TY - THES A1 - Franke, Christian T1 - Advancing Single-Molecule Localization Microscopy: Quantitative Analyses and Photometric Three-Dimensional Imaging T1 - Weiterentwicklung von Einzel-Molekül Lokalisations-Mikroskopie: Quantitative Analysen und photometrische drei-dimensionale Bildgebung N2 - Since its first experimental implementation in 2005, single-molecule localization microscopy (SMLM) emerged as a versatile and powerful imaging tool for biological structures with nanometer resolution. By now, SMLM has compiled an extensive track-record of novel insights in sub- and inter- cellular organization.\\ Moreover, since all SMLM techniques rely on the analysis of emission patterns from isolated fluorophores, they inherently allocate molecular information $per$ $definitionem$.\\ Consequently, SMLM transitioned from its origin as pure high-resolution imaging instrument towards quantitative microscopy, where the key information medium is no longer the highly resolved image itself, but the raw localization data set.\\ The work presented in this thesis is part of the ongoing effort to translate those $per$ $se$ molecular information gained by SMLM imaging to insights into the structural organization of the targeted protein or even beyond. Although largely consistent in their objectives, the general distinction between global or segmentation clustering approaches on one side and particle averaging or meta-analyses techniques on the other is usually made.\\ During the course of my thesis, I designed, implemented and employed numerous quantitative approaches with varying degrees of complexity and fields of application.\\ \\ In my first major project, I analyzed the localization distribution of the integral protein gp210 of the nuclear pore complex (NPC) with an iterative \textit{k}-means algorithm. Relating the distinct localization statistics of separated gp210 domains to isolated fluorescent signals led, among others, to the conclusion that the anchoring ring of the NPC consists of 8 homo-dimers of gp210.\\ This is of particular significance, both because it answered a decades long standing question about the nature of the gp210 ring and it showcased the possibility to gain structural information well beyond the resolution capabilities of SMLM by crafty quantification approaches.\\ \\ The second major project reported comprises an extensive study of the synaptonemal complex (SNC) and linked cohesin complexes. Here, I employed a multi-level meta-analysis of the localization sets of various SNC proteins to facilitate the compilation of a novel model of the molecular organization of the major SNC components with so far unmatched extend and detail with isotropic three-dimensional resolution.\\ In a second venture, the two murine cohesin components SMC3 and STAG3 connected to the SNC were analyzed. Applying an adapted algorithm, considering the disperse nature of cohesins, led to the realization that there is an apparent polarization of those cohesin complexes in the SNC, as well as a possible sub-structure of STAG3 beyond the resolution capabilities of SMLM.\\ \\ Other minor projects connected to localization quantification included the study of plasma membrane glycans regarding their overall localization distribution and particular homogeneity as well as the investigation of two flotillin proteins in the membrane of bacteria, forming clusters of distinct shapes and sizes.\\ \\ Finally, a novel approach to three-dimensional SMLM is presented, employing the precise quantification of single molecule emitter intensities. This method, named TRABI, relies on the principles of aperture photometry which were improved for SMLM.