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Regulation of late cell cycle genes and control of proliferation and cell death
No full textThe differential expression of proteins within the cell cycle is a major control mechanism of cell division. Several hundreds of genes are transcriptionally regulated to be expressed in the early or late cell cycle phases, respectively. The MuvB-based DREAM/MMB complexes are key regulators of late cell cycle genes.
By analyzing the function of A-MYB in cell cycle regulation, I could show that A-MYB and B-MYB can form two alternative variants of the MMB complex, A-MMB and B-MMB, and that they are essential for the activation of late cell cycle genes through cell cycle-dependent binding to CHR promoter sites. I validated these results in various cancer-derived and untransformed cell lines. Interestingly, when A-MYB alone is lost, late cell cycle gene expression is unperturbed, as B-MYB expression generally is very high. However, the impact of B-MYB deficiency on late cell cycle gene expression is highly dependent on the expression level of A-MYB that is expressed on lower levels than B-MYB. Only when both A-MYB and B-MYB are depleted, the expression of late cell cycle genes consistently diminishes. The proteins expressed under the control of A-MYB and B-MYB represent major regulators and executors of mitosis, such as Cyclins, PLKs, and Ki-67, a tumor marker of high clinical relevance. Consequently, the expression of A-MYB or B-MYB are required for a successful cell division. Meanwhile the cell cycle in cells that solely express A-MYB is only delayed, loss of both proteins leads to cell cycle arrest and apoptosis, even though a considerable proportion of cells escaped cell cycle arrest and thereby apoptosis through endoreplication. Overall, this study shifts the focus of A MYB as a master regulator of meiosis towards its additional regulatory function in mitosis and its oncogenic potential.
To capture the cooperating effects of cell division, cell cycle control, and apoptosis on proliferation after A-MYB and B-MYB loss, I developed a new assay system. The CeDaD assay detects cell division activity and cell death on a single cell level by combining two well established flow cytometric assays. For broader applicability, I validated its utility on other cell culture setups. The CeDaD assay can compare the proliferation dynamics of different cell conditions offering a more detailed insight into the mechanisms of proliferation than classic proliferation assays. Altogether, this assay provides an easily implementable tool for preclinical and basic research.:1 Introduction 1
1.1 The cell cycle 1
1.2 Cell cycle gene regulation 2
1.2.1 Cell cycle gene regulation through RB-E2F and DREAM 3
1.2.2 DREAM, MMB and FOXM1-MuvB regulate late cell cycle genes 4
1.3 The interconnection between cell division and cell death 5
1.3.1 p53-p21-DREAM/RB signaling 6
1.3.2 Small molecule cell cycle inhibition 7
1.3.3 Current methods to analyze cell death and cell division 8
1.4 Aim of this thesis 9
2 Publications 10
2.1 Uxa, S., Castillo-Binder, P., Kohler, R., Stangner, K., Müller, G. A., Engeland, K. Ki-67 gene expression. Cell death and differentiation 28, 3357–3370 (2021). 10
2.2 Kohler, R. & Engeland, K. A-MYB substitutes for B-MYB in activating cell cycle genes and in stimulating proliferation. Nucleic acids research 52, 6830–6849 (2024). 10
2.3 Nöltner, L., Engeland, K. & Kohler, R. CeDaD-a novel assay for simultaneous tracking of cell death and division in a single population. Cell death discovery 11, 86 (2025). 10
3 Discussion 52
3.1 Ki-67 is regulated through DREAM/MMB 52
3.2 A-MYB and B-MYB are essential for the expression of central mitotic regulators and for proliferation 52
3.3 CeDaD tracks proliferation by measuring cell division and cell death simultaneously 54
4 Summary 55
5 Zusammenfassung 56
References 57
Publications 72
Selbstständigkeitserklärung 73
Danksagung 74
Nachweis über Anteile der Co-Autoren 7
Performance of serum neurofilament light chain in a wide spectrum of clinical courses of amyotrophic lateral sclerosis: a cross-sectional multicenter study
No full textBackground and purpose: The objective was to assess the performance of serum neuro-
filament light chain (sNfL) in amyotrophic lateral sclerosis (ALS) in a wide range of disease
courses, in terms of progression, duration and tracheostomy invasive ventilation (TIV).
Methods: A prospective cross-sectional study at 12 ALS centers in Germany was per-
formed. sNfL concentrations were age adjusted using sNfL Z scores expressing the num-
ber of standard deviations from the mean of a control reference database and correlated
to ALS duration and ALS progression rate (ALS-PR), defined by the decline of the ALS
Functional Rating Scale.
