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- Graduate School of Life Sciences (69) (entfernen)
Sonstige beteiligte Institutionen
- Bio-Imaging Center Würzburg (1)
- CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - the development agency of the Brazilian Federal Government (1)
- DAAD - Deutscher Akademischer Austauschdienst (1)
- Institute for Biochemistry I, University of Cologne (1)
- Medizinische Klinik und Poliklinik 1, Abteilung Kardiologie (1)
- Medizinische Klinik und Poliklinik 1, Abteilung Nephrologie (1)
Die Phosphoglykolat-Phosphatase PGP (früher auch als AUM bezeichnet) wurde in unserem Labor als Mitglied der HAD-Typ-Phosphatasen identifiziert. Die genetische Inaktivierung des Enzyms im gesamten Mausorganismus führt ab E8.5 zu einer Wachstumsverzögerung muriner Embryonen und bis E12.5 schließlich zu deren Tod. Im Gegensatz dazu sind Mäuse mit einer PGP-Inaktivierung in hämatopoetischen Zellen und im Endothel lebensfähig und phänotypisch unauffällig. Neue Erkenntnisse schreiben dem Enzym neben einer Aktivität gegenüber Phosphoglykolat auch Aktivitäten gegenüber Glycerin-3-phosphat (G3P), P-Erythronat und P-Lactat zu. Da diese Phosphatase-Aktivitäten Auswirkungen auf den Lipidstoffwechsel nahelegen, wurde in der vorliegenden Arbeit mittels massenspektrometrischer Methoden der Einfluss der Phosphoglykolat-Phosphatase auf den Metabolismus von Signal-, Membran- und Speicherlipiden in murinen Embryonen und Lymphozyten untersucht.
Nach Inaktivierung der PGP im gesamten Organismus wurden in E8.5-Embryonen erhöhte Diacylglycerin (DG)-, Triacylglycerin (TG)- und Sphingomyelin (SM)-Spiegel gemessen, während niedrigere Phosphatidylcholin (PC)-Level vorlagen.
In PGP-inaktivierten Lymphozyten waren G3P-, DG-, TG-, PC- und SM-Level nicht verändert. Dafür kam es zu signifikanten Erhöhungen der Phosphatidylglycerol (PG*)- und Cardiolipin (CL)-Spiegel.
Zusammenfassend konnte gezeigt werden, dass die PGP in unterschiedlichen Geweben differenzielle Effekte auf die Spiegel verschiedener Lipide hat. Dies deckt neue Funktionen der PGP für die Regulation des Lipidmetabolismus auf. Die vorliegende Arbeit stellt somit die Grundlage für weitere Untersuchungen über die genauen Ursachen und Folgen dieser Regulation dar und lässt auf eine wichtige Rolle der PGP als metabolische Phosphatase im Organismus schließen.
Das Multiple Myelom muss trotz stetiger Fortschritte im Hinblick auf die verfügbaren Therapieoptionen und die Krankheitsprognose weiterhin im Wesentlichen als eine unheilbare Erkrankung angesehen werden. Dies kann vor allem auf die große inter- und intraindividuelle Heterogenität des MM zurückgeführt werden, welche die Entwicklung gezielter molekularer Therapiestrategien erheblich erschwert. Hierbei stellen loss-of-function- Experimente, welche die Identifikation einzelner oder mehrerer potenziell therapeutisch relevanter Zielstrukturen durch die (kombinierte) Depletion von Proteinen ermöglichen, eine wichtige Säule dar, für deren Durchführung verschiedene Systeme mit jeweils eigenen Vor- und Nachteilen zur Verfügung stehen. Im Rahmen dieser Arbeit konnte die Etablierung eines auf RNA-Interferenz basierenden stabilen und induzierbaren Knockdownsystems durch Elektroporation von MM Zelllinien mit Einzel- und Mehrfach-shRNA-Vektoren abgeschlossen werden. Die Transfektion von tet-Repressor-exprimierenden Zellinien mit einer oder mehreren shRNAExpressionskassetten innerhalb eines Plasmidvektors ermöglicht durch die vollständige Repression der shRNA-Transkription im nicht-induzierten Zustand die Selektion erfolgreich transponierter Zellen ohne Effekt-vermittelte Bias und die Generierung großer Zellmengen für Versuchsreihen in vergleichsweise kurzer Zeit. Die Induktion der verschiedenen in dieser Arbeit evaluierten Einzel- und Mehrfach-shRNA-Konstrukte gegen (Kombinationen von) Zielstrukturen im Ras/MAPK- sowie im NFκB-Signalsystem mittels Doxyzyklin als Induktionsagens zeigte durchweg deutliche und den Erwartungen aus transienten Experimenten entsprechende Knockdownergebnisse. Auch die Resultate hinsichtlich funktioneller Readouts und zellphysiologischer Effekte der induzierten