\\ With TRABI it was shown, that widely used Gaussian fitting based localization software underestimates photon counts significantly. This mismatch was utilized as a $z$-dependent parameter, enabling the conversion of 2D SMLM data to a virtual 3D space. Furthermore it was demonstrated, that TRABI can be combined beneficially with a multi-plane detection scheme, resulting in superior performance regarding axial localization precision and resolution.\\ Additionally, TRABI has been subsequently employed to photometrically characterize a novel dye for SMLM, revealing superior photo-physical properties at the single-molecule level.\\ Following the conclusion of this thesis, the TRABI method and its applications remains subject of diverse ongoing research. N2 - Seit ihrer ersten experimentellen Umsetzung in 2005 hat sich die Einzel-Molekül Lokalisations-Mikroskopie (\textit{engl.} single-molecule localization microscopy (SMLM)) als vielseitig einsetzbares Verfahren in der biologischen Bildgebung etabliert, vor allem aufgrund ihres hohen Auflösungsvermögens im Nanometer Bereich. Bis heute wurde eine Reihe neuer Erkenntnisse bezüglich der sub- und inter- zellulären Organisation durch den Einsatz der SMLM erlangt.\\ Aufgrund der Tatsache, dass alle SMLM Techniken auf dem Prinzip basieren, isolierte Fluorophore zu detektieren und zu analysieren, beinhalten SMLM Daten $per$ $definitionem$ molekulare Informationen.\\ Folgerichtig entwickelte sich das Feld der SMLM vom reinen Bildgebungsinstrument mit Nanometer-Auflösung hin zu quantitativer Mikroskopie, bei welcher der Fokus nicht länger vornehmlich auf dem hochaufgelöstem Bild, sondern vielmehr auf den Lokalisationsdaten liegt.\\ Die vorliegende Arbeit ist als Teil der anhaltenden Bestrebungen zu sehen, aus den $per$ $se$ molekularen Informationen der SMLM weiterführende Erkenntnisse über die strukturelle Organisation der markierten Proteine zu gewinnen. Obwohl mit der gleichen prinzipiellen Zielsetzung versehen, unterscheiden sich hierbei globale oder Segmentierungs- Clusteranalysen von Lokalisations-Meta-Analysen oder so genannten \textit{particle averaging} Ansätzen.\\ Während meiner Doktorarbeit habe ich verschiedene Quantifizierungs Ansätze entworfen, implementiert und angewendet, mit unterschiedlichen Graden an Komplexität und Breite des Anwendungsgebietes.\\ \\ In meinem ersten wesentlichem Projekt analysierte ich mit einem iterativen \textit{k}-means Algorithmus die Lokalisationsverteilung des integralen Proteins gp210, welches Teil des Kernporenkomplexes ist (\textit{engl.} nuclear pore complex (NPC)). Durch den Vergleich der charakteristischen Lokalisations-Statistik von separierten gp210 Domänen mit isolierten Fluoreszenzmarkern konnte unter anderem festgestellt werden, dass der Verankerungsring des NPC aus acht gp210 Homodimeren bestehen muss.\\ Diese Erkenntnis beantwortet zum einen eine jahrzehntealte Frage nach der Zusammensetzung des gp210 Rings und zum anderen liefert sie ein Beispiel dafür, dass durch eine geschickte Analyse der Lokalisationsstatistik strukturelle Informationen erlangt werden können, die jenseits des räumlichen Auflösungsvermögens von SMLM liegen.\\ \\ Das zweite hier vorgestellte wesentliche Projekt beinhaltet eine umfassende Studie des Synaptonemalen Komplexes (\textit{engl.} synaptonemal complex (SNC)) und damit verbundenen Cohesin Komplexen. Um die molekulare Organisation des SNC zu untersuchen, implementierte ich eine multi-level Meta-Analyse der Lokalisationsdaten mehrerer SNC Komponenten. Aus dessen Ergebnissen konnte ein neues drei dimensionales molekulares Modell des SNC erstellt werden.\\ Nachfolgend wurden die beiden murinen Cohesine SMC3 und STAG3 mit adaptierter Methodik untersucht. Hierbei musste die starke intrinsische Dispersion der Cohesin-Signale berücksichtigt werden. Die Analyse ergab deutliche Hinweise auf eine Polarisation der Cohesine innerhalb des SNC. Zudem zeigte sich eine mögliche Substruktur in der Organisation von STAG3, die unterhalb der Auflösungsgrenze von SMLM liegt.