570 Biowissenschaften; Biologie
Refine
Has Fulltext
- yes (139)
Is part of the Bibliography
- yes (139)
Year of publication
- 2019 (139) (remove)
Document Type
- Journal article (78)
- Doctoral Thesis (60)
- Preprint (1)
Keywords
- Tissue Engineering (7)
- Candida albicans (3)
- Genexpression (3)
- In vitro (3)
- Signaltransduktion (3)
- cancer (3)
- leukemic cells (3)
- metagenomics (3)
- 3D Tumormodell (2)
- 3D tissue model (2)
Institute
- Theodor-Boveri-Institut für Biowissenschaften (68)
- Graduate School of Life Sciences (33)
- Julius-von-Sachs-Institut für Biowissenschaften (10)
- Lehrstuhl für Tissue Engineering und Regenerative Medizin (9)
- Rudolf-Virchow-Zentrum (8)
- Fakultät für Biologie (4)
- Institut für Hygiene und Mikrobiologie (4)
- Institut für Molekulare Infektionsbiologie (4)
- Institut für Pharmakologie und Toxikologie (4)
- Institut für Virologie und Immunbiologie (3)
- Lehrstuhl für Biochemie (3)
- Medizinische Klinik und Poliklinik II (3)
- Abteilung für Molekulare Innere Medizin (in der Medizinischen Klinik und Poliklinik II) (2)
- Center for Computational and Theoretical Biology (2)
- Deutsches Zentrum für Herzinsuffizienz (DZHI) (2)
- Institut für Anatomie und Zellbiologie (2)
- Institut für Humangenetik (2)
- Institut für Medizinische Strahlenkunde und Zellforschung (2)
- Klinik und Poliklinik für Psychiatrie, Psychosomatik und Psychotherapie (2)
- Pathologisches Institut (2)
- Abteilung für Funktionswerkstoffe der Medizin und der Zahnheilkunde (1)
- Comprehensive Cancer Center Mainfranken (1)
- Institut für Experimentelle Biomedizin (1)
- Institut für Geographie und Geologie (1)
- Institut für Klinische Epidemiologie und Biometrie (1)
- Institut für Klinische Neurobiologie (1)
- Institut für Pharmazie und Lebensmittelchemie (1)
- Institut für Systemimmunologie (1)
- Klinik und Poliklinik für Dermatologie, Venerologie und Allergologie (1)
- Klinik und Poliklinik für Kinder- und Jugendpsychiatrie, Psychosomatik und Psychotherapie (1)
- Klinik und Poliklinik für Nuklearmedizin (1)
- Lehrstuhl für Molekulare Psychiatrie (1)
- Medizinische Klinik und Poliklinik I (1)
- Urologische Klinik und Poliklinik (1)
Sonstige beteiligte Institutionen
Morphological and Functional Ultrashort Echo Time (UTE) Magnetic Resonance Imaging of the Human Lung
(2019)
In this thesis, a 3D Ultrashort echo time (3D-UTE) sequence was introduced in the Self-gated Non-Contrast-Enhanced Functional Lung Imaging (SENCEFUL) framework. The sequence was developed and implemented on a 3 Tesla MR scanner. The 3D-UTE technique consisted of a nonselective RF pulse followed by a koosh ball quasi-random sampling order of the k-space. Measurements in free-breathing and without contrast agent were performed in healthy subjects and a patient with lung cancer.
A gating technique, using a combination of different coils with high signal correlation, was evaluated in-vivo and compared with a manual approach of coil selection. The gating signal offered an estimation of the breathing motion during measurement and was used as a reference to segment the acquired data into different breathing phases.
Gradient delays and trajectory errors were corrected during post-processing using the Gradient Impulse Response Function. Iterative SENSE was then applied to determine the fully sampled data.
In order to eliminate signal changes caused by motion, a 3D image registration was employed, and the results were compared to a 2D image registration method.
Ventilation was assessed in 3D and regionally quantified by monitoring the signal changes in the lung parenchyma. Finally, image quality and quantitative ventilation values were compared to the standard 2D-SENCEFUL technique.
3D-UTE, combined with an automatic gating technique and SENCEFUL MRI, offered ventilation maps with high spatial resolution and SNR. Compared to the 2D method, UTE-SENCEFUL greatly improved the clinical quality of the structural images and the visualization of the lung parenchyma.
Through‐plane motion, partial volume effects and ventilation artifacts were also reduced with a three-dimensional method for image registration.
UTE-SENCEFUL was also able to quantify regional ventilation and presented similar results to previous studies.
Protein quality control systems are critical for cellular proteostasis and survival under stress conditions. The ubiquitin proteasome system (UPS) plays a pivotal role in proteostasis by eliminating misfolded and damaged proteins. However, exposure to the environmental toxin arsenite results in the accumulation of polyubiquitylated proteins, indicating an overload of the UPS. Arsenite stress induces the rapid formation of stress granules (SGs), which are cytoplasmic assemblies of mRNPs stalled in translation initiation. The mammalian proteins ZFAND2A/B (also known as AIRAP and AIRAPL, respectively) bind to the 26S proteasome, and ZFAND2A has been shown to adapt proteasome activity to arsenite stress. They belong to a small subfamily of AN1 type zinc finger containing proteins that also comprises the unexplored mammalian member ZFAND1 and its yeast homolog Cuz1.
In this thesis, the cellular function of Cuz1 and ZFAND1 was investigated. Cuz1/ZFAND1 was found to interact with the ubiquitin-selective, chaperone-like ATPase Cdc48/p97 and with the 26S proteasome. The interaction between Cuz1/ZFAND1 and Cdc48/p97 requires a predicted ubiquitin-like domain of Cuz1/ZFAND1. In vivo, this interaction was strongly dependent on acute arsenite stress, suggesting that it is a part of the cellular arsenite stress response. Lack of Cuz1/ZFAND1 caused a defect in the clearance of arsenite induced SG clearance. ZFAND1 recruits both, the 26S proteasome and p97, to arsenite-induced SGs for their normal clearance. In the absence of ZFAND1, SGs lack the 26S proteasome and p97, accumulate defective ribosomal products and become aberrant. These aberrant SGs persist after arsenite removal and undergo degradation via autophagy. ZFAND1 depletion is epistatic to the expression of pathogenic mutant p97 with respect to SG clearance, suggesting that ZFAND1 function is relevant to the multisystem degenerative disorder, inclusion body myopathy associated with Paget’s disease of bone and frontotemporal dementia and amyotrophic lateral sclerosis (IBMPFD/ALS).
In this work models for molecular networks consisting of ordinary differential equations are extended by terms that include the interaction of the corresponding molecular network with the environment that the molecular network is embedded in. These terms model the effects of the external stimuli on the molecular network. The usability of this extension is demonstrated with a model of a circadian clock that is extended with certain terms and reproduces data from several experiments at the same time.
Once the model including external stimuli is set up, a framework is developed in order to calculate external stimuli that have a predefined desired effect on the molecular network. For this purpose the task of finding appropriate external stimuli is formulated as a mathematical optimal control problem for which in order to solve it a lot of mathematical methods are available. Several methods are discussed and worked out in order to calculate a solution for the corresponding optimal control problem. The application of the framework to find pharmacological intervention points or effective drug combinations is pointed out and discussed. Furthermore the framework is related to existing network analysis tools and their combination for network analysis in order to find dedicated external stimuli is discussed.