Knockouts stehen im Einklang mit vorangegangenen Experimenten und bestätigen somit die Äquivalenz des stabilen induzierbaren Systems zu transienten Ansätzen auf RNAi-Basis oder zu pharmakologischen Inhibitoren. Der hierbei erzielte hypomorphe Phänotyp innerhalb einer polyklonalen Zellpopulation bildet die Realität einer medikamentösen Blockade einer oder weniger Zielstrukturen einer heterogenen MM Tumorpopulation näherungsweise ab, weshalb das vorgestellte System ein hilfreiches, kosteneffizientes und leicht zu handhabendes Werkzeug für die Identifikation potenziell relevanter Zielstrukturen für molekulare Therapieansätze im Multiplen Myelom darstellt
Development and application of computational tools for RNA-Seq based transcriptome annotations
(2019)
In order to understand the regulation of gene expression in organisms, precise genome annotation is essential. In recent years, RNA-Seq has become a potent method for generating and improving genome annotations. However, this Approach is time consuming and often inconsistently performed when done manually. In particular, the discovery of non-coding RNAs benefits strongly from the application of RNA-Seq data but requires significant amounts of expert knowledge and is labor-intensive. As a part of my doctoral study, I developed a modular tool called ANNOgesic that can detect numerous transcribed genomic features, including non-coding RNAs, based on RNA-Seq data in a precise and automatic fashion with a focus on bacterial and achaeal species. The software performs numerous analyses and generates several visualizations. It can generate annotations of high-Resolution that are hard to produce using traditional annotation tools that are based only on genome sequences. ANNOgesic can detect numerous novel genomic Features like UTR-derived small non-coding RNAs for which no other tool has been developed before. ANNOgesic is available under an open source license (ISCL) at https://github.com/Sung-Huan/ANNOgesic.
My doctoral work not only includes the development of ANNOgesic but also its application to annotate the transcriptome of Staphylococcus aureus HG003 - a strain which has been a insightful model in infection biology. Despite its potential as a model, a complete genome sequence and annotations have been lacking for HG003. In order to fill this gap, the annotations of this strain, including sRNAs and their functions, were generated using ANNOgesic by analyzing differential RNA-Seq data from 14 different samples (two media conditions with seven time points), as well as RNA-Seq data generated after transcript fragmentation. ANNOgesic was
also applied to annotate several bacterial and archaeal genomes, and as part of this its high performance was demonstrated. In summary, ANNOgesic is a powerful computational tool for RNA-Seq based annotations and has been successfully applied to several species.
Bei der Multiplen Sklerose (MS) handelt es sich um eine Autoimmunerkrankung des zentralen Nervensystems (ZNS). Abhängig von der betroffenen ZNS-Region kann es zu vielfältigen Symptomen kommen. Neben neurologischen Symptomen verursacht durch ZNS-Läsionen leidet ein Großteil der MS-Patienten auch unter gastrointestinalen Funktionsstörungen. Diese gastrointestinalen Symptome wurden bisher eher auf Läsionen im Rückenmark zurückgeführt und nicht direkt in Verbindung mit der autoimmunen Ätiologie der Erkrankung gebracht.
In dieser Studie wurde das enterische Nervensystem (ENS) in einem B-Zell- und Antikörper-abhängigen Mausmodell der MS untersucht. Dafür wurde der Autoimmunprozess durch Immunisierung mit MP4, einem Fusionsprotein aus dem Myelin-Basischen-Protein (MBP) und dem Proteolipid-Protein (PLP), ausgelöst. Das ZNS und ENS wurden in den unterschiedlichen Erkrankungsstadien immunhistochemisch und elektronenmikroskopisch analysiert. Neben der Immunpathologie des ZNS konnte dabei eine Degeneration des ENS schon vor dem Einsetzen der ersten neurologischen Defizite nachgewiesen werden. Die ENS-Pathologie war antikörper-mediiert und ging einher mit einer verringerten gastrointestinalen Motilität sowie mit einer Gliose und Neurodegeneration des ENS.
