Verlauf der Serumspiegel des „Pregnancy associated plasma protein-A“ im ersten Trimenon bei schwangeren Patientinnen nach Spontankonzeption sowie nach assistierter Reproduktion

In der vorliegenden Arbeit wurden retrospektiv die Daten und Serumproben von 103 Patientinnen der Fertilitätssprechstunde der Universitäts-Frauenklinik Bonn von 2007 bis zum Frühjahr 2010 bezüglich der Pregnancy associated Plasma Protein-A-Serumspiegel (PAPP-A) im Schwanger-schaftsverlauf bei unterschiedlicher Stimulationsstärke und in Hinblick auf mögliche beeinflus-sende Parameter hin analysiert. Ziel dieser Arbeit war es den Verlauf der PAPP-A-Serumkonzentration zum Zeitpunkt der Ovula-tionsinduktion bis zum 52. Schwangerschaftstag nach Konzeption zu untersuchen und mögliche Unterschiede zwischen den Stimulationsstärken (ohne -, low-Dose-, hohe hormonelle Stimula-tion) herauszuarbeiten. Der Parameter humanes Choriongonadotropin (hCG), dessen Verlauf im Serum während der Schwangerschaft bereits gut untersucht ist, diente in der vorliegenden Arbeit als Vergleichspara-meter zum PAPP-A, um die Validität unseres Beobachtungskollektivs zu dokumentieren. Die Messung von PAPP-A aus den Serum-Reihen (~15./~25./~35. Tag p.c.) der Patientinnen er-folgte durch einen neuen, hochsensitiven Immunoassay, der im Gegensatz zu früheren Testsys-temen auch freies PAPP-A bestimmt. Insgesamt konnten wir bei 85 % der Patientinnen zum Zeitpunkt der Ovulationsinduktion eine PAPP-A-Serumkonzentration nachweisen. Verglichen mit den PAPP-A-Serumspiegeln 12-17 Tage nach Konzeption, zeigte sich kein signifikanter Unterschied. Zwischen den PAPP-A- und Östradiol-Serumspiegeln ergab sich eine negative Korrelation auf signifikantem Niveau. Die periovulatorischen Östradiol-Serumspiegel unterschieden sich in Abhängigkeit von der Stimula-tionsstärke signifikant. Im Verlauf der Frühschwangerschaft waren ein exponentieller Anstieg der PAPP-A- sowie ein exponentieller Anstieg des hCG-Serumspiegel zu beobachten. Ab einer hCG- Konzentration von über 5000 IU/L wurde eine enge positive Korrelation zum PAPP-A-Serumspiegel deutlich. Die Multiple of Median-Werte (MoM) beider Parameter waren bei Abortschwangerschaften sig-nifikant niedriger. Ebenso zeigten sich in Abhängigkeit von der Stimulationsstärke für beide Pa-rameter niedrigere MoM-Werte unter hochdosierter hormoneller Stimulation, jedoch konnte nur für das hCG das Signifikanzniveau erreicht werden. Die Auswertung fertilitätsbeeinflussender Nebendiagnosen zeigte sowohl für PAPP-A als auch für hCG signifikant niedrigere MoM-Werte bei Raucherinnen

PCR-based analysis of microbial communities during the EuroGeoMars campaign at Mars Desert Research Station, Utah