Results: In the total ALS cohort (n = 1378) the sNfL Zscore was elevated (3.04; 2.46–3.43;
99.88th percentile). There was a strong correlation of sNfL Z score with ALS-PR (p < 0.001).
In patients with long (5–10 years, n = 167) or very long ALS duration ( >10 years, n = 94)
the sNfL Z score was significantly lower compared to the typical ALS duration of <5 years
(n = 1059) (p < 0.001). Furthermore, in patients with TIV, decreasing sNfL Z scores were
found in correlation with TIV duration and ALS-PR (p = 0.002; p < 0.001).
Conclusions: The finding of moderate sNfL elevation in patients with long ALS duration
underlined the favorable prognosis of low sNfL. The strong correlation of sNfL Z score
with ALS-PR strengthened its value as progression marker in clinical management and
research. The lowering of sNfL in correlation with long TIV duration could reflect a re-
duction either in disease activity or in the neuroaxonal substrate of biomarker formation
during the protracted course of ALS
Increased plasma level of terminal complement complex in AMD patients: potential functional consequences for RPE cells
No full textPurpose: Polymorphisms in complement genes are risk-associated for age-related macular degeneration (AMD). Functional analysis revealed a common deficiency to control the alternative complement pathway by risk-associated gene polymorphisms. Thus, we investigated the levels of terminal complement complex (TCC) in the plasma of wet AMD patients with defined genotypes and the impact of the complement activation of their plasma on second-messenger signaling, gene expression, and cytokine/chemokine secretion in retinal pigment epithelium (RPE) cells.
Design: Collection of plasma from patients with wet AMD (n = 87: 62% female and 38% male; median age 77 years) and controls (n = 86: 39% female and 61% male; median age 58 years), grouped for risk factor smoking and genetic risk alleles CFH 402HH and ARMS2 rs3750846, determination of TCC levels in the plasma, in vitro analysis on RPE function during exposure to patients' or control plasma as a complement source.
Methods: Genotyping, measurement of TCC concentrations, ARPE-19 cell culture, Ca2+ imaging, gene expression by qPCR, secretion by multiplex bead analysis of cell culture supernatants.
Main outcome measures: TCC concentration in plasma, intracellular free Ca2+, relative mRNA levels, cytokine secretion.
Results: TCC levels in the plasma of AMD patients were five times higher than in non-AMD controls but did not differ in plasma from carriers of the two risk alleles. Complement-evoked Ca2+ elevations in RPE cells differed between patients and controls with a significant correlation between TCC levels and peak amplitudes. Comparing the Ca2+ signals, only between the plasma of smokers and non-smokers, as well as heterozygous (CFH 402YH) and CFH 402HH patients, revealed differences in the late phase. Pre-stimulation with complement patients' plasma led to sensitization for complement reactions by RPE cells. Gene expression for surface molecules protective against TCC and pro-inflammatory cytokines increased after exposure to patients' plasma. Patients' plasma stimulated the secretion of pro-inflammatory cytokines in the RPE.
Conclusion: TCC levels were higher in AMD patients but did not depend on genetic risk factors. The Ca2+ responses to patients' plasma as second-messenger represent a shift of RPE cells to a pro-inflammatory phenotype and protection against TCC. We conclude a substantial role of high TCC plasma levels in AMD pathology
Experiencing (Shared) Decision Making: Results from a Qualitative Study of People with Mental Illness and Their Family Members
No full text(1) Background: There is a fundamental shift in healthcare toward shared decision making (SDM). This study explores SDM from the perspective of individuals affected by mental illness and their family members and investigates factors which promote and hinder the process. (2) Methods: We conducted N = 15 telephone interviews (n = 4 adults affected by mental illness, n = 5 family members, n = 6 both applicable, the majority reporting experiences with affective and anxiety disorders). Data were recorded, transcribed, and analyzed according to procedures established by Mayring. (3) Results: Individuals affected by mental illness and their family members have a strong desire to be involved in treatment decisions and to participate in finding a diagnosis. Often these stakeholders are denied the opportunity to participate; sometimes enabling behaviors impede participation. The stigmatization of mental illnesses is a major barrier. There are also structural barriers to SDM within the healthcare system. Peer support, self-help associations, and psychosocial counseling services are important to empowering individuals and promoting SDM. (4) Conclusions: SDM has the potential to improve the quality of mental healthcare. Barriers can be mitigated and new approaches for interventions in the psychiatric sector have been identified. This study has also shown the importance of understanding SDM as a process that should begin at the diagnostic phase
