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Die nukleare Translokation des Kalziumbindenden-Proteins Calbindin-1 und dessen Rolle bei der Kalziumhomöostase in den distalen Tubuluszellen der Niere.
No full textThe nuclear translocation of the calcium binding protein calbindin-1 and its role in the calcium-homeostasis in kidney distal tubule cells
Calcium (Ca2+) plays a key role in many physiological processes like hormone secretion, muscle contraction, bone formation, nerve conduction, exocytosis, and (in)activation of enzymes. The body Ca2+ homeostasis is tightly regulated and disturbances in its balance may lead to pathogenesis. Kidney especially renal epithelium as the site of Ca2+ reabsorption plays an important role in the body Ca2+ homeostasis. The Ca2+-reabsorption in the kidney can be paracellular or transcellular. The latest takes place in distal convoluted tubule (DCT) cells and is regulated by PTH and VD3 [1,2], and plays a role in the fine-tuning of whole-body calcium homeostasis. Increase in cytosolic calcium concentration has been associated with cellular dysfunctions in a variety of conditions such as diabetes mellitus, hypertension, hyperparathyroidism and chronic kidney diseases (CKD) [3,4].
In DCT, Ca2+ enters cells across the apical part through the Ca2+ selective channel TRPV5, then binds to calbindin-1 (Calb-1), the major intracellular calcium binding proteins in DCT, and diffuses to the basolateral membrane, where Ca2+ is extruded via the Na/Ca exchanger (Ncx1) or plasma membrane calcium-transporting ATPase 1 (PMCA1) [1]. In this process of transcellular Ca2+ reabsorption, the Calb1 plays a key role Calb-1 as an intracellular Ca2+- buffer and -transit protein from the apical side to the basolateral side without significant modification in the intracellular Ca2+ [1]. Our mice model for chronic kidney disease (Calreticulin heterozygous mice, Calr+/-) suffers from disturbance in Ca2+-homeostasis and cellular calcium toxicity. Histoimmunological investigations of kidney tissues revealed a nuclear translocation of Calb-1 in the DCT-cells from Calr+/- mice. Our preliminary observations shed light on potential still unknown function of Calb-1 in DCT-cells. The aim of the present study is to clear the role, if there is any, of the nuclear translocation of Calb-1 in DCT-cells.
The following questions will be addressed:
1. Does Calbindin-1 undergo a nuclear translocation and which conditions are responsible of the cellular compartmental translocation of Calb-1? For this purpose, renal tissue form Calr+/- and Calr+/+ will be stained with antibody against Calb-1 and confocal microscopy analysis will be performed to confirm the nuclear localization of Calb-1 in Calr+/- kidney DCT-cells. Furthermore, established DCT-cell lines (MDCK and MDBK) will be used to investigated the conditions of the nuclear translocation of Calb-1.
2. Does the nuclear translocation of Calb-1 have any impact on its role in Ca2+-buffering and transition in DCT-cells? DCT-cell lines will be subjected to conditions that trigger the Cabl-1 nuclear translocation and the cytosolic free Ca2+ concentration will be investigated using Fura-2 as the indicator of intracellular calcium concentration. Furthermore, similar experiments will be performed under condition of Calb-1 overexpression (plasmid) or down-regulation (siRNA).
3. Which role play the nuclear Calb-1? Does the nuclear Calb-1 plays any role in gene expression regulation?2026-02-1
The role of Tc-foxQ2 in insect neural development analyzed by novel genetic tools
No full textThe insect brain serves as a powerful model for deciphering the principles of complex neural architecture. This dissertation investigates the genetic and cellular mechanisms underlying brain development and function in the red flour beetle, Tribolium castaneum, with a specific focus on the highly conserved transcription factor Tc-foxQ2.
Our results reveal that Tc-foxQ2 functions as a developmental stage-specific regulator. During embryogenesis, it acts as a fundamental patterning factor essential for gross brain formation and neuronal survival. In contrast, during metamorphosis, its role shifts toward a more refined regulatory function; while its knockdown does not alter the gross brain structure, it causes minor defects in neuronal positioning.
By integrating 3D reconstruction with advanced imaging, I characterized the cellular landscape of Tc-foxQ2 in the adult brain, identifying nine distinct neuronal clusters. Remarkably, Tc-foxQ2 marks 71.2% of all dopaminergic neurons. I discovered a unique Tc-foxQ2-positive cluster that connects the mushroom body (MB) and the central complex (CX), providing the first evidence of a transcription factor labeling neurons that bridge these two higher-order centers. Functional analyses through RNAi-mediated knockdown demonstrate that Tc-foxQ2 is essential for the development of these dopaminergic neurons and the regulation of locomotor activity, as its depletion leads to hyperactivity and specific cell loss.
