Medicalgenomics.org : an open source database and retrieval system for biomedical analysis

Die Molekularbiologie von Menschen ist ein hochkomplexes und vielfältiges Themengebiet, in dem in vielen Bereichen geforscht wird. Der Fokus liegt hier insbesondere auf den Bereichen der Genomik, Proteomik, Transkriptomik und Metabolomik, und Jahre der Forschung haben große Mengen an wertvollen Daten zusammengetragen. Diese Ansammlung wächst stetig und auch für die Zukunft ist keine Stagnation absehbar. Mittlerweile aber hat diese permanente Informationsflut wertvolles Wissen in unüberschaubaren, digitalen Datenbergen begraben und das Sammeln von forschungsspezifischen und zuverlässigen Informationen zu einer großen Herausforderung werden lassen. Die in dieser Dissertation präsentierte Arbeit hat ein umfassendes Kompendium von humanen Geweben für biomedizinische Analysen generiert. Es trägt den Namen medicalgenomics.org und hat diverse biomedizinische Probleme auf der Suche nach spezifischem Wissen in zahlreichen Datenbanken gelöst. Das Kompendium ist das erste seiner Art und sein gewonnenes Wissen wird Wissenschaftlern helfen, einen besseren systematischen Überblick über spezifische Gene oder funktionaler Profile, mit Sicht auf Regulation sowie pathologische und physiologische Bedingungen, zu bekommen. Darüber hinaus ermöglichen verschiedene Abfragemethoden eine effiziente Analyse von signalgebenden Ereignissen, metabolischen Stoffwechselwegen sowie das Studieren der Gene auf der Expressionsebene. Die gesamte Vielfalt dieser Abfrageoptionen ermöglicht den Wissenschaftlern hoch spezialisierte, genetische Straßenkarten zu erstellen, mit deren Hilfe zukünftige Experimente genauer geplant werden können. Infolgedessen können wertvolle Ressourcen und Zeit eingespart werden, bei steigenden Erfolgsaussichten. Des Weiteren kann das umfassende Wissen des Kompendiums genutzt werden, um biomedizinische Hypothesen zu generieren und zu überprüfen.

Development of a system measuring adhesion forces in powder collectives

Fine powders commonly have poor flowability and dispersibility due to interparticle adhesion that leads to formation of agglomerates. Knowing about adhesion in particle collectives is indispensable to gain a deeper fundamental understanding of particle behavior in powders. Especially in pharmaceutical industry a control of adhesion forces in powders is mandatory to improve the performance of inhalation products. Typically the size of inhalable particles is in the range of 1 - 5 µm. In this thesis, a new method was developed to measure adhesion forces of particles as an alternative to the established colloidal probe and centrifuge technique, which are both experimentally demanding, time consuming and of limited practical applicability. The new method is based on detachment of individual particles from a surface due to their inertia. The required acceleration in the order of 500 000 g is provided by a Hopkinson bar shock excitation system and measured via laser vibrometry. Particle detachment events are detected on-line by optical video microscopy. Subsequent automated data evaluation allows obtaining a statistical distribution of particle adhesion forces. To validate the new method, adhesion forces for ensembles of single polystyrene and silica microspheres on a polystyrene coated steel surface were measured under ambient conditions. It was possible to investigate more than 150 individual particles in one experiment and obtain adhesion values of particles in a diameter range of 3 - 13 µm. This enables a statistical evaluation while measuring effort and time are considerably lower compared to the established techniques. Measured adhesion forces of smaller particles agreed well with values from colloidal probe measurements and theoretical predictions. However, for the larger particles a stronger increase of adhesion with diameter was observed. This discrepancy might be induced by surface roughness and heterogeneity that influence small and large particles differently. By measuring adhesion forces of corrugated dextran particles with sizes down to 2 µm it was demonstrated that the Hopkinson bar method can be used to characterize more complex sample systems as well. Thus, the new device will be applicable to study a broad variety of different particle-surface combinations on a routine basis, including strongly cohesive powders like pharmaceutical drugs for inhalation.

