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Evaluation der Clinical Frailty Scale (CFS) als Messinstrument von Frailty in einer geriatrisch-kardiologischen Kohorte: Vergleich zu Tests der Leistungsfähigkeit und Alltagsfunktionalität
No full textZusammenfassung
Hintergrund: Die alternde Bevölkerung in Deutschland stellt das Gesundheitssystem vor neue Herausforderungen wie Frailty, ein Konzept mit erhöhtem Risiko für unerwünschte klinische Outcomes. Während der Frailty-Index und das Phänotypmodell mehrere Domänen erfassen, sind sie zeit- und kostenaufwendig. Die Clinical Frailty Scale bietet eine schnellere Alternative, konzentriert sich jedoch nur auf körperliche Leistungsfähigkeit und Alltagsfunktionalität
Fragestellung: Es stellt sich die Frage, ob die Clinical Frailty Scale allein ausreicht, um Frailty als multidimensionales Konzept qualitativ gut in Studien zu erfassen.
Methodik: In einer quantitativen Querschnittserhebung urden 101 Patient*innen (≥75 Jahre) nach einem Herz-Eingriff in Rehakliniken in Niedersachsen oder Hessen untersucht. Es wurden eine explorative Faktorenanalyse und Spearman-Korrelationen zwischen der Clinical Frailty Scale, Leistungstests (6 min Walking Test, Short Physical Performance Battery, Handkraft) und Alltagsfunktionalität (Katz-Index, Pflegegrad) durchgeführt. Zudem wurden Chi-Quadrat-Tests verwendet, um Zusammenhänge mit soziodemografischen (Alleinleben) und klinischen Merkmalen (Komorbiditäten/Polypharmazie) zu prüfen. Auch die Testdurchführungszeit wurde erfasst
Ergebnisse: Die Faktorenanalyse identifizierte zwei Hauptfaktoren: körperliche Leistungsfähigkeit und Alltagsfunktionalität. Die Clinical Frailty Scale korrelierte gut mit den Leistungstests und spiegelte die körperliche Leistungsfähigkeit wider, war jedoch in der Beurteilung der Alltagsfunktionalität und Polypharmazie/Komorbidität begrenzt. Psychosoziale Faktoren wie Alleinleben wurden nicht erfasst. Der 6 min Walking Test war zeitaufwändiger, zeigte jedoch die höchste Qualität, während die Clinical Frailty Scale am schnellsten durchführbar war.
Diskussion: Die Clinical Frailty Scale eignet sich gut zur Bewertung körperlicher Frailty für Studienzwecke. Für eine umfassendere Erfassung von Frailty sollten jedoch komplexere Instrumente wie der Frailty-Index in Betracht gezogen werden. Längere Tests zeigten oft höhere Qualität, wobei die Wahl des Instruments von der körperlichen Verfassung und Grunderkrankung der Population abhängt.
Schlussfolgerung: Die Clinical Frailty Scale ist geeignet, körperliche Leistungsfähigkeit in Studien abzubilden. Für andere Frailty-Domänen sollten zusätzliche Instrumente verwendet werden.Summary
Background:
The aging population in Germany poses new challenges to the healthcare system, including frailty, a concept associated with an increased risk of adverse clinical outcomes. While the Frailty Index and the Fried Frailty Phenotype assess multiple domains, they are time- and cost-intensive. The Clinical Frailty Scale offers a quicker alternative but focuses primarily on physical performance and functional independence in activities of daily living.
Research Question:
This study investigates whether the Clinical Frailty Scale alone is sufficient to adequately capture frailty as a multidimensional concept in clinical research.
Methods:
In a quantitative cross-sectional study, 101 patients aged 75 years and older were assessed following a cardiac intervention in rehabilitation clinics in Lower Saxony or Hesse. Exploratory factor analysis and Spearman correlations were performed between the Clinical Frailty Scale, physical performance measures (6-Minute Walk Test, Short Physical Performance Battery, Handgrip Strength), and functional status (Katz Index, level of care dependency). Chi-square tests were used to examine associations with sociodemographic factors (living alone) and clinical characteristics (comorbidities and polypharmacy). Test administration time was also recorded.
Results:
Factor analysis identified two main factors: physical performance and functional status. The Clinical Frailty Scale showed strong correlations with physical performance measures and adequately reflected physical frailty. However, it was limited in assessing functional status, polypharmacy, and comorbidities. Psychosocial factors such as living alone were not captured. The 6-Minute Walk Test required the most time but demonstrated the highest assessment quality, while the Clinical Frailty Scale was the quickest to administer.
Discussion:
The Clinical Frailty Scale is well suited for assessing physical frailty in research settings. However, for a more comprehensive evaluation of frailty, more complex instruments such as the Frailty Index should be considered. Longer assessments often demonstrated higher quality, and the choice of instrument should depend on the physical condition and underlying disease of the study population.
