Funktionelle Untersuchung von genetischen Faktoren mit prognostischer Relevanz in der Onkologie

Lungenkarzinome sind im Vergleich zu anderen Krebsarten die häufigste Todesursache weltweit. Aufgrund der späten Diagnose und einer hohen Rate an Therapieresistenzen liegt die 5-Jahres-Überlebensraten bei nur 13% und ist damit eine der niedrigsten überhaupt. Die Identifikation von potentiellen Biomarkern für eine Verbesserung der Therapieansätze rückt dabei zunehmend in den Vordergrund. Epigenetische Veränderungen spielen bei der Progression von Tumoren eine elementare Rolle, wobei diese mit der Karzinogenese und der Ausprägung einer Therapiereistenz in Verbindung stehen. Epigenetische Modifikatoren wie die H3K4-Methyltransferase KMT2D und das Chromatin-Bindeprotein (Reader) BRD4 sind häufig in verschiedenen Krebsarten wie z.B. in NSCLC mutiert. Im Rahmen dieser Doktorarbeit sollten in verschiedenen Lungenkrebszelllinien unter Verwendung von CRISPR CAS9 definierte Mutationen in den Kandidatengenen KMT2D und BRD4 induziert werden, um funktionelle Mechanismen eingehender zu untersuchen, die mit einer Tumorprogression assoziiert werden. Die Deletionen im Kandidatengen KMT2D unterscheiden sich dadurch, dass bei einer der Deletionen ein Verlust der SET-Domäne erfolgt (Zelllinie B6), wohingegen bei der anderen Deletion das Leseraster C-terminal und damit auch die SET-Domäne erhalten bleibt (Zelllinie A76). In BRD4 wird eine Deletion des gesamten Gens eingefügt. In ChIP-Seq –Analysen der Parental-Zelllinien erfolgte zunächst die Identifikation spezifisch angereicherter Signalwege, die in Prozessen der zellulären Stressantwort, dem Spleißprozess und der epigenetischen Regulation eine Rolle spielten. Zudem konnte eine starke Anreicherung im EGFR-Signalweg festgestellt werden. Um die mögliche Auswirkung der induzierten Mutationen in diesen identifizierten Signalwegen eingehender zu betrachten, wurden diese in Expressions-, Proliferations- und methylierungsspezifischen Experimenten analysiert. Des Weiteren wurde der Einfluss von KMT2D auf eine epigenetisch bedingte Therapieresistenz gegen die EGFR-wirkenden Chemotherapeutika Cetuximab und Erlotinib untersucht. Die induzierten Mutationen führten zu einer verminderten RNA- und Proteinexpression der Kandidatengene. Bei der KMT2D-Zelllinie A76 konnten deutliche morphologische Veränderungen beobachtet werden. Die Zellproliferation war in der Analyse verglichen zur Parental-Zelllinie HCC827 deutlich in den KMT2D-mutierten Zelllinien vermindert. In den Western Blot Analysen führten die induzierten KMT2D- und BRD4-Mutationen zu Veränderung der gesamten Histon-Mengen, die sich in einer differenziellen H3K4- und H3K27-Methylierung in den mutierten Zelllinien widerspiegelten. Durch einen Hitzestress konnte eine Stabilisierung der methylierten Histon-Mengen beobachtet werden. Zudem wurden Unterschiede in der Zellviabilität nach der Induktion durch einen Hitzestress ermittelt. In den BRD4-mutierten Zelllinien konnten hingegen keine Unterschiede in der Zellviabilität nach einem Hitzeschock im Vergleich zur parentalen Zelllinie Wi38 festgestellt werden. Die KMT2D-Mutationen resultierten in einer veränderten RNA-Expression von H3K4- und H3K27-relevanter Methyltransferasen und Demethylasen. Zudem konnten Veränderungen in der Expressionsanalyse von U2 snRNP Spleiß-Faktoren in den KMT2D- und BRD4-mutierten Zelllinien festgestellt werden. Bei der KMT2D-Mutante A76 konnte zudem in Zellviabilitätsassays eine erhöhte Sensitivität gegenüber Cetuximab und Erlotinib beobachtet werden

Kern-, Eineltern- und Stieffamilien in Europa: eine Analyse ihrer Häufigkeiten und Einbindung in haushaltsübergreifende Strukturen