Mithilfe von Immunpräzipitation und Massenspektrometrie konnten im ENS vier mögliche Zielstrukturen des Autoimmunprozesses identifiziert werden, was auf sog. epitope spreading hindeutet. Auch im Plasma von MS-Patienten konnten Antikörper gegen drei dieser Antigene nachgewiesen werden. Des Weiteren zeigten sich in Kolon-Resektaten von MS-Patienten erste Ansätze einer Neurodegeneration und Gliose des ENS.
In dieser Studie wurde zum ersten Mal ein direkter Zusammenhang zwischen der Autoimmunreaktion gegen das ZNS und einer simultanen Reaktion gegen das ENS gezeigt. Dies kann einen Paradigmenwechsel im Verständnis der Immunpathogenese der MS anstoßen und neue therapeutische und diagnostische Ansätze initiieren.
The role of multicellularity as the predominant microbial lifestyle has been affirmed by studies on the genetic regulation of biofilms and the conditions driving their formation. Biofilms are of prime importance for the pathology of chronic infections of the opportunistic human pathogen Staphylococcus aureus.
The recent development of a macrocolony biofilm model in S. aureus opened new opportunities to study evolution and physiological specialization in biofilm communities in this organism. In the macrocolony biofilm model, bacteria form complex aggregates with a sophisticated spatial organization on the micro- and macroscale. The central positive and negative regulators of this organization in S. aureus are the alternative sigma factor σB and the quorum sensing system Agr, respectively. Nevertheless, nothing is known on additional factors controlling the macrocolony morphogenesis.
In this work, the genome of S. aureus was screened for novel factors that are required for the development of the macrocolony architecture. A central role for basic metabolic pathways was demonstrated in this context as the macrocolony architecture was strongly altered by the disruption of nucleotide and carbohydrate synthesis. Environmental signals further modulate macrocolony morphogenesis as illustrated by the role of an oxygen-sensitive gene regulator, which is required for the formation of complex surface structures. A further application of the macrocolony biofilm model was demonstrated in the study of interstrain interactions. The integrity of macrocolony communities was macroscopically visibly disturbed by competitive interactions between clinical isolates of S. aureus.
The results of this work contribute to the characterization of the macrocolony biofilm model and improve our understanding of developmental processes relevant in staphylococcal infections. The identification of anti-biofilm effects exercised through competitive interactions could lead to the design of novel antimicrobial strategies targeting multicellular bacterial communities.
The Myb-MuvB (MMB) multiprotein complex is a master regulator of cell cycle-dependent gene expression. Target genes of MMB are expressed at elevated levels in several different cancer types and are included in the chromosomal instability (CIN) signature of lung, brain, and breast tumors.
This doctoral thesis showed that the complete loss of the MMB core subunit LIN9 leads to strong proliferation defects and nuclear abnormalities in primary lung adenocarcinoma cells. Transcriptome profiling and genome-wide DNA-binding analyses of MMB in lung adenocarcinoma cells revealed that MMB drives the expression of genes linked to cell cycle progression, mitosis, and chromosome segregation by direct binding to promoters of these genes. Unexpectedly, a previously unknown overlap between MMB-dependent genes and several signatures of YAP-regulated genes was identified. YAP is a transcriptional co-activator acting downstream of the Hippo signaling pathway, which is deregulated in many tumor types. Here, MMB and YAP were found to physically interact and co-regulate a set of mitotic and cytokinetic target genes, which are important in cancer. Furthermore, the activation of mitotic genes and the induction of entry into mitosis by YAP were strongly dependent on MMB. By ChIP-seq and 4C-seq, the genome-wide binding of MMB upon YAP overexpression was analyzed and long-range chromatin interaction sites of selected MMB target gene promoters were identified. Strikingly, YAP strongly promoted chromatin-association of B-MYB through binding to distal enhancer elements that interact with MMB-regulated promoters through chromatin looping.
Together, the findings of this thesis provide a so far unknown molecular mechanism by which YAP and MMB cooperate to regulate mitotic gene expression and suggest a link between two cancer-relevant signaling pathways.
