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Stability of Synuclein Proteins
No full textVorsprung Promotionskolleg der Klinik für Neurologie der Universitätsmedizin GöttingenDas Protein α-Synuklein, das neben β- und γ-Synuklein Mitglied der Synuklein-Familie ist, trägt entscheidend zur Pathogenese der Parkinson Erkrankung bei. Auch β-Synuklein steht durch zwei Mutationen in ätiopathologischer Verbindung mit der Entstehung einer Synuk-leinopathie, der DLB. Demgegenüber steht γ-Synuklein nicht mit neurodegenerativen Er-krankungen im Zusammenhang und zeigt nur unter speziellen experimentellen Konditionen die Fähigkeit in unlösliche Aggregate überzugehen. Das Aggregationspotenzial der drei Pro-teine mit Bildung toxischer Spezies wird durch verschiedene Faktoren bestimmt, dabei spielt die Proteinkonzentration, beeinflusst durch z. B. Überexpression im experimentellen Kon-text oder Duplikationen und Triplikationen des SNCA-Gens bei früh einsetzender PD, eine entscheidende Rolle. Neben der Regulation der Synthese wird das Proteingleichgewicht durch die Degradationsmodalitäten aufrechterhalten. Unterschiede im Abbau und damit in der Stabilität der Proteine wurden als Ursache für das unterschiedliche Aggregationspotenzial und damit die unterschiedliche Neurotoxizität der drei Synuklein-Proteine angenommen. In den hier durchgeführten Untersuchungen wurde die Degradationskinetik für die drei Prote-ine auf Basis eines Zellkulturmodells in embryonalen Kortexneuronen der Ratte mit trans-gener Überexpression der drei Synukleine durch Adeno-assoziierte virale Vektoren ermittelt. Die drei Synuklein-Proteine zeigten eine sehr ähnliche Degradationskinetik, die sich als ex-ponentieller Zerfall darstellte. Initial ließ sich eine rasche Degradation mit Reduktion der Proteinkonzentration um 70 % über die ersten sieben Tage nach Expressionsstopp zeigen, mit im Anschluss verlangsamter Degradation, bei der die Proteinkonzentration über die nächsten 7 Tage gegen null ging. Auf Basis dieser Untersuchungen ist von einer ähnlichen Degradationskinetik für die drei Synuklein-Proteine auszugehen. Unterschiede in der Stabi-lität scheinen kein entscheidender Faktor für die unterschiedlichen Neurotoxizität von α-, β- und γ-Synuklein zu sein.The protein α-synuclein, which belongs to the synuclein family along with β- and γ-synuclein, plays a crucial role in the pathogenesis of Parkinson's disease. β-synuclein is also etiopathologically linked to the development of a synucleinopathy, dementia with Lewy bodies (DLB), due to two mutations. In contrast, γ-synuclein is not associated with neurodegenerative diseases and only shows the ability to form insoluble aggregates under specific experimental conditions. The aggregation potential of the three proteins, leading to the formation of toxic species, is determined by various factors, with protein concentration playing a decisive role. This concentration can be influenced by factors such as overexpression in experimental contexts or duplications and triplications of the SNCA gene in early-onset Parkinson's disease (PD). Besides the regulation of synthesis, protein balance is maintained by degradation mechanisms. Differences in degradation and thus protein stability have been suggested as reasons for the varying aggregation potentials and neurotoxicities of the three synuclein proteins.
In the present study, the degradation kinetics of the three proteins were examined using a cell culture model in embryonic rat cortex neurons with transgenic overexpression of the three synucleins through adeno-associated viral vectors. The three synuclein proteins exhibited very similar degradation kinetics, characterized by exponential decay. Initially, a rapid degradation was observed, with a reduction in protein concentration by 70% during the first seven days after expression cessation, followed by a slower degradation phase in which protein concentration approached zero over the next seven days. Based on these findings, the three synuclein proteins appear to share similar degradation kinetics. Differences in protein stability do not seem to be a decisive factor in the varying neurotoxicities of α-, β-, and γ-synuclein2025-03-0
Analyzing Pedicle Screw Strength with Finite Element Method in Relation to Vertebral Bone Properties
No full textVertebral fractures are increasingly prevalent in our aging population. Conservative treatments and minimally invasive surgeries are standard, but posterior fixation systems like pedicle-screw-rod constructs face failure risks such as screw loosening, primarily caused by age-related diminished bone quality and osteoporosis. This poses significant challenges for individuals and healthcare systems. This study developed simplified models of healthy and osteoporotic vertebrae, characterized by a 50% reduction in cortical thickness and a 4-fold decrease in E-modulus in osteoporotic bone. Finite element analysis (FEA) was employed to examine the impact of screw size and bone quality on stability, measuring loads at a 5.4 mm displacement threshold, a key parameter for loosening. Results demonstrated that cortical thickness had a greater influence on screw stability than biomechanical properties and increasing screw diameter improved stability more effectively than increasing screw length, particularly in osteoporotic bone. Subcortical bone properties contributed minimally. Validation against existing literature supports the conclusion that effective pedicle screw anchoring requires adequate cortical bone and quality in the pedicle region. Future research should refine subcortical bone properties in models and incorporate morphological changes from aging to improve simulation accuracy.2025-02-1