The total framework is verified with biological examples by comparing the calculated results with data from literature. For this purpose platelet aggregation is investigated based on a corresponding gene regulatory network and associated receptors are detected. Furthermore a transition from one to another type of T-helper cell is analyzed in a tumor setting where missing agents are calculated to induce the corresponding switch in vitro. Next a gene regulatory network of a myocardiocyte is investigated where it is shown how the presented framework can be used to compare different treatment strategies with respect to their beneficial effects and side effects quantitatively. Moreover a constitutively activated signaling pathway, which thus causes maleficent effects, is modeled and intervention points with corresponding treatment strategies are determined that steer the gene regulatory network from a pathological expression pattern to physiological one again.
Die verfügbaren in vitro Genotoxizitätstests weisen hinsichtlich ihrer Spezifität und ihres Informationsgehalts zum vorliegenden Wirkmechanismus (Mode of Action, MoA) Einschränkungen auf. Um diese Mängel zu überwinden, wurden in dieser Arbeit zwei Ziele verfolgt, die zu der Entwicklung und Etablierung neuer in vitro Methoden zur Prüfung auf Genotoxizität in der Arzneimittelentwicklung beitragen.
1. Etablierung und Bewertung einer neuen in vitro Genotoxizitätsmethode (MultiFlow Methode)
Die MultiFlow Methode basiert auf DNA-schadensassoziierten Proteinantworten von γH2AX (DNA-Doppelstrangbrüche), phosphorylierten H3 (S10) (mitotische Zellen), nukleären Protein p53 (Genotoxizität) und cleaved PARP1 (Apoptose) in TK6-Zellen. Insgesamt wurden 31 Modellsubstanzen mit dem MultiFlow Assay und ergänzend mit dem etablierten Mikrokerntest (MicroFlow MNT), auf ihre Fähigkeit verschiedene MoA-Gruppen (Aneugene/Klastogene/Nicht-Genotoxine) zu differenzieren, untersucht. Die Performance der „neuen“ gegenüber der „alten“ Methode führte zu einer verbesserten Sensitivität von 95% gegenüber 90%, Spezifität von 90% gegenüber 72% und einer MoA-Klassifizierungsrate von 85% gegenüber 45% (Aneugen vs. Klastogen).
2. Identifizierung mechanistischer Biomarker zur Klassifizierung genotoxischer Substanzen
Die Analyse 67 ausgewählter DNA-schadensassoziierter Gene in der QuantiGene Plex Methode zeigte, dass mehrere Gene gleichzeitig zur MoA-Klassifizierung beitragen können. Die Kombination der höchstrangierten Marker BIK, KIF20A, TP53I3, DDB2 und OGG1 ermöglichte die beste Identifizierungsrate der Modellsubstanzen. Das synergetische Modell kategorisierte 16 von 16 Substanzen korrekt in Aneugene, Klastogene und Nicht-Genotoxine. Unter Verwendung der Leave-One-Out-Kreuzvalidierung wurde das Modell evaluiert und erreichte eine Sensitivität, Spezifität und Prädiktivität von 86%, 83% und 85%. Ergebnisse der traditionellen qPCR Methode zeigten, dass Genotoxizität mit TP53I3, Klastogenität mit ATR und RAD17 und oxidativer Stress mit NFE2L2 detektiert werden kann.
Durch die Untersuchungen von posttranslationalen Modifikationen unter Verwendung der High-Content-Imaging-Technologie wurden mechanistische Assoziationen für BubR1 (S670) und pH3 (S28) mit Aneugenität, 53BP1 (S1778) und FANCD2 (S1404) mit Klastogenität, p53 (K373) mit Genotoxizität und Nrf2 (S40) mit oxidativem Stress identifiziert.
Diese Arbeit zeigt, dass (Geno)toxine unterschiedliche Gen- und Proteinveränderungen in TK6-Zellen induzieren, die zur Erfassung mechanistischer Aktivitäten und Einteilung (geno)toxischer MoA-Gruppen (Aneugen/Klastogen/ Reaktive Sauerstoffspezies) eingesetzt werden können und daher eine bessere Risikobewertung von Wirkstoffkandidaten ermöglichen.
Das Sprouty-related, EVH1 domain containing protein 2 (SPRED2) ist ein
inhibitorisches, downstream von Ras wirkendes Protein des MAP-Kinase Signalwegs,
welches entscheidenden Einfluss auf die Regulation von Proliferation, Expression von
Proteinen und der zellulären Homöostase hat. Der kardiale Phänotyp von SPRED2-
defizienten Mäusen zeigt nicht nur eine deutliche linksventrikuläre Hypertrophie,
sondern auch eine erhöhte Fibrosierung des Herzgewebes. Zellulär wird die SPRED2-
Defizienz durch die Akkumulation von vesikulären Strukturen innerhalb der Zelle,
sowie eine markant erhöhte Anzahl von Vesikeln entlang der longitudinalen Reihen
der Mitochondrien gekennzeichnet.
Ziel dieser Arbeit war es, den Charakter dieser vesikulären Strukturen näher zu
beleuchten und festzustellen, in welchem Zusammenhang die subzellulär veränderte
Architektur mit der Hypertrophie der SPRED2-defizienten Tiere steht. Um diese
Fragestellung zu beantworten, wurde zunächst nach einem vesikulären
Degradationsmechanismus gesucht, der in SPRED2-/--Cardiomyocyten betroffen sein
könnte. Die Macroautophagie, im folgenden Autophagie bezeichnet, ist ein solcher
Degradationsmechanismus, bei dem selektiv langlebige Proteine und Zellorganellen
abgebaut werden. Es konnten signifikante Veränderung der Protein-Level an
Schlüsselpositionen der Autophagie identifiziert werden. Das Ubiquitin-aktivierende
(E1) Enzym Homolog Atg7 sowie die Cystein-Protease Atg4B zeigen sich im SPRED2-
KO deutlich reduziert. Ebenso Atg16L, das als essentieller Bestandteil des Atg5-
Atg12-Atg16-Konjugationssystems bei der Konjugation von MAPLC3-II an das
Phospholipid Phosphatidylethanolamin beteiligt ist. Die Autophagie-Rate als
Verhältnis von konjugiertem zu unkonjugiertem MAPLC3 ist ebenfalls reduziert. Die
Akkumulation der autophagischen Vesikel zeigt sich kongruent zu dem erhöhten
Protein-Level der autophagischen Cargo-Rezeptoren SQSTM1 und NBR1, sowie des
lysosomalen Markers CathepsinD. Außer der verringerten Autophagie-Rate zeigt sich
in Einklang mit der Fibrosierung des Herzgewebes eine erhöht aktive Caspase-3 als
Marker für Apoptose. Um die mitochondriale Integrität näher zu beleuchten, wurde die
Menge an reaktiven Sauerstoffspezies (ROS) in Wildtyp und SPRED2-KO untersucht.
Hierbei zeigte sich eine erhöhte Menge an ROS im KO, was ein Hinweis auf eine
Beeinträchtigung der Mitochondrien darstellt.
Letztlich wurde die Hypothese überprüft, ob ein gestörter Transport der Vesikel
durch eine Beeinträchtigung der Motorproteine Dynein und Kinesin vorliegt. In der Tat
zeigte sich die Aktivität der Dynein-ATPase verringert in der Abwesenheit von
SPRED2. Diese Beobachtung wird durch die erhöhten Mengen des vSNARE-Proteins
VTI1b unterstützt, was letztlich die Akkumulation der autophagischen Vesikel mit einer
verringerten Fähigkeit zur Membranfusion und dem ineffizienteren Transport der
Vesikel in Einklang bringt.
