128 research outputs found

    Detection of UV-induced gene activation in mammalian cells by means of a GFP vector stably integrated into the genome

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    Titelblatt, Inhaltsverzeichnis, Selbstandigkeitserklarung, Lebenslauf, Danksagung 1\. Einleitung 2\. Material und Methoden, Teil 1 2\. Material und Methoden, Teil 2 3\. Ergebnisse, Teil 1 3\. Ergebnisse, Teil 2 3\. Ergebnisse, Teil 3 4\. Diskussion 5\. Zusammenfassung 6\. Summary 7\. Abkurzungen und Glossar 8\. LiteraturUm die Vorteile der Cofaktor-unabhangigen Expression von ýGreen Fluorescent Proteiný (GFP) in heterologen Systemen und der Nachweisbarkeit von GFP in lebenden Zellen in der Untersuchung der Wirkungen ultravioletter (UV-) Strahlung auf Saugerzellen zu nutzen, sollte eine stabile Zellinie hergestellt werden, mit deren Hilfe UV-induzierte Genaktivierung in Saugerzellen anhand der grunen Fluoreszenz des Reporterproteins Enhanced GFP (EGFP) gemessen werden kann. Dazu wurden das an Saugerzellen adaptierte EGFP und seine destabilisierte Variante, d2EGFP, auf ihre Einsatzfahigkeit als Reporter fur die Promotoraktivitat in Saugerzellen untersucht. Im Vergeich zu den Ausgangszellinien zeigten stabil transfizierte, konstitutiv EGFP- oder d2EGFP- exprimierende CHO-Zellinien keine Veranderung des Wachstumsverhaltens und der Empfindlichkeit gegenuber Rontgen- und UVC-Strahlung. EGFP konnte in lebenden und Formaldehyd-fixierten Zellen durch FACS-Analyse und im Fluoreszenzmikroskop in jeder einzelnen Zelle, und zeitsparend im MTP- Fluorimeter nachgewiesen werden. Dabei besteht eine lineare Abhangigkeit der Fluoreszenzintensitat von der Anzahl der Zellen. Nach stabiler Transfektion kann das Wachtum dieser Zellen nach Behandlung mit einem zytotoxischen Agens anhand der EGFP-Fluoreszenz im MTP-Fluorimeter gemessen werden. Konstitutiv exprimiertes d2EGFP, das eine Halbwertszeit von 3 h in CHO-Zellen hat, konnte dagegen nur durch FACS-Analyse und im Fluoreszenzmikroskop sichtbar gemacht werden. Zur Untersuchung der UV-induzierbaren Genexpression wurde die stabil transfizierte Zellinie HEK-pNF-kappaB/Neo hergestellt, bei der das Reportergen d2EGFP unter Kontrolle eines synthetischen Promotors steht, der vier NF- kappaB-Bindungsstellen und den minimalen Thymidinkinase-Promotor enthalt. In dieser humanen embryonalen Nierenzellinie kann UVA-induzierte Genaktivierung anhand der d2EGFP-Fluoreszenz gemessen werden. TNF-alpha loste in bis zu 90 % der Zellen dieser Zellinie d2EGFP-Expression aus und wurde deshalb als Positivkontrolle der Induktion NF-kappaB-abhangiger Genexpression verwendet. UVC- und UVB-Strahlung losten keine erhohte d2EGFP-Expression aus, Rontgenstrahlung nur in hohen Dosen. Behandlung mit dem Tumorpromotor PMA verursachte d2EGFP-Expression