Conclusion:
The Clinical Frailty Scale is appropriate for assessing physical performance in clinical studies. Additional instruments are required to capture other domains of frailty.10000-01-01Diese Dateien sind bis zum Abschluß der Staatsexamensprüfung gesperrt.These files are not accessible until the state exam has been completed
Subcellular mechanisms of Dysferlin-mediated tubular membrane biogenesis in cardiac hypertrophy
No full textDeutsche Gesellschaft für Kardiologie (DGK)Deutsches Zentrum für Herz-Kreislauf-Forschung (DZHK)Studienstiftung des Deutschen VolkesDysferlin is a 238 kDa calcium-binding transmembrane protein out of the ferlin family which is expressed in striated muscle cells. Dysferlin-gene mutations are associated with skeletal muscle disorders known as Dysferlinopathies. In skeletal muscle cells, Dysferlin is involved in calcium-dependent membrane repair. Further, its involvement in tubule membrane biogenesis of the transverse axial tubule (TAT) network, whose reorganization is associated with limitations in myocyte function, is also being discussed. Dysferlinopathie-patients also develop a mild form of dilated cardiomyopathy. In the heart, Dysferlin is most strongly expressed in the ventricle, and its expression increases significantly in the context of pressure overload induced hypertrophic remodeling processes. However, the mechanisms of Dysferlin-mediated maladaptive tubule reorganization in pressure overload induced hypertrophy and the associated development of heart failure are not yet fully understood. The aim of this dissertation is to investigate in more detail Dysferlin-mediated subcellular mechanisms of cell membrane repair and tubule membrane biogenesis in cardiac hypertrophy.
For experiments, a Dysferlin knockout mouse line was used. A general characterization revealed no impairment of the mice in terms of overall survival, but a gender-specific analysis showed increased mortality in knockout male mice. However, histological examinations failed to reveal any changes in cell morphology or fibrosis as a cause of increased mortality. Transverse aortic constriction (TAC) surgery was performed to induce cardiac pressure overload, and myocardial infarction (MI) surgery was performed to induce an anterior wall infarction. However, post-TAC, the Dysferlin-deficient males did not show increased mortality, which can be explained by a protective aspect of reduced maladaptive remodeling due to Dysferlin deficiency. Both, echocardiographic measurements and analyses of single ventricular cardiomyocytes (VM) showed a reduction in pressure overload induced cardiac hypertrophy in Dysferlin deficiency. In wildtype mice, Dysferlin expression is already increased up to 143.2 ± 9.857% one week post-TAC. Using stimulated emission depletion nanoscopy, more accurate localization analyses of Dysferlin and possible interacting proteins could be performed.
Here, it was shown that Dysferlin is localized along the transverse axial tubule (TAT) network and is particularly close to proteins of the calcium release units (CRU). Dysferlin may stabilize the CRU, thereby preventing arrhythmogenic potentials that contribute to the development of heart failure. Post-TAC, Dysferlin is increasingly localized along newly formed axial tubules in hypertrophied wildtype VM. Our results show for the first time that Dysferlin is involved in axial tubule biogenesis, thus suggesting that Dysferlin plays an important role in hypertrophic remodeling undergoing pressure overload.
To ensure rapid cell membrane repair, it seems reasonable to assume that Dysferlin is stored intracellularly, from where, for example, calcium-triggered Dysferlin can be recruited for membrane repair. To investigate this hypothesis, this dissertation quantified sarcolemmal-expressed proteins, which we were able to establish for the first time in living VM. Using a membrane-impermeable crosslinker, proteins with extracellular lysines were linked to complexes with a higher molecular weight and thus quantitatively determined. Our experiments showed Dysferlin expression on the cell membrane of 22.14 ± 0.6156% under baseline conditions. Provoked by hypertrophic remodeling, Dysferlin expression on the cell membrane increased significantly, suggesting storage of Dysferlin within the membrane of intracellular vesicles. Dysferlin is recruited to the cell membrane surface, where it may work together with intracellular membrane reservoirs to repair and remodel the cellular membrane. In the context of myocardial infarction events, initial studies have detected an increase in Dysferlin expression, particularly along the infarct scar, but also globally in the left ventricular myocardium. Analogous to the role of Dysferlin in pressure overload induced remodeling, we suspect that Dysferlin is also involved in the stabilization and reorganization of surviving, postmitotic VM. Dysferlin may protect VM from mechanical overload and the resulting cell death. STED studies also showed a colocalization of Dysferlin and CRU proteins in ischemic events. More detailed studies of Dysferlin in myocardial infarction events need to be focussed in future experiments.