Da die gesamten Experimente in einem globalen SPRED2-KO System
durchgeführt wurden, können eventuelle Auswirkungen der beeinflussten hormonellen
Situation der SPRED2-KO Tiere auf den Herzphänotyp nicht final ausgeschlossen
werden. Um die genaue Wirkung einer SPRED2-Defizienz auf das Herzgewebe und
das Herz als Organ zu untersuchen, wurde im Rahmen dieser Arbeit eine SPRED2-
defiziente knockout Mauslinie mit konditionalem Potential generiert, die eine
gesteuerte Deletion von SPRED2 im Herzgewebe erlaubt.
Das Multiple Myelom (MM) ist eine maligne B-Zell-Erkrankung, welche von einer großen Heterogenität auf der biologischen und klinischen Ebene sowie in der Therapieantwort geprägt ist. Durch die biologische Interpretation von whole exome sequencing (WES)-Daten der Tumor- und Normalproben von fünf MM-Patienten und sechs MM-Zelllinien (ZL) sowie dem Einbezug von publizierten next generation sequencing (NGS)-Daten von 38 MM-Patienten konnten in dieser Dissertation sowohl somatische tumorrelevante Mutationen identifiziert als auch ein MM-spezifisches Signaltransduktionsnetzwerk definiert werden. Interessanterweise wurde in fast 100 % der MM-Patienten mindestens eine Mutation und in ~50 % der MM-Patienten sogar mehr als eine Mutation innerhalb dieses Netzwerkes beobachtet, was auf eine inter- und intra-individuelle Signalweg-Redundanz hinweist, die für die individuelle Therapieentscheidung möglicherweise von Bedeutung sein könnte. Außerdem konnte bestätigt werden, dass identische, positionsspezifische und genspezifische Mutationen im MM selten wiederholt auftreten. Als häufig mutierte Gene im MM konnten KRAS, NRAS, LRP1B, FAM46C, WHSC1, ALOX12B, DIS3 und PKHD1 identifiziert werden. Interessanterweise wurde die DIS3-Mutation in der MM-ZL OPM2 gemeinsam mit einer copy neutral loss of heterozygosity (CNLOH) im DIS3-Lokus detektiert, und in der MM-ZL AMO1 wurde eine noch nicht näher charakterisierte KRAS-Mutation in Exon 4 in Verbindung mit einem copy number (CN)-Zugewinn und einer erhöhten KRAS-Genexpression gefunden. DIS3 ist ein enzymatisch aktiver Teil des humanen RNA-Exosom-Komplexes und KRAS ein zentrales Protein im RTK-Signalweg, wodurch genetische Aberrationen in diesen Genen möglicherweise in der Entstehung oder Progression des MMs eine zentrale Rolle spielen. Daher wurde die gesamte coding sequence (CDS) der Gene DIS3 und KRAS an Tumorproben eines einheitlich behandelten Patientensets der DSMM-XI-Studie mit einem Amplikon-Tiefen-Sequenzierungsansatz untersucht. Das Patientenset bestand aus 81 MM-Patienten mit verfügbaren zytogenetischen und klinischen Daten. Dies ergab Aufschluss über die Verteilung der Mutationen innerhalb der Gene und dem Vorkommen der Mutationen in Haupt- und Nebenklonen des Tumors. Des Weiteren wurde die Assoziation der Mutationen mit weiteren klassischen zytogenetischen Alterationen (z.B. Deletion von Chr 13q14, t(4;14)-Translokation) untersucht und der Einfluss der Mutationen in Haupt- und Nebenklonen auf den klinischen Verlauf und die Therapieantwort bestimmt. Besonders hervorzuheben war dabei die Entdeckung von sieben neuen Mutationen sowie drei zuvor unbeschriebenen hot spot-Mutationen an den Aminosäure (AS)-Positionen p.D488, p.E665 und p.R780 in DIS3. Es wurde des Weiteren die Assoziation von DIS3-Mutationen mit einer Chr 13q14-Deletion und mit IGH-Translokationen bestätigt. Interessanterweise wurde ein niedrigeres medianes overall survival (OS) für MM-Patienten mit einer DIS3-Mutation sowie auch eine schlechtere Therapieantwort für MM-Patienten mit einer DIS3-Mutation im Nebenklon im Vergleich zum Hauptklon beobachtet. In KRAS konnten die bereits publizierten Mutationen bestätigt und keine Auswirkungen der KRAS-Mutationen in Haupt- oder Nebenklon auf den klinischen Verlauf oder die Therapieantwort erkannt werden. Erste siRNA vermittelte knockdown-Experimente von KRAS und Überexpressionsexperimente von KRAS-Wildtyp (WT) und der KRAS-Mutationen p.G12A, p.A146T und p.A146V mittels lentiviraler Transfektion zeigten eine Abhängigkeit der Phosphorylierung von MEK1/2 und ERK1/2 von dem KRAS-Mutationsstatus.
Zusammenfassend liefert die vorliegende Dissertation einen detaillierten Einblick in die molekularen Strukturen des MMs, vor allem im Hinblick auf die Rolle von DIS3 und KRAS bei der Tumorentwicklung und dem klinischen Verlauf.
Studies on the role of platelet serotonin in platelet function, hemostasis, thrombosis and stroke
(2019)
Platelet activation and aggregation are important processes in hemostasis resulting in reduction of blood loss upon vessel wall injury. However, platelet activation can lead to thrombotic events causing myocardial infarction and stroke. A more detailed understanding of the regulation of platelet activation and the subsequent formation of thrombi is essential to prevent thrombosis and ischemic stroke. Cations, platelet surface receptors, cytoskeletal rearrangements, activation of the coagulation cas-cade and intracellular signaling molecules are important in platelet activation and thrombus formation. One such important molecule is serotonin (5 hydroxytryptamin, 5 HT), an indolamine platelet agonist, biochemically derived from tryptophan. 5 HT is secreted from the enterochromaffin cells into the gastrointestinal tract (GI) and blood. Blood borne 5 HT has been proposed to regulate hemostasis by acting as a vaso-constrictor and by triggering platelet signaling through 5 HT2A receptor. Although platelets do not synthetize 5 HT, they take it up from the blood and store it in their dense granules which are secreted upon platelet activation. To identify the molecu-lar composite of the 5 HT uptake system in platelets and elucidate the role of platelet released 5-HT in thrombosis and ischemic stroke, 5 HT transporter knock out mice (5Htt / ) were analyzed in different in vitro and in vivo assays and in a model of is-chemic stroke. In 5Htt / platelets, 5 HT uptake from the blood was completely abol-ished and agonist-induced Ca2+ influx through store operated Ca2+ entry (SOCE), integrin activation, degranulation and aggregation responses to glycoprotein (GP) VI and C type lectin-like receptor 2 (CLEC 2) were reduced. These observed in vitro defects in 5Htt / platelets could be normalized by the addition of exogenous 5 HT. Moreover, reduced 5 HT levels in the plasma, an increased bleeding time and the formation of unstable thrombi were observed ex vivo under flow and in vivo in the abdominal aorta and carotid artery of 5Htt / mice. Surprisingly, in the transient middle cerebral artery occlusion model (tMCAO) of ischemic stroke 5Htt / mice showed near-ly normal infarct volumes and a neurological outcome comparable to control mice. Although secreted platelet 5 HT does not appear to play a crucial role in the devel-opment of reperfusion injury after stroke, it is essential to amplify the second phase of platelet activation through SOCE and thus plays an important role in thrombus stabilization.
To further investigate the role of cations, granules and their contents and regulation of integrin activation in the process of thrombus formation, genetically modified mice were analyzed in the different in vivo thrombosis models. Whereas Tph1 / mice (lacking the enzyme responsible for the production of 5 HT in the periphery), Trpm7KI (point mu-tation in the kinase domain of Trpm7 channel, lacking kinase activity) and Unc13d / /Nbeal2 / mice (lacking α granules and the release machinery of dense granules) showed a delayed thrombus formation in vivo, MagT1y/ mice (lacking a specific Mg2+ transporter) displayed a pro thrombotic phenotype in vivo. Trpm7fl/fl Pf4Cre (lacking the non specific Mg2+ channel) and RIAM / mice (lacking a potential linker protein in integrin “inside out” signaling) showed no alterations in thrombus formation upon injury of the vessel wall.