in bis 40 % der Zellen einer Population. Der Nachweis dieser induzierten d2EGFP-Expression erforderte die FACS-Analyse oder das Betrachten im Fluoreszenzmikroskop, eine Messung im MTP-Fluorimeter war aufgrund der geringen Fluoreszenzintensitat nicht moglich.In order to benefit from the advantages of the cofactor-independent expression of ýGreen Fluorescent Proteiný (GFP) in heterologous systems and its detection in living cells for the exploration of the effects of ultraviolet (UV) radiation in mammalian cells, a stable cell line was established, by means of which UV induced gene activation can be measured as green fluorescence of the reporter protein ýEnhanced GFPý (EGFP). For this purpose, the suitability of EGFP and its destabilized variant, d2EGFP, as reporter of promoter activity in mammalian cells was examined. By means of stably transfected, constitutively EGFP or d2EGFP expressing CHO cells, different detection methods were tested. Compared to the parental cell lines, these stably transfected cell lines showed no change in growth behaviour and in the sensitivity towards X-rays and UVC radiation. EGFP was either monitored in living and in formaldehyde fixed cells by FACS analysis and in the fluorescence microscope in each individual cell, or by a time- saving procedure using a microplate reader. A linear correlation between fluorescence intensity and cell number per well was found. Using these stably transfected cells, the effect of cytotoxic agents on cell growth can be measured as a reduced EGFP fluorescence increase in the microplate reader. Constitutively expressed d2EGFP, which shows a half-life of 3 h in CHO cells, can only be monitored by FACS analysis and in the fluorescence microscope, but not in the microplate reader. For the examination of UV induced gene expression, the stably transfected cell line HEK-pNF-kappaB/Neo was generated, in which the reporter gene d2EGFP is under the control of a synthetic promoter, which consists of four NF-kappaB binding sites and the minimal thymidin kinase promoter. In this human embryonic kidney (HEK) cell line, UVA induced gene expression can be measured as an increase of EGFP fluorescence. TNF-alpha treatment of cells gave rise to substantial d2EGFP expression in up to 90 % of the cells and was therefore used as a positive control of induction of NF-kappaB dependent gene expression. UVC and UVB radiation caused no increase in d2EGFP expression, X-rays did so only at high doses. Treatment of cells with the tumour promoter PMA produced d2EGFP expression in up to 40 % of the population. The detection of this induced d2EGFP expression required FACS analysis or inspection in the fluorescence microscope, measurement in the microplate reader was impossible due to the low fluorescence intensity

