| dc.contributor.advisor | Gruber, Jens Dr. | |
| dc.contributor.author | Böker, Kai Oliver | |
| dc.date.accessioned | 2017-04-11T08:31:32Z | |
| dc.date.available | 2017-04-11T08:31:32Z | |
| dc.date.issued | 2017-04-11 | |
| dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-0023-3E0D-6 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-6240 | |
| dc.language.iso | eng | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 570 | de |
| dc.title | Functional characterization of npcRNAs - Intercellular trafficking of generegulatory components via exosomes | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Gruber, Jens Dr. | |
| dc.date.examination | 2016-12-15 | |
| dc.description.abstracteng | Exosomes are small extracellular vesicles (EVs) of endocytic origin and get released by a multitude of cell types. These vesicles have been found in a variety of body fluids and more recently an important role in exchanging genetic information between cells was postulated for exosomes.
The aims of this thesis were to investigate the npcRNA content of exosomes and analyse their putative role in cell-cell communication. I established three different exosome isolation techniques yielding EVs in the appropriate size range determined by nanoparticle tracking analysis (NTA) and detected three exosomal marker proteins, i.e. Alix, TSG101 and CD9, by immunoblot. Furthermore, the identity of exosomes was confirmed by electron microscopy. Stable cell lines were generated to investigate the effect of the individual marker proteins on exosome production and characteristics. The tetraspanin CD9 positively influenced exosome and microvesicle production, while overexpression of Alix or TSG reduced the amount of EVs. In the context of lentiviral vector production a role in membrane fusion was identified for CD9, indicating a direct participation in fusion of exosomes with target cell membranes. However, extracellular CD9-enriched and WT vesicles comprise similar npcRNA content, suggesting no active role of CD9 in sorting of npcRNAs to exosomes.
The extracellular npcRNA content of four human cell lines was successfully examined via Next Generation Sequencing. Data analysis was performed using the DESeq package in R with self-made, question-adjusted, scripts. NGS data analysis revealed secretion (exosome) and retention (cell) motifs for miRNAs, an important finding for further cell-cell communication studies. Furthermore, I identified differentially expressed miRNAs after Jurkat exosome treatment in Raji cells by NGS and in silico analysis revealed two target genes (DNMT1 and Bcl-2) for these candidates. Extracellular vesicles derived from T-lymphocytes transferred sufficient amounts of functional miRNAs to induce a significant knockdown of DNMT1 as was confirmed by qPCR analysis. We generated a reporter gene assay that enabled detection of the horizontal transfer of synthetic siRNA (siGL-2) in exosomes. The results indicated a direct and functional communication via extracellular vesicles. In contact-inhibiting co-cultures we were able to confirm an EV mediated transfer of fluorescent labelled siRNAs between human B-lymphocytes and colon carcinoma cells, reflecting the cellular communication in the tumor microenvironment. Modification of cells with npcRNAs to study EV-functions is difficult in some cell types and hard to adapt for in vivo research. We utilized a gene delivery technology based on virus-like particles (VLP) that facilitates modification of cells in vivo. Feasibility was confirmed in a rat animal model by systemic delivery of functional siRNAs. The directed in vivo manipulation of cells opens new prospects for the exosome research and has to be expanded in the future. | de |
| dc.contributor.coReferee | Jahn, Reinhard Prof. Dr. | |
| dc.subject.eng | Exosomes, miRNA, RNAi, VLPs, Extracellular Vesicles | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-11858/00-1735-0000-0023-3E0D-6-9 | |
| dc.affiliation.institute | Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB) | de |
| dc.subject.gokfull | Biologie (PPN619462639) | de |
| dc.identifier.ppn | 884378861 | |