Inefficient vascularisation of solid tumours leads to the formation of oxygen and nutrient gradients. In order to mimic this specific feature of the tumour microenvironment, a multicellular tumour spheroid (SPH) culture system was used. These experiments were implemented in p53 isogenic colon cancer cell lines (HCT116 p53 +/+ and HCT116 p53-/-) since Tp53 has important regulatory functions in tumour metabolism. First, the characteristics of the cells cultured as monolayers and as spheroids were investigated by using RNA sequencing and metabolomics to compare gene expression and metabolic features of cells grown in different conditions. This analysis showed that certain features of gene expression found in tumours are also present in spheroids but not in monolayer cultures, including reduced proliferation and induction of hypoxia related genes. Moreover, comparison between the different genotypes revealed that the expression of genes involved in cholesterol homeostasis is induced in p53 deficient cells compared to p53 wild type cells and this difference was only detected in spheroids and tumour samples but not in monolayer cultures. In addition, it was established that loss of p53 leads to the induction of enzymes of the mevalonate pathway via activation of the transcription factor SREBP2, resulting in a metabolic rewiring that supports the generation of ubiquinone (coenzyme Q10). An adequate supply of ubiquinone was essential to support mitochondrial electron transport and pyrimidine biosynthesis in p53 deficient cancer cells under conditions of metabolic stress. Moreover, inhibition of the mevalonate pathway using statins selectively induced oxidative stress and apoptosis in p53 deficient colon cancer cells exposed to oxygen and nutrient deprivation. This was caused by ubiquinone being required for electron transfer by dihydroorotate dehydrogenase, an essential enzyme of the pyrimidine nucleotide biosynthesis pathway. Supplementation with exogenous nucleosides relieved the demand for electron transfer and restored viability of p53 deficient cancer cells under metabolic stress. Moreover, the mevalonate pathway was also essential for the synthesis of ubiquinone for nucleotide biosynthesis to support growth of intestinal tumour organoids. Together, these findings highlight the importance of the mevalonate pathway in cancer cells and provide molecular evidence for an enhanced sensitivity towards the inhibition of mitochondrial electron transfer in tumour-like metabolic environments.
The FDA approval of targeted therapy with BRAFV600E inhibitors like vemurafenib and dabrafenib in 2011 has been the first major breakthrough in the treatment of metastatic melanoma since almost three decades. Despite increased progression free survival and elevated overall survival rates, complete responses are scarce due to resistance development approximately six months after the initial drug treatment. It was previously shown in our group that melanoma cells under vemurafenib pressure in vitro and in vivo exhibit features of drug-induced senescence. It is known that some cell types, which undergo this cell cycle arrest, develop a so-called senescence associated secretome and it has been reported that melanoma cell lines also upregulate the expression of different factors after senescence induction. This work describes the effect of the vemurafenib-induced secretome on cells. Conditioned supernatants of vemurafenib-treated cells increased the viability of naive fibroblast and melanoma cell lines. RNA analysis of donor melanoma cells revealed elevated transcriptional levels of FGF1, MMP2 and CCL2 in the majority of tested cell lines under vemurafenib pressure, and I could confirm the secretion of functional proteins. Similar observations were also done after MEK inhibition as well as in a combined BRAF and MEK inhibitor treatment situation. Interestingly, the transcription of other FGF ligands (FGF7, FGF17) was also elevated after MEK/ERK1/2 inhibition. As FGF receptors are therapeutically relevant, I focused on the analysis of FGFR-dependent processes in response to BRAF inhibition. Recombinant FGF1 increased the survival rate of melanoma cells under vemurafenib pressure, while inhibition of the FGFR pathway diminished the viability of melanoma cells in combination with vemurafenib and blocked the stimulatory effect of vemurafenib conditioned medium. The BRAF inhibitor induced secretome is regulated by active PI3K/AKT signaling, and the joint inhibition of mTor and BRAFV600E led to decreased senescence induction and to a diminished induction of the secretome-associated genes. In parallel, combined inhibition of MEK and PI3K also drastically decreased mRNA levels of the relevant secretome components back to basal levels.
In summary, I could demonstrate that BRAF inhibitor treated melanoma cell lines acquire a specific PI3K/AKT dependent secretome, which is characterized by FGF1, CCL2 and MMP2. This secretome is able to stimulate other cells such as naive melanoma cells and fibroblasts and contributes to a better survival under drug pressure. These data are therapeutically highly relevant, as they imply the usage of novel drug combinations, especially specific FGFR inhibitors, with BRAF inhibitors in the clinic.
The plasma membrane is one of the most thoroughly studied and at the same time most complex, diverse, and least understood cellular structures. Its function is determined by the molecular composition as well as the spatial arrangement of its components. Even after decades of extensive membrane research and the proposal of dozens of models and theories, the structural organization of plasma membranes remains largely unknown. Modern imaging tools such as super-resolution fluorescence microscopy are one of the most efficient techniques in life sciences and are widely used to study the spatial arrangement and quantitative behavior of biomolecules in fixed and living cells. In this work, direct stochastic optical reconstruction microscopy (dSTORM) was used to investigate the structural distribution of mem-brane components with virtually molecular resolution. Key issues are different preparation and staining strategies for membrane imaging as well as localization-based quantitative analyses of membrane molecules.
An essential precondition for the spatial and quantitative analysis of membrane components is the prevention of photoswitching artifacts in reconstructed localization microscopy images. Therefore, the impact of irradiation intensity, label density and photoswitching behavior on the distribution of plasma membrane and mitochondrial membrane proteins in dSTORM images was investigated. It is demonstrated that the combination of densely labeled plasma membranes and inappropriate photoswitching rates induces artificial membrane clusters. Moreover, inhomogeneous localization distributions induced by projections of three-dimensional membrane structures such as microvilli and vesicles are prone to generate artifacts in images of biological membranes. Alternative imaging techniques and ways to prevent artifacts in single-molecule localization microscopy are presented and extensively discussed.
Another central topic addresses the spatial organization of glycosylated components covering the cell membrane. It is shown that a bioorthogonal chemical reporter system consisting of modified monosaccharide precursors and organic fluorophores can be used for specific labeling of membrane-associated glycoproteins and –lipids. The distribution of glycans was visualized by dSTORM showing a homogeneous molecule distribution on different mammalian cell lines without the presence of clusters. An absolute number of around five million glycans per cell was estimated and the results show that the combination of metabolic labeling, click chemistry, and single-molecule localization microscopy can be efficiently used to study cell surface glycoconjugates.
In a third project, dSTORM was performed to investigate low-expressing receptors on cancer cells which can act as targets in personalized immunotherapy. Primary multiple myeloma cells derived from the bone marrow of several patients were analyzed for CD19 expression as potential target for chimeric antigen receptor (CAR)-modified T cells. Depending on the patient, 60–1,600 CD19 molecules per cell were quantified and functional in vitro tests demonstrate that the threshold for CD19 CAR T recognition is below 100 CD19 molecules per target cell. Results are compared with flow cytometry data, and the important roles of efficient labeling and appropriate control experiments are discussed.