    Kabinenluftqualität mit Schwerpunkt auf Geruchsereignissen – Eine Literaturstudie

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    Einleitung: Während eines Fume and Smell Events (FSE) können Dämpfe oder Gerüche ins Cockpit oder in die Flugzeugkabine gelangen. Insbesondere Kontaminationen der Zapfluft mit Triebwerks- oder Hydrauliköl werden mit vom fliegenden Personal berichteten vor allem neurologischen und/oder respiratorischen Symptomen während und / oder nach einem FSE in Verbindung gebracht. Zudem besteht Besorgnis über eine mögliche chronische Exposition mit toxischen Substanzen in der Kabinenluft. Fragestellungen: In dieser Studie wurde wissenschaftliche Literatur zu Ursachen von Gerüchen, Dampf- und Rauchentwicklung in Flugzeugen, zu chemischen Substanzen, die bei FSE in die Kabine gelangen könnten, zu einer möglichen Exposition von fliegendem Personal bei FSE, und zu akuten und chronischen Symptomen, die nach FSE-Exposition von fliegendem Personal im Kontext mit dem „aerotoxischen Syndrom“ beschrieben wurden und möglichen Ursachen, recherchiert und ausgewertet. Methodik: Durch eine orientierende Literaturrecherche wurde Suchstrings definiert und für eine systematische Recherche in den Literaturdatenbanken Medline, Web of Science und Scopus verwendet. Nach Import in eine Literaturdatenbank folgte ein Abstract- und Volltextscreening, eine Zuordnung zu den Fragestellungen und eine Bewertung der Evidenzklasse der Publikationen, die in Evidenztabellen mit einer zusammenfassenden Bewertung der Evidenzlage aufgelistet wurden. Ergebnisse: Die Häufigkeit von Kabinenluftereignissen wurde auf 1 Ereignis pro 2000-15.000 Flüge geschätzt. FSE können sich in allen Flugphasen ereignen, mit einer Häufung während Steig- und Reiseflug. Als Ursachen wurden u.a. Probleme mit der „Auxiliary Power Unit“ (APU), den Triebwerken, der Klimaversorgung/Environmental Control System (ECS), elektrischen Systemen und Küchengeräten, Kaffeemaschinen und Öfen identifiziert. Messflüge ergaben, CO, Ozon, volatile organic compounds (VOCs) und semi-volatile organic compounds (SVOCs) und Organophosphate im Normalbetrieb und bei vereinzelten Geruchsereignissen aktuelle Richt- oder Grenzwerte nicht überschreiten. Erhöhte CO₂-Konzentrationen und Partikelzählraten traten in der Taxiphase auf. Während des Reiseflugs sind die Konzentrationen von Luftkontaminanten aufgrund der hohen Luftwechselrate in der Flugzeugkabine meist gering und steigen gelegentlich durch Service und Passagieraktivitäten an. Die Beschreibung des durch FSE-ausgelösten Symptomkomplexes beruht im Wesentlichen auf Fall- und Fragebogen-Studien. Die in Expositionsstudien und nach versehentlicher Exposition beschriebenen Symptome zeigen eine gute Übereinstimmung mit einem Teil der durch Fragebogenstudien ermittelten akuten Symptome. Biomarker-Studien ergaben keinen Hinweis auf eine Organophosphatvergiftung. Genetische Variationen bei der Entgiftung wurden in einer Risikoabschätzung mit einem Faktor von 4000 als theoretischer maximaler Unterschied zwischen empfindlichen und unempfindlichen Individuen berücksichtigt. Die berichteten chronischen Symptome sind unspezifisch und divers und es bleibt unklar, ob die Symptome beim fliegenden Personal häufiger vorkommen als in der Allgemeinbevölkerung oder in anderen Berufsgruppen. Wenige Studien wiesen eine Kontrollgruppe auf und zeigten, dass Müdigkeit, Schlafstörungen und Depressionen von Flugbegleiterinnen häufiger berichtet wurden als von der Kontrollgruppe. Vereinzelte Studien mit neurologischer Untersuchung und psychometrischen Tests zeigten eine geringfügige Verschlechterung weniger Parameter. Des Weiteren wurden in kleinen Studien bei Betroffenen Veränderungen des Gehirns mit bildgebenden Verfahren gefunden, oder eine Erhöhung Nervensystem-spezifischer Auto-Antikörper im Serum. Die Bedeutung dieser morphologischen Veränderungen und der Auto-Antikörper im Krankheitsprozess und für die Diagnostik ist noch unklar. Schlussfolgerungen: Es lässt sich vermuten, dass in seltenen Fällen einer starken Kontamination der Zapfluft mit (pyrolysierten) Ölen CO, CO₂, Aldehyde und Feinstaub und ggf. weitere Substanzen ansteigen und akute Symptome wie Augen-, Nasen- und Rachenreizung, Schmerzen / Engegefühl in der Brust, Schwindel, Übelkeit, Erbrechen und Kopfschmerzen direkt oder indirekt verursachen können. Die Datenlage lässt eine Kausalitätsbewertung für mögliche chronische Symptome nicht zu. Dafür wäre u.a. ein Expositionsmonitoring des fliegenden Personals nach Identifikation geeigneter Markersubstanzen im Vergleich zu einer Kontrollgruppe ohne Exposition gegenüber Kabinenluftkontaminanten erforderlich. Danksagung: Die Literaturstudie erfolgte mit freundlicher Unterstützung der BG Verkehr

    Validation of biological recognition elements for signal transduction as first step in the development of whole cell biosensors