Balloon-borne observations of the transitions between typical states of the Arctic atmospheric boundary layer at Station Nord
No full textWithin the framework of (AC)3, a measurement campaign was conducted
at the Villum Research Station, Station Nord (Greenland) from 19 March 2024 to 18
April 2024. Our objective was to characterize the lower Arctic boundary layer, particularly
its temporal evolution under transitions such as polar day-to-night shifts, cloud
cover changes, precipitation onsets, and the formation of low-level jets. We employed
radiosondes and a tethered balloon system, which enabled high-resolution profiling of
wind fields, temperature, humidity, and radiative fluxes. Despite harsh conditions, with
temperatures averaging -35◦C early in the campaign, we successfully conducted daily
measurements. These were complemented by standard meteorological data from the
station, aerosol measurements from a Helikite system, and a remote-sensing station
including radar and lidar systems. The campaign provided valuable insights into the
dynamics of the Arctic boundary layer and contributes to the improvement of modelling
approaches, which represents the next step in this project.Im Rahmen von (AC)3 wurde vom 19. März 2024 bis 18. April
2024 eine Messkampagne an der Villum Research Station, Station Nord (Grönland),
durchgeführt. Unser Ziel war es, die untere arktische Grenzschicht zu charakterisieren,
insbesondere ihre zeitliche Entwicklung während Übergängen wie dem Wechsel vom
Polartag zur Polarnacht, Änderungen der Bewölkung, Einsetzen von Niederschlag und
der Bildung von Low-Level-Jets. Zum Einsatz kamen Radiosonden sowie ein Fesselballonsystem,
welches die Messung hochaufgelöster Profile von Windfeldern, Temperatur,
Feuchtigkeit und Strahlungsflüssen ermöglichte. Trotz herausfordernder Bedingungen
mit Durchschnittstemperaturen von -35◦C zu Beginn der Kampagne konnten
die täglichen Messungen erfolgreich durchgeführt werden. Ergänzt wurden diese durch
standardmeteorologische Daten der Station, Aerosolmessungen mittels eines Helikite-
Systems sowie Fernerkundungsdaten aus Radar- und Lidar-Messungen. Die Kampagne
lieferte wertvolle Erkenntnisse zur Dynamik der arktischen Grenzschicht und trägt zur
Verbesserung von Modellierungsansätzen bei, was den nächsten Schritt in diesem Projekt
darstellt
In Vitro Properties and Pharmacokinetics of Temporarily PEGylated Onc72 Prodrugs
No full textThe favorable properties of antimicrobial peptides (AMPs) to rapidly kill
pathogens are often limited by unfavorable pharmacokinetics due to fast
degradation and renal clearance rates. Here, a prodrug strategy linking
proline-rich AMP Onc72 to polyethylene glycol (PEGs) with average molecular
weights of 5 and 20 kDa via a peptide linker containing a protease cleavage
site is tested for the first time in vivo. Onc72 is released from these 5k- and
20k-prodrugs in mouse serum with half-life times (t1/2 ) of 8 and 14 h,
respectively. Importantly, PEGylation protects Onc72 from proteolytic
degradation providing a prolonged release of Onc72, balancing the
degradation of free Onc72, and leading to relatively stable Onc72
concentrations and high antibacterial activities. The prodrugs are not
hemolytic on human erythrocytes and show only slight cytotoxic effects on
human cell lines indicating promising safety margins. When administered
subcutaneously to female CD-1 mice, the prodrugs elimination t1/2 are 66 min
and ≈5.5 h, respectively, compared to 43 min of free Onc72. The maximal
Onc72 plasma levels are obtained ≈1 and ≈8 h postadministration,
respectively. In conclusion, the prodrugs provide extended elimination t1/2
and a constant release of Onc72 in mice, potentially limiting adverse effects
and increasing efficacy
Oxidative stress, hormones, and effects of natural antioxidants on intestinal inflammation in inflammatory bowel disease
No full textInflammatory bowel disease (IBD) is a chronic, relapsing gastrointestinal (GI)
disorder characterized by intestinal inflammation. The etiology of IBD is
multifactorial and results from a complex interplay between mucosal
immunity, environmental factors, and host genetics. Future therapeutics for GI
disorders, including IBD, that are driven by oxidative stress require a greater
understanding of the cellular and molecular mechanisms mediated by reactive
oxygen species (ROS). In the GI tract, oxidative stressors include infections and
pro-inflammatory responses, which boost ROS generation by promoting the
production of pro-inflammatory cytokines. Nuclear factor kappa B (NF-kB) and
nuclear factor erythroid 2–related factor 2 (Nrf2) represent two important
signaling pathways in intestinal immune cells that regulate numerous
physiological processes, including anti-inflammatory and antioxidant activities.