Detrimental effects of exuberant and restrained immune responses against the central nervous system in the context of multiple sclerosis and glioblastoma multiforme

Glioblastoma multiforme (GBM) is the most common and most aggressive astrocytic tumor of the central nervous system (CNS) in adults. The standard treatment consisting of surgery, followed by a combinatorial radio- and chemotherapy, is only palliative and prolongs patient median survival to 12 to 15 months. The tumor subpopulation of stem cell-like glioma-initiating cells (GICs) shows resistance against radiation as well as chemotherapy, and has been suggested to be responsible for relapses of more aggressive tumors after therapy. The efficacy of immunotherapies, which exploit the immune system to specifically recognize and eliminate malignant cells, is limited due to strong immunosuppressive activities of the GICs and the generation of a specialized protective microenvironment. The molecular mechanisms underlying the therapy resistance of GICs are largely unknown. rnThe first aim of this study was to identify immune evasion mechanisms in GICs triggered by radiation. A model was used in which patient-derived GICs were treated in vitro with fractionated ionizing radiation (2.5 Gy in 7 consecutive passages) to select for a more radio-resistant phenotype. In the model cell line 1080, this selection process resulted in increased proliferative but diminished migratory capacities in comparison to untreated control GICs. Furthermore, radio-selected GICs downregulated various proteins involved in antigen processing and presentation, resulting in decreased expression of MHC class I molecules on the cellular surface and diminished recognition potential by cytotoxic CD8+ T cells. Thus, sub-lethal fractionated radiation can promote immune evasion and hamper the success of adjuvant immunotherapy. Among several immune-associated proteins, interferon-induced transmembrane protein 3 (IFITM3) was found to be upregulated in radio-selected GICs. While high expression of IFITM3 was associated with a worse overall survival of GBM patients (TCGA database) and increased proliferation and migration of differentiated glioma cell lines, a strong contribution of IFITM3 to proliferation in vitro as well as tumor growth and invasiveness in a xenograft model could not be observed. rnMultiple sclerosis (MS) is the most common autoimmune disease of the CNS in young adults of the Western World, which leads to progressive disability in genetically susceptible individuals, possibly triggered by environmental factors. It is assumed that self-reactive, myelin-specific T helper cell 1 (Th1) and Th17 cells, which have escaped the control mechanisms of the immune system, are critical in the pathogenesis of the human disease and its animal model experimental autoimmune encephalomyelitis (EAE). It was observed that in vitro differentiated interleukin 17 (IL-17) producing Th17 cells co-expressed the Th1-phenotypic cytokine Interferon-gamma (IFN-γ) in combination with the two respective lineage-associated transcription factors RORγt and T-bet after re-isolation from the CNS of diseased mice. Pathogenic molecular mechanisms that render a CD4+ T cell encephalitogenic have scarcely been investigated up to date. rnIn the second part of the thesis, whole transcriptional changes occurring in in vitro differentiated Th17 cells in the course of EAE were analyzed. Evaluation of signaling networks revealed an overrepresentation of genes involved in communication between the innate and adaptive immune system and metabolic alterations including cholesterol biosynthesis. The transcription factors Cebpa, Fos, Klf4, Nfatc1 and Spi1, associated with thymocyte development and naïve T cells were upregulated in encephalitogenic CNS-isolated CD4+ T cells, proposing a contribution to T cell plasticity. Correlation of the murine T-cell gene expression dataset to putative MS risk genes, which were selected based on their proximity (± 500 kb; ensembl database, release 75) to the MS risk single nucleotide polymorphisms (SNPs) proposed by the most recent multiple sclerosis GWAS in 2011, revealed that 67.3% of the MS risk genes were differentially expressed in EAE. Expression patterns of Bach2, Il2ra, Irf8, Mertk, Odf3b, Plek, Rgs1, Slc30a7, and Thada were confirmed in independent experiments, suggesting a contribution to T cell pathogenicity. Functional analysis of Nfatc1 revealed that Nfatc1-deficient CD4+ T cells were restrained in their ability to induce clinical signs of EAE. Nfatc1-deficiency allowed proper T cell activation, but diminished their potential to fully differentiate into Th17 cells and to express high amounts of lineage cytokines. As the inducible Nfatc1/αA transcript is distinct from the other family members, it could represent an interesting target for therapeutic intervention in MS.rn