Herzschrittmachersysteme sind eine weitverbreitete Möglichkeit Herz-Kreislauf-Erkrankungen zu behandeln. Wegen der natürlichen Reaktion des Immunsystems auf Fremdkörper, erfolgt aber eine fortschreitende Verkapselung der Herzschrittmacherelektrode. Die Folge ist eine ansteigende Verminderung der Stimulationseffizienz durch Erhöhung der Anregungsschwelle. Die Integration der Elektrode in das Gewebe ist dabei mangelhaft und wird bestimmt durch Implantateigenschaften wie Größe, Flexibilität und Dimensionalität. Um die Integration zu verbessern, stellen dreidimensionale (3D) bzw. gewebeartige Elektroden eine Alternative zu den derzeit verwendeten planaren Metallelektroden dar. Zur Entwicklung einer leitfähigen, 3D und faserförmigen Elektrode wurden in dieser Arbeit Kohlenstoff-Nanofaser-Scaffolds über Elektrospinnen hergestellt. Durch die Modifikation des Fasergerüstes mit Natriumchlorid (NaCl) während der Scaffoldherstellung, konnte das Fasernetzwerk aufgelockert und Poren generiert werden. Die Kohlenstofffaser-Elektroden zeigten einen effizienten Energieübertrag, welcher vergleichbar mit heutigen Titannitrid (TiN) -Elektroden ist. Die Auflockerung des Fasergewebes hatte eine verbesserte Flexibilität des Faserscaffolds zu Folge. Neben der Flexibilität, konnte auch die Infiltration von Zellen in das poröse Faserscaffold erheblich verbessert werden. Dabei konnten Fibroblasten durch das gesamte Scaffold migrieren. Die Kompatibilität mit kardialen Zellen, die Grundvoraussetzung von Herzschrittmacherelektroden, wurde in vitro nachgewiesen. Durch die Kombination aus dem 3D-Elektrodengerüst mit einer Co-Kultur aus humanen Kardiomyozyten, mesenchymalen Stammzellen und Fibroblasten, erfolgte eine Einbettung der Elektrode in funktionelles kardiales Gewebe. Dadurch konnte ein lebender Gewebe-Elektroden-Hybrid generiert werden, welcher möglicherweise die Elektrode vor Immunzellen in vivo abschirmen kann. Eine Zusammenführung der hybriden Elektrode mit einen Tissue-Engineerten humanen kardialen Patch in vitro, führte zu Bildung einer nahtlosen Elektronik-Gewebe-Schnittstelle. Die fusionierte Einheit wurde abschließend auf ihre mechanische Belastbarkeit getestet und konnte über einen Elektroden-Anschluss elektrisch stimuliert werden.
Chronic Kidney Disease as an Important Co-morbid Condition in Coronary Heart Disease Patients
(2019)
In patients with coronary heart disease (CHD) the control of the modifiable “traditional” cardiovascular risk factors such as hypertension, dyslipidemia, diabetes, achieving/maintaining normal body weight and smoking cessation is of major importance to improve prognosis. Guideline recommendations for secondary CHD prevention include specific treatment targets for blood pressure, lipid levels, and markers of glucose metabolism for both younger and older patients. Chronic kidney disease (CKD) has been identified as a “non-traditional” risk factor for worse outcome in CHD patients, as it is associated with a markedly increased risk for subsequent CV events and mortality.
The specific objectives of the current thesis-project are to investigate (a) the quality of care in a recent sample of German CHD patients and to investigate variation of risk factor control between younger and elder patients (≤70 versus >70 years), (b) to analyze the prevalence of CKD across Europe in stable CHD patients in the outpatient setting and during a hospital stay for CHD, (c) to investigate the level of awareness of CKD in German CHD patients and their treating physicians.
Data from the European-wide EUROASPIRE IV study were used that include data on 7998 CHD patients in the ambulatory setting (study visit) and during a hospital stay for CHD (index). The German EUROASPIRE IV study center in Würzburg recruited 536 patients in 2012-2013. Risk factor control was compared against the current recommendations of the European Society of Cardiology. CKD was described by stages of glomerular filtration rate (eGFR) and albuminuria. German patients were asked in an additional kidney specific module whether they have ever been told by a physician about renal impairment. The fact that CKD or acute kidney injury (AKI) was mentioned in prominent parts of the hospital discharge letter as well as correct ICD-coding of CKD or AKI served as a proxy for physician’s awareness of CKD.
The majority of German CHD patients was treated with the recommended drug therapies including e.g. β-blockers, anti-platelets and statins. However, treatment targets for blood pressure and LDL-cholesterol levels were not achieved in many patients (45% and 53%, respectively) and glycemic control in diabetic CHD patients with HbA1-levels <7% was insufficient (61%). A minority of patients reported on current smoking (10%), but unhealthy life-styles e.g. overweight/obesity (85%/37%) were frequent. Patterns of care differed between younger and older CHD patients while older patients were less likely to receive the recommended medical CHD-therapy, were more likely to have uncontrolled blood pressure and also to be diabetic. However, a greater proportion of diabetic patients >70 years was achieving the HbA1c target, and less elder patients were current smokers or were obese. About 17% of patients on average had CKD (eGFR< 60 ml/min/1.73m²) in the entire European sample at the study visit, and an additional 10% had albuminuria despite preserved eGFR, with considerable variation among countries. Impaired kidney function was observed in every fifth patient admitted for CHD in the entire European dataset of the EUROASPIRE IV study. Of the German CHD patients with CKD at the study visit, only a third were aware of their renal impairment. A minority of these patients was being seen by nephrologists, however, with a higher likelihood of CKD awareness and specialist care in more advanced stages of CKD. About a third of patients admitted for CHD showed either CKD or AKI during the hospital stay, but the discharge letter mentioned chronic or acute kidney disease only in every fifth of these patients. In contrast, correct ICD coding of CKD or AKI was more complete, but still suboptimal.