Modulation of insulin-induced genotoxicity in vitro and genomic damage in gestational diabetes
(2019)
Diabetes mellitus is a global health problem, where the risk of diabetes increases rapidly
due to the lifestyle changes. Patients with type II diabetes have many complications
with increased risk of morbidity and mortality. High levels of insulin may lead to DNA
oxidation and damage. Several studies proposed that hyperinsulinemia may be an
important risk factor for various types of cancer. To investigate insulin signaling
pathway inducing oxidative stress and genomic damage, pharmaceutical and natural
compounds which can interfere with the insulin pathway including PI3K inhibitors,
resveratrol, lovastatin, and RAD-001 were selected due to their beneficial effects
against metabolic disorder. Thus, the anti-genotoxic potential of these compounds
regarding insulin-mediated oxidative stress were investigated in normal rat kidney cells
in vitro. Our compounds showed protective effect against genotoxic damage and
significantly decreased reactive oxygen specious after treatment of cells with insulin
with different mechanisms of protection between the compounds. Thus, these
compounds may be attractive candidates for future support of diabetes mellitus therapy.
Next, we explored the link between gestational diabetes mellitus and genomic damage
in cells derived from human blood. Moreover, we investigated the influence of
estradiol, progesterone, adrenaline and triiodothyronine on insulin-induced genomic
damage in vitro. First, we studied the effect of these hormones in human promyelocytic
leukemia cells and next ex vivo with non-stimulated and stimulated peripheral blood
mononuclear cells. In parallel, we also measured the basal genomic damage using three
conditions (whole blood, non-stimulated and stimulated peripheral blood mononuclear
cells) in a small patient study including non-pregnant controls with/without hormonal
contraceptives, with a subgroup of obese women, pregnant women, and gestational
diabetes affected women. A second-time point after delivery was also applied for
analysis of the blood samples. Our results showed that GDM subjects and obese
individuals exhibited higher basal DNA damage compared to lower weight nonpregnant
or healthy pregnant women in stimulated peripheral blood mononuclear cells
in both comet and micronucleus assays. On the other hand, the DNA damage in GDM
women had decreased at two months after birth. Moreover, the applied hormones also
showed an influence in vitro in the enhancement of the genomic damage in cells of the control and pregnant groups but this damage did not exceed the damage which existed
in obese and gestational diabetes mellitus patients with high level of genomic damage.
In conclusion, insulin can induce genomic damage in cultured cells, which can be
modulated by pharmaceutical and naturals substances. This may be for future use in the
protection of diabetic patients, who suffer from hyperinsulinemia during certain disease
stages. A particular form of diabetes, GDM, was shown to lead to elevated DNA
damage in affected women, which is reduced again after delivery. Cells of affected
women do not show an enhanced, but rather a reduced sensitivity for further DNA
damage induction by hormonal treatment in vitro. A potential reason may be an
existence of a maximally inducible damage by hormonal influences.
Neurodevelopmental disorders, including attention-deficit/hyperactivity disorder (ADHD) and autism spectrum disorder (ASD) are disorders of mostly unknown etiopathogenesis, for which both genetic and environmental influences are expected to contribute to the phenotype observed in patients. Changes at all levels of brain function, from network connectivity between brain areas, over neuronal survival, synaptic connectivity and axonal growth, down to molecular changes and epigenetic modifications are suspected to play a key roles in these diseases, resulting in life-long behavioural changes.
Genome-wide association as well as copy-number variation studies have linked cadherin-13 (CDH13) as a novel genetic risk factor to neuropsychiatric and neurodevelopmental disorders. CDH13 is highly expressed during embryonic brain development, as well as in the adult brain, where it is present in regions including the hippocampus, striatum and thalamus (among others) and is upregulated in response to chronic stress exposure. It is however unclear how CDH13 interacts with environmentally relevant cues, including stressful triggers, in the formation of long-lasting behavioural and molecular changes. It is currently unknown how the environment influences CDH13 and which long term changes in behaviour and gene expression are caused by their interaction. This work therefore investigates the interaction between CDH13 deficiency and neonatal maternal separation (MS) in mice with the aim to elucidate the function of CDH13 and its role in the response to early-life stress (ELS).
For this purpose, mixed litters of wild-type (Cdh13+/+), heterozygous (Cdh13+/-) and homozygous knockout (Cdh13-/-) mice were maternally separated from postnatal day 1 (PN1) to postnatal day 14 (PN14) for 3 hours each day (180MS; PN1-PN14). In a first series of experiments, these mice were subjected to a battery of behavioural tests starting at 8 weeks of age in order to assess motor activity, memory functions as well as measures of anxiety. Subsequently, expression of RNA in various brain regions was measured using quantitativ real-time polymerase chain reaction (qRT-PCR). A second cohort of mice was exposed to the same MS procedure, but was not behaviourally tested, to assess molecular changes in hippocampus using RNA sequencing.
Behavioural analysis revealed that MS had an overall anxiolytic-like effect, with mice after MS spending more time in the open arms of the elevated-plus-maze (EPM) and the light compartment in the light-dark box (LDB). As a notable exception, Cdh13-/- mice did not show an increase of time spent in the light compartment after MS compared to Cdh13+/+ and Cdh13+/- MS mice. During the Barnes-maze learning task, mice of most groups showed a similar ability in learning the location of the escape hole, both in terms of primary latency and primary errors. Cdh13-/- control (CTRL) mice however committed more primary errors than Cdh13-/- MS mice. In the contextual fear conditioning (cFC) test, Cdh13-/- mice showed more freezing responses during the extinction recall, indicating a reduced extinction of fear memory. In the step-down test, an impulsivity task, Cdh13-/- mice had a tendency to wait longer before stepping down from the platform, indicative of more hesitant behaviour. In the same animals, qRT-PCR of several brain areas revealed changes in the GABAergic and glutamatergic systems, while also highlighting changes in the gatekeeper enzyme Glykogensynthase-Kinase 3 (Gsk3a), both in relation to Cdh13 deficiency and MS. Results from the RNA sequencing study and subsequent gene-set enrichment analysis revealed changes in adhesion and developmental genes due to Cdh13 deficiency, while also highlighting a strong link between CDH13 and endoplasmatic reticulum function. In addition, some results suggest that MS increased pro-survival pathways, while a gene x environment analysis showed alterations in apoptotic pathways and migration, as well as immune factors and membrane metabolism. An analysis of the overlap between gene and environment, as well as their interaction, highlighted an effect on cell adhesion factors, underscoring their importance for adaptation to the environment.
Overall, the stress model resulted in increased stress resilience in Cdh13+/+ and Cdh13+/- mice, a change absent in Cdh13-/- mice, suggesting a role of CDH13 during programming and adaptation to early-life experiences, that can results in long-lasting consequences on brain functions and associated behaviours. These changes were also visible in the RNA sequencing, where key pathways for cell-cell adhesion, neuronal survival and cell-stress adaptation were altered. In conclusion, these findings further highlight the role of CDH13 during brain development, while also shedding light on its function in the adaptation and response during (early life) environmental challenges.
FinO domain proteins such as ProQ of the model pathogen Salmonella enterica have emerged as a new class of major RNA-binding proteins in bacteria. ProQ has been shown to target hundreds of transcripts, including mRNAs from many virulence regions, but its role, if any, in bacterial pathogenesis has not been studied. Here, using a Dual RNA-seq approach to profile ProQ-dependent gene expression changes as Salmonella infects human cells, we reveal dysregulation of bacterial motility, chemotaxis, and virulence genes which is accompanied by altered MAPK (mitogen-activated protein kinase) signaling in the host. Comparison with the other major RNA chaperone in Salmonella, Hfq, reinforces the notion that these two global RNA-binding proteins work in parallel to ensure full virulence. Of newly discovered infection-associated ProQ-bound small noncoding RNAs (sRNAs), we show that the 3′UTR-derived sRNA STnc540 is capable of repressing an infection-induced magnesium transporter mRNA in a ProQ-dependent manner. Together, this comprehensive study uncovers the relevance of ProQ for Salmonella pathogenesis and highlights the importance of RNA-binding proteins in regulating bacterial virulence programs.