The search for evidence of past or present life on Mars will require the detection of markers that indicate the presence of life. Because deoxyribonucleic acid (DNA) is found in all known living organisms, it is considered to be a ‘biosignature' of life. The main function of DNA is the long-term storage of genetic information, which is passed on from generation to generation as hereditary material. The Polymerase Chain Reaction (PCR) is a revolutionary technique which allows a single fragment or a small number of fragments of a DNA molecule to be amplified millions of times, making it possible to detect minimal traces of DNA. The compactness of the contemporary PCR instruments makes routine sample analysis possible with a minimum amount of laboratory space. Furthermore the technique is effective, robust and straightforward. Our goal was to establish a routine for the detection of DNA from micro-organisms using the PCR technique during the EuroGeoMars simulation campaign. This took place at the Mars Society's Mars Desert Research Station (MDRS) in Utah in February 2009 (organized with the support of the International Lunar Exploration Working Group (ILEWG), NASA Ames and the European Space Research and Technology Centre (ESTEC)). During the MDRS simulation, we showed that it is possible to establish a minimal molecular biology lab in the habitat for the immediate on-site analysis of samples by PCR after sample collection. Soil and water samples were taken at different locations and soil depths. The sample analysis was started immediately after the crew returned to the habitat laboratory. DNA was isolated from micro-organisms and used as a template for PCR analysis of the highly conserved ribosomal DNA to identify representatives of the different groups of micro-organisms (bacteria, archaea and eukarya). The PCR products were visualized by agarose gel electrophoresis and documented by transillumination and digital imaging. The microbial diversity in the collected samples was analysed with respect to sampling depth and the presence or absence of vegetation. For the first time, we have demonstrated that it is possible to perform direct on-site DNA analysis by PCR at MDRS, a simulated planetary habitat in an extreme environment that serves as a model for preparation and optimization of techniques to be used for future Mars exploratio

Comparative transcription map of the wobbler critical region on mouse chromosome 11 and the homologous region on human chromosome 2p13-14

BACKGROUND: To support the positional cloning of the mouse mutation wobbler (wr) the corresponding regions on human Chr2p13-14 and mouse Chr11 were analyzed in detail and compared with respect to gene content, order, and orientation. RESULTS: The gene content of the investigated regions was highly conserved between the two species: 20 orthologous genes were identified on our BAC/YAC contig comprising 4.5 Mb between REL/Rel and RAB1A/Rab1a. Exceptions were pseudogenes ELP and PX19 whose mouse counterparts were not located within the analyzed region. Two independently isolated genomic clones indicate an inversion between man and mouse with the inverted segment being identical to the wobbler critical interval. We investigated the wobbler critical region by extensive STS/EST mapping and genomic sequencing. Additionally, the full-length cDNA sequences of four newly mapped genes as well as the previously mapped gene Otx1 were established and subjected to mutation analysis. Our data indicate that all genes in the wr critical region have been identified. CONCLUSION: Unexpectedly, neither mutation analysis of cDNAs nor levels of mRNAs indicated which of the candidate genes might be affected by the wr mutation. The possibility arises that there might be hitherto unknown effects of mutations, in addition to structural changes of the mRNA or regulatory abnormalities

Post-Transcriptional Dynamics is Involved in Rapid Adaptation to Hypergravity in Jurkat T Cells

The transcriptome of human immune cells rapidly reacts to altered gravity in a highly dynamic way. We could show in previous experiments that transcriptional patterns show profound adaption after seconds to minutes of altered gravity. To gain further insight into these transcriptional alteration and adaption dynamics, we conducted a highly standardized RNA-Seq experiment with human Jurkat T cells exposed to 9xg hypergravity for 3 and 15 min, respectively. We investigated the frequency with which individual exons were used during transcription and discovered that differential exon usage broadly appeared after 3 min and became less pronounced after 15 min. Additionally, we observed a shift in the transcript pool from coding towards non-coding transcripts. Thus, adaption of gravity-sensitive differentially expressed genes followed a dynamic transcriptional rebound effect. The general dynamics were compatible with previous studies on the transcriptional effects of short hypergravity on human immune cells and suggest that initial up-regulatory changes mostly result from increased elongation rates. The shift correlated with a general downregulation of the affected genes. All chromosome bands carried homogenous numbers of gravity-sensitive genes but showed a specific tendency towards up- or downregulation. Altered gravity affected transcriptional regulation throughout the entire genome, whereby the direction of differential expression was strongly dependent on the structural location in the genome. A correlation analysis with potential mediators of the early transcriptional response identified a link between initially upregulated genes with certain transcription factors. Based on these findings, we have been able to further develop our model of the transcriptional response to altered gravity

Expression of hypoxia-inducible genes is suppressed in altered gravity due to impaired nuclear HIF1α accumulation

