Studien der Genexpressionsänderung während der Entwicklung von Drosophila melanogaster mittels DNA-Microarrays

Die Fruchtfliege Drosophila melanogaster ist einer der am besten untersuchten Modellorganismen in der Entwicklungsbiologie. Die Kenntnis des Genoms und die Zahl der annotierten protein-kodierenden Gene von Drosophila sind jedoch Schwankungen unterworfen, da sie meist auf Computerberechnungen bereits bekannter Gene und nur teilweise auf experimentellen Nachweisen beruhen. Um das Transkriptom von Drosophila im Verlauf der Entwicklung zu untersuchen und neue potentielle Gene zu identifizieren wurde im Rahmen dieser Arbeit ein DNA-Microarray entwickelt, mit dem Veränderungen in der mRNA-Expression analysiert wurden. Auf der Grundlage einer neuentwickelten ab initio Genvorhersage im Rahmen einer Koorporation wurden spezifische Primer generiert, die eine Amplifikation der potentiellen Gene ermöglichten. Mit einem zweistufigen PCR-Protokoll konnten 20.948 (98%) PCR-Fragmente von 21.396 vorhergesagten Genen amplifiziert werden. Diese wurden als Basis für die Herstellung des Microarrays verwendet. So war es möglich die Vorteile computergestützter in silico- Berechnungen der möglichen Gene mit experimentellen Analysen zu kombinieren, um die Expression von Genen zu detektieren. Durch Optimierung der Protokolle und Methoden in der DNA-Chip-Konstruktion wurde ein hochdichter Microarray hergestellt, der nahezu das gesamte Transkriptom von Drosophila repräsentiert. Die Analyse differentiell exprimierter Gene erfolgte jeweils mit zwei RNA-Populationen die während der reversen Transkription mit unterschiedlichen Fluoreszenzfarbstoffen markiert und anschließend auf einem Microarray co-hybridisiert wurden. Es wurden Hybridisierungen mit der RNA aus 9 unterschiedlichen Entwicklungsstadien von Drosophila melanogaster durchgeführt, die den gesamten Lebenszyklus der Fliege umfassten. Nach der Quantifizierung der detektierten Signalintensitäten erfolgte die bioinformatische Datenanalyse mit unterschiedlichen Softwareprogrammen. Die Genexpressionsanalysen ergaben das 13.812 Gene im Verlauf der Entwicklung von Drosophila exprimiert werden, wovon ungefähr 2.500 bisher unbekannten Genen entsprechen. Es konnte außerdem gezeigt werden, daß die Entwicklungsstadien spezifische Regulationen von Genaktivitäten aufweisen, die mit morphologischen Veränderungen während der Entwicklung korrelieren und daß Gengruppen ähnlicher biochemischer oder physiologischer Funktion gemeinsam reguliert werden

Hybrid Simulators for Product Service-Systems : Innovation potential demonstrated on urban bike mobility

One major goal of the Rethinking Prototyping project is to bring scientists from different domains like engineering and arts to explore collaboratively new approaches of development and testing of Product Service Systems (PSS). PSS combine products, services, and infrastructure to fulfil individual customer needs. Therefore, the development of PSS is an extension of traditional engineering design process, which mainly refers to purely tangible products or intangible services into an integrated development process of products and services. The basis is a new technology called Smart Hybrid Prototyping (SHP), a joint development by Fraunhofer IPK and the TU Berlin. SHP is an innovative technology for a multimodal interdisciplinary evaluation of virtual prototypes in early development stages. It is based upon methods of Mixed Reality extended by modern industrial technologies to allow natural interaction with virtual prototypes of mechanical or mechatronic systems. It serves as a bridge between physical reality and digital virtuality. The use cases in this paper are based on urban bike mobility. Therefore, three concepts have been worked out to specify main requirements for an urban hybrid bike simulator. The first use case is from the perspective of a bicycle rental, where rental services for the users can be developed, validated, and optimized. The second use case provides the integration of mobile devices like smartphones and tablets for the development and validation of mobile services for bicyclists. The third use case is oriented on development and validation of new bicycles and urban mobility concepts like e-bikes, pedelecs, tripelecs and sharing services. Based on these generic use cases the requirements on a hybrid bicycle simulator were derived. Why a bicycle simulator? Well, we are firmly convinced that the future of urban mobility is determined from trends such as ecological rethinking and the desire for sports and healthy life. Furthermore, it is one of the most competitive and agile markets using most innovative materials and manufacturing technologies