Die vorliegende Studie befasst sich mit den Anteilen von Eineltern- und Stieffamilien sowie haushaltsübergreifenden Familienstrukturen in verschiedenen europäischen Ländern. Die Frage, wie viele Eineltern- und Stieffamilien es in den verschiedenen Ländern Europas tatsächlich gibt, konnte bislang nicht zufriedenstellend beantwortet werden. Für viele Länder existieren lediglich Schätzungen unter Rückgriff auf Scheidungsraten. Analysen auf Basis der Surveys des 'Generations and Gender Programme' (GGP), die mittlerweile für 16 Staaten Europas (Belgien, Bulgarien, Deutschland, Estland, Frankreich, Georgien, Italien, Litauen, Niederlande, Norwegen, Österreich, Polen, Rumänien, Tschechische Republik, Ungarn, inklusive Russland) verfügbar sind, ermöglichen es allerdings, komplexe Familienstrukturen, wie sie bei Scheidungs- und Trennungsfamilien vorliegen, zu analysieren. Insgesamt werden 55.350 Familienhaushalte berücksichtigt, in denen minderjährige Kinder leben. Die Ergebnisse zeigen, dass die Anteile von Scheidungs- und Trennungsfamilien in den vorliegenden Ländern zwischen 7 und 30 Prozent variieren. Dabei wird ein Nord-Süd-Gefälle deutlich: Während der Anteil an Scheidungs- und Trennungsfamilien in den nordeuropäischen Ländern eher hoch ausfällt, ist er in den südeuropäischen Ländern deutlich geringer. Darüber hinaus ist der Anteil an Scheidungs- und Trennungsfamilien in einigen osteuropäischen Ländern besonders niedrig, in anderen jedoch außerordentlich hoch. Neben der Verteilung von Eineltern- und Stieffamilien hinweg, werden auch haushaltsübergreifende Konstellationen berücksichtigt. Die Analysen zeigen, dass man die Diversität von Familien deutlich unterschätzt, wenn auf der Haushaltsebene verharrt wird.This study deals with the prevalence of single-parent- and stepfamilies in Europe and their linkages in cross-household constellations. The question about the prevalence of these family constellations could not be answered satisfactory in the past. Noumerous studies revert to rough estimations based on crude divorce rates. The analysis presents color-coded descriptive statistics from official sources as well as from the surveys of the "Generations and Gender Program" (GGP), allowing visual comparisons of the prevalence of complex family structures that emerge from divorce and separation as predicted by crude divorce rates and as present in nationally representative survey samples. Data are now available for 16 European states (Austria, Belgium, Bulgaria, the Czech Republic, Estonia, France, Georgia, Germany, Hungary, Italy, Lithuania, the Netherlands, Norway, Poland, Romania, and Russia) and include a total of 55,350 family households with non-adult children. Single-parent families and stepfamilies constituted between 7 and 30 percent of the national samples. A north-south divide is clearly evident such that the share of single-parent families and stepfamilies is much higher in Europe's northern countries. Eastern Europe, however, includes countries in which the share of single-parent- and stepfamilies was extraordinarily high and countries in which the share was extraordinarily low. Beside the prevalence of single-parent- and stepfamilies cross-household constellations are considered. Analyes emphasize an underestimation of complex family structures because of the persistent neclection of crosshousehold constellations

Insights from Multimodal Preclinical Imaging in Immunocompetent Nude Mice

Hydrogels based on gelatin have evolved as promising multifunctional biomaterials. Gelatin is crosslinked with lysine diisocyanate ethyl ester (LDI) and the molar ratio of gelatin and LDI in the starting material mixture determines elastic properties of the resulting hydrogel. In order to investigate the clinical potential of these biopolymers, hydrogels with different ratios of gelatin and diisocyanate (3-fold (G10_LNCO3) and 8-fold (G10_LNCO8) molar excess of isocyanate groups) were subcutaneously implanted in mice (uni- or bilateral implantation). Degradation and biomaterial-tissue- interaction were investigated in vivo (MRI, optical imaging, PET) and ex vivo (autoradiography, histology, serum analysis). Multimodal imaging revealed that the number of covalent net points correlates well with degradation time, which allows for targeted modification of hydrogels based on properties of the tissue to be replaced. Importantly, the degradation time was also dependent on the number of implants per animal. Despite local mechanisms of tissue remodeling no adverse tissue responses could be observed neither locally nor systemically. Finally, this preclinical investigation in immunocompetent mice clearly demonstrated a complete restoration of the original healthy tissue