Extravehicular activities, the backbone of manned space exploration programs, set astronauts into mild hypoxia. Unfortunately, microgravity aggravates threatening symptoms of hypoxia such as vision impairment and brain edema. Hypoxia-inducible factors (HIFs) sense cellular hypoxia and, subsequently, change the cells’ expression profile instantaneously by rapidly translocating—most likely cytoskeleton-dependently—into the nucleus and subsequently forming transcription complexes with other proteins. We tested the hypothesis that this fundamental process could be altered by sudden changes in gravitational forces in parabolic flights using a newly developed pocket-size cell culture lab that deoxygenizes cells within 15 min. Sudden gravity changes (SGCs 1g–1.8g–0g–1.8g–1g) during hypoxic exposure suppressed expression of the HIF1α-dependent genes investigated as compared with hypoxia at constant 1g. Normoxic cells subjected to SGCs showed reduced nuclear but not cytoplasmatic HIF1α signal and appeared to have disturbed cytoskeleton architecture. Inhibition of the actin-dependent intracellular transport using a combination of myosin V and VI inhibitors during hypoxia mimicked the suppression of the HIF1α-dependent genes observed during hypoxic exposure during SGCs. Thus, SGCs seem to disrupt the cellular response to hypoxia by impairing the actin-dependent translocation of HIF1α into the nucleus

Rapid Downregulation of H3K4me3 Binding to Immunoregulatory Genes in Altered Gravity in Primary Human M1 Macrophages

The sensitivity of human immune system cells to gravity changes has been investigated in numerous studies. Human macrophages mediate innate and thus rapid immune defense on the one hand and activate T- and B-cell-based adaptive immune response on the other hand. In this process they finally act as immunoeffector cells, and are essential for tissue regeneration and remodeling. Recently, we demonstrated in the human Jurkat T cell line that genes are differentially regulated in cluster structures under altered gravity. In order to study an in vivo near system of immunologically relevant human cells under physically real microgravity, we performed parabolic flight experiments with primary human M1 macrophages under highly standardized conditions and performed chromatin immunoprecipitation DNA sequencing (ChIP-Seq) for whole-genome epigenetic detection of the DNA-binding loci of the main transcription complex RNA polymerase II and the transcription-associated epigenetic chromatin modification H3K4me3. We identified an overall downregulation of H3K4me3 binding loci in altered gravity, which were unequally distributed inter- and intrachromosomally throughout the genome. Three-quarters of all affected loci were located on the p arm of the chromosomes chr5, chr6, chr9, and chr19. The genomic distribution of the downregulated H3K4me3 loci corresponds to a substantial extent to immunoregulatory genes. In microgravity, analysis of RNA polymerase II binding showed increased binding to multiple loci at coding sequences but decreased binding to central noncoding regions. Detection of altered DNA binding of RNA polymerase II provided direct evidence that gravity changes can lead to altered transcription. Based on this study, we hypothesize that the rapid transcriptional response to changing gravitational forces is specifically encoded in the epigenetic organization of chromatin

Cytoskeletal stability and metabolic alterations in primary human macrophages in long-term microgravity

The immune system is one of the most affected systems of the human body during space flight. The cells of the immune system are exceptionally sensitive to microgravity. Thus, serious concerns arise, whether space flight associated weakening of the immune system ultimately precludes the expansion of human presence beyond the Earth's orbit. For human space flight, it is an urgent need to understand the cellular and molecular mechanisms by which altered gravity influences and changes the functions of immune cells. The CELLBOX-PRIME (= CellBox-Primary Human Macrophages in Microgravity Environment) experiment investigated for the first time microgravity-associated long-term alterations in primary human macrophages, one of the most important effector cells of the immune system. The experiment was conducted in the U.S. National Laboratory on board of the International Space Station ISS using the NanoRacks laboratory and Biorack type I standard CELLBOX EUE type IV containers. Upload and download were performed with the SpaceX CRS-3 and the Dragon spaceship on April 18th, 2014 / May 18th, 2014. Surprisingly, primary human macrophages exhibited neither quantitative nor structural changes of the actin and vimentin cytoskeleton after 11 days in microgravity when compared to 1g controls. Neither CD18 or CD14 surface expression were altered in microgravity, however ICAM-1 expression was reduced. The analysis of 74 metabolites in the cell culture supernatant by GC-TOF-MS, revealed eight metabolites with significantly different quantities when compared to 1g controls. In particular, the significant increase of free fucose in the cell culture supernatant was associated with a significant decrease of cell surface-bound fucose. The reduced ICAM-1 expression and the loss of cell surface-bound fucose may contribute to functional impairments, e.g. the activation of T cells, migration and activation of the innate immune response. We assume that the surprisingly small and non-significant cytoskeletal alterations represent a stable "steady state" after adaptive processes are initiated in the new microgravity environment. Due to the utmost importance of the human macrophage system for the elimination of pathogens and the clearance of apoptotic cells, its apparent robustness to a low gravity environment is crucial for human health and performance during long-term space missions

