Entwicklung von rangbasierten Kriterien und Methoden zur Optimierung der Normalisierung von Genexpressionsexperimenten am Beispiel membranbasierter cDNA-Arrays

Die Genexpressionsmessung mit DNA-Arrays ist eine sehr komplexe und dadurch fehleranfällige Methode. Jeder der notwendigen Einzelschritte der Messung beeinflußt das letztendliche Meßergebnis durch verschiedene Parameter in signifikanter Weise. Im Gegensatz zu herkömmlichen statistischen Daten mit vielen Messungen und wenig Meßparametern beinhalten die Arraydaten sehr viele Meßparameter und nur wenig Messungen. Der Normalisierung kommt damit eine zentrale Rolle in der Datenanalyse zu. Diese soll die Meßparameter berücksichtigen und die Meßdaten auf die zumessende Größe „Anzahl einer bestimmten mRNA-Spezies in einer definierten Probe“ zurückführen oder zumindestens eine weitgehende Vergleichbarkeit zwischen Experimenten herstellen. Im Rahmen dieser Arbeit wurde ein einfaches Hybridisierungsmodell entwickelt, um Einflüsse von Meßparametern auf die Signalfunktion abzuschätzen. Durch das Hybridisierungsmodell kann der zentrale Meßschritt der Hybridisierung der immoblisierten DNASonde mit der in Lösung befindlichen cDNA-Probe simuliert werden. Hiermit können die Art und Stärke der Haupteinflußgrößen abgeschätzt werden. Für quantitative Aussagen sind die dem Modell zugrunde liegenden Annahmen zu einfach und die verfügbaren Informationen zu unvollständig. Qualitative Vorhersagen sind dagegen möglich. So kann z.B. mengeninduzierte Kreuzhybridisierung auf Oligoarrays beschrieben und verstanden werden. Der zweite und Hauptteil dieser Arbeit beschäftigt sich mit der Normalisierung von cDNA-Filterarrays. Dazu wurde mehrere Skalierungsmethoden und eine nichtlineare adaptive Methode miteinander verglichen. Dabei konnte gezeigt werden, daß die vom Autor entwickelte Normalisierungsmethode des asymmetrisch gestutzten Mittels bei geeigneter Parametrisierung in Bezug auf Testdaten den geringsten Normalisierungsfehler verursacht. Für die Realdaten stellte sich ein leichter Vorteil der Normalisierung durch den Spezialfall des 2%igen oberen gestutzten Mittels heraus, da es damit möglich ist den Einfluß von Sättigungseffekten etc. auf den Skalierungsquotienten zu minimieren. Ausführlich wurden dabei die Grundlagen der rangbasierten Normalisierungsmethoden erörtert und der Fehlereinfluß der nichtlinearen Rangwertnormalisierung diskutiert. Für die Qualitätsbeurteilung und Normalisierungskontrolle erwiesen sich zwei rangbasierte Kriterien als sehr nützlich: die Rang-Intensitäts-Kurven und die davon abgeleitete relative Rang-Intensitäts-Standardabweichung. Beide Kriterien wurden dabei erstmals für die Genexpressionsanalyse angewandt. Ein weiterer Aspekt der zugehörigen Visualisierungen lieferte die Grundlage für die retrospektive Fehlerabschätzung aus realen Genexpressionsdaten. Es wurde gezeigt, daß die mittleren negativen Werte der Rang-Intensitäts-Verteilungen sich proportional zur Standardabweichung des statistischen Rauschanteils verhalten