\\ \\ Weitere Nebenprojekte im Zusammenhang mit quantitativer Lokalisationsanalyse umfassten die Untersuchung der Lokalisationsverteilung von Plasma-Membran Glykanen, sowie zweier Flotillin Proteine in den Membranen von Bakterien, welche Cluster unterschiedlicher Form und Größe aufzeigten.\\ \\ Schließlich wird ein neuartiger Ansatz für dreidimensionale SMLM vorge-stellt, die auf der genauen Bestimmung von Einzel-Molekül Intensitäten basiert. Diese Methode, genannt TRABI, stützt sich auf die Prinzipien der Apertur Photometrie, welche für die SMLM angepasst und verbessert wurden.\\ Mit TRABI konnte gezeigt werden, dass weit verbreitete Lokalisations-Software, die auf $Gaussian-Fitting$ basiert, die Photonenzahl von Emittern oftmals stark unterschätzt. Diese Diskrepanz kann als $z$-abhängiger Parameter verwendet werden um z.B. einen 2D SMLM Datenatz in einen virtuellen 3D Raum zu überführen. Außerdem wird gezeigt, dass TRABI vorteilhaft mit einem multi-plane Detektionsschema kombiniert werden kann und dabei höhere axiale Lokalisationsgenauigkeiten und Auflösungen er-reicht.\\ Zudem wurde TRABI eingesetzt, um einen neuen Fluoreszenzfarbstoff für SMLM zu charakterisieren und dessen verbesserte photo-physikalische Eigenschaften auf Einzel-Molekül Basis zu demonstrieren.\\ Auch nach Abschluss dieser Arbeit ist die TRABI Methode und deren Anwendung weiterhin Gegenstand diverser Forschungen. KW - Einzelmolekülmikroskopie KW - Quantitative Mikroskopie KW - dSTORM Y1 - 2019 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-156355 ER - TY - JOUR A1 - Franke, Katharina A1 - Vilne, Baiba A1 - da Costa, Olivia Prazeres A1 - Rudelius, Martina A1 - Peschel, Christian A1 - Oostendorp, Robert A. J. A1 - Keller, Ulrich T1 - In vivo hematopoietic Myc activation directs a transcriptional signature in endothelial cells within the bone marrow microenvironment JF - Oncotarget N2 - Cancer pathogenesis involves tumor-intrinsic genomic aberrations and tumor-cell extrinsic mechanisms such as failure of immunosurveillance and structural and functional changes in the microenvironment. Using Myc as a model oncogene we established a conditional mouse bone marrow transduction/transplantation model where the conditional activation of the oncoprotein Myc expressed in the hematopoietic system could be assessed for influencing the host microenvironment. Constitutive ectopic expression of Myc resulted in rapid onset of a lethal myeloproliferative disorder with a median survival of 21 days. In contrast, brief 4-day Myc activation by means of the estrogen receptor (ER) agonist tamoxifen did not result in gross changes in the percentage/frequency of hematopoietic lineages or hematopoietic stem/progenitor cell (HSPC) subsets, nor did Myc activation significantly change the composition of the non-hematopoietic microenvironment defined by phenotyping for CD31, ALCAM, and Sca-1 expression. Transcriptome analysis of endothelial CD45-Ter119-cells from tamoxifen-treated MycER bone marrow graft recipients revealed a gene expression signature characterized by specific changes in the Rho subfamily pathway members, in the transcription-translation-machinery and in angiogenesis. In conclusion, intra-hematopoietic Myc activation results in significant transcriptome alterations that can be attributed to oncogene-induced signals from hematopoietic cells towards the microenvironment, e. g. endothelial cells, supporting the idea that even pre-leukemic HSPC highjack components of the niche which then could protect and support the cancer-initiating population. KW - stem-cells KW - mutations KW - C-Myc KW - Rho-GTPases KW - niche KW - leukemia KW - target KW - growth KW - cycle KW - apoptosis, Myc KW - microenvironment KW - endothelial cells Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-145844 VL - 