This dissertation demonstrated the relevant role of Dysferlin in sarcolemmal reorganization and the repair mechanisms of VM, particularly in the context of pressure-induced cardiac hypertrophy. Cumulative membrane damage and maladaptive remodeling influence the cell membrane structure and action potential conduction, leading to the development of heart failure. A more detailed understanding of these processes can help prevent the development of heart failure before cardiac decompensation, which is associated with enormous limitations in the quality of life of patients, occurs. Therapeutic goals could be (1) to stabilize the connections between the tubule and sarcoplasmic reticulum for adequate electromechanical stimulus conduction, (2) promoting the regeneration of the TAT membrane in heart failure, and (3) controlling the remodeling of the TAT membrane and the growth of the VM in pressure overload induced cardiac hypertrophy. However, further studies are necessary for clinical implementation, particularly with regard to the activation and regulation of Dysferlin.10000-01-01Diese Dateien sind bis zum Abschluß der Staatsexamensprüfung gesperrt. !Es liegt auch ein Embargo vor!These files are not accessible until the state exam has been completed
Interaction between ATG2, ATG9, and WIPI4 and their role in autophagosome membrane formation.
No full textAutophagy is a cellular recycling mechanism involved in various physiological functions, such as homeostasis, cellular stress response, and nutrient deprivation. A key step in autophagy is the formation of the autophagosome membrane. Autophagy-related gene protein (ATG)2 is a lipid transfer protein that plays a crucial role in membrane formation by supplying lipids. In this process, ATG2 interacts with other proteins, such as WD-repeat protein Interacting with PhosphoInositides (WIPI)4 and ATG9. WIPI4 acts as a membrane anchor for ATG2 and helps bind the C-terminal end of ATG2 to the membrane. No function of WIPI4 independent of protein-protein interaction is currently known. ATG9 is the only known transmembrane protein involved in the autophagy process. In recent years, it has been demonstrated that ATG9 possesses a lipid-scrambling function. Additionally, a direct interaction between ATG9 and ATG2 has been identified. It is therefore hypothesized that ATG9 may accelerate lipid transfer by ATG2. ATG9 is believed to balance membrane asymmetry between the outer and inner membrane layers, which arises from lipid transfer to the outer membrane layer.
Using the giant unilamellar vesicle (GUV) assay, I was able to demonstrate the protein binding of WIPI4 and ATG2 to membranes and the phosphatidylinositol 3-phosphate (PI3P)-dependence of the process. Furthermore, I showed ATG2-induced clustering of lipid vesicles. I observed that ATG2 accumulates in a ring-like structure at the contact site between two membranes and draws the membranes closer together. Additionally, I microscopically demonstrated the interaction of ATG9-containing large unilamellar vesicles (LUVs) with PI3P-containing GUVs in the presence of ATG2. With this, I have laid the foundation for further investigations in this field, aiming to visually represent autophagosomal membrane formation.
To study how ATG9 promotes autophagosome formation, I measured the effect of ATG9 on lipid transfer by ATG2 in a lipid transfer assay. I found that ATG9 increases the lipid transfer rate by a factor of 2.38. This confirms the important role of ATG9 in membrane expansion of the autophagosome. The exact mechanism by which ATG9 accelerates lipid transfer by ATG2 remains unclear. Possible factors include direct interaction, lipid-scrambling activity, and membrane-bending properties of ATG9. To enhance the effect of ATG9, additional ATG proteins such as WIPI4 and ATG13/101 can be included. To better understand the process of autophagosome formation, further ATG proteins can be incorporated into the lipid transfer assay in the future.10000-01-01Diese Dateien sind bis zum Abschluß der Staatsexamensprüfung gesperrt.These files are not accessible until the state exam has been completed
The importance of the immunoproteasome in the treatment of thymic epithelial tumors
No full textEpitheliale Thymustumoren sind seltene neoplastische Erkrankungen des Thymus und werden anhand ihrer histopathologischen Eigenschaften in Thymome und Thymuskarzinome unterteilt. Therapeutisch stellt die chirurgische Tumorresektion die erste Wahl dar und kann durch eine Radiotherapie sowie konventionelle Chemotherapeutika ergänzt werden. Rezidive treten häufig auf, doch aufgrund der geringen Mutationslast dieser Tumoren konnten bisher nur wenige molekulare Targets für eine zielgerichtete Zweitlinienbehandlung identifiziert werden. Aktuelle Beobachtungen weisen auf einen relevanten Zusammenhang zwischen dem Metabolismus epithelialer Thymustumoren und der Aktivität des Ubiquitin-Proteasom-Systems hin. Basierend auf der molekularen Zusammensetzung der katalytischen Untereinheiten werden verschiedene Proteasom-Subtypen unterschieden, wobei das Immunoproteasom (IP) in den Fokus dieser Arbeit geriet. Anhand von Frischgewebsproben und Zellkulturen wurde untersucht, inwiefern das IP in epithelialen Thymustumoren therapeutisch genutzt werden kann. Immunhistochemische Färbungen sowie die Auswertung genomischer Datensätze