Swiss Parabolic Flights: Development of a Non-Governmental Parabolic Flight Program in Switzerland Based on the Airbus A310 ZERO-G

Parabolic flights are one of the most important pillars for research, development, and applications in space. Accordingly, we developed the world’s first non-governmental parabolic flight program using Novespace’s Airbus A310 ZERO-G. Through the flexible combination of academic research with industrial experiments, as well as with the support of private persons and low administrative efforts, we achieved a highly cost-efficient small-scale campaign concept, which is located at the Air Base Dübendorf in Switzerland. The program was very successful, and it resulted in 31 experiments and tests conducted by Universities and organizations in the industry in microgravity, culminating in many scientific publications and in larger subsequent projects for all users. We describe here how we designed, developed, tested, and built up this program. We also discuss the difficulties, problems, and success factors of a project that—for the first time—was successfully built from the “bottom-up”, and which was a large-scale flight research platform by scientists for scientists on a voluntary, non-governmental, and non-commercial basis

Rapid alterations of cell cycle control proteins in human T lymphocytes in microgravity

In our study we aimed to identify rapidly reacting gravity-responsive mechanisms in mammalian cells in order to understand if and how altered gravity is translated into a cellular response. In a combination of experiments using "functional weightlessness" provided by 2D-clinostats and real microgravity provided by several parabolic flight campaigns and compared to in-flight-1g-controls, we identified rapid gravity-responsive reactions inside the cell cycle regulatory machinery of human T lymphocytes. In response to 2D clinorotation, we detected an enhanced expression of p21 Waf1/Cip1 protein within minutes, less cdc25C protein expression and enhanced Ser147-phosphorylation of cyclinB1 after CD3/CD28 stimulation. Additionally, during 2D clinorotation, Tyr-15-phosphorylation occurred later and was shorter than in the 1 g controls. In CD3/CD28-stimulated primary human T cells, mRNA expression of the cell cycle arrest protein p21 increased 4.1-fold after 20s real microgravity in primary CD4+ T cells and 2.9-fold in Jurkat T cells, compared to 1 g in-flight controls after CD3/CD28 stimulation. The histone acetyltransferase (HAT) inhibitor curcumin was able to abrogate microgravity-induced p21 mRNA expression, whereas expression was enhanced by a histone deacetylase (HDAC) inhibitor. Therefore, we suppose that cell cycle progression in human T lymphocytes requires Earth gravity and that the disturbed expression of cell cycle regulatory proteins could contribute to the breakdown of the human immune system in space

Zwischen Fürsorge und Forschungszielen : ethische Leitlinien für die Forschung mit Kindern zu sensiblen Themenbereichen

Das vorliegende forschungsethische Konzept schafft die Grundlagen für eine ethisch reflektierte Forschung mit Kindern in sensiblen Themenbereichen wie der zivilen Sicherheitsforschung. Es versteht sich als ein dezidiert forschungsethisches Konzept, das interdisziplinär und unter Einbezug rechtlicher Aspekte ansetzt. Um neu aufkommenden Sicherheitsgefährdungen auf digitaler Ebene entgegenzutreten, bedarf es forschungsethisch fundierten Ergebnissen und Projekten, welche Kinder schützen und am Selbstschutz beteiligen werden. Kinder als handelnde Subjekte anzuerkennen, impliziert, dass die entstehenden Projekte deutlich aussagekräftiger und zielführender sein werden.The present research ethics concept lays the foundations for ethically research with children in sensitive areas such as civil security research. security research. It is understood as a decidedly research-ethical concept, that is interdisciplinary and includes legal aspects. In order to counter emerging security threats on a digital level, research-ethically based results and projects are needed that will protect children and involve them in self-protection. Recognizing children as acting subjects implies that the emerging projects will be much more meaningful and purposeful