Influence of contrast agent administration on the measurement of bone mineral density using quantitative computed tomography in spine and proximal femur
No full textZiel: In der Routine erstellte Computertomographie (CT) -Aufnahmen erfolgen häufig unter Gabe eines Kontrastmittels (KM). Sie beinhalten viele bislang ungenutzte diagnostische Informationen. In dieser Arbeit sollte untersucht werden, ob man Aufnahmen, welche in der Routine häufig unter einer KM-Gabe erfolgen, ebenfalls für die Bone mineral density (BMD)-Messung verwenden kann. Messungen der bislang hierfür verwendeten nativen Aufnahmen sollten dabei mit den Ergebnissen der KM-Aufnahmen verglichen werden. Der Vergleich wurde für die Wirbelsäule und das proximale Femur durchgeführt. Material und Methoden: Untersucht wurden insgesamt 130 Patienten, 60 hiervon mit ausschließlich Wirbelsäulenaufnahmen, 3 mit Aufnahmen des proximalen Femurs sowie 67 mit sowohl Wirbelsäulenaufnahmen als auch Aufnahmen des proximalen Femurs. Es wurden lediglich Patienten mit einbezogen, welche in einem maximalen Abstand von 6 Monaten sowohl eine CT-Bildgebung mit und ohne KM erhielten. Die Messungen wurden mittels der Software CliniQCT (Mindways Inc. TX, USA) an den KM-gestützten und den nativen Aufnahmen durchgeführt. Es wurden monatliche asynchrone Kalibrierungen aller CT-Geräte der Universitätsmedizin Göttingen für die QCT-Messung durchgeführt. Zusätzliche Parameter wie Alter, Geschlecht, Größe, Gewicht und applizierte KM-Menge wurden erhoben. Ergebnisse: Es zeigten sich signifikante Korrelationen zwischen den Messungen mit und ohne KM an der Wirbelsäule und am proximalen Femur. Die BMD-Messungen unterschieden sich signifikant jeweils für beide Lokalisationen. Es zeigte sich ein unterschiedlich großer Einfluss des KMs auf die Messunterschiede in beiden Lokalisationen (19,4 mg/cm3 für die Wirbelsäule vs. 5,4 mg/cm3 für das proximale Femur). Es konnten für beide Lokalisationen Umrechnungsformeln formuliert werden. Der zusätzlich erhobene Parameter BMI zeigte eine negative Korrelation. Schlussfolgerung: Die BMD-Messwerte nativ und mit KM unterscheiden sich signifikant. Es zeigt sich ein unterschiedlich großer Effekt des KMs auf beide Lokalisationen. Messwerte des proximalen Femurs weisen geringere mittlere Differenzen zwischen den Messwerten KM und nativ auf als an der Wirbelsäule. Die KM-gestützten BMD-Messwerte sollten daher nicht direkt verwendet, sondern mittels einer Lokalisations-spezifischen Umrechnungsformel in die nativen Werte überführt werden. Somit können auch KM-gestützte Aufnahmen zu der Verbesserung der Osteoporose-Diagnostik und -Therapie sowie der Planung von Wirbelsäulen-Operationen auch in Notfallsituationen beitragen.Purpose: Computed tomography (CT) images taken in clinical routine are often obtained under the administration of a contrast agent (CA). These images provide a considerable amount of currently unused diagnostic information. The aim of this study was to investigate whether these images can also be used for bone mineral density (BMD) measurement. Measurements of the native images previously used for this purpose were compared with the results of the contrast-enhanced images. The measurements were performed for the spine and the proximal femur. Material and methods: A total of 130 patients were examined, 60 had only spinal images, 3 had images of the proximal femur and 67 had both spinal images and images of the proximal femur. Only patients who received both CT imaging with and without CA at a maximum interval of 6 months were included. Quantitative computed tomography (QCT) measurements were performed using CliniQCT (Mindways Inc. TX, USA). Monthly asynchronous calibrations of all CT scanners at the University Medical Center Göttingen were performed for accurate QCT measurement. Additional parameters such as age, gender, height, weight and amount of KM applied were recorded. Results: There were significant correlations between the measurements with and without CA at both the spine and the proximal femur. The BMD measurements differed significantly for both locations. The influence of the BMD on the measurement differences in both locations differed (19.4 mg/cm3 for the spine vs. 5.4 mg/cm3 for the proximal femur). Conversion equations could be derived for both locations. The parameter BMI showed a negative correlation, whereby a higher BMI resulted in a lower influence of the CA on the BMD-measurement. Conclusion: The BMD measurements natively and with CA differ significantly. There is a different effect of the CA on both localizations. Smaller mean differences were found between the contrast-enhanced and native results in the proximal femur compared to the spine. The BMD measurements based on CA should therefore not be used directly, but can be converted using a localization-specific conversion equation. Therefore CA-based images can also contribute to the improvement of osteoporosis diagnostics and treatment as well as the planning of spinal surgery, even in emergency situations.2025-03-0
Traumatic subaxial facet fractures – problems and opportunities of the current AOSpine classification
No full textHintergrund. Facettengelenkverletzungen werden erstmals in der neuen AOSpine Klassifikation für die subaxiale Halswirbelsäule genauer untersucht und klassifiziert. Ziel dieser Beobachtungsstudie ist die pathomorphologische Untersuchung und kritische Überprüfung von Facettengelenkverletzungen im Hinblick auf Stabilität und die neue Klassifikation. Zusätzlich wurden im Rahmen der Klassifikation erstmals auch Modifikatoren eingeführt. Insbesondere der Modifikator M1 (Verletzung des posterio-ligamentären-Komplexes (PLC)) wurde bei gleichzeitigem Auftreten einer Facettengelenkverletzung untersucht.