Natural antioxidant compounds exhibit ROS scavenging and increase antioxidant
defense capacity to inhibit pro-oxidative enzymes, which may be useful in IBD
treatment. In this review, we discuss various polyphenolic substances (such as
resveratrol, curcumin, quercetin, green tea flavonoids, caffeic acid phenethyl
ester, luteolin, xanthohumol, genistein, alpinetin, proanthocyanidins,
anthocyanins, silymarin), phenolic compounds including thymol, alkaloids such
as berberine, storage polysaccharides such as tamarind xyloglucan, and other
phytochemicals represented by isothiocyanate sulforaphane and food/spices
(such as ginger, flaxseed oil), as well as antioxidant hormones like melatonin that
target cellular signaling pathways to reduce intestinal inflammation occurring
with IBD
Single-cell and genetic multi-omics analysis combined with experiments confirmed the signature and potential targets of cuproptosis in hepatocellular carcinoma
No full textBackground: Cuproptosis, as a recently discovered type of programmed cell
death, occupies a very important role in hepatocellular carcinoma (HCC) and
provides new methods for immunotherapy; however, the functions of cuproptosis
in HCC are still unclear.
Methods: We first analyzed the transcriptome data and clinical information of 526
HCC patients using multiple algorithms in R language and extensively described
the copy number variation, prognostic and immune infiltration characteristics of
cuproptosis related genes (CRGs). Then, the hub CRG related genes associated
with prognosis through LASSO and Cox regression analyses and constructed a
prognostic prediction model including multiple molecular markers and
clinicopathological parameters through training cohorts, then this model was
verified by test cohorts. On the basis of the model, the clinicopathological
indicators, immune infiltration and tumor microenvironment characteristics of
HCC patients were further explored via bioinformation analysis. Then, We further
explored the key gene biological function by single-cell analysis, cell viability and
transwell experiments. Meantime, we also explored the molecular docking of the
hub genes.
Results: We have screened 5 hub genes associated with HCC prognosis and
constructed a prognosis prediction scoring model. And the model results showed
that patients in the high-risk group had poor prognosis and the expression levels of
multiple immune markers, including PD-L1, CD276 and CTLA4, were higher than
those patients in the low-risk group. We found a significant correlation between
risk score and M0 macrophages and memory CD4+ T cells. And the single-cell
analysis and molecular experiments showed that BEX1 were higher expressed in
HCC tissues and deletion inhibited the proliferation, invasion and migration and
EMT pathway of HCC cells. Finally, it was observed that BEX1 could bind to
sorafenib to form a stable conformation.
Conclusion: The study not only revealed the multiomics characteristics of CRGs in
HCC but also constructed a new high-accuracy prognostic prediction model.
Meanwhile, BEX1 were also identified as hub genes that can mediate the
cuproptosis of hepatocytes as potential therapeutic targets for HCC
Die Anosognosie für die homonyme Hemianopsie und die Selbstüberschätzung: eine klinische Verhaltensstudie zum Umgang mit Unsicherheit
No full textHintergrund: Fehlendes Störungsbewusstsein für eine homonyme Hemianopsie ist ein häufig auftretendes Defizit nach einem Schlaganfall. Ein neues Defizit, zum Beispiel die homonyme Hemianopsie, kann als ein Zustand der Unsicherheit verstanden werden, welcher zur Exploration, zum Erkennen und schließlich zum Einbinden des Defizits in ein aktualisiertes Selbstbild führen müsste. Die Unfähigkeit, in Situationen von Unsicherheit neue Überzeugungen zu entwickeln, trägt nachweislich zur Anosognosie der Hemiplegie bei.
Ziele/ Fragestellung: In dieser Studie soll untersucht werden, ob Patienten mit Anosognosie für homonyme Hemianopsie ebenfalls übermäßig zuversichtlich sind und Schwierigkeiten haben, frühere Überzeugungen an neue Umstände anzupassen. Wir verwenden dazu das gleiche Rätselparadigma, welches zuvor für die Anosognosie der Hemiplegie verwendet wurde. Ziel dieser Arbeit ist es zu untersuchen, ob es sich bei der Selbstüberschätzung um einen supramodalen Faktor handelt, der zur Entstehung der Anosognosie beiträgt.