Cell Type-Specific Role of RNA Nuclease SMG6 in Neurogenesis

SMG6 is an endonuclease, which cleaves mRNAs during nonsense-mediated mRNA decay (NMD), thereby regulating gene expression and controling mRNA quality. SMG6 has been shown as a differentiation license factor of totipotent embryonic stem cells. To investigate whether it controls the differentiation of lineage-specific pluripotent progenitor cells, we inactivated Smg6 in murine embryonic neural stem cells. Nestin-Cre-mediated deletion of Smg6 in mouse neuroprogenitor cells (NPCs) caused perinatal lethality. Mutant mice brains showed normal structure at E14.5 but great reduction of the cortical NPCs and late-born cortical neurons during later stages of neurogenesis (i.e., E18.5). Smg6 inactivation led to dramatic cell death in ganglionic eminence (GE) and a reduction of interneurons at E14.5. Interestingly, neurosphere assays showed self-renewal defects specifically in interneuron progenitors but not in cortical NPCs. RT-qPCR analysis revealed that the interneuron differentiation regulators Dlx1 and Dlx2 were reduced after Smg6 deletion. Intriguingly, when Smg6 was deleted specifically in cortical and hippocampal progenitors, the mutant mice were viable and showed normal size and architecture of the cortex at E18.5. Thus, SMG6 regulates cell fate in a cell type-specific manner and is more important for neuroprogenitors originating from the GE than for progenitors from the cortex

Automated detection of residual cells after sex-mismatched stem-cell transplantation – evidence for presence of disease-marker negative residual cells

<p>Abstract</p> <p>Background</p> <p>A new chimerism analysis based on automated interphase fluorescence in situ hybridization (FISH) evaluation was established to detect residual cells after allogene sex-mismatched bone marrow or blood stem-cell transplantation.</p> <p>Cells of 58 patients were characterized as disease-associated due to presence of a bcr/abl-gene-fusion or a trisomy 8 and/or a simultaneous hybridization of gonosome-specific centromeric probes. The automatic slide scanning platform Metafer with its module MetaCyte was used to analyse 3,000 cells per sample.</p> <p>Results</p> <p>Overall 454 assays of 58 patients were analyzed. 13 of 58 patients showed residual recipient cells at one stage of more than 4% and 12 of 58 showed residual recipient cells less than 4%, respectively. As to be expected, patients of the latter group were associated with a higher survival rate (48 vs. 34 month). In only two of seven patients with disease-marker positive residual cells between 0.1–1.3% a relapse was observed. Besides, disease-marker negative residual cells were found in two patients without relapse at a rate of 2.8% and 3.3%, respectively.</p> <p>Conclusion</p> <p>The definite origin and meaning of disease-marker negative residual cells is still unclear. Overall, with the presented automatic chimerism analysis of interphase FISH slides, a sensitive method for detection of disease-marker positive residual cells is on hand.</p

RHAMM deficiency disrupts folliculogenesis resulting in female hypofertility

The postnatal mammalian ovary contains the primary follicles, each comprising an immature oocyte surrounded by a layer of somatic granulosa cells. Oocytes reach meiotic and developmental competence via folliculogenesis. During this process, the granulosa cells proliferate massively around the oocyte, form an extensive extracellular matrix (ECM) and differentiate into cumulus cells. As the ECM component hyaluronic acid (HA) is thought to form the backbone of the oocyte-granulosa cell complex, we deleted the relevant domain of the Receptor for HA Mediated Motility (RHAMM) gene in the mouse. This resulted in folliculogenesis defects and female hypofertility, although HA-induced signalling was not affected. We report that wild-type RHAMM localises at the mitotic spindle of granulosa cells, surrounding the oocyte. Deletion of the RHAMM C-terminus in vivo abolishes its spindle association, resulting in impaired spindle orientation in the dividing granulosa cells, folliculogenesis defects and subsequent female hypofertility. These data reveal the first identified physiological function for RHAMM, during oogenesis, and the importance of this spindle-associated function for female fertility