bestätigten, dass epitheliale Thymustumoren zusätzlich zum konstitutiven Proteasom auch das IP exprimieren. Proteinchemischen Analysen zufolge scheinen dabei insbesondere Thymuskarzinome ein hohes IP-Expressionsniveau aufzuweisen und sich diesbezüglich von Tumoren anderer Entitäten abzugrenzen. Eine spezifische Inhibition des IPs durch ONX-0914 oder KZR-504 zeigte im Zellkulturmodell keine effektive Viabilitätsminderung, während Carfilzomib als nicht-selektiver Proteasominhibitor in beiden untersuchten Thymuskarzinom-Zelllinien eine gute antitumorale Wirkung erzielte. Knockdowns der einzelnen katalytischen Untereinheiten des IPs wiesen nach, dass eine starke IP-Expression mit einem signifikant verbesserten Ansprechen von Thymuskarzinomzellen auf eine Carfilzomib-Behandlung einhergeht. Indem Korrelationsanalysen zwischen den IP-Expressionsniveaus verschiedener Tumorentitäten und den zugehörigen IC50-Werten für Carfilzomib durchgeführt wurden, konnte insbesondere für die β5i-Untereinheit des IPs auch entitätsübergreifend eine expressionsabhängige Vulnerabilität der Zellen gegenüber Carfilzomib bestätigt werden. Zusammenfassend verdeutlichen die Ergebnisse dieser Arbeit somit das therapeutische Potenzial einer Behandlung epithelialer Thymusneoplasien mit Carfilzomib in Abhängigkeit vom spezifischen IP-Expressionsniveau des Tumors.Thymic epithelial tumors are rare neoplastic diseases of the thymus and are divided into thymomas and thymic carcinomas based on their histopathological characteristics. Surgical tumor resection is the first choice of treatment and can be supplemented by radiotherapy and conventional chemotherapy. Recurrences occur frequently, but due to the low mutational burden of these tumors, only a few molecular targets for targeted second-line treatment have been identified to date. Current observations indicate a relevant connection between the metabolism of thymic epithelial tumors and the activity of the ubiquitin-proteasome system. Based on the molecular composition of the catalytic subunits, different proteasome subtypes are distinguished, with the immunoproteasome (IP) becoming the focus of this work. Fresh tissue samples and cell cultures were used to investigate how the IP can be used therapeutically in thymic epithelial tumors. Immunohistochemical staining and the evaluation of genomic data sets confirmed that thymic epithelial tumors express the IP in addition to the constitutive proteasome. According to protein chemical analyses, thymic carcinomas in particular appear to have a high level of IP expression and are therefore different from tumors of other entities. Specific inhibition of the IP by ONX-0914 or KZR-504 did not show any effective reduction in viability in the cell culture model, while carfilzomib, as a non-selective proteasome inhibitor, achieved a good antitumor effect in both thymic carcinoma cell lines examined. Knockdowns of the individual catalytic subunits of the IP demonstrated that strong IP expression is associated with a significantly improved response of thymic carcinoma cells to carfilzomib treatment. By performing correlation analyses between the IP expression levels of different tumor entities and the corresponding IC50 values for carfilzomib, an expression-dependent vulnerability of the cells to carfilzomib could be confirmed across entities, particularly for the β5i subunit of the IP. In summary, the results of this study illustrate the therapeutic potential of treating thymic epithelial neoplasms with carfilzomib depending on the specific IP expression level of the tumor.10000-01-01Diese Dateien sind bis zum Abschluß der Staatsexamensprüfung gesperrt.These files are not accessible until the state exam has been completed
AMPK inhibitor-based combination therapies in pancreatic ductal adenocarcinoma
No full textRegarding the dismal prognosis of pancreatic ductal adenocarcinoma, this thesis aimed to identify a new therapeutic option by inhibiting the adenosine monophosphate-activated protein kinase (AMPK). Since cancer cells grow uncontrollably and require more nutrients and oxygen relatively to healthy tissue, they are forced to undergo metabolic reprogramming. Thus, intervening in energy homeostasis by targeting the central metabolic regulator AMPK seemed a promising approach for cancer therapy.
This project foremost used murine pancreatic cancer cells which are either wildtype or contain a genetic knockout of Prkaa1, the gene encoding for the α-subunit of AMPK.
Conducting a drug screen in wildtype and knockout cell lines identified Erastin, a ferroptosis inducing agent, to be more effective in AMPK-deficient compared to AMPK-proficient cell lines.
Additionally, Erastin applied together with the potential AMPK inhibitor PF-3758309 was discovered to be a synergistic combination, which was also confirmed in patient-derived cell lines and organoids.
Further findings revealed a stronger increase of caspase activity and oxidative stress upon Erastin treatment in conditions of genetically or pharmacologically inhibited AMPK.
Notably, the voltage-dependent anion channel 1 (VDAC1), an important regulator of mitochondrial homeostasis in the outer membrane, was a stronger upregulated in AMPK knockouts in comparison to wildtype cell lines upon Erastin treatment, indicating that loss of AMPK leads to some sort of homeostatic dysregulation.