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    Choosing the proper combination of receptor element, cell type and measurable signal requires major consideration for developing cell-based biosensors. In order to use physiologically relevant cellular responses towards (geno)toxic conditions, information on the mechanism of action and of the expected outcome of exposure needs to be considered

    Growth and biofilm formation of Penicillium chrysogenum in simulated microgravity

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    Penicillium sp. are one of the main fungal genera detected on board the Russian Space Station (MIR) and the International Space Station (ISS), demonstrating its ability to grow on the space stations´ walls and to maintain growth under microgravity (1-3). As a spore-forming microorganism, Penicillium sp. poses a concern for planetary protection and to human/astronaut health, as its spores, associated with respiratory diseases, can be dispersed through the air (4). Fungal growth on the ISS has shown to promote biodegradation of the spacecraft materials, compromising their integrity. Biofilms are groups of organisms adhered to each other by self-synthesized extracellular polymeric substances, and are ubiquitous in industrial and natural environments (5). It has been reported that Penicillium sp. forms biofilms, which are associated with higher tolerance/resistance to adverse conditions (6). Therefore, biofilm formed on the ISS may have deleterious effects on astronaut’s health and/or on ISS materials. To gain valuable knowledge to control biofilm during long duration spaceflight missions, the NASA-funded project “Characterization of Biofilm Formation, Growth, and Gene Expression on Different Materials and Environmental Conditions in Microgravity” is currently being prepared. Pre-flight testing include: defining and optimizing the growth medium and culturing conditions of P. chrysogenum DSM 1075; characterizing the morphological response of P. chrysogenum growth under simulated microgravity; assessing biofilm formation by P. chrysogenum under different conditions. The study of this fungal strain represents the beginning of a new line of research on board ISS. The knowledge gained can be applicable to a) the safety and maintenance of crewed spacecraft, b) planetary protection, c) mitigation of biofilm-associated illnesses on the crew, as well as on the Earth. Besides, P. chrysogenum is of major medical and historical importance, as it presents the original and present-day industrial source of the antibiotic penicillin, and as an important producer of antifungal proteins and other relevant enzymes

    Radiation Response of Murine Embryonic Stem Cells

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    To understand the mechanisms of disturbed differentiation and development by radiation, murine CGR8 embryonic stem cells (mESCs) were exposed to ionizing radiation and differentiated by forming embryoid bodies (EBs). The colony forming ability test was applied for survival and the MTT test for viability determination after X-irradiation. Cell cycle progression was determined by flow cytometry of propidium iodide-stained cells, and DNA double strand break (DSB) induction and repair by γH2AX immunofluorescence. The radiosensitivity of mESCs was slightly higher compared to the murine osteoblast cell line OCT-1. The viability 72 h after X-irradiation decreased dose-dependently and was higher in the presence of leukemia inhibitory factor (LIF). Cells exposed to 2 or 7 Gy underwent a transient G2 arrest. X-irradiation induced γH2AX foci and they disappeared within 72 h. After 72 h of X-ray exposure, RNA was isolated and analyzed using genome-wide microarrays. The gene expression analysis revealed amongst others a regulation of developmental genes (Ada, Baz1a, Calcoco2, Htra1, Nefh, S100a6 and Rassf6), downregulation of genes involved in glycolysis and pyruvate metabolism whereas upregulation of genes related to the p53 signaling pathway. X-irradiated mESCs formed EBs and differentiated toward cardiomyocytes but their beating frequencies were lower compared to EBs from unirradiated cells. These results suggest that X-irradiation of mESCs deregulate genes related to the developmental process. The most significant biological processes found to be altered by X-irradiation in mESCs were the development of cardiovascular, nervous, circulatory and renal system. These results may explain the X-irradiation induced-embryonic lethality and malformations observed in animal studies