6 IS - 26 SP - 21827 EP - 21839 ER - TY - JOUR A1 - Schneider, Johannes A1 - Klein, Teresa A1 - Mielich-Süss, Benjamin A1 - Koch, Gudrun A1 - Franke, Christian A1 - Kuipers, Oskar P. A1 - Kovács, Ákos T. A1 - Sauer, Markus A1 - Lopez, Daniel T1 - Spatio-temporal Remodeling of Functional Membrane Microdomains Organizes the Signaling Networks of a Bacterium JF - PLoS Genetics N2 - Lipid rafts are membrane microdomains specialized in the regulation of numerous cellular processes related to membrane organization, as diverse as signal transduction, protein sorting, membrane trafficking or pathogen invasion. It has been proposed that this functional diversity would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known. We recently discovered that bacterial membranes organize their signal transduction pathways in functional membrane microdomains (FMMs) that are structurally and functionally similar to the eukaryotic lipid rafts. In this report, we took advantage of the tractability of the prokaryotic model Bacillus subtilis to provide evidence for the coexistence of two distinct families of FMMs in bacterial membranes, displaying a distinctive distribution of proteins specialized in different biological processes. One family of microdomains harbors the scaffolding flotillin protein FloA that selectively tethers proteins specialized in regulating cell envelope turnover and primary metabolism. A second population of microdomains containing the two scaffolding flotillins, FloA and FloT, arises exclusively at later stages of cell growth and specializes in adaptation of cells to stationary phase. Importantly, the diversification of membrane microdomains does not occur arbitrarily. We discovered that bacterial cells control the spatio-temporal remodeling of microdomains by restricting the activation of FloT expression to stationary phase. This regulation ensures a sequential assembly of functionally specialized membrane microdomains to strategically organize signaling networks at the right time during the lifespan of a bacterium. KW - membrane proteins KW - gene expression KW - bacillus subtilis KW - fluorescence microscopy KW - cell fusion KW - signal transduction KW - gene regulation KW - lipids Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-125577 VL - 11 IS - 4 ER - TY - JOUR A1 - Marenholz, Ingo A1 - Esparza-Gordillo, Jorge A1 - Rüschendorf, Franz A1 - Bauerfeind, Anja A1 - Strachan, David P. A1 - Spycher, Ben D. A1 - Baurecht, Hansjörg A1 - Magaritte-Jeannin, Patricia A1 - Sääf, Annika A1 - Kerkhof, Marjan A1 - Ege, Markus A1 - Baltic, Svetlana A1 - Matheson, Melanie C. A1 - Li, Jin A1 - Michel, Sven A1 - Ang, Wei Q. A1 - McArdle, Wendy A1 - Arnold, Andreas A1 - Homuth, Georg A1 - Demenais, Florence A1 - Bouzigon, Emmanuelle A1 - Söderhäll, Cilla A1 - Pershagen, Göran A1 - de Jongste, Johan C. A1 - Postma, Dirkje S. A1 - Braun-Fahrländer, Charlotte A1 - Horak, Elisabeth A1 - Ogorodova, Ludmila M. A1 - Puzyrev, Valery P. A1 - Bragina, Elena Yu A1 - Hudson, Thomas J. A1 - Morin, Charles A1 - Duffy, David L. A1 - Marks, Guy B. A1 - Robertson, Colin F. A1 - Montgomery, Grant W. A1 - Musk, Bill A1 - Thompson, Philip J. A1 - Martin, Nicholas G. A1 - James, Alan A1 - Sleiman, Patrick A1 - Toskala, Elina A1 - Rodriguez, Elke A1 - Fölster-Holst, Regina A1 - Franke, Andre A1 - Lieb, Wolfgang A1 - Gieger, Christian A1 - Heinzmann, Andrea A1 - Rietschel, Ernst A1 - Keil, Thomas A1 - Cichon, Sven A1 - Nöthen, Markus M. A1 - Pennel, Craig E. A1 - Sly, Peter D. A1 - Schmidt, Carsten O. A1 - Matanovic, Anja A1 - Schneider, Valentin A1 - Heinig, Matthias A1 - Hübner, Norbert A1 - Holt, Patrick G. A1 - Lau, Susanne A1 - Kabesch, Michael A1 - Weidinger, Stefan A1 - Hakonarson, Hakon A1 - Ferreira, Manuel A. R. A1 - Laprise, Catherine A1 - Freidin, Maxim B. A1 - Genuneit, Jon A1 - Koppelman, Gerard