This project underlines the crucial role of AMPK in cancer and considers AMPK inhibitor-centred combination therapies to be worth further research to establish this therapeutic approach in a clinical context to treat cancer patients.10000-01-01Diese Dateien sind bis zum Abschluß der Staatsexamensprüfung gesperrt.These files are not accessible until the state exam has been completed
Relevance of G-Protein-Coupled Estrogen Receptor 1 (GPER1) for the Progression of Vulvar Carcinoma
No full textDas Vulvakarzinom ist ein gynäkologischer Tumor, welcher vor allem Frauen im höheren Lebensalter betrifft. Überwiegend handelt es sich um Plattenepithelkarzinome. Therapeutisch wird i. d. R. eine Tripel-Therapie aus Bestrahlung, Chemotherapie und Operation genutzt. Trotzdem kommen Rezidive mit etwa 20 % recht häufig vor.
Der G-Protein-gekoppelte Estrogen-Rezeptor 1 (GPER1) ist in diverse zelluläre Signalwege eingebunden. Es wird angenommen, dass er eine suppressive oder fördernde Rolle in der Karzinogenese diverser Neoplasien spielt, wobei die genaue Funktion oftmals umstritten und vom jeweiligen Gewebetyp abhängig ist. Der GPER1 konnte im Zytoplasma und Zellkern der Vulvakarzinomzellen A431 und CAL-39 mittels Immunzytologie nachgewiesen werden. Anhand von Tissue Microarrays kann eine Korrelation zwischen Tumorgrad und GPER1-Expression angenommen werden.
Um die Auswirkungen des GPER1 in Vulvakarzinomzellen zu verstehen, wurde dieser mit G1 aktiviert oder mit G36 inhibiert, bevor die Experimente durchgeführt wurden. Die durch Western Blots überprüfte Proteinexpression von GPER1, Estrogenrezeptor α und Estrogenrezeptor β veränderte sich nicht signifikant durch die Behandlungen mit G1 und G36. Nach GPER1-Aktivierung konnte eine dosisabhängige Reduktion der Proliferationsfähigkeit und Zellvitalität mittels Resazurin- und BrdU-Testung nachgewiesen werden. In der zellmorphologischen Beobachtung zeigten sich zudem Zeichen der Nekrose nach G1-Behandlung. Im Rahmen der Gap-Closure-Testung wurde eine dosisabhängige Hemmung der Migration nach GPER1-Aktivierung bei beiden Zelllinien beobachtet. Mittels Kolonie-Formations-Testung wurde die Koloniebildungsfähigkeit der Vulvakarzinomzellen untersucht. GPER1-Aktivierung mit G1 hemmte die Kolonie-Größe und senkte deren Anzahl in beiden Zelllinien. Nach G36-Behandlung und somit Antagonisierung von GPER1 nahm die Kolonie-Größe zu, wobei deren Anzahl sich nicht signifikant veränderte. Auch die durch die Tumorsphären-Formations-Testung überprüfte Größe der Tumorsphären von A431 nahm nach G1-Behandlung ab und nach G36-Behandlung zu, wobei sich die Anzahl der Tumorsphären nicht veränderte. Bei CAL-39 zeigten beide Behandlungen keinen Einfluss auf die Tumorsphären-Größe bzw. -Anzahl, vermutlich bedingt durch den Tumorsphärenzerfall in non-adhärenter Umgebung.