IMPORTANCE
The protein ProQ has recently been discovered as the centerpiece of a previously overlooked “third domain” of small RNA-mediated control of gene expression in bacteria. As in vitro work continues to reveal molecular mechanisms, it is also important to understand how ProQ affects the life cycle of bacterial pathogens as these pathogens infect eukaryotic cells. Here, we have determined how ProQ shapes Salmonella virulence and how the activities of this RNA-binding protein compare with those of Hfq, another central protein in RNA-based gene regulation in this and other bacteria. To this end, we apply global transcriptomics of pathogen and host cells during infection. In doing so, we reveal ProQ-dependent transcript changes in key virulence and host immune pathways. Moreover, we differentiate the roles of ProQ from those of Hfq during infection, for both coding and noncoding transcripts, and provide an important resource for those interested in ProQ-dependent small RNAs in enteric bacteria.
Measles is an extremely contagious vaccine-preventable disease responsible
for more than 90000 deaths worldwide annually. The number of deaths has
declined from 8 million in the pre-vaccination era to few thousands every year due
to the highly efficacious vaccine. However, this effective vaccine is still unreachable
in many developing countries due to lack of infrastructure, while in developed
countries too many people refuse vaccination. Specific antiviral compounds are not
yet available. In the current situation, only an extensive vaccination approach
along with effective antivirals could help to have a measles-free future. To develop
an effective antiviral, detailed knowledge of viral-host interaction is required.
This study was undertaken to understand the interaction between MV and
the innate host restriction factor APOBEC3G (A3G), which is well-known for its
activity against human immunodeficiency virus (HIV). Restriction of MV
replication was not attributed to the cytidine deaminase function of A3G, instead,
we identified a novel role of A3G in regulating cellular gene functions. Among two
of the A3G regulated host factors, we found that REDD1 reduced MV replication,
whereas, KDELR2 hampered MV haemagglutinin (H) surface transport thereby
affecting viral release. REDD1, a negative regulator of mTORC1 signalling
impaired MV replication by inhibiting mTORC1. A3G regulated REDD1
expression was demonstrated to inversely correlate with MV replication. siRNA
mediated silencing of A3G in primary human blood lymphocytes (PBL) reduced
REDD1 levels and simultaneously increased MV titres. Also, direct depletion of
REDD1 improved MV replication in PBL, indicating its role in A3G mediated
restriction of MV. Based on these finding, a new role of rapamycin, a
pharmacological inhibitor of mTORC1, was uncovered in successfully diminishing
MV replication in Vero as well as in human PBL. The ER and Golgi resident
receptor KDELR2 indirectly affected MV by competing with MV-H for cellular
chaperones. Due to the sequestering of chaperones by KDELR2, they can no longer
assist in MV-H folding and subsequent surface expression. Taken together, the two
A3G-regulated host factors REDD1 and KDELR2 are mainly responsible for
mediating its antiviral activity against MV.
The nuclear envelope serves as important messenger RNA (mRNA) surveillance system. In yeast and human, several control systems act in parallel to prevent nuclear export of unprocessed mRNAs. Trypanosomes lack homologues to most of the involved proteins and their nuclear mRNA metabolism is non-conventional exemplified by polycistronic transcription and mRNA processing by trans-splicing. We here visualized nuclear export in trypanosomes by intra- and intermolecular multi-colour single molecule FISH. We found that, in striking contrast to other eukaryotes, the initiation of nuclear export requires neither the completion of transcription nor splicing. Nevertheless, we show that unspliced mRNAs are mostly prevented from reaching the nucleus-distant cytoplasm and instead accumulate at the nuclear periphery in cytoplasmic nuclear periphery granules (NPGs). Further characterization of NPGs by electron microscopy and proteomics revealed that the granules are located at the cytoplasmic site of the nuclear pores and contain most cytoplasmic RNA-binding proteins but none of the major translation initiation factors, consistent with a function in preventing faulty mRNAs from reaching translation. Our data indicate that trypanosomes regulate the completion of nuclear export, rather than the initiation. Nuclear export control remains poorly understood, in any organism, and the described way of control may not be restricted to trypanosomes.
Proteine können aufgrund ihrer biochemischen Vielfalt eine Vielzahl von Interaktionen
mit anderen Proteinen oder chemischen Verbindungen eingehen. Im ersten Teil dieser
Arbeit wurden Protein-Protein Interaktionen mittels chemischen Quervernetzens
untersucht. Das Ziel war, neue und verbesserte Methoden zu entwickeln, um
Interaktionsnetzwerke zu erstellen. Im zweiten Teil wurden die Interaktionen von
Proteinen mit niedermolekularen Verbindungen untersucht, um Drug Targets zu
identifizieren und zu validieren.
Die Untersuchung von Protein-Protein Interaktionen mittels Massenspektrometrie (MS)
ist eine leistungsfähige Methode, um alle potentiellen Interaktionen eines Proteins nach
einer Anreicherung (Co-IP) aus einem Zelllysat zu detektieren. Durch das zusätzliche
Quervernetzen dieser Proteine und anschließender MS kann ein Interaktionsnetzwerk
erstellt werden, um direkte von indirekten Interaktionen unterscheiden zu können
(Topology Mapping). Zur Methodenetablierung wurden kommerzielle Crosslinker und
rekombinante Proteine von bekannten Interaktionspartnern mit niedriger Komplexität
verwendet. Die beiden Interaktionspartner NPL4 und UFD1 konnten mit dem Crosslinker
BS3 erfolgreich quervernetzt und anhand der vernetzten Peptide identifiziert werden. Im
nächsten Schritt wurde dieser Arbeitsablauf auf eine Co-IP des Mediatorkomplexes aus
Hefe angewendet. Die Probenkomplexität ist hierbei 500 - 1000-fach höher als bei der
Verwendung von rekombinanten Proteinen. Nach der erfolgreichen Quervernetzung
konnte innerhalb des Komplexes ein Interaktionsnetzwerk erstellt werden. Diese Daten
passen zu dem bereits bekannten Modell des Mediatorkomplexes. Interaktionen zu
bekannten Interaktionspartnern, wie der RNA-Pol II, konnten aufgrund deren
substöchiometrischen Anreicherung nicht identifiziert werden.
Aufgrund der genannten Limitationen beim Quervernetzen von Proteinen wurden
folgende neue und verbesserte Methoden entwickelt:
1. Verwendung des spaltbaren Crosslinkers (DSSO), der während der Messung selektiv
durch niedrige Kollisionsenergie gespalten werden kann, um die Datenbanksuche zu
vereinfachen. Die Funktionalität der DSSO-Strategie konnte erfolgreich am Protein
Cytochrom C getestet werden. Bei der ersten Fragmentierung wird der Linker gespalten, anschließend können die getrennten Peptide separat fragmentiert werden. Die erzeugten
Daten sind mit einer Standarddatenbanksuche kompatibel, was bei gemischten Spektren
von zwei Peptiden nicht der Fall wäre. Beim Quervernetzen der rekombinanten
Interaktionspartner UBX und p97N mit DSSO konnte der zu bestätigende Crosslink
zwischen zwei Lysinen nicht identifziert werden. Grund hierfür könnte eine zu kurze
Linkerlänge von DSSO sein. Diese Versuche brachten jedoch einige Limitationen des
Ansatzes zum Vorschein, wie die Beschränkung auf die Protease Trypsin, aufgrund der
positiven Ladung am C-Terminus und die Notwendigkeit von großen Proteinmengen, da
das Spalten des Linkers einen zusätzlichen Intensitätsverlust für die folgende
Identifizierung der Peptide mit sich bringt.