Methodik. Retrospektiv wurden Patienten- und Bilddaten an einem Wirbelsäulenzentrum Level I über einen Zeitraum von Januar 2013 bis August 2021 nachuntersucht. Die einzelnen Subtypen der Facettengelenkverletzungen (F1-F4) wurden hinsichtlich Stabilität und pathomorphologsicher Merkmale im CT und Röntgen miteinander verglichen. Vorhandene MRT-Aufnahmen wurden ausgewertet und die einzelnen Strukturen des PLC´s separat bewertet.
Ergebnisse. Insgesamt wurden n=74 Verletzungen in die Studie eingeschlossen, von denen > 94% (n=70) einen Subtyp zugeordnet werden. F1 und F2 Verletzungen unterschieden sich nicht in Bezug auf Stabilität (p=1,00), obgleich die Klassifikation davon ausgeht. Für das Abweichen vom Wirbelkörper C7 (p=0,003), das Vorliegen eines losen Fragments (p p<0,001) konnten statistisch signifikante Unterschiede als Inzidenz für eine instabile Verletzung herausgearbeitet werden. Ein weiter Gelenkspalt (p=0,01) zeigte ebenso statistisch signifikante Unterschiede, ohne dass hier ein kritischer Schwellenwert errechnet werden konnte.
Bezogen auf den Modifikator M1 wurden n=36 Patienten mit vollständigen Bilddatensätzen eingeschlossen werden. Mit Ausnahme der Gelenkkapseln konnten alle Strukturen eindeutig dargestellt werden. Der Modifikator M1 konnte in 80,6% der Fälle attestiert werden. Es zeigte sich eine Verletzungshierarchie in der das Ligamentum flavum (LF) eine Leitrolle einnimmt. Es zeigte sich ein statistisch signifikanter Zusammenhang (p=0,008) zwischen dem Vorliegen einer LF-Verletzung und einer knöchernen Instabilität.
Diskussion. Trotz guter klinischer Anwendbarkeit zeigt die neue AOSpine Klassifikation für die subaxiale Halswirbelsäule Schwächen bei der Beurteilung von Facettengelenkverletzungen. In der aktuellen Fassung (F1-F2) ist sie bei der Stabilitätsbeurteilung als ein wesentliches Entscheidungskriterium für das Ableiten der Therapieempfehlung nur von geringem klinischem Nutzen, da keine sichere Aussage über die Stabilität getroffen werden kann. Alternativ stehen weitere bildmorphologische Differenzierungskriterien (Höhe der Verletzung, knöcherne Fragmente, Gelenkspaltweite) zur Verfügung, die besser geeignet sind, zu entscheiden, ob eine Verletzung konservativ therapiert (stabil) oder chirurgisch stabilisiert (instabil) werden soll.
In der jetzigen Form liefert der Modifikator M1 für die klinische Entscheidungsfindung keinen nennenswerten Informationsgewinn, wenngleich die Bedeutung des PLCs unbestritten ist. Diese Arbeit beschreibt das LF an der Spitze einer Verletzungshierarchie. Es konnte gezeigt werden, dass vom Zustand des LF Rückschlüsse auf die knöcherne Stabilität gezogen werden können.Background. One of the latest developments in the field of traumatic spinal injuries is the new AOSpine injury classification of the subaxial cervical spine. Based on the classification of the thoracolumbar spine, facet joint injuries are also integrated for the first time. The overall aim of this study is to check the classification for applicability and clinical benefit. Furthermore, modifiers were introduced, which will also be examined in the context of this work. In particular the modifier M1 (injury to the posterio-ligamentous-complex (PLC)) was investegated in the simultaneous presence of a facet joint injury.