EDF1 coordinates cellular responses to ribosome collisions

Translation of aberrant mRNAs induces ribosomal collisions, thereby triggering pathways for mRNA and nascent peptide degradation and ribosomal rescue. Here we use sucrose gradient fractionation combined with quantitative proteomics to systematically identify proteins associated with collided ribosomes. This approach identified Endothelial differentiation-related factor 1 (EDF1) as a novel protein recruited to collided ribosomes during translational distress. Cryo-electron microscopic analyses of EDF1 and its yeast homolog Mbf1 revealed a conserved 40S ribosomal subunit binding site at the mRNA entry channel near the collision interface. EDF1 recruits the translational repressors GIGYF2 and EIF4E2 to collided ribosomes to initiate a negative-feedback loop that prevents new ribosomes from translating defective mRNAs. Further, EDF1 regulates an immediate-early transcriptional response to ribosomal collisions. Our results uncover mechanisms through which EDF1 coordinates multiple responses of the ribosome-mediated quality control pathway and provide novel insights into the intersection of ribosome-mediated quality control with global transcriptional regulation

Use of Complex DNA and Antibody Microarrays as Tools in Functional Analyses

While the deciphering of basic sequence information on a genomic scale is yielding complete genomic sequences in ever-shorter intervals, experimental procedures for elucidating the cellular effects and consequences of the DNA-encoded information become critical for further analyses. In recent years, DNA microarray technology has emerged as a prime candidate for the performance of many such functional assays. Technically, array technology has come a long way since its conception some 15 years ago, initially designed as a means for large-scale mapping and sequencing

Circulating Micro-RNAs as Potential Blood-Based Markers for Early Stage Breast Cancer Detection

INTRODUCTION: MicroRNAs (miRNAs, miRs) are a class of small, non-coding RNA molecules with relevance as regulators of gene expression thereby affecting crucial processes in cancer development. MiRNAs offer great potential as biomarkers for cancer detection due to their remarkable stability in blood and their characteristic expression in many different diseases. We investigated whether microarray-based miRNA profiling on whole blood could discriminate between early stage breast cancer patients and healthy controls. METHODS: We performed microarray-based miRNA profiling on whole blood of 48 early stage breast cancer patients at diagnosis along with 57 healthy individuals as controls. This was followed by a real-time semi-quantitative Polymerase Chain Reaction (RT-qPCR) validation in a separate cohort of 24 early stage breast cancer patients from a breast cancer screening unit and 24 age matched controls using two differentially expressed miRNAs (miR-202, miR-718). RESULTS: Using the significance level of p<0.05, we found that 59 miRNAs were differentially expressed in whole blood of early stage breast cancer patients compared to healthy controls. 13 significantly up-regulated miRNAs and 46 significantly down-regulated miRNAs in our microarray panel of 1100 miRNAs and miRNA star sequences could be detected. A set of 240 miRNAs that was evaluated by radial basis function kernel support vector machines and 10-fold cross validation yielded a specificity of 78.8%, and a sensitivity of 92.5%, as well as an accuracy of 85.6%. Two miRNAs were validated by RT-qPCR in an independent cohort. The relative fold changes of the RT-qPCR validation were in line with the microarray data for both miRNAs, and statistically significant differences in miRNA-expression were found for miR-202. CONCLUSIONS: MiRNA profiling in whole blood has potential as a novel method for early stage breast cancer detection, but there are still challenges that need to be addressed to establish these new biomarkers in clinical use

Fine-mapping of the HNF1B multicancer locus identifies candidate variants that mediate endometrial cancer risk.