2. Da die niedrige Abundanz von quervernetzten Peptiden das Hauptproblem bei deren
Identifizierung ist, wurde eine Methode entwickelt, um während der Messung direkt nach
diesen niedrig abundanten Spezies zu suchen. Entscheidendes Kriterium hierfür war, dass
quervernetzte Peptide zwei C-Termini haben. Diese wurden zur Hälfte enzymatisch mit
18O bzw. 16O markiert und wieder vereinigt. Der resultierende Massenunterschied von 8
Da (4 x 18O) kommt ausschließlich bei zwei quervernetzten Peptiden vor und kann
während der Messung direkt gesucht werden. Die vollständige Markierung von Peptiden
mit 18O wurde zunächst am Protein Beta-Galaktosidase getestet. Bereits hier stellte sich
heraus, dass der enzymatische Rücktausch von 18O zu 16O ein Problem darstellt und die
Markierungseffizienz von Aminosäuren beeinflusst wird, die sich C-terminal nach der
Spaltstelle befinden. Mit dieser Strategie ließ sich somit keine vollständige Markierung
für alle Peptide erreichen, was für diese Strategie essentiell gewesen wäre.
3. Um alle Probleme zu umgehen, die bei der Identifizierung von quervernetzten Peptiden
auftreten, wurde eine Methode entwickelt, um quervernetzte Proteine anhand von Profilen
nach einer Auftrennung im Polyacrylamidgel (SDS-PAGE) zu identifizieren. Durch das
Quervernetzen von Proteinen entstehen zusätzliche Proteinbanden nach einer SDSPAGE,
die im Gel nach oben verschoben sind. Alle Proteine in diesen neu erzeugten
Bereichen stellen somit potentielle Interaktionspartner dar. Als Modellsystem wurde der
Mediatorkomplex verwendet. Er wurde aus einem Zelllysat mittels Co-IP angereichert
und anschließend quervernetzt. Aus den mittels LC-MS/MS gemessenen Gelfraktionen wurden Proteinprofile erstellt und miteinander verglichen. Die Intensitätsmaxima der
Proteine des Mediatorkomplexes konnten in bestimmten zusätzlichen Fraktionen
gefunden werden, was den indirekten Nachweis für eine Interaktion darstellt. Die
Funktionalität der Strategie konnte somit bestätigt werden. Ein verbleibender Nachteil ist
jedoch die zu geringe Trennleistung von Polyacrylamidgelen. Befinden sich mehr als 50
Proteine in einer Fraktion, können potentielle Interaktionspartner nicht eindeutig zu einer
Untereinheit eines Komplexes zugeordnet werden.
Im zweiten Teil der Arbeit wurde im Rahmen der Klinischen Forschergruppe 216
(CRU216) Interaktionen von Proteinen mit verschiedenen niedermolekularen
Verbindungen massenspektrometrisch untersucht, um potentielle Drug Targets zu
identifizieren. Diese Versuche sind vergleichbar mit Co-IP Experimenten, da sich der
Arbeitsablauf nur durch die Anreicherung mittels chemischer Verbindung unterscheidet.
Hierzu wurden biotinylierte Verbindungen immobilisiert und potentielle Drug Targets
aus einem komplexen Zelllysat angereichert. Die Identifzierung der echten
Bindungspartner wurde über quantitive Massenspektrometrie erreicht. Dabei wurden die
angereicherten Proteine, die an die niedermolekularen Substanzen binden mit einer
geeigneten Kontrollanreicherung verglichen. Mit den getesteten α-acyl
Aminocarboxamiden konnten verschiedene Proteinkomplexe und interagierende Proteine
spezifisch angereichert werden. Hierbei waren die vier Kinasen DNA-PK, ATM, ATR
und mTOR besonders interessant, da sie mit onkogenem Signalling und
Überlebensmechanismen wie der Hitzeschockantwort in Zellen des Multiplen Myeloms
(MM) in Verbidnung stehen. Die Inhibition der DNA-PK, ATM, ATR und mTOR mit α-
acyl Aminocarboxamiden stellt somit einen möglichen Therapieansatz dar, wenn er
zusammen mit hitzestressauslösenden Inhibitoren verwendet wird. Weiterhin konnte
gezeigt werden, dass die Armadillodomäne innerhalb der potentiellen Drug targets
signifkant angereichert wurde. Sie stellt damit eine potentielle Bindestelle der α-acyl
Aminocarboxamide dar.
Abschließend wurden Proteine mit biotinylierten Naphtylisochinolinen aus einem MMZelllysat
angereichert, deren Vorläufersubstanzen eine Wirkung auf Tumorzellen und den Malariaparasit Plasmodium falciparum gezeigt hatten. Hierbei konnten vor allem RNAbindende-
und mRNA-Splicing Proteine identifiziert werden, die zum Teil essentiell für
das Spleißen in-vivo sind. Hierzu gehören mehrere Untereinheiten der Splicing Factoren
3A und 3B. Die Veränderung der transkriptionellen Regulation und der resultierende
Effekt auf Krebszellen konnte bereits in anderen Studien mit dem Inhibitor Spliceostatin
A gezeigt werden, der das Spleißen beeinflusst.
The skeletal system forms the mechanical structure of the body and consists of bone, which is hard connective tissue. The tasks the skeleton and bones take over are of mechanical, metabolic and synthetic nature. Lastly, bones enable the production of blood cells by housing the bone marrow. Bone has a scarless self-healing capacity to a certain degree. Injuries exceeding this capacity caused by trauma, surgical removal of infected or tumoral bone or as a result from treatment-related osteonecrosis, will not heal. Critical size bone defects that will not heal by themselves are still object of comprehensive clinical investigation. The conventional treatments often result in therapies including burdening methods as for example the harvesting of autologous bone material. The aim of this thesis was the creation of a prevascularized bone implant employing minimally invasive methods in order to minimize inconvenience for patients and surgical site morbidity. The basis for the implant was a decellularized, naturally derived vascular scaffold (BioVaSc-TERM®) providing functional vessel structures after reseeding with autologous endothelial cells. The bone compartment was built by the combination of the aforementioned scaffold with synthetic β-tricalcium phosphate. In vitro culture for tissue maturation was performed using bioreactor technology before the testing of the regenerative potential of the implant in large animal experiments in sheep. A tibia defect was treated without the anastomosis of the implant’s innate vasculature to the host’s circulatory system and in a second study, with anastomosis of the vessel system in a mandibular defect. While the non-anastomosed implant revealed a mostly osteoconductive effect, the implants that were anastomosed achieved formation of bony islands evenly distributed over the defect.
In order to prepare preconditions for a rapid approval of an implant making use of this vascularization strategy, the manufacturing of the BioVaSc-TERM® as vascularizing scaffold was adjusted to GMP requirements.
Die essenzielle, Ubiquitin-selektive ATPase p97 reguliert eine Vielzahl unterschiedlicher Prozesse in Eukaryoten. Dazu zählen Proteinqualitätskontrolle, DNA-Reparatur, Signaltransduktion, Zellzykluskontrolle, Autophagie sowie das endolysosomale System. Diese unterschiedlichen Funktionen von p97 werden durch die Bindung von Kofaktoren engmaschig gesteuert und kontrolliert. Die größte und am besten untersuchte Gruppe von p97-Kofaktoren sind die Proteine der UBX Familie. Diese zeichnen sich durch den Besitz einer UBX-Domäne aus, welche die Bindung an p97 vermittelt. Das in höheren Eukaryoten konservierte Familienmitglied UBXD1 besitzt darüber hinaus mit einer PUB-Domäne und einem VIM-Motiv noch mindestens zwei weitere p97-Bindemodule. UBXD1 kann an Vesikel des endolysosomalen Degradationssytems lokalisieren, seine genauen zellulären Funktionen sind jedoch noch weitgehend unbekannt.