Materials and methods. Retrospective review of cervical spine CTs and MRT´s on all patients who presented to our trauma clinic between January 2013 and August 2021. The individual subtypes of facet joint injuries (F1-F4) were compared in terms of stability and morphological characteristics.
All structures of the PLC has been evauated individualy. The apperance of the modificator M1 was compared in terms of stability and sequence of injured components oft he PLC.
Results. A total of n=74 facet injuries were included in the study, of which >94% (n=70) were assigned a subtype. It was noticeable that F1 and F2 injuries did not differ from each other in terms of their stability, contrary to the basic assumption of the classification (p=1.00). The deviation of the C7 vertebral body (p=0.003) and the presence of a loose fragment (p<0.001) were identified as indications of an unstable injury. The width of the joint gap (p=0.010) was also shown to be significantly different in both groups (stable vs. unstable). Eventough a threshold value could not be determined.
Regarding modifier M1 a total of n=36 patients were included. With the exception of the joint capsules, all structures could be clearly examined. The modifier M1 could be attested in 80.6% of cases. An injury hierarchy was revealed in which the ligamentum flavum (LF) plays a leading role. There was a statistically significant relationship (p=0.008) between the presence of an LF injury and bony instability.
Conclusion. Despite its good applicability, the classification proves to be unsuitable in terms of clinical benefit when making therapy decisions because no statement can be made about stability. Alternatively, this study provides differentiation criteria that not only indicate instability but are also less error-prone and clearer than the original criteria.
Although the importance of the PLC is undisputed, the M1 modifier does not provide any significant information for clinical decision-making if there is a facet injury. This work describes the LF at the top of an injury hierarchy. The status of the LF might be used to get an idea about stability of the injury.2026-02-0
Stellenwert der Leberbiopsie zur Klärung der Ätiologie unklarer chronischer Leberwerterhöhung
No full textElevated liver enzymes are common and require thorough investigation to enable timely treatment and prevent fibrosis progression. Advances in diagnostics have reduced the need for liver biopsies, but they remain valuable in unclear cases. This dissertation retrospectively analyzed clinical, serological, imaging, and histopathological data from 84 patients biopsied at UMG between 2015 and 2021 for chronically unclear elevated liver enzymes or cryptogenic cirrhosis. The aim was to evaluate diagnostic clarification rates and underlying causes.This study found that biopsies clarified the cause in under half of cases, influencing treatment particularly through immunosuppressive therapy and lifestyle changes. Clinical data lacked predictive value for etiological clarification, likely due to heterogeneity among diagnostic groups and nonspecific predictors.
Given the clarification rate, therapeutic impact, and complication rates within established reference values, liver biopsy remains a justified diagnostic tool for chronically unclear elevated liver enzymes.2025-04-2
Urinary Dickkopf‐3 as a Potential Marker for Estimated Glomerular Filtration Rate Decline in Patients With Heart Failure
No full textChronische Herzinsuffizienz führt in 50% der Fälle zu einer chronischen Nierenerkrankung. Insbesondere im Sinne eines kardiorenalen Syndroms. Bisher gibt es wenige prädiktive Möglichkeiten, um Patienten mit einem erhöhten renalen Risiko bei bestehender Herzinsuffizienz zu identifizieren.
Dickkopf-3 (uDKK-3) ist ein von Tubulusepithelzellen unter Stress produziertes und sezerniertes Glykoprotein, das möglicherweise auch bei Patienten mit Herzinsuffizienz erhöht ist und mit einem erhöhten Risiko für renalen Funktionsverlust einhergeht.
In der Arbeit wurde an einem Studienkollektiv von 488 asymptomatischen Herzinsuffizienten Patienten und 45 Herzgesunden Probanden untersucht, inwieweit sich in der kranken Kohorte erhöhte uDKK-3 Spiegel finden. Zudem wurde untersucht ob eine Korrelation zwischen der Änderung der eGFR und den initialen uDKK-3 Spiegeln besteht. Es standen für die Herzinsuffizienten Probanden Follow-up Daten von 1, 4 und 5 Jahren zur Verfügung. Die uDKK-3 Spiegel wurden mittels eines kommerziellen ELISA-Test-Kits gemessen.
Es zeigten sich erhöhte uDKK-3 Spiegeln bei Patienten mit Herzinsuffizienz im Vergleich zu den Herzgesunden Probanden (256,9 pg/mg Kreatinin [IQR 119,2-509,4 pg/mg Kreatinin] vs. 107,5 pg/mg Kreatinin [60,5-181,2 pg/mg Kreatinin]). In einem Regressionsmodell zeigte sich zudem eine Assoziation von log(uDKK-3) zur eGFR-Änderung in einem medianen Beobachtungszeitraum von 13 Monaten (erwartete höherer eGFR-Verlust von 0,8039 mL/min/1,73m² pro Jahr [95%-KI: 0,002-1,606 ml/min/1,73m²/Jahr, p=0,049; OR 1,345 [95%-KI 1,049-1,741], p=0,021).