Methoden: In einer prospektiven Studie wurden Patienten mit homonymer Hemianopsie aufgrund eines erstmaligen Schlaganfalls im Versorgungsgebiet der Arteria cerebri posterior und eine altersgleiche Kontrollgruppe eingeschlossen. Die zehn Rätsel sind so konzipiert, dass sie eine Situation der Ungewissheit schaffen, die innerhalb von fünf aufeinanderfolgenden Hinweisen aufgelöst wird, da diese das Zielwort zunehmend eingrenzen. Nach jedem Hinweis müssen die Patienten das Zielwort erraten und ihr Vertrauen in die Richtigkeit ihrer Antwort einschätzen. Die Patienten wurden einmalig während ihres Krankenhausaufenthalts (Median 4,0 Tage nach dem Schlaganfall) getestet.
Ergebnisse: Entsprechend des modifizierten Bisiach-Scores hatten 12 von 29 Patienten (41%) eine Anosognosie für die homonyme Hemianopsie. Alle Patienten mit Anosognosie hatten linksseitige Gesichtsfelddefekte. Patienten mit Anosognosie unterschieden sich nicht in Bezug auf Läsionsgröße, globale kognitive Fähigkeiten, mentale Flexibilität und ihre Gedächtnisfunktion. Patienten mit Anosognosie zeigten bei den ersten beiden Hinweisen (Situation der Unsicherheit) tatsächlich höhere Vertrauenswerte als Patienten ohne Anosognosie oder Kontrollen. Dies ließ sich durch einen signifikanten Interaktionseffekt in einer gemischten ANOVA mit den Faktoren Gruppe (Anosognosie, Nosognosie, Kontrollen) und Hinweise (Rätsel 1 - 5) demonstrieren. Es fand sich hingegen kein Hinweis darauf, dass die Patienten mit Anosognosie übermäßig stark an bereits falsifizierten früheren Überzeugungen festhalten. Eine Läsionsanalyse weist für Patienten mit Anosognosie eine klare Tendenz für das Vorliegen rechtshemisphärieller Läsionen auf, welche in Subtraktionsanalysen eine mehr anterior-temporale Verteilung zeigten.
Schlussfolgerungen: Ein übermäßiges Gefühl der Sicherheit in Situationen der Ungewissheit scheint zur Anosognosie bei Hemianopsie beizutragen. Hierbei könnte es sich um einen supramodalen Faktor handeln, der zur Nichtwahrnehmung von Defiziten führt, ähnlich wie bei der Anosognosie der Hemiplegie.:1. Einführung 1
1.1. Grundlagen des Störungsbewusstseins 1
1.2. Die Homonyme Hemianopsie 2
1.3. Die Testung der Anosognosie 3
1.4. Einflussfaktoren auf die Anosognosie der Hemianopsie 4
1.5. Theorien zur Entstehung der Anosognosie 6
1.6. Hirnschädigung und Anosognosie 8
2. Herleitung der Fragestellung und Hypothesen 9
3. Wissenschaftliche Publikation 11
4. Zusammenfassung der Arbeit 23
5. Literaturverzeichnis 27
A Anlagen 33
A.1 Supplemental Materials 34
A.2 Darstellung des eigenen Beitrags 45
A.3 Erklärung über die eigenständige Abfassung der Arbeit 46
A.4 Lebenslauf 47
A.5 Publikationen 48
A.6 Danksagung 4
A Photoreceptor-Based Hydrogel with Red Light-Responsive Reversible Sol-Gel Transition as Transient Cellular Matrix
No full textHydrogels with adjustable mechanical properties have been engineered as
matrices for mammalian cells and allow the dynamic, mechano-responsive
manipulation of cell fate and function. Recent research yields hydrogels,
where biological photoreceptors translated optical signals into a reversible
and adjustable change in hydrogel mechanics. While their initial application
provides important insights into mechanobiology, broader implementation is
limited by a small dynamic range of addressable stiffness. Herein, this
limitation is overcome by developing a photoreceptor-based hydrogel with
reversibly adjustable stiffness from ≈800 Pa to the sol state. The hydrogel is
based on star-shaped polyethylene glycol, functionalized with the red/far-red
light photoreceptor phytochrome B (PhyB), or phytochrome-interacting factor
6 (PIF6). Upon illumination with red light, PhyB heterodimerizes with PIF6,
thus crosslinking the polymers and resulting in gelation. However, upon
illumination with far-red light, the proteins dissociate and trigger a complete
gel-to-sol transition. The hydrogel’s light-responsive mechanical properties
are comprehensively characterized and it is applied as a reversible
extracellular matrix for the spatiotemporally controlled deposition of
mammalian cells within a microfluidic chip. It is anticipated that this
technology will open new avenues for the site- and time-specific positioning
of cells and will contribute to overcome spatial restrictions