    UNRAVELING ASTROCYTE BEHAVIOUR IN THE SPACE BRAIN: RADIATION RESPONSE OF PRIMARY ASTROCYTES

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    Exposure to ionizing radiation as part of space radiation, is a major limiting factor for crewed space exploration. Astronauts will encounter different types of space radiation, which may cause cognitive damage causing detrimental effects on learning and attention, elevated anxiety and depression. Due to its limited regenerative potential, especially the central nervous system (CNS) is very vulnerable towards radiation-induced damage. Astrocytes, the most abundant glial cells of the CNS, have different crucial functions in the CNS, e.g. maintaining normal brain function. In this work, the response of astrocytes towards low linear energy transfer (LET) X-rays and high-LET carbon ions was compared to unravel possible specific effects of space-relevant high-LET radiation. [...

    Towards sustainable human space exploration—priorities for radiation research to quantify and mitigate radiation risks

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    Human spaceflight is entering a new era of sustainable human space exploration. By 2030 humans will regularly fly to the Moon’s orbit, return to the Moon’s surface and preparations for crewed Mars missions will intensify. In planning these undertakings, several challenges will need to be addressed in order to ensure the safety of astronauts during their space travels. One of the important challenges to overcome, that could be a major showstopper of the space endeavor, is the exposure to the space radiation environment. There is an urgent need for quantifying, managing and limiting the detrimental health risks and electronics damage induced by space radiation exposure. Such risks raise key priority topics for space research programs. Risk limitation involves obtaining a better understanding of space weather phenomena and the complex radiation environment in spaceflight, as well as developing and applying accurate dosimetric instruments, understanding related short- and long-term health risks, and strategies for effective countermeasures to minimize both exposure to space radiation and the remaining effects post exposure. The ESA/SciSpacE Space Radiation White Paper identifies those topics and underlines priorities for future research and development, to enable safe human and robotic exploration of space beyond Low Earth Orbit

    The impact of microgravity and gravitational countermeasures on the gut microbiome of humans enrolled in the AGBRESA study

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    The Artificial Gravity Bed Rest Study – AGBRESA – was the first joint study conducted by DLR, ESA and NASA to simulate the effects of microgravity on healthy subjects. Moreover, the study included the use of artificial gravity protocols in a short-arm human centrifuge as a measure to counteract the negative effects of weightlessness. The health of the gut translates into the overall wellbeing since the disruption of the gut symbiotic networks – dysbiosis – could be due to either diet, antibiotic ingestion, sleep disturbance, physical activity or psychological stresses. In recent times, the gut microbiome has changed from being a complementary addition to our digestive tract to a potentially life-changing role by directly being the source of stimuli which revealed to impact neurochemistry, behavior and overall physiological status. Combined, microbial fluctuations could alter the intestinal microbiota composition and bacterial metabolite production, or more severely, in the disruption of host intestinal barrier integrity and the immune system activity, triggering intestinal inflammation syndromes and making the gut a very relevant organ to be studied in the context of spaceflight. Thus, 12 subjects, 8 males, were subjected to bed rest at negative 6-degree inclination for a period of 60 days with a preceding baseline of 15 days and posterior recovery period of 14 days. In other to characterize the gut microenvironment of healthy humans in simulated microgravity, fecal samples were collected during the baseline stage (once), during the head-down tilt treatment (at days 10, 30, and 50) and during the recovery period (once), and the samples were then processed for 16S rRNA sequencing and taxonomic analysis of the gut microenvironment. The characterization of the prokaryote flora was conducted 1) throughout time in contrast to the baseline reference and 2) in the context of the gravitational countermeasure vs the bed-rest-only control. The analysis revealed the detection of commensal microorganisms described to positively impact the gut such as Bifidobacterium spp., Lactobacillus spp., Akkermansia spp. and Enterococus spp.. Interestingly, we were able to detect pathogens like Campylobacter hominis which has been linked to severe bowel diseases ulcerative colitis and Crohn's disease. Also, opportunistic microorganisms such as Fusobacterium spp., Prevotella spp., Pseudomonas spp., Staphylococcus and Streptococcus spp., could potentially indicate an imbalance of the microbial networks and be a good an indicator of dysbiosis. Additionally, we set aside samples to undergo proteomic and metabolite analysis to improve the characterization of the gut microenvironment under microgravity simulation and the extent of the gravitational countermeasure recovery on bowel condition. Overall, the microgravity simulation performed on the AGBRESA study did not impact dramatically the fitness of the participants. Nonetheless, the analysis of the gut provides important insights on the triggers that occur during the adaptation of human physiology to long term exposure to spaceflight conditions and whether these relate to the described complications associated with gut disease