Patienten mit einer uDKK3-Konzentration ≥354 pg/mg Kreatinin waren mit einem signifikant höheren Risiko für einen eGFR-Verlust beim 1-Jahres Folow-up assoziiert (erwarteter höherer eGFR-Verlust 4,358 ml/min/1,73m² [95%-KI: 1,482-9,593 ml/min/1,73m²; p=0,004].
Auch Patienten ohne bestehende chronische Nierenschädigung zeigten erhöhe uDKK3 Werte im Vergleich zur gesunden Kontrollgruppe (233,4 pg/mg Kreatinin [109,0-436,9 pg/mg Kreatinin] vs. 107,5 pg/mg Kreatinin [60,5-181,2 pg/mg Kreatinin], p<0,001).
Die Ergebnisse zeigen das Potential von uDKK-3 als renaler Prognoseparameter auch bei Patienten mit einer Herzinsuffizienz. Vor allem auch bei Patienten, die noch keine chronische Nierenschädigung aufweisen. Nichtsdestotrotz zeigte sich das die Prognosefähigkeit auf einen kurzen Zeitraum beschränkt ist und es daher empfehlenswert wäre, uDKK3 in der klinischen Praxis in regelmäßigen Abständen zu messen. Die Ergebnisse bieten zudem eine Grundlage für die weitere Erforschung der Rolle von uDKK-3 als renalem Prognoseparameter in verschiedenen Kontexten.Chronic heart failure leads to chronic kidney disease in 50% of cases, particularly in the context of cardio-renal syndrome. In clinical routine, there are few predictive methods to identify patients with an increased renal risk in the presence of heart failure.
Dickkopf-3 (uDKK-3) is a stress-induced, tubuloepitheliar secreted glycoprotein, which may also be elevated in patients with heart failure and might be associated with an increased risk of eGFR loss.
In a cohort of 488 asymptomatic heart failure (HF) patients and 45 healthy controls uDKK3 were measured, using a commercial ELISA-Test-Kit, to determine whether elevated uDKK-3 levels could be found in the HF cohort. Additionally, the correlation between the change in eGFR and initial uDKK-3 levels was investigated. Follow-up data were available for heart failure patients at 1, 4, and 5 years.
Increased uDKK-3 levels were found in patients with heart failure compared to healthy controls (256.9 pg/mg creatinine [IQR 119.2-509.4 pg/mg creatinine] vs. 107.5 pg/mg creatinine [60.5-181.2 pg/mg creatinine]). In a regression model, an association between log(uDKK-3) and the change in eGFR was observed over a median observation period of 13 months (expected higher eGFR loss of 0.8039 mL/min/1.73m² per year [95% CI: 0.002-1.606 mL/min/1.73m²/year, p=0.049; OR 1.345 [95% CI 1.049-1.741], p=0.021).
Patients with a uDKK-3 concentration ≥354 pg/mg creatinine were associated with a significantly higher risk of eGFR loss at 1-year follow-up (expected higher eGFR loss of 4.358 mL/min/1.73m² [95% CI: 1.482-9.593 mL/min/1.73m²; p=0.004]).
Even patients without existing chronic kidney damage showed elevated uDKK-3 values compared to control group (233.4 pg/mg creatinine [109.0-436.9 pg/mg creatinine] vs. 107.5 pg/mg creatinine [60.5-181.2 pg/mg creatinine], p<0.001).
The results highlight the potential of uDKK-3 as a renal prognostic marker, even in patients with heart failure, particularly in those who do not yet have chronic kidney damage. Nevertheless, it was shown that the prognostic ability is limited to a short period, and therefore it would be advisable to measure uDKK-3 at regular intervals in clinical practice. These findings also provide a basis for further research on the role of uDKK-3 as a renal prognostic marker in different contexts.2026-02-1
Establishing Standardized Incisor Inclinations - An Examination of Potentially Relevant Factors for Achieving Cephalometric Target Values
No full textGemäß den erzielten Ergebnissen führte weder die Verwendung von Schneidezahnbrackets mit Torqueunterschieden von 10° - (Roth versus Ricketts-Brackets) noch die alternative Verwendung von 0,017x0,025 CNA Abschlussbögen anstelle von 0,016x0,022 CNA Bögen zu signifikant unterschiedlichen Veränderungen der Schneidezahninklinationen. Es mag deshalb nicht weiter überraschen, dass vermeintlich kleinere Abweichungen, wie sie bedingt durch Hersteller- oder Materialunterschiede bestanden haben könnten, klinisch nicht offenkundig wurden. Stattdessen dominierte gruppenübergreifend eine behandlungsbedingte signifikante Tendenz zur Proklination der Schneidezähne. Diese war dem Ziel, durch kieferorthopädische Behandlungen morphologisch begründete anerkannte normgerechte Inklinationen zu erreichen, entgegengerichtet, entsprach aber in Qualität (Oberkiefer), beziehungsweise Qualität und Quantität (Unterkiefer) einem Vorhersagemodell auf mathematisch – geometrischen Grundlagen.