Common variants in the hepatocyte nuclear factor 1 homeobox B (HNF1B) gene are associated with the risk of Type II diabetes and multiple cancers. Evidence to date indicates that cancer risk may be mediated via genetic or epigenetic effects on HNF1B gene expression. We previously found single-nucleotide polymorphisms (SNPs) at the HNF1B locus to be associated with endometrial cancer, and now report extensive fine-mapping and in silico and laboratory analyses of this locus. Analysis of 1184 genotyped and imputed SNPs in 6608 Caucasian cases and 37 925 controls, and 895 Asian cases and 1968 controls, revealed the best signal of association for SNP rs11263763 (P = 8.4 × 10(-14), odds ratio = 0.86, 95% confidence interval = 0.82-0.89), located within HNF1B intron 1. Haplotype analysis and conditional analyses provide no evidence of further independent endometrial cancer risk variants at this locus. SNP rs11263763 genotype was associated with HNF1B mRNA expression but not with HNF1B methylation in endometrial tumor samples from The Cancer Genome Atlas. Genetic analyses prioritized rs11263763 and four other SNPs in high-to-moderate linkage disequilibrium as the most likely causal SNPs. Three of these SNPs map to the extended HNF1B promoter based on chromatin marks extending from the minimal promoter region. Reporter assays demonstrated that this extended region reduces activity in combination with the minimal HNF1B promoter, and that the minor alleles of rs11263763 or rs8064454 are associated with decreased HNF1B promoter activity. Our findings provide evidence for a single signal associated with endometrial cancer risk at the HNF1B locus, and that risk is likely mediated via altered HNF1B gene expression

Atrial septal defect in adults is associated with airway hyperresponsiveness

Objective: The association between secundum atrial septal defects (ASD) and asthma-like dyspnea with consequent long-term pulmonary inhalant use, is poorly understood in adult ASD patients. Airway hyperresponsiveness is suggested to be the underlying mechanism of cardiac asthma from mitral valve disease and ischemic cardiomyopathy. We hypothesized that airway hyperresponsiveness may also be found in adult ASD patients. Our aim was to study airway responsiveness in adult ASD patients before percutaneous closure and at short-and long-term postprocedural follow-up. Methods: This prospective study included 31 ASD patients (65% female, mean age 49 ± 15y) who underwent spirometry and bronchoprovocation testing pre-and six-month postprocedurally, with additional bronchoprovocation at 2-year follow-up. Airway hyperresponsiveness was defined as ≥20% fall of forced expiratory volume in 1-second (FEV1) following <8.0 mg/mL of inhaled methacholine. Results: Airway hyperresponsiveness was found in 19/30 patients (63%[95%CI 45%-81%]; post hoc statistical power = 89%). Asthma-like symptoms wheezing, chest tightness, and cough were more frequently reported in airway hyperresponsive patients. Airway responsiveness was not influenced by successful percutaneous ASD closure, corresponding to persistence of asthma-like symptoms postclosure. Regardless of airway responsiveness, postprocedural right-sided reverse remodeling significantly improved dyspnea and pulmonary function. Conclusions: This study is the first to report a high prevalence of airway hyperresponsiveness in a cohort of unrepaired adult ASD patients, and confirms the association between asthma-like symptoms and ASD in adults. Attention to symptoms and pulmonary function should be given during clinical follow-up of adult ASD patients, both before and long after repair

Tema 14: Aglomerados de estrelas

We present observations of the symbiotic star CH Cyg with a new JHK-band beam combiner mounted to the IOTA interferometer. The new beam combiner consists of an anamorphic cylindrical lens system and a grism, and allows the simultaneous recording of spectrally dispersed J-, H- and K-band Michelson interferograms. The observations of CH Cyg were conducted on 5, 6, 8 and 11 June 2001 using baselines of 17m to 25m. From the interferograms of CH Cyg, J-, H-, and K-band visibility functions can be determined. Uniform-disk fits to the visibilities give, e.g., stellar diameters of (7.8 ± 0.6) mas and (8.7 ± 0.8) mas in H and K, respectively. Angular stellar filter radii and Rosseland radii are derived from the measured visibilities by fitting theoretical center-to-limb intensity variations (CLVs) of Mira star models. The available HIPPARCOS parallax of CH Cyg allows us to determine linear radii. For example, on the basis of the K-band visibility, Rosseland radii in the range of 214 to 243 solar radii can be derived utilizing CLVs of different fundamental mode Mira models as fit functions. These radii agree well within the error bars with the corresponding theoretical model Rosseland radii of 230 to 282 solar radii. Models of first overtone pulsators are not in good agreement with the observations. The wavelength dependence of the stellar diameter can be well studied by using visibility ratios V(λ1)/V(λ2) since ratios of visibilities of different spectral channels can be measured with higher precision than absolute visibilities. We found that the 2.03 μm uniform disk diameter of CH Cyg is approximately 1.1 times larger than the 2.15 μm and 2.26 μm uniform-disk diameter