Ziel dieser Arbeit war die funktionelle Charakterisierung von humanem UBXD1. Dafür wurden Kandidaten eines zuvor durchgeführten Yeast-Two-Hybrid-Screens auf ihre Two Hybrid-Interaktion mit unterschiedlichen UBXD1-Varianten getestet. Darüber hinaus wurde durch Immunpräzipitationsexperimente untersucht, ob die Kandidatenproteine auch in Säugerzellen mit UBXD1 interagieren. Als vielversprechende neue Bindungspartner von UBXD1 wurden so die Ubiquitin-Ligase TRIAD3A und das Ubiquitin-editierende Protein A20 identifiziert. Desweiteren konnte gezeigt werden, dass die Interaktion zwischen UBXD1 und A20 von einer funktionellen PUB Domäne und dem siebten Zinkfinger Motiv von A20 abhängig ist.
Da sowohl TRIAD3A als auch A20 negative Regulatoren des NF B Signalweges sind, wurde daraufhin untersucht, ob auch UBXD1 eine Funktion in diesem Signalweg besitzt. Tatsächlich war in UBXD1-depletierten HeLa 57A-Zellen die NF B-abhängige Expression eines Reportgens nach Aktivierung des Signalweges durch TNF, IL-1, Doxorubicin und H2O2 stark reduziert. Dabei spricht die verringerte Aktivierung nach unterschiedlichen Stimuli für eine generelle Rolle von UBXD1 im NF B Signalweg. Durch quantitative Echtzeit-PCR konnte gezeigt werden, dass in HeLa- und HEK293T-Zellen nach UBXD1-Depletion auch die Expression endogener NF B Zielgene verringert ist. Da in UBXD1-depletierten Zellen nach Stimulation mit TNF oder IL-1 bereits die Kerntranslokation des NF B-Transkriptionsfaktor p65 reduziert ist, ist davon auszugehen, dass UBXD1 an einer früheren Phase der Aktivierung des Signalweges beteiligt ist. Möglicherweise ist dies darauf zurückzuführen, dass UBXD1 bekannte Funktionen von A20 reguliert und etwa die Bindung von A20 an Vesikel des endolysosomalen Systems oder an lineare Ubiquitinketten beeinflusst. Diese Arbeit beschreibt somit eine neue Funktion des p97-Kofaktors UBXD1 im NF B-Signalweg.
Development and proof of concept of a biological vascularized cell‐based drug delivery system
(2019)
A major therapeutic challenge is the increasing incidence of chronic disorders.
The persistent impairment or loss of tissue function requires constitutive on‐demand
drug availability optimally achieved by a drug delivery system ideally directly connected
to the blood circulation of the patient. However, despite the efforts and achievements in
cell‐based therapies and the generation of complex and customized cell‐specific
microenvironments, the generation of functional tissue is still unaccomplished.
This study demonstrates the capability to generate a vascularized platform technology to
potentially overcome the supply restraints for graft development and clinical application
with immediate anastomosis to the blood circulation.
The ability to decellularize segments of the rat intestine while preserving the ECM for
subsequent reendothelialization was proven. The reestablishment of a functional
arteriovenous perfusion circuit enabled the supply of co‐cultured cells capable to replace
the function of damaged tissue or to serve as a drug delivery system. During in vitro
studies, the applicability of the developed miniaturized biological vascularized scaffold
(mBioVaSc‐TERM®) was demonstrated. While indicating promising results in short term
in vivo studies, long term implantations revealed current limitations for the translation
into clinical application. The gained insights will impact further improvements of quality
and performance of this promising platform technology for future regenerative therapies.
Myocardial infarction (MI) is a major cause of health problems and is among the leading deadly ending diseases. Accordingly, regenerating functional myocardial tissue and/or cardiac repair by stem cells is one of the most desired aims worldwide. Indeed, the human heart serves as an ideal target for regenerative intervention, because the capacity of the adult myocardium to restore itself after injury or infarct is limited. Thus, identifying new sources of tissue resident adult stem or progenitor cells with cardiovascular potential would help to establish more sophisticated therapies in order to either prevent cardiac failure or to achieve a functional repair. Ongoing research worldwide in this field is focusing on a) induced pluripotent stem (iPS) cells, b) embryonic stem (ES) cells and c) adult stem cells (e. g. mesenchymal stem cells) as well as cardiac fibroblasts or myofibroblasts. However, thus far, these efforts did not result in therapeutic strategies that were transferable into the clinical management of MI and heart failure. Hence, identifying endogenous and more cardiac-related sources of stem cells capable of differentiating into mature cardiomyocytes would open promising new therapeutic opportunities. The working hypothesis of this thesis is that the vascular wall serves as a niche for cardiogenic stem cells. In recent years, various groups have identified different types of progenitors or mesenchymal stem cell-like cells in the adventitia and sub-endothelial zone of the adult vessel wall, the so called vessel wall-resident stem cells (VW-SCs). Considering the fact that heart muscle tissue contains blood vessels in very high density, the physiological relevance of VW-SCs for the myocardium can as yet only be assumed. The aim of the present work is to study whether a subset of VW-SCs might have the capacity to differentiate into cardiomyocyte-like cells. This assumption was challenged using adult mouse aorta-derived cells cultivated in different media and treated with selected factors. The presented results reveal the generation of spontaneously beating cardiomyocyte-like cells using specific media conditions without any genetic manipulation. The cells reproducibly started beating at culture days 8-10. Further analyses revealed that in contrast to several publications reporting the Sca-1+ cells as cardiac progenitors the Sca-1- fraction of aortic wall-derived VW-SCs reproducibly delivered beating cells in culture. Similar to mature cardiomyocytes the beating cells developed sarcomeric structures indicated by the typical cross striated staining pattern upon immunofluorescence analysis detecting α-sarcomeric actinin (α-SRA) and electron microscopic analysis. These analyses also showed the formation of sarcoplasmic reticulum which serves as calcium store. Correspondingly, the aortic wall-derived beating cardiomyocyte-like cells (Ao-bCMs) exhibited calcium oscillations. This differentiation seems to be dependent on an inflammatory microenvironment since depletion of VW-SC-derived macrophages by treatment with clodronate liposomes in vitro stopped the generation of Ao bCMs. These locally generated F4/80+ macrophages exhibit high levels of VEGF (vascular endothelial growth factor). To a great majority, VW-SCs were found to be positive for VEGFR-2 and blocking this receptor also stopped the generation VW-SC-derived beating cells in vitro. Furthermore, the treatment of aortic wall-derived cells with the ß-receptor agonist isoproterenol or the antagonist propranolol resulted in a significant increase or decrease of beating frequency. Finally, fluorescently labeled aortic wall-derived cells were implanted into the developing chick embryo heart field where they became positive for α-SRA two days after implantation. The current data strongly suggest that VW-SCs resident in the vascular adventitia deliver both progenitors for an inflammatory microenvironment and beating cells. The present study identifies that the Sca-1- rather than Sca-1+ fraction of mouse aortic wall-derived cells harbors VW-SCs differentiating into cardiomyocyte-like cells and reveals an essential role of VW-SCs-derived inflammatory macrophages and VEGF-signaling in this process. Furthermore, this study demonstrates the cardiogenic capacity of aortic VW-SCs in vivo using a chimeric chick embryonic model.