Auch veränderten sich die Standardabweichungen in den Untersuchungsgruppen durch die Behandlungen inhomogen, jedoch war keine einheitliche Tendenz zur Verkleinerung der Wertestreuungen erkennbar. In der Konsequenz muss davon ausgegangen werden, dass es unter den besonderen Umständen dieser Studie (0,018 - Slot, Bogenstärken, Bogenmaterial, u.a.) keine Anzeichen für die bzgl. der Schneidezahninklination erwartete Bogen-/Bracketdominanz gibt. Stattdessen ist wahrscheinlich, dass andere Faktoren, wie z.B. die individuelle basale Konfiguration der Patienten, die initiale Platzbilanz, die Schneidezahnmorphologie, u.a. einen relevanten Einfluss auf die Veränderung der Schneidezahninklinationen während einer kieferorthopädischen MB - Behandlung haben. Im Rahmen zukünftiger Untersuchungen sollte verifiziert werden, ob diese Ergebnisse dem Anspruch einer Allgemeingültigkeit genügen.According to the results obtained in this retrospective in vivo study, neither the use of incisor brackets with torque differences of 10° (Roth vs. Ricketts brackets) nor the alternative use of 0.017x0.025 CNA finishing wires instead of 0.016x0.022 CNA ones led to significantly different changes in incisor inclinations. It was therefore not surprising that also potentially minor deviations, as might have been expected due to manufacturing differences were not clinically evident. This was the case when steel brackets with equal torque values from different providers (Forestadent vs. Dentaurum) were compared and, in the same way, when different bracket materials (steel vs. ceramic) were tested. Instead, a treatment-induced significant tendency toward incisor proclination was observed across all groups. Such a proclination, being detected in both arches, opposed the objective of morphologically aspired standardized inclinations to be achieved by orthodontic treatment. However, the observed proclination corresponded in quality (upper arch) or in both quality and quantity (lower arch) to a predictive model based on mathematical and geometric calculations.
Additionally, the standard deviations within the study groups changed inconsistently during treatment, with no uniform trend toward a reduced dispersion of values. As a consequence, it can be concluded that there was no evidence for the expected wire/bracket dominance regarding post – treatment incisor inclination. This holds true at least under the specific conditions of this study (slot and wire dimensions, wire materials, etc.). Instead, other factors seemed to have a more significant impact on changes of incisor inclinations during orthodontic MB treatment. According to the literature, there is some evidence existing that these factors might be the initial space conditions, the incisor morphology, the initial incisor inclinations and the individual basal configuration of the patients. Future studies should verify whether these results meet the criteria for general validity regardless of the specific conditions of the fixed appliance system being used.2025-04-0
Molecular dynamics simulations of ion permeation and gating in potassium channels
No full textPotassium (K+) channels are transmembrane ion channels that conduct K+ ions with exceptional selectivity and permeation rates. K+ channels participate in a plethora of important cellular processes, including termination of action potentials in excitable cells. This thesis addresses the two main phenomena associated with K+ channels - mechanisms of ion permeation and gating of K+ channel activity - using molecular dynamics (MD) simulations.
In studies of K+ channels, the use of both experimental and computational methods has significantly contributed to our understanding of K+ channel structure and function. Among these methods, MD simulations allow studying the processes relevant to K+ channels at time and space resolutions often inaccessible to other approaches. In the present thesis, first the physical basis and practical aspects of using MD simulations are described, along with strengths and limitations of the method. Then, I focus on K+ channels, and discuss the two most common models of K+ permeation, soft and direct knock-on, along with the available experimental and computational data addressing the two models. I conclude the introduction to the thesis by addressing the gating mechanisms - regulation of the equilibrium between conductive and non-conductive states of a channel, together with the associated changes in the channel conformation - described for various K+ channel families.
In this thesis, ion permeation mechanisms in K+ channels are studied specifically on the example of a model K+ channel, KcsA, and its selectivity filter (SF) mutants. The SF is the narrowest part of the ion permeation pathway, largely conserved between different K+ channel families. Generally, most structures of K+ channels exhibit a direct knock-on-like ion configuration in the SF, characterized by the absence of water molecules and presence of close ion-ion contacts. Certain point mutations in the SF, however, allow to isolate soft knock-on-like configurations, where neighboring ions are separated by water molecules. This, coupled with the specific effects these mutations have on the ion occupancy in non-adjacent K+ binding sites, was proposed to favor the soft knock-on mechanism in the wild type channels as well. Here, we use MD simulations with an applied electric field to study ion permeation in the WT and mutant KcsA. By testing various combinations of force fields, mutations and voltages, we show not only that ion permeation mechanisms are fundamentally different in mutant and WT channels, but also that the complete loss of the ion solvation shell upon entry and close ion-ion contacts (direct knock-on mechanism) in the SF are crucial to establish the hallmark high conductance and ion selectivity in K+ channels.