Insgesamt zeigte die Aktivierung von GPER1 mit dem Agonisten G1 eine Reduzierung des tumorerzeugenden Potentials und somit eine tumorsuppressive Wirkung auf die Vulvakarzinomzellen A431 und CAL-39. Dementsprechend ist die weitere Erforschung der Funktionsweise des GPER1 im Vulvakarzinom bedeutend, um den Rezeptor als künftiges Target in der Therapie des Karzinoms nutzen zu können.The vulvar carcinoma is a gynecological tumor that primarily affects older women. It mostly comprises squamous cell carcinomas. The standard treatment typically involves a triple therapy of radiation, chemotherapy and surgery. Nevertheless, recurrences are relatively common, occurring in about 20% of cases. The G-protein-coupled estrogen receptor 1 (GPER1) is involved in various cellular signaling pathways. It is believed to play either a suppressive or promotive role in the carcinogenesis of various neoplasms, although its exact function is often disputed and dependent on the tissue type. GPER1 has been detected in the cytoplasm and nucleus of vulvar carcinoma cells A431 and CAL-39 through immunocytology. A correlation between tumor grade and GPER1 expression has been suggested based on tissue microarrays. To understand the effects of GPER1 in vulvar carcinoma cells, it was activated with G1 or inhibited with G36 before conducting experiments. The protein expression of GPER1, Estrogen Receptor α and Estrogen Receptor β, verified by Western Blots, did not significantly change with G1 and G36 treatments. After GPER1 activation, a dose-dependent reduction in proliferation ability and cell viability was observed through Resazurin and BrdU assays. Morphological observation also showed signs of necrosis after G1 treatment. In the Gap Closure assay, a dose-dependent inhibition of migration was observed in both cell lines following GPER1 activation. Colony Formation assays indicated that GPER1 activation with G1 reduced colony size and number in both cell lines. After G36 treatment, which antagonizes GPER1, colony size increased, while the number did not significantly change. The size of tumorspheres from A431, assessed by Tumorsphere Formation assay, decreased after G1 treatment and increased after G36 treatment, although the number of tumorspheres remained unchanged. In CAL-39, both treatments had no impact on tumorsphere size or number, likely due to tumorsphere disintegration in a non-adherent environment. Overall, GPER1 activation with the agonist G1 demonstrated a reduction in the tumorigenic potential, indicating a tumor-suppressive effect on vulvar carcinoma cells A431 and CAL-39. Therefore, further investigation into the function of GPER1 in vulvar carcinoma is important to potentially utilize this receptor as a therapeutic target in future cancer treatments.10000-01-01Diese Dateien sind bis zum Abschluß der Staatsexamensprüfung gesperrt.These files are not accessible until the state exam has been completed
The influence of microfluidics on retinal ganglion cell neurite outgrowth
No full textPromotionskolleg der Universitätsmedizin Göttingen (Jacob-Henle-Programm)Mittels Stammzelltransplantation könnten in Zukunft retinale Ganglienzellen (RGZ) in einem degenerierten Nervus opticus ersetzt werden. Hierbei könnte das auswachsende Axon extrazellulären Flüssigkeitsströmen ausgesetzt sein, deren Auswirkungen auf das Neuritenwachstum von RGZ bislang kaum untersucht sind. Primäre Kulturen von RGZ und – zum Vergleich – kortikaler Neurone wurden in Flusskammern für 24 Stunden verschiedenen Flussraten ausgesetzt, die als Scherkräfte (10⁻⁷ bis 10⁻³ dyn/cm²) auf die Zellen wirkten. Die Parameter Zelldichte, Länge des längsten Neuriten, Länge aller anderen Neuriten, Anzahl der Neuriten sowie die Wachstumsrichtung wurden ausgewertet. Die Flow/Non-Flow-Ratio wurde für jeden Parameter bestimmt und statistisch gegen den Wert 1 (keine Veränderung) getestet. Ebenso wurden die mechanosensitiven Calciumkanäle TRPV1 und TRPV4 sowie Piezo1/2 pharmakologisch inhibiert. Mittels BAPTA-AM wurde intrazelluläres Calcium gebunden.
Die Länge des längsten Neuriten von RGZ wurde durch geringe, mikrofluidische Scherkräfte (10⁻⁶ dyn/cm²) signifikant gesteigert, sowie das Wachstum aller anderen außer des längsten Neuriten reduziert. Bei höheren Scherkräften (10⁻³ dyn/cm²) zeigte sich hingegen ein neurodegenerativer Effekt mit Verringerung der Länge des längsten Neuriten sowie der Zelldichte. Kortikale Neurone reagierten weder auf mikrofluidische noch auf erhöhte RGZ-degenerative Scherkräfte. Sowohl der neuritenwachstumssteigernde Effekt geringer Scherkräfte als auch der degenerative Effekt höherer Scherkräfte wurden durch die Inhibition mechanosensitiver Calciumkanäle an RGZ (vor allem TRPV1 und TRPV4, weniger oder nicht durch Piezo1/2-Inhibition) sowie dosisabhängig durch den Calciumchelator BAPTA-AM gehemmt.
Zusammengefasst reagieren RGZ sensibel auf extrazelluläre Flüssigkeitsströme der getesteten Scherkraftstärken. Geringere Scherkräfte unterstützten das Auswachsen des späteren Axons und hemmten das Wachstum späterer Dendriten. Höhere Scherkräfte induzierten eine Neurodegeneration. Dieser Effekt wurde vermutlich über einen Calciumeinstrom durch mechansosensitive Calciumkanäle vermittelt und könnte für die Entwicklung von Stammzelltherapien relevant werden.Replacing retinal ganglion cells (RGCs) in a degenerated optic nerve is not yet possible but may become feasible in the future through stem cell transplantation. Microfluidic fluid streams, presumably present in the optic nerve, are among the potential cues influencing the speed and direction of axon outgrowth toward its target. The largely unknown effect of microfluidics on RGC neurite outgrowth was studied.