H. A1 - Melén, Erik A1 - Dizier, Marie-Hélène A1 - Henderson, A. John A1 - Lee, Young Ae T1 - Meta-analysis identifies seven susceptibility loci involved in the atopic march JF - Nature Communications N2 - Eczema often precedes the development of asthma in a disease course called the 'atopic march'. To unravel the genes underlying this characteristic pattern of allergic disease, we conduct a multi-stage genome-wide association study on infantile eczema followed by childhood asthma in 12 populations including 2,428 cases and 17,034 controls. Here we report two novel loci specific for the combined eczema plus asthma phenotype, which are associated with allergic disease for the first time; rs9357733 located in EFHC1 on chromosome 6p12.3 (OR 1.27; P = 2.1 x 10(-8)) and rs993226 between TMTC2 and SLC6A15 on chromosome 12q21.3 (OR 1.58; P = 5.3 x 10(-9)). Additional susceptibility loci identified at genome-wide significance are FLG (1q21.3), IL4/KIF3A (5q31.1), AP5B1/OVOL1 (11q13.1), C11orf30/LRRC32 (11q13.5) and IKZF3 (17q21). We show that predominantly eczema loci increase the risk for the atopic march. Our findings suggest that eczema may play an important role in the development of asthma after eczema. KW - chromosome 11Q13 KW - risk KW - genomewide association KW - hay fever KW - birth cohort KW - filaggrin mutations KW - food allergy KW - juvenile myoclonic epilepsy KW - childhood asthma KW - dermatitis Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-139835 VL - 6 IS - 8804 ER - TY - JOUR A1 - Freitag‐Wolf, Sandra A1 - Munz, Matthias A1 - Junge, Olaf A1 - Graetz, Christian A1 - Jockel‐Schneider, Yvonne A1 - Staufenbiel, Ingmar A1 - Bruckmann, Corinna A1 - Lieb, Wolfgang A1 - Franke, Andre A1 - Loos, Bruno G. A1 - Jepsen, Søren A1 - Dommisch, Henrik A1 - Schaefer, Arne S. T1 - Sex‐specific genetic factors affect the risk of early‐onset periodontitis in Europeans JF - Journal of Clinical Periodontology N2 - Aims Various studies have reported that young European women are more likely to develop early‐onset periodontitis compared to men. A potential explanation for the observed variations in sex and age of disease onset is the natural genetic variation within the autosomal genomes. We hypothesized that genotype‐by‐sex (G × S) interactions contribute to the increased prevalence and severity. Materials and methods Using the case‐only design, we tested for differences in genetic effects between men and women in 896 North‐West European early‐onset cases, using imputed genotypes from the OmniExpress genotyping array. Population‐representative 6823 controls were used to verify that the interacting variables G and S were uncorrelated in the general population. Results In total, 20 loci indicated G × S associations (P < 0.0005), 3 of which were previously suggested as risk genes for periodontitis (ABLIM2, CDH13, and NELL1). We also found independent G × S interactions of the related gene paralogs MACROD1/FLRT1 (chr11) and MACROD2/FLRT3 (chr20). G × S‐associated SNPs at CPEB4, CDH13, MACROD1, and MECOM were genome‐wide‐associated with heel bone mineral density (CPEB4, MECOM), waist‐to‐hip ratio (CPEB4, MACROD1), and blood pressure (CPEB4, CDH13). Conclusions Our results indicate that natural genetic variation affects the different heritability of periodontitis among sexes and suggest genes that contribute to inter‐sex phenotypic variation in early‐onset periodontitis. KW - alveolar bone loss KW - gene × sex interaction KW - genetic risk KW - heritability KW - inflammation Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-262445 VL - 48 IS - 11 SP - 1404 EP - 1413 ER - TY - JOUR A1 - Philipp-Abbrederis, Kathrin A1 - Herrmann, Ken A1 - Knop, Stefan A1 - Schottelius, Margret A1 - Eiber, Matthias A1 - Lückerath, Katharina A1 - Pietschmann, Elke A1 - Habringer, Stefan A1 - Gerngroß, Carlos