31 the receptor residence time correlate with structural rearrangements observed in both 32 structures and simulations. Ultimately, our findings reveal why nature has conserved 33 the capacity to open up this region and highlight how differences in the details of the 34 ligand entry process result in differential evolutionary constraints across the steroid 35 receptors.This study was supported by The European Research Council (2009-Adg25027-535 PELE) to V.G and by the SEV-2011-00067 grant of the Severo Ochoa Program. We 536 would like to acknowledge our AstraZeneca colleagues J. Hartleib, R.Unwin and 537 R.Knöll for helpful discussions. We also thank N. Blomberg (ELIXIR) and R. Neutze 538 (University of Gothenburg) for careful reading of the manuscript.Peer ReviewedPostprint (author's final draft

The ATP-Binding Cassette Proteins of the Deep-Branching Protozoan Parasite Trichomonas vaginalis

The ATP binding cassette (ABC) proteins are a family of membrane transporters and regulatory proteins responsible for diverse and critical cellular process in all organisms. To date, there has been no attempt to investigate this class of proteins in the infectious parasite Trichomonas vaginalis. We have utilized a combination of bioinformatics, gene sequence analysis, gene expression and confocal microscopy to investigate the ABC proteins of T. vaginalis. We demonstrate that, uniquely among eukaryotes, T. vaginalis possesses no intact full-length ABC transporters and has undergone a dramatic expansion of some ABC protein sub-families. Furthermore, we provide preliminary evidence that T. vaginalis is able to read through in-frame stop codons to express ABC transporter components from gene pairs in a head-to-tail orientation. Finally, with confocal microscopy we demonstrate the expression and endoplasmic reticulum localization of a number of T. vaginalis ABC transporters

Zinc Finger Nuclease mediated knockout of ADP dependent Glucokinase in Cancer cell lines: Effects on cell survival and Mitochondrial Oxidative Metabolism

<div><p>Zinc finger nucleases (ZFN) are powerful tools for editing genes in cells. Here we use ZFNs to interrogate the biological function of <i>ADPGK</i>, which encodes an ADP-dependent glucokinase (ADPGK), in human tumour cell lines. The hypothesis we tested is that ADPGK utilises ADP to phosphorylate glucose under conditions where ATP becomes limiting, such as hypoxia. We characterised two ZFN knockout clones in each of two lines (H460 and HCT116). All four clones had frameshift mutations in all alleles at the target site in exon 1 of <i>ADPGK,</i> and were ADPGK-null by immunoblotting. <i>ADPGK</i> knockout had little or no effect on cell proliferation, but compromised the ability of H460 cells to survive siRNA silencing of hexokinase-2 under oxic conditions, with clonogenic survival falling from 21±3% for the parental line to 6.4±0.8% (p = 0.002) and 4.3±0.8% (p = 0.001) for the two knockouts. A similar increased sensitivity to clonogenic cell killing was observed under anoxia. No such changes were found when <i>ADPGK</i> was knocked out in HCT116 cells, for which the parental line was less sensitive than H460 to anoxia and to hexokinase-2 silencing. While knockout of <i>ADPGK</i> in HCT116 cells caused few changes in global gene expression, knockout of <i>ADPGK</i> in H460 cells caused notable up-regulation of mRNAs encoding cell adhesion proteins. Surprisingly, we could discern no consistent effect on glycolysis as measured by glucose consumption or lactate formation under anoxia, or extracellular acidification rate (Seahorse XF analyser) under oxic conditions in a variety of media. However, oxygen consumption rates were generally lower in the <i>ADPGK</i> knockouts, in some cases markedly so. Collectively, the results demonstrate that <i>ADPGK</i> can contribute to tumour cell survival under conditions of high glycolytic dependence, but the phenotype resulting from knockout of <i>ADPGK</i> is cell line dependent and appears to be unrelated to priming of glycolysis in these lines.</p></div