RHAMM deficiency disrupts folliculogenesis resulting in female hypofertility

The postnatal mammalian ovary contains the primary follicles, each comprising an immature oocyte surrounded by a layer of somatic granulosa cells. Oocytes reach meiotic and developmental competence via folliculogenesis. During this process, the granulosa cells proliferate massively around the oocyte, form an extensive extracellular matrix (ECM) and differentiate into cumulus cells. As the ECM component hyaluronic acid (HA) is thought to form the backbone of the oocyte-granulosa cell complex, we deleted the relevant domain of the Receptor for HA Mediated Motility (RHAMM) gene in the mouse. This resulted in folliculogenesis defects and female hypofertility, although HA-induced signalling was not affected. We report that wild-type RHAMM localises at the mitotic spindle of granulosa cells, surrounding the oocyte. Deletion of the RHAMM C-terminus in vivo abolishes its spindle association, resulting in impaired spindle orientation in the dividing granulosa cells, folliculogenesis defects and subsequent female hypofertility. These data reveal the first identified physiological function for RHAMM, during oogenesis, and the importance of this spindle-associated function for female fertility

Scientific drilling projects in ancient lakes: integrating geological and biological histories

Sedimentary sequences in ancient or long-lived lakes can reach several thousands of meters in thickness and often provide an unrivalled perspective of the lake's regional climatic, environmental, and biological history. Over the last few years, deep drilling projects in ancient lakes became increasingly multi- and interdisciplinary, as, among others, seismological, sedimentological, biogeochemical, climatic, environmental, paleontological, and evolutionary information can be obtained from sediment cores. However, these multi- and interdisciplinary projects pose several challenges. The scientists involved typically approach problems from different scientific perspectives and backgrounds, and setting up the program requires clear communication and the alignment of interests. One of the most challenging tasks, besides the actual drilling operation, is to link diverse datasets with varying resolution, data quality, and age uncertainties to answer interdisciplinary questions synthetically and coherently. These problems are especially relevant when secondary data, i.e., datasets obtained independently of the drilling operation, are incorporated in analyses. Nonetheless, the inclusion of secondary information, such as isotopic data from fossils found in outcrops or genetic data from extant species, may help to achieve synthetic answers. Recent technological and methodological advances in paleolimnology are likely to increase the possibilities of integrating secondary information, e.g., through molecular dating of molecular phylogenies. Some of the new approaches have started to revolutionize scientific drilling in ancient lakes, but at the same time, they also add a new layer of complexity to the generation and analysis of sediment core data. The enhanced opportunities presented by new scientific approaches to study the paleolimnological history of these lakes, therefore, come at the expense of higher logistic, communication, and analytical efforts. Here we review types of data that can be obtained in ancient lake drilling projects and the analytical approaches that can be applied to empirically and statistically link diverse datasets for creating an integrative perspective on geological and biological data. In doing so, we highlight strengths and potential weaknesses of new methods and analyses, and provide recommendations for future interdisciplinary deep drilling projects

Search for dark matter produced in association with bottom or top quarks in √s = 13 TeV pp collisions with the ATLAS detector

A search for weakly interacting massive particle dark matter produced in association with bottom or top quarks is presented. Final states containing third-generation quarks and miss- ing transverse momentum are considered. The analysis uses 36.1 fb−1 of proton–proton collision data recorded by the ATLAS experiment at √s = 13 TeV in 2015 and 2016. No significant excess of events above the estimated backgrounds is observed. The results are in- terpreted in the framework of simplified models of spin-0 dark-matter mediators. For colour- neutral spin-0 mediators produced in association with top quarks and decaying into a pair of dark-matter particles, mediator masses below 50 GeV are excluded assuming a dark-matter candidate mass of 1 GeV and unitary couplings. For scalar and pseudoscalar mediators produced in association with bottom quarks, the search sets limits on the production cross- section of 300 times the predicted rate for mediators with masses between 10 and 50 GeV and assuming a dark-matter mass of 1 GeV and unitary coupling. Constraints on colour- charged scalar simplified models are also presented. Assuming a dark-matter particle mass of 35 GeV, mediator particles with mass below 1.1 TeV are excluded for couplings yielding a dark-matter relic density consistent with measurements