In summary, quality of secondary prevention in German CHD patients indicates considerably room for improvement, with life-style modifications may become an even greater factor in prevention campaigns than medical treatment into certain target ranges. Preventive therapies should also consider different needs in older individuals acknowledging physical and mental potential, other comorbidities and drug-interactions with co-medication. CKD is common in CHD patients, not only in the elderly. Since CHD and CKD affect each other and impact on worse prognosis of each other, raising the awareness of CKD among patients and physicians and considering CKD in medical therapy may improve prognosis and slow disease progression of CHD as well as CKD.
Hematopoietic stem cell transplantation is a curative therapy for malignant diseases of the haematopoietic system. The patients first undergo chemotherapy or irradiation therapy which depletes the majority of tumour cells before they receive the transplant, consisting of haematopoietic stem cells and mature T cells from a healthy donor. The donor T cells kill malignant cells that have not been eliminated by the conditioning therapy (graft versus leukaemia effect, GvL), and, therefore, are crucially required to prevent relapse of the tumour. However, the donor T cells may also severely damage the patient’s organs causing acute graft versus host disease (aGvHD). In mice, aGvHD can be prevented by interfering with the co-stimulatory CD28 signal on donor T cells. However, experimental models using conventional CD28 knockout mice as T cell donors or αCD28 antibodies have some disadvantages, i.e. impaired T cell development in the thymus of CD28 knockout mice and systemic CD28 blockade with αCD28 antibodies. Thus, it remains unclear how CD28 co-stimulation on different donor T cell subsets contributes to the GvL effect and aGvHD, respectively.
We developed mouse models of aGvHD and the GvL effect that allowed to selectively delete CD28 on certain donor T cell populations or on all donor T cells. CD4+ conventional T cells (Tconv cells), regulatory T cells (Treg cells) or CD8+ T cells were isolated from either Tamoxifen-inducible CD28 knockout (iCD28KO) mice or their wild type (wt) littermates. Allogeneic recipient mice were then transplanted with T cell depleted bone marrow cells and different combinations of iCD28KO and wt T cell subsets. Tamoxifen treatment of the recipients caused irreversible CD28 deletion on the iCD28KO donor T cell population. In order to study the GvL response, BCL-1 tumour cells were injected into the mice shortly before transfer of the T cells.
CD4+ Tconv mediated aGvHD was efficiently inhibited when wt Treg cells were co-transplanted. In contrast, after selective CD28 deletion on donor Treg cells, the mice developed a late and lethal flare of aGvHD, i.e. late-onset aGvHD. This was associated with a decline in iCD28KO Treg cell numbers around day 20 after transplantation. CD28 ablation on either donor CD4+ Tconv cells or CD8+ T cells reduced but did not abrogate aGvHD. Moreover, iCD28KO and wt CD8+ T cells were equally capable of killing allogeneic target cells in vivo and in vitro. Due to this sufficient anti-tumour activity of iCD28KO CD8+ T cells, they had a therapeutic effect in our GvL model and 25% of the mice survived until the end of the experiment (day 120) without any sign of the malignant disease. Similarly, CD28 deletion on all donor T cells induced long-term survival. This was not the case when all donor T cells were isolated from wt donor mice. In contrast to the beneficial outcome after CD28 deletion on all donor T cells or only CD8+ T cells, selective CD28 deletion on donor CD4+ Tconv cells completely abrogated the GvL effect due to insufficient CD4+ T cell help from iCD28KO CD4+ Tconv cells.
This study demonstrates that therapeutic inhibition of the co-stimulatory CD28 signal in either all donor T cells or only in CD8+ T cells might protect patients from aGvHD without increasing the risk of relapse of the underlying disease. Moreover, deletion of CD28 on donor Treg cells constitutes a mouse model of late-onset aGvHD which can be a useful tool in aGvHD research.
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).