HTAP3 fires: towards a multi-model, multi-pollutant study of fire impacts

Open biomass burning has major impacts globally and regionally on atmospheric composition. Fire emissions include particulate matter, tropospheric ozone precursors, greenhouse gases, as well as persistent organic pollutants, mercury and other metals. Fire frequency, intensity, duration, and location are changing as the climate warms, and modelling these fires and their impacts is becoming more and more critical to inform climate adaptation and mitigation, as well as land management. Indeed, the air pollution from fires can reverse the progress made by emission controls on industry and transportation. At the same time, nearly all aspects of fire modelling – such as emissions, plume injection height, long-range transport, and plume chemistry – are highly uncertain. This paper outlines a multi-model, multi-pollutant, multi-regional study to improve the understanding of the uncertainties and variability in fire atmospheric science, models, and fires’ impacts, in addition to providing quantitative estimates of the air pollution and radiative impacts of biomass burning. Coordinated under the auspices of the Task Force on Hemispheric Transport of Air Pollution, the international atmospheric modelling and fire science communities are working towards the common goal of improving global fire modelling and using this multi-model experiment to provide estimates of fire pollution for impact studies. This paper outlines the research needs, opportunities, and options for the fire-focused multi-model experiments and provides guidance for these modelling experiments, outputs, and analysis that are to be pursued over the next 3 to 5 years. It proposes a plan for delivering specific products at key points over this period to meet important milestones relevant to science and policy audiences

Research and Design of a Routing Protocol in Large-Scale Wireless Sensor Networks

无线传感器网络，作为全球未来十大技术之一，集成了传感器技术、嵌入式计算技术、分布式信息处理和自组织网技术，可实时感知、采集、处理、传输网络分布区域内的各种信息数据，在军事国防、生物医疗、环境监测、抢险救灾、防恐反恐、危险区域远程控制等领域具有十分广阔的应用前景。 本文研究分析了无线传感器网络的已有路由协议，并针对大规模的无线传感器网络设计了一种树状路由协议，它根据节点地址信息来形成路由，从而简化了复杂繁冗的路由表查找和维护，节省了不必要的开销，提高了路由效率，实现了快速有效的数据传输。 为支持此路由协议本文提出了一种自适应动态地址分配算——ADAR（AdaptiveDynamicAddre...As one of the ten high technologies in the future, wireless sensor network, which is the integration of micro-sensors, embedded computing, modern network and Ad Hoc technologies, can apperceive, collect, process and transmit various information data within the region. It can be used in military defense, biomedical, environmental monitoring, disaster relief, counter-terrorism, remote control of haz...学位：工学硕士院系专业：信息科学与技术学院通信工程系_通信与信息系统学号：2332007115216

CYK4 Promotes Antiparallel Microtubule Bundling by Optimizing MKLP1 Neck Conformation

Centralspindlin, a constitutive 2:2 heterotetramer of MKLP1 (a kinesin-6) and the non-motor subunit CYK4, plays important roles in cytokinesis. It is crucial for the formation of central spindle microtubule bundle structure. Its accumulation at the central antiparallel overlap zone is key for recruitment and regulation of downstream cytokinesis factors and for stable anchoring of the plasma membrane at the midbody. Both MKLP1 and CYK4 are required for efficient microtubule bundling. However, the mechanism by which CYK4 contributes to this is unclear. Here we performed structural and functional analyses of centralspindlin using high-speed atomic force microscopy, Fӧrster resonance energy transfer analysis, and in vitro reconstitution. Our data reveal that CYK4 binds to a globular mass in the atypically long MKLP1 neck domain between the catalytic core and the coiled coil and thereby reconfigures the two motor domains in the MKLP1 dimer to be suitable for antiparallel microtubule bundling. Our work provides insights into the microtubule bundling during cytokinesis and into the working mechanisms of the kinesins with non-canonical neck structures

A Strategic AI Procedure Model For Implementing Artificial Intelligence

For most industries, Artificial Intelligence (AI) holds substantial potentials. In the last decades, the extent of data created worldwide is exponentially increasing, and this trend is likely to continue. However, despite the prospects, many companies are not yet using AI at all or not generating added value. Often, an AI project does not exceed its pilot phase and is not scaled up. The problems to create value from AI applications in companies are manifold, especially since AI itself is diverse and there is no ‘one size fits all’ approach. One often stated obstacle, why many AI projects fail, is a missing AI strategy. This leads to isolated solutions, which do not consider synergies, scalability and seldom result in added value for the company. To create a company-specific AI strategy with a top-down approach, a generic but holistic framework is needed. This paper proposes a strategic AI procedure model that enables companies to define a specific AI strategy for successfully implementing AI solutions. In addition, we demonstrate in this paper how we apply the introduced strategic AI procedure model on an AI-based flexible monitoring and regulation system for power distribution grid operators in the context of an ongoing research project