A1 - Franke, Katharina A1 - Rudelius, Martina A1 - Schirbel, Andreas A1 - Lapa, Constantin A1 - Schwamborn, Kristina A1 - Steidle, Sabine A1 - Hartmann, Elena A1 - Rosenwald, Andreas A1 - Kropf, Saskia A1 - Beer, Ambros J A1 - Peschel, Christian A1 - Einsele, Hermann A1 - Buck, Andreas K A1 - Schwaiger, Markus A1 - Götze, Katharina A1 - Wester, Hans-Jürgen A1 - Keller, Ulrich T1 - In vivo molecular imaging of chemokine receptor CXCR4 expression in patients with advanced multiple myeloma JF - EMBO Molecular Medicine N2 - CXCR4 is a G-protein-coupled receptor that mediates recruitment of blood cells toward its ligand SDF-1. In cancer, high CXCR4 expression is frequently associated with tumor dissemination andpoor prognosis. We evaluated the novel CXCR4 probe [\(^{68}\)Ga]Pentixafor for invivo mapping of CXCR4 expression density in mice xenografted with human CXCR4-positive MM cell lines and patients with advanced MM by means of positron emission tomography (PET). [\(^{68}\)Ga]Pentixafor PET provided images with excellent specificity and contrast. In 10 of 14 patients with advanced MM [\(^{68}\)Ga]Pentixafor PET/CT scans revealed MM manifestations, whereas only nine of 14 standard [\(^{18}\)F]fluorodeoxyglucose PET/CT scans were rated visually positive. Assessment of blood counts and standard CD34\(^{+}\) flow cytometry did not reveal significant blood count changes associated with tracer application. Based on these highly encouraging data on clinical PET imaging of CXCR4 expression in a cohort of MM patients, we conclude that [\(^{68}\)Ga]Pentixafor PET opens a broad field for clinical investigations on CXCR4 expression and for CXCR4-directed therapeutic approaches in MM and other diseases. KW - FDG PET/CT KW - cells KW - CXCR4/SDF-1 KW - CXCR4 KW - multiple myeloma KW - positron emission tomography KW - chemokine receptor KW - in vivo imaging KW - malignancies KW - involvement KW - microenvironment KW - survival KW - cancer KW - autologous transplantation KW - bone disease Y1 - 2015 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-148738 VL - 7 IS - 4 ER - TY - JOUR A1 - Brevik, Erlend J A1 - van Donkelaar, Marjolein M. J. A1 - Weber, Heike A1 - Sánchez-Mora, Cristina A1 - Jacob, Christian A1 - Rivero, Olga A1 - Kittel-Schneider, Sarah A1 - Garcia-martinez, Iris A1 - Aebi, Marcel A1 - van Hulzen, Kimm A1 - Cormand, Bru A1 - Ramos-Quiroga, Josep A A1 - Lesch, Klaus-Peter A1 - Reif, Andreas A1 - Ribases, Marta A1 - Franke, Barbara A1 - Posserud, Maj-Britt A1 - Johansson, Stefan A1 - Lundervold, Astri J. A1 - Haavik, Jan A1 - Zayats, Tetyana T1 - Genome-wide analyses of aggressiveness in attention-deficit hyperactivity disorder JF - American Journal of Medical Genetics Part B-Neuropsychiatric Genetics N2 - Aggressiveness is a behavioral trait that has the potential to be harmful to individuals and society. With an estimated heritability of about 40%, genetics is important in its development. We performed an exploratory genome-wide association (GWA) analysis of childhood aggressiveness in attention deficit hyperactivity disorder (ADHD) to gain insight into the underlying biological processes associated with this trait. Our primary sample consisted of 1,060 adult ADHD patients (aADHD). To further explore the genetic architecture of childhood aggressiveness, we performed enrichment analyses of suggestive genome-wide associations observed in aADHD among GWA signals of dimensions of oppositionality (defiant/vindictive and irritable dimensions) in childhood ADHD (cADHD). No single polymorphism reached genome-wide significance (P<5.00E-08). The strongest signal in aADHD was observed at rs10826548, within a long noncoding RNA gene (beta = -1.66, standard error (SE) = 0.34, P = 1.07E-06), closely followed by rs35974940 in the neurotrimin gene (beta = 3.23, SE = 0.67, P = 1.26E-06). The