The second project is focused on gating in BK channels. BK channels are activated by membrane depolarization and binding of Ca2+, which is facilitated by a complex structure featuring the pore, voltage- and Ca2+-sensing domains. Despite several CryoEM structures capturing the full-length BK in activating and deactivating conditions, the mechanism by which ion permeation is stopped in the closed state of the channel is unclear, as the pore in deactivating conditions lacks any physical constrictions often present in other K+ channels. By performing atomistic simulations of the full-length BK, we suggest that occupancy of the pore by lipids defines the closed, non-conductive state of the channel. In the corresponding part of the thesis, I describe the mechanisms by which lipid entry into the pore occurred, as well as the associated effects on the channel’s conductance. Then, I address the effect of membrane composition on BK activity. It was shown that, generally, negatively charged lipids increase both the open probability and single-channel current in BK. In good agreement with the experimental data, our coarse-grained and atomistic MD simulations suggest a multi-modal mechanism, in which the negative charge on the lipid headgroups leads to reduced lipid entry, increased local K+ concentration in the pore and increased conformational stability of the open-state channel.
Finally, in ‘Conclusions and outlook’ I summarize the findings of the two projects, and discuss the approaches to further validate our conclusions, and expand upon the corresponding research questions.2026-01-2
Quantifizierung spontaner Calcium-Freisetzungen in humanen atrialen Kardiomyozyten aus induzierten pluripotenten Stammzellen (iPSC)
No full textAtrial fibrillation (AF) is a cardiac disorder characterized by disorganized electrical excitation of the atria. AF is the most common cardiac arrhythmia in clinical practice and is associated with increased cardiovascular mortality and morbidity. It has been shown that increased spontaneous diastolic Ca²⁺ releases from the sarcoplasmic reticulum (SR), known as the Ca²⁺ leak, play a significant role in the onset and maintenance of AF.
A major challenge in AF research and the development of therapeutic approaches is the lack of an appropriate model. Recent studies have therefore proposed the use of cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs). This study evaluated the Ca²⁺ leak in the form of Ca²⁺ sparks to answer questions regarding the Ca²⁺ homeostasis of iPSC-CMs and to further establish this cell model.
For this purpose, iPSC-CMs were loaded with the fluorescent Ca²⁺ indicator Fluo-4-acetoxymethyl ester (Fluo-4 AM) and analyzed using confocal microscopy. The resulting images were used to qualitatively and quantitatively assess Ca²⁺ sparks with the software ImageJ®.
A frequently discussed characteristic of iPSC-CMs is their developmental stage and lack of maturity. Therefore, this study examined changes in the Ca²⁺ leak during their maturation process. The Ca²⁺ leak of atrial and ventricular iPSC-CMs was measured and compared over a period of 150 days, starting from differentiation. The study demonstrated that spontaneous diastolic Ca²⁺ leakage decreased over time in both atrial and ventricular iPSC-CMs. These findings support the hypothesis that iPSC-CMs develop a more mature electromechanical coupling mechanism with age.
Another part of the study investigated whether in vitro tachypacing could induce changes in spontaneous diastolic Ca²⁺ release from the SR, similar to those observed in persistent AF. To this end, atrial and ventricular iPSC-CMs were electrically stimulated for 24 hours at frequencies of 1 and 3 Hz, after which the Ca²⁺ leak was measured. The results showed that in both atrial and ventricular iPSC-CMs, spontaneous diastolic Ca²⁺ leakage decreased with increasing pacing frequency. The reasons for this decrease despite higher pacing frequencies are discussed in this study. It is suggested that tachypacing may have initiated a maturation process in iPSC-CMs. Furthermore, the duration of tachypacing may not have been sufficient to observe the expected changes in the Ca²⁺ leak.
To mimic the pathophysiology of AF, iPSC-CMs were subjected to arrhythmic stimulation in further experiments. The arrhythmia was examined separately from tachycardia at the molecular and electrophysiological levels and led to a significant increase in the Ca²⁺ leak in ventricular iPSC-CMs. However, this effect was not observed in atrial iPSC-CMs.
Additional experiments in this study investigated the effects of Ivabradine on iPSC-CMs. The aim was to analyze how Ivabradine affects the Ca²⁺ leak and the spontaneous contraction frequency of iPSC-CMs. In atrial iPSC-CMs, Ivabradine treatment did not alter spontaneous contraction frequency or Ca²⁺ leakage. In contrast, Ivabradine appeared to have an arrhythmogenic effect in ventricular iPSC-CMs. This observation cannot be explained by the molecular mechanism of Ivabradine. The observed effects may be due to the immaturity of the cells or an unspecific action of Ivabradine.