Primary cell cultures of postnatal rat RGCs and cortical neurons were used. Cells were grown for 48 hours inside flow chambers and exposed for 24 hours to different flow rates, inducing shear stress ranging from 10⁻⁷ to 10⁻³ dyn/cm². Cell density, length of the longest neurite, length of all but the longest neurite, number of neurites, and growth direction were assessed. The flow-to-non-flow ratio of each parameter was calculated and statistically tested with reference to the value 1 (no change). To inhibit mechanosensitive calcium channels, I-RTX (TRPV1), HC-067047 (TRPV4), or GsMTx4 (Piezo1 & 2) were added. BAPTA-AM was used as an intracellular calcium chelator.
Lower shear stress (10⁻⁶ dyn/cm²) significantly increased the length of the longest neurite while inhibiting the length of all other neurites. Higher shear stress (10⁻³ dyn/cm²) caused significant degeneration, with reduced length of the longest neurite and decreased cell density. However, no change was observed in cortical neurons at either level of shear stress. Assuming the effects were mediated by mechanosensitive channels, the above-mentioned inhibitors were tested under both low and high shear stress conditions. The growth-promoting effect of low shear stress on the longest neurite was inhibited by all mechanosensitive channel inhibitors. The degenerative effects of higher shear stress on neurite length and cell density were significantly reduced by TRPV1 and TRPV4 inhibition, but not by Piezo1/2 inhibition, and showed a dose-dependent response to BAPTA-AM.
In conclusion, RGCs appear to be highly sensitive to extracellular microfluidic flow. Lower shear stress promotes outgrowth of the presumed future axon while inhibiting elongation of future dendrites, whereas higher shear stress induces neurodegeneration. These effects may be mediated by mechanosensitive channels.2025-05-2210000-01-01Diese Dateien sind bis zum Abschluß der Staatsexamensprüfung gesperrt.These files are not accessible until the state exam has been completed
Functional apoptosis profiling for the further development of targeted therapies for adult T-cell acute lymphoblastic leukemia (T-ALL)
No full textT-cell acute lymphoblastic leukaemia is a very aggressive haematological neoplasia characterised by malignantly transformed precursor T-lymphocytes. Considering the poor prognosis and limited treatment options for affected patients, there is an urgent need to identify and test new treatment options. The growing understanding of the molecular properties of T-ALL cells, in addition to the investigation of apoptotic mechanisms and their therapeutic targets, is a relevant treatment option for patients and is the central topic of this work. To identify potential therapeutics addressing genetic alterations in T-ALL, recently published genetic datasets from patients with T-ALL were analyzed with a bioinformatic approach that systematically queries public databases to report evidence for therapeutics targeting distinct genetic alterations in cancer. This analysis identified several potential therapeutics for T-ALL that were subsequently tested for their cytotoxic activity in T-ALL cell lines. Notably, inhibitors of the PI3K/mTOR pathway showed promising preclinical activity. However, the heterogenity of cellular responses prompted additional experiments dissecting specific effects of PI3K/mTOR inhibitors on the regulation of mitochondrial apoptosis using BH3 profiling. These experiments provided a rationale for drug combination strategies of PI3K/mTOR inhibitors and BH3 mimetics as selective inhibitors of anti-apoptotic BCL-2 family members. Based on these findings, the effects of simultaneous inhibition of the PI3K/mTOR signaling pathway and specific BH3-mimetic were investigated and heterogenous, cell line-specific effects of drug combinations were observed. These results underline the need for a precise, functional characterization of the specific impact of targeted therapeutics on the regulation of mitochondrial apoptosis as a basis for personalized therapeutic strategies incorporating specific BH3-mimetics. Here, dynamic BH3 profiling could be used as an integrative functional tool to establish personalized drug combinations for T-ALL patients.10000-01-01Diese Dateien sind bis zum Abschluß der Staatsexamensprüfung gesperrt.These files are not accessible until the state exam has been completed
The influence of histone H3 modifications on darolutamide resistance in prostate cancer
No full textProstate cancer is one of the most frequently diagnosed malignancies in men worldwide and accounts for a substantial proportion of cancer-related deaths. With the introduction of darolutamide, a next-generation androgen receptor (AR) antagonist, a novel therapeutic option with promising clinical results became available in 2020. However, with the expanded clinical use of darolutamide, an increasing number of resistant cases has been observed. Previous studies have identified epigenetic alterations as key drivers in the development and progression of prostate cancer. This study investigated the role of histone H3 lysine 27 trimethylation (H3K27me3) and its writer protein, Enhancer of Zeste Homolog 2 (EZH2), in the development of resistance to darolutamide. VCaP cells were used as an in vitro model of castration-resistant prostate cancer (CRPC) and were treated with increasing concentrations of darolutamide to induce resistance. Western blot analyses revealed elevated protein levels of the repressive histone modification H3K27me3 and EZH2 in darolutamide-resistant VCaP cells (VCaP Daro). These changes correlated with reduced transcription of H3K27me3 target genes, including insulin-like growth factor-binding protein 3 (IGFBP3), homeobox D1 (HOXD1), AR splice variant V7 (ARV7), and AR full-length (Arfl). EZH2 knockdown resulted in decreased H3K27me3 levels and increased transcription of target genes. Similar effects were observed after treatment with EPZ-6438, a selective inhibitor of EZH2 methyltransferase activity. Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) revealed a global, genome-wide increase in H3K27me3 occupancy in VCaP Daro, accompanied by a redistribution of H3K27me3 peaks toward gene promoter regions, potentially associated with altered AR binding. Subsequent RNA sequencing identified the sonic hedgehog (SHH) signaling pathway as the most strongly upregulated pathway in VCaP Daro, suggesting a central role in the resistance mechanism. Increased transcription of GLI Family Zinc Finger proteins 1 and 2 (GLI1, GLI2) served as readouts for Hedgehog pathway activation. Hedgehog reporter assays demonstrated SHH ligand secretion in VCaP Daro but not in wild-type VCaP cells (VCaP Wt), indicating possible autocrine or paracrine activation. Furthermore, treatment with the Smoothened inhibitor vismodegib demonstrated that Hedgehog signaling contributes to increased EZH2 expression. Both EPZ-6438 and vismodegib significantly reduced cell viability in both cell lines, as determined by MTS assays. Notably, the inhibitory effects were significantly stronger in VCaP Daro than in VCaP Wt, with vismodegib exhibiting greater cytotoxicity than EPZ-6438. In conclusion, these results suggest that EZH2 and SHH represent promising therapeutic targets for the treatment of darolutamide-resistant CRPC. As corresponding inhibitors are already clinically available, there is substantial translational potential. Further studies are required to elucidate the underlying molecular mechanisms.10000-01-01Diese Dateien sind bis zum Abschluß der Staatsexamensprüfung gesperrt.These files are not accessible until the state exam has been completed
Mode of action of direct KRAS inhibition and development of KRAS inhibitor-based combination therapies
No full textAs pancreatic ductal adenocarcinoma (PDAC) has still one of the least favourable survival rates among cancers, better treatment is urgently needed. This thesis aimed to make use of a new class of targeted therapies, the Kirsten Rat Sarcoma virus (KRAS)G12C inhibitors and to evaluate their selectivity and mechanisms in context of PDAC cell lines. Because these inhibitors are also known to be susceptible to resistance development, potential combination therapies were sought and evaluated.
First of all, different KRASG12C inhibitors were tested on various PDAC cell lines with and without the KRASG12C mutation. Sotorasib, Adagrasib and MRTX-1257 revealed a selective reduction of cell viability in both Clonogenic and CellTiter-Glo® Assays specifically in KRASG12C mutated MiaPaCa2 cells. By the testing, Sotorasib was prioritized for the following experiments due to the specificity and the authorization for clinical use before this work has started. Further experiments were mainly done on human 2D cell lines which were either commercially available (MiaPaCa2) or generated from patient-derived organoids (PAN51T-2D).
To comprehend the impact of KRASG12C inhibitors on signalling pathways, Western blot analysis was performed on cell lysates obtained from MiaPaCa2 and PAN51T-2D cells following different treatment durations of Sotorasib. On-target activity of Sotorasib was detected in both cell lines after all time points. In addition, a downregulation of downstream targets of KRAS was observed after a treatment time of 24 hours. However, after incubation with Sotorasib for 48 hours and 72 hours, a dose-dependent recovery of ERK phosphorylation was exhibited, suggesting that downstream pathways were reactivated, besides KRAS blockade.
To overcome these resistance mechanisms, a Sotorasib-anchored unbiased drug screen with 126 different drugs, ranging from clinical approved drugs to preclinical compounds, was performed on MiaPaCa2 cells. Nine potential candidates were identified, of which three could be validated by Clonogenic, CellTiter-Glo® Assays, and the calculation of a Bliss-synergy score. These were the SHP2 inhibitor TNO155, the Son of Sevenless KRAS interaction inhibitor BI-3406, and the tyrosine kinase inhibitor Nintedanib.
Ultimately, Western blot analysis was performed once more after treating both cell lines with Sotorasib and TNO155 in combination for 72 hours. This time, the pERK recovery could be prevented in the PAN51T-2D cell line, while the combined treatment had only little effect on the MiaPaCa2 cell line. Additionally, cell cycle analysis was performed via fluorescence-activated cell sorting, which found that a combination treatment led to an increased fraction of MiaPaCa2 cells entering G1 cell cycle arrest. Contrary, the treatment of PAN51T-2D cells with Sotorasib and TNO155 did not lead to a G1 cell cycle arrest.
In summary, this work has identified the SHP2 inhibitor TNO155 as a potential synergistic partner for Sotorasib, with the ability to overcome potential resistance mechanisms. Nevertheless, more research is needed to fully understand the divergent
cellular responses after the combination treatment.10000-01-01Diese Dateien sind bis zum Abschluß der Staatsexamensprüfung gesperrt.These files are not accessible until the state exam has been completed