The effect of dynamic recrystallisation on the rheology and microstructures of partially molten rocks

This work was founded by the joint project “Rheology of the continental crust in collision”, funded by the Procope scheme of PHC Egide in France and by the DAAD PPP scheme in Germany. M-GL acknowledges the support of the Juan de la Cierva programme of the Government of Spain’s Ministry for Science, Innovation and Universities. EGR acknowledges the support of the Beatriu de Pinós programme of the Government of Catalonia's Secretariat for Universities and Research of the Department of Economy and Knowledge (2016 BP 00208). This work benefited from discussions with Pi L. Jolivet and E. Burov within the ERC project RHEOLITH. We thank Elisabetta Mariani and Marcin Dabrowski for their helpful comments, together with the editorial guidance of Dave Healy and Bill Dunne.Peer reviewedPostprin

Human cytomegalovirus infection in tumor cells of the nervous system is not detectable with standardized pathologico-virological diagnostics.

BACKGROUND Experimental findings have suggested that human cytomegalovirus (HCMV) infection of tumor cells may exert oncomodulatory effects that enhance tumor malignancy. However, controversial findings have been published on the presence of HCMV in malignant tumors. Here, we present the first study that systematically investigates HCMV infection in human nervous system tumors by highly sensitive immunohistochemistry in correlation with the HCMV serostatus of the patients. METHODS Immunohistochemical and quantitative PCR-based methods to detect different HCMV antigens and genomic HCMV DNA were optimized prior to the investigation of pathological samples. Moreover, the pathological results were matched with the HCMV serostatus of the patients. RESULTS HCMV immediate-early, late, and pp65 antigens could be detected in single cells from HCMV strain Hi91-infected UKF-NB-4 neuroblastoma cells after 1:1024 dilution with noninfected UKF-NB-4 cells. Genomic HCMV DNA could be detected in copy numbers as low as 430 copies/mL. However, we did not detect HCMV in tumors from a cohort of 123 glioblastoma, medulloblastoma, or neuroblastoma patients. Notably, we detected nonspecifically positive staining in tumor tissues of HCMV seropositive and seronegative glioblastoma patients. The HCMV serostatus of 67 glioblastoma patients matched the general epidemiological prevalence data for Western countries (72% of female and 57% of male glioblastoma patients were HCMV seropositive). Median survival was not significantly different in HCMV seropositive versus seronegative glioblastoma patients. CONCLUSIONS The prevalence of HCMV-infected tumor cells may be much lower than previously reported based on highly sensitive detection methods

Applying FAIR Principles to plant phenotypic data management in GnpIS

GnpIS is a data repository for plant phenomics that stores whole field and greenhouse experimental data including environment measures. It allows long-term access to datasets following the FAIR principles: Findable, Accessible, Interoperable, and Reusable, by using a flexible and original approach. It is based on a generic and ontology driven data model and an innovative software architecture that uncouples data integration, storage, and querying. It takes advantage of international standards including the Crop Ontology, MIAPPE, and the Breeding API. GnpIS allows handling data for a wide range of species and experiment types, including multiannual perennial plants experimental network or annual plant trials with either raw data, i.e., direct measures, or computed traits. It also ensures the integration and the interoperability among phenotyping datasets and with genotyping data. This is achieved through a careful curation and annotation of the key resources conducted in close collaboration with the communities providing data. Our repository follows the Open Science data publication principles by ensuring citability of each dataset. Finally, GnpIS compliance with international standards enables its interoperability with other data repositories hence allowing data links between phenotype and other data types. GnpIS can therefore contribute to emerging international federations of information systems

Inhibition of insulin-like growth factor-1 receptor signaling enhances growth-inhibitory and proapoptotic effects of gefitinib (Iressa) in human breast cancer cells