top GWA SNPs observed in aADHD showed significant enrichment of signals from both the defiant/vindictive dimension (Fisher's P-value = 2.28E-06) and the irritable dimension in cADHD (Fisher's P-value = 0.0061). In sum, our results identify a number of biologically interesting markers possibly underlying childhood aggressiveness and provide targets for further genetic exploration of aggressiveness across psychiatric disorders. KW - Large multicenter ADHD KW - Antisocial behavior KW - Diagnostic approach KW - Rating scale KW - Gene KW - Deficit/hyperactivity disorder KW - Susceptibility loci KW - Conduct disorder KW - Association KW - Adult KW - ADHD KW - Aggression KW - GWAS Y1 - 2016 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-188116 VL - 171B IS - 5 ER - TY - THES A1 - Franke, Christian T1 - Gelenkknorpelintegration im Tissue Engineering: Untersuchung von Polyethylenglykol- und Hyaluronsäure-Komponenten für ein Adhäsivum und Etablierung eines biomechanischen Versuchsmodells T1 - Articular cartilage integration in tissue engineering: Investigation of polyethylene glycol and hyaluronic acid components for an adhesive and establishment of a biomechanical test model N2 - Gelenkknorpel besitzt aufgrund seiner avaskulären Natur und der fehlenden mitotischen Aktivität der Chondrozyten bei Schäden kaum Potential zur Selbstheilung. Traumatische Läsionen und degenerative Veränderungen münden im Krankheitsbild der Osteoarthrose, welches mit dem Untergang des Gelenkknorpels einhergeht. Ein neuerer Therapieansatz ist das Tissue Engineering von Gelenkknorpel, wobei jedoch die laterale Integration der Implantate mit dem nativen Knorpelgewebe problematisch bleibt. Ein Adhäsivum kann neben einer adäquaten Sofortadhäsion die Langzeitintegration fördern. In dieser Arbeit wurden verschiedene Polyethylenglykol (PEG)-basierte Zweikomponentenkleber, ausgehend vom kommerziell erhältlichen Gewebekleber CoSeal™, auf ihre Eignung für Gelenkknorpel untersucht. Dabei wurde Hyaluronsäure (HA) als physiologischer Bestandteil von Gelenkknorpel in thiolierter Form (HA-SH) als Komponente verwendet und auf seine prointegrativen Eigenschaften untersucht. Der den CoSeal™-Komponenten entsprechende 4-Succinimidyl-Glutarat/4-Thiol-PEG (4SG/4T-PEG)-Kleber hatte sich trotz seiner hohen Sofortadhäsionskraft auch nach der Substitution des 4T-PEG mit HA-SH als zu schnell in flüssiger Umgebung degradierend gezeigt, um eine suffiziente Langzeitintegration zu erreichen. Durch die Verwendung der langsamer degradierenden funktionellen 4-Succinimidyl-Carbonat-PEG (4C-PEG)-Komponente konnte die Langzeitadhäsionskraft in Kombination mit 4-Amin-PEG (4A-PEG) durch die stabilere Amid-Bindung zum einen und in Kombination mit HA-SH zum anderen signifikant gesteigert werden. Immunhistochemisch konnten bei beiden HA-haltigen Klebern Zeichen von Knorpelintegration nachgewiesen werden, während der 4C/4A-PEG-Kleber keine Integrationszeichen aufwies. Im 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bromid (MTT)-Assay war bei keinem Adhäsivum eine zytotoxische Wirkung zu erkennen. Insgesamt bieten die untersuchten PEG-basierten Adhäsiva im Vergleich zu den weitverbreiteten Fibrinklebern eine deutlich höhere Sofortadhäsion, welche vergleichbar mit glutaraldehydbasierten Klebern ist. Allerdings können die initialen adhäsiven Kräfte, trotz histologisch nachweisbaren Integrationszeichen bei Inkorporation von HA, nicht langfristig aufrechterhalten werden, so dass Fibrinkleber weiterhin die Spitzengruppe in Sachen Langzeitadhäsion bilden. Da PEG eine ausgezeichnete Biokompatibilität, einfache Anwendbarkeit und zahlreiche weitere chemische Anpassungsmöglichkeiten zur Feinabstimmung der Degradationseigenschaften bietet, ist in Zukunft ein erfolgreicher Einsatz auch im Bereich von Gelenkknorpel denkbar. Für die experimentelle