    The Use of ProteoTuner Technology to Study Nuclear Factor κB Activation by Heavy Ions

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    Nuclear factor κB (NF-κB) activation might be central to heavy ion-induced detrimental processes such as cancer promotion and progression and sustained inflammatory responses. A sensitive detection system is crucial to better understand its involvement in these processes. Therefore, a DD-tdTomato fluorescent protein-based reporter system was previously constructed with human embryonic kidney (HEK) cells expressing DD-tdTomato as a reporter under the control of a promoter containing NF-κB binding sites (HEK-pNFκB-DD-tdTomato-C8). Using this reporter cell line, NF-κB activation after exposure to different energetic heavy ions (¹⁶O, 95 MeV/n, linear energy transfer—LET 51 keV/µm; ¹²C, 95 MeV/n, LET 73 keV/μm; ³⁶Ar, 95 MeV/n, LET 272 keV/µm) was quantified considering the dose and number of heavy ions hits per cell nucleus that double NF-κB-dependent DD-tdTomato expression. Approximately 44 hits of ¹⁶O ions and ≈45 hits of ¹²C ions per cell nucleus were required to double the NF-κB-dependent DD-tdTomato expression, whereas only ≈3 hits of ³⁶Ar ions were sufficient. In the presence of Shield-1, a synthetic molecule that stabilizes DD-tdTomato, even a single particle hit of ³⁶Ar ions doubled NF-κB-dependent DD-tdTomato expression. In conclusion, stabilization of the reporter protein can increase the sensitivity for NF-κB activation detection by a factor of three, allowing the detection of single particle hits’ effects

    Radiation Type- and Dose-Specific Transcriptional Responses across Healthy and Diseased Mammalian Tissues

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    Ionizing radiation (IR) is a genuine genotoxic agent and a major modality in cancer treatment. IR disrupts DNA sequences and exerts mutagenic and/or cytotoxic properties that not only alter critical cellular functions but also impact tissues proximal and distal to the irradiated site. Unveiling the molecular events governing the diverse effects of IR at the cellular and organismal levels is relevant for both radiotherapy and radiation protection. Herein, we address changes in the expression of mammalian genes induced after the exposure of a wide range of tissues to various radiation types with distinct biophysical characteristics. First, we constructed a publicly available database, termed RadBioBase, which will be updated at regular intervals. RadBioBase includes comprehensive transcriptomes of mammalian cells across healthy and diseased tissues that respond to a range of radiation types and doses. Pertinent information was derived from a hybrid analysis based on stringent literature mining and transcriptomic studies. An integrative bioinformatics methodology, including functional enrichment analysis and machine learning techniques, was employed to unveil the characteristic biological pathways related to specific radiation types and their association with various diseases. We found that the effects of high linear energy transfer (LET) radiation on cell transcriptomes significantly differ from those caused by low LET and are consistent with immunomodulation, inflammation, oxidative stress responses and cell death. The transcriptome changes also depend on the dose since low doses up to 0.5 Gy are related with cytokine cascades, while higher doses with ROS metabolism. We additionally identified distinct gene signatures for different types of radiation. Overall, our data suggest that different radiation types and doses can trigger distinct trajectories of cell-intrinsic and cell-extrinsic pathways that hold promise to be manipulated toward improving radiotherapy efficiency and reducing systemic radiotoxicities
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