INTRODUCTION: Gefitinib (Iressa, ZD 1839, AstraZeneca) blocks the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) and inhibits proliferation of several human cancer cell types including breast cancer. Phase II clinical trials with gefitinib monotherapy showed an objective response of 9 to 19% in non-small-cell lung cancer patients and less than 10% for breast cancer, and phase III results have indicated no benefit of gefitinib in combination with chemotherapy over chemotherapy alone. In order to improve the antineoplastic activity of gefitinib, we investigated the effects of blocking the signalling of the insulin-like growth factor 1 receptor (IGF-1R), a tyrosine kinase with a crucial role in malignancy that is coexpressed with EGFR in most human primary breast carcinomas. METHODS: AG1024 (an inhibitor of IGF-1R) was used with gefitinib for treatment of MDA468, MDA231, SK-BR-3, and MCF-7 breast cancer lines, which express similar levels of IGF-1R but varying levels of EGFR. Proliferation assays, apoptosis induction studies, and Western blot analyses were conducted with cells treated with AG1024 and gefitinib as single agents and in combination. RESULTS: Gefitinib and AG1024 reduced proliferation in all lines when used as single agents, and when used in combination revealed an additive-to-synergistic effect on cell growth inhibition. Flow cytometry measurements of cells stained with annexin V-propidium iodide and cells stained for caspase-3 activation indicated that adding an IGF-1R-targeting strategy to gefitinib results in higher levels of apoptosis than are achieved with gefitinib alone. Gefitinib either reduced or completely inhibited p42/p44 Erk kinase phosphorylation, depending on the cell line, while Akt phosphorylation was reduced by a combination of the two agents. Overexpression of IGF-1R in SK-BR-3 cells was sufficient to cause a marked enhancement in gefitinib resistance. CONCLUSION: These results indicate that IGF-1R signaling reduces the antiproliferative effects of gefitinib in several breast cancer cell lines, and that the addition of an anti-IGF-1R strategy to gefitinib treatment may be more effective than a single-agent approach

The Effects of Age on Inflammatory and Coagulation-Fibrinolysis Response in Patients Hospitalized for Pneumonia

Objective: To determine whether inflammatory and hemostasis response in patients hospitalized for pneumonia varies by age and whether these differences explain higher mortality in the elderly. Methods: In an observational cohort of subjects with community-acquired pneumonia (CAP) recruited from emergency departments (ED) in 28 hospitals, we divided subjects into 5 age groups (85% subjects, older subjects had modestly increased hemostasis markers and IL-6 levels (p,0.01). Conclusions: Modest age-related increases in coagulation response occur during hospitalization for CAP; however these differences do not explain the large differences in mortality. Despite clinical recovery, immune resolution may be delayed in older adults at discharge. © 2010 Kale et al

Making maps of cosmic microwave background polarization for B-mode studies: The POLARBEAR example

Analysis of cosmic microwave background (CMB) datasets typically requires some filtering of the raw time-ordered data. For instance, in the context of ground-based observations, filtering is frequently used to minimize the impact of low frequency noise, atmospheric contributions and/or scan synchronous signals on the resulting maps. In this work we have explicitly constructed a general filtering operator, which can unambiguously remove any set of unwanted modes in the data, and then amend the map-making procedure in order to incorporate and correct for it. We show that such an approach is mathematically equivalent to the solution of a problem in which the sky signal and unwanted modes are estimated simultaneously and the latter are marginalized over. We investigated the conditions under which this amended map-making procedure can render an unbiased estimate of the sky signal in realistic circumstances. We then discuss the potential implications of these observations on the choice of map-making and power spectrum estimation approaches in the context of B-mode polarization studies. Specifically, we have studied the effects of time-domain filtering on the noise correlation structure in the map domain, as well as impact it may haveon the performance of the popular pseudo-spectrum estimators. We conclude that although maps produced by the proposed estimators arguably provide the most faithful representation of the sky possible given the data, they may not straightforwardly lead to the best constraints on the power spectra of the underlying sky signal and special care may need to be taken to ensure this is the case. By contrast, simplified map-makers which do not explicitly correct for time-domain filtering, but leave it to subsequent steps in the data analysis, may perform equally well and be easier and faster to implement. We focused on polarization-sensitive measurements targeting the B-mode component of the CMB signal and apply the proposed methods to realistic simulations based on characteristics of an actual CMB polarization experiment, POLARBEAR. Our analysis and conclusions are however more generally applicable. \ua9 ESO, 2017