Untersuchung von Adhäsiva und Gelenkknorpel werden biomechanische Versuchsmodelle benötigt. Der Tensile-Test des Sandwich-Modells konnte im Rahmen dieser Arbeit erfolgreich etabliert und ein Protokoll festgelegt werden. In einem vergleichenden Versuch mit dem Push-Out-Test des Disc-Ring-Modells, welches als Referenzmodell dient, konnte in Bezug auf die Reproduzierbarkeit und Qualität der Messergebnisse die Gleichwertigkeit gezeigt werden. Insgesamt bietet er eine gute Alternative zum Push-Out-Test, um weiterführende Fragestellung, wie z.B. extrinsische Kraftwirkungen auf das Konstrukt, zu untersuchen. N2 - Articular cartilage has little potential for self-healing due to its avascular nature and the lack of mitotic activity of chondrocytes in case of damage. Traumatic injuries and degenerative changes lead to the development of osteoarthritis, which is characterized by the destruction of articular cartilage. A more recent therapeutic approach is tissue engineering of articular cartilage, but the lateral integration of implants with native cartilage tissue remains problematic. An adhesive can promote long-term integration in addition to adequate immediate adhesion. In this thesis, various polyethylene glycol (PEG)-based two-component adhesives, derived from the commercially available tissue adhesive CoSeal™, were investigated for their suitability for articular cartilage. Hyaluronic acid (HA), a physiological component of articular cartilage, was used as a component in its thiolated form (HA-SH) and examined for its pro-integrative properties. Despite its high immediate adhesive strength, the 4-succinimidyl-glutarate/4-thiol-PEG (4SG/4T-PEG) adhesive, whose components correspond to the CoSeal™ components, showed rapid degradation in a liquid environment even after substituting the 4T-PEG-component with HA-SH, which hindered sufficient long-term integration. By using the slower degrading functional 4-succinimidyl-carbonate-PEG (4C-PEG) component, the long-term adhesive strength was significantly increased in combination with 4-amine-PEG (4A-PEG) due to the resulting more stable amide bond an in combination with HA-SH. Immunohistochemical analysis showed signs of cartilage integration for both HA-containing adhesives, while the 4C/4A-PEG-adhesive showed no signs of integration. In the 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, none of the adhesives exhibited cytotoxic effects. Overall, the investigated PEG-based adhesives offer a significantly higher immediate adhesive strength compared to the widely used fibrin glues, which is comparable to glutaraldehyde-based adhesives. However, despite histologically detectable signs of integration when HA was incorporated, the initial adhesive forces cannot be maintained in the long term, so fibrin glue continues to be at the forefront in terms of long-term adhesion. Since PEG offers excellent biocompatibility, easy applicability, and numerous other chemical customization options for fine-tuning degradation properties, successful use in the field of articular cartilage is conceivable in the future. For the experimental investigation of adhesives and articular cartilage, biomechanical test models are required. The tensile test of the sandwich model including a corresponding protocol was successfully established in this thesis. In a comparative experiment with the push-out test of the disc-ring model, which serves as a reference model, equivalence was demonstrated in terms of reproducibility and quality of test results. Overall, it provides a good alternative to the push-out test for investigating further questions, such as extrinsic force effects on the construct. KW - Knorpel KW - Hyaluronsäure KW - Gewebekleber KW - Polyethylenglykol KW - Knorpelintegration Y1 - 2023 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-323375 ER -