To examine how the Ca²⁺ leak can be modulated by interventions in Ca²⁺ homeostasis, further experiments involved the inhibition of protein kinases CaMKII and PKA using Carbachol (CCh) and H-89. CCh significantly reduced diastolic Ca²⁺ leakage in both atrial and ventricular iPSC-CMs, leading to a pronounced antiarrhythmic effect. H-89 treatment also reduced diastolic Ca²⁺ leakage in atrial iPSC-CMs. However, no effect was observed in ventricular iPSC-CMs. These experiments demonstrate that the Ca²⁺ homeostasis of iPSC-CMs can be modulated in a similar manner to that of adult primary human cells. The fact that both cell types rely on the same mechanisms highlights their similarities. This represents a significant opportunity for cardiovascular disease research using the iPSC-CM model.
Although iPSC-CMs resemble primary human cardiomyocytes, they are not entirely comparable. While iPSC-CMs exhibit significant similarities to adult cells, their immature characteristics mean that not all findings can be directly extrapolated.
Overall, this study contributes to a deeper understanding of the mechanisms involved in the Ca²⁺ homeostasis of iPSC-CMs and provides a valuable foundation for future research into cardiac Ca²⁺ homeostasis. Furthermore, this work enhances the electrophysiological characterization of iPSC-CMs and supports their establishment as a model for AF research2025-04-0
Die Etablierung einer neuen NGS-basierten Panelanalyse zur Identifikation der genetischen Ursachen isolierter und syndromaler Formen der Mikrophthalmie
No full textMicrophthalmia and anophthalmie are part of the spectrum of rare diseases. Microphthalmia is defined as a developmental disorder of the eye, in which the globe of the eye is more than two standard deviations below the population mean, whereas anophthalmia is defined as the complete absence of visual tissue with, in some cases, some residual neuroectodermal tissue still being detectable. Both, microphthalmia as well as anophthalmia, can occur as isolated findings or they can be associated with additional phenotypic features as part of a syndromic disorder. In case of microphthalmia, clinical manifestation can be highly variable ranging from restricted view up to complete amaurosis. High clinical variability often complicates the identification of underlying causes in affected patient. Still, this is of vital importance for not only resolving the diagnostic odyssey of affected patients, but it also plays a role in the management of treatment options in these patients and underlines the necessity of a multidisciplinary team with ophthalmologists, human geneticist, ocularists and pediatricians in treatment of these patients.
The major aim of my thesis was the genetic analysis and characterization of patients with microphthalmia/anophthalmia using next generation sequencing technologies.
In the first part of my thesis, a NGS-based multigene-panel was designed, which in total encompasses 53 genes that are associated with isolated and syndromic forms of microphthalmia/anophthalmia. I established and validated this multigene-panel for routine diagnostic testing and used it for identification of the underlying genetic cause in a patient cohort with an ocular phenotype.
Secondly, selected cases of this patient cohort that could not be solved by multigene-panel analysis, were subjected to whole-exome sequencing to detect the underlying genetic cause and possibly identify new genes that are involved in the pathogenesis of microphthalmia/anophthalmia. Detected variants were analyzed regarding their functional effect, classified in terms of pathogenicity and specific variants were tested for cosegregation in the respective families. Based on this strategy, I was able to identify pathogenic and likely pathogenic in four patients that were subjected to exome sequencing.
Based on this strategy, I was able to identify a pathogenic hemizygous variant in MED12 (c.886C>T) in a patient with a complex ocular malformation and additional phenotypical features. Using bioinformatic and molecular genetic approaches, I was able to provide evidence for causality of this variant, and thereby I could expand the spectrum of pathogenic variants in MED12. As a result, MED12 was included in the multigene-panel for microphthalmia/anophthalmia that was designed in course of this thesis. In a second case, I was able to identify a homozygous variant in NCAPH (c.391C>T) by using exome sequencing and subsequent bioinformatic analysis of homozygous variants. So far, only a single case with ocular malformations has been described in the literature carrying a pathogenic variant in this gene (recessive inheritance, homozygous variant on position c.728C>T in NCAPH). The identification of pathogenic variants in NCAPH in a second patient with an overlapping phenotype supports the results of the previously reported case and introduced NCAPH as a causative gene for a Mendelian disorder in humans.
In summary, the data presented in this thesis established new diagnostic tools for the genetic examination of patients with isolated and syndromic forms of microphthalmia/ anophthalmia. Additionally, using exome sequencing, I was able to identify the underlying genetic cause in patients with ocular malformations. These results will support reliable identification of causative variants in affected patients, thereby directly having an impact on patients and their families affected by undiagnosed disorders and providing new perspective to therapeutic approaches.2025-04-2