Furthermore, to facilitate high-resolution neural imaging, I joined an effort led by another lab to develop and validate a cross-species toolkit of membrane-localizing tags. Tested across ten species from seven phyla, this toolkit provides a robust methodology for labeling cell boundaries. Within Tribolium, the KRas and KRas6R tags were identified as superior tools for precisely localizing fluorescent proteins to the plasma membrane. Collectively, this work identified functional roles for Tc-foxQ2 in regulating motor activity and details of neural circuit formation. These findings provide novel insights into the molecular mechanisms governing insect brain development.2026-02-2
Parthenogenetic Stem Cells from Unfertilized Porcine Oocytes
No full textMammalian parthenogenesis remains significantly understudied due to fundamental biological limitations arising from genomic imprinting, where the absence of paternally imprinted genes leads to developmental abnormalities and embryonic arrest. While parthenogenetic stem cells have been derived from mouse and human sources, extending this research to translationally relevant large animal models like pigs represents a critical knowledge gap, despite their physiological and anatomical similarities to humans making them ideal for regenerative medicine applications. This thesis aimed to establish efficient protocols for generating and characterising parthenogenetic stem cells from unfertilised porcine oocytes, focusing on optimising activation methods, culture conditions, and comprehensive characterisation of the derived stem cells. Additionally, it aimed to develop protocols for the cardiac differentiation of porcine parthenogenetic stem cells to establish the basis for subsequent applications in regenerative medicine.
In this thesis, novel porcine parthenogenetic stem cell lines (pPSCs) were generated (n=24 from n=2,578 oocytes) and characterized, which included a comparison to porcine embryonic stem cell (pESCs) lines (n=10 from n=2,917 in vitro fertilized oocytes) and induced pluripotent stem cell (piPSC) lines (n=1). Molecular and immunofluorescence characterisation confirmed authentic pluripotent markers, including OCT4A, NANOG, and SALL4, while maintaining normal diploid karyotypes (2n = 38) across all examined cell lines (n=5 pPSC lines, n=5 pESC lines, and n=1 piPSC line). Bulk RNA sequencing analysis revealed distinct transcriptional profiles in the tested pluripotent stem cell lines, demonstrating particularly strong signatures of naïve pluripotency in pPSCs. Finally, first evidence for cardiac differentiation of porcine parthenogenetic stem cells is presented, demonstrating robust expression of cardiac-specific markers (cTnT, α-actinin) with characteristic cross-striated patterns and spontaneous contractile activity.
The use of pPSC derivatives offers clear advantages in regenerative medicine applications, because of their largely homozygous genome, including haploidentity of the coding sequences for the immunologically highly relevant multi-histocompatibility complex (MHC) proteins. This trait offers opportunities for "off-the-shelf” therapeutic use with minimal immunosuppression or no immune suppression, the latter being a major limitation in classical allograft approaches. The pPSC model is considered a stepping stone to enable pivotal preclinical stepping stones on the way to clinical translation.2026-09-2
Besteht ein Zusammenhang zwischen dem Nachweis von Bacteroides species und Enterococcus faecalis im Blut von traumatologischen Intensivpatienten und der Entstehung einer Sepsis?
No full textUnser Mikrobiom besteht aus einer Vielzahl von Bakterien, die als Kommensale unserer
Darmflora unserem Organismus nützlich sind. Viele von ihnen sind jedoch Pathogene,
sobald sie in unseren Blutkreislauf translozieren. Dies zu verhindern ist Aufgabe der
Darmbarriere, welche unter dem Einfluss der brain-gut-axis steht. Ziel dieser Dissertation
war es zu untersuchen, ob eine signifikante Assoziation zwischen bakterieller
Translokation und Sepsis besteht und ob sich Enterozyten- bzw. tight-junction-
Bestandteile als Biomarker für Translokation eignen. Der Translokationsnachweis
erfolgte mittels PCR aus Patientenblut zweier exemplarischer Kommensale mit fakultativ
pathogenen Eigenschaften: Bacteroides spp. und Enterococcus faecalis. Des Weiteren erfolgte
die serielle Bestimmung von iFABP und Claudin 3 als Permeabilitätsmarker der
Darmbarriere, der Laborparameter zur Bestimmung des SOFA-Scores zur Diagnostik
der Sepsis, sowie der gängigen Parameter der Inflammation, Nierenfunktion und
gastrointestinalen Funktion. Zwar konnte bei einigen Patienten eine Translokation
nachgewiesen werden, eine Korrelation mit erhöhten Permeabilitätsmarkern konnte
jedoch nicht beobachtet werden. Eine im Vergleich leicht erhöhte Translokationsrate bei
den septischen Patienten war statistisch nicht signifikant.Our microbiome consists of a multitude of bacteria that are beneficial to our organism as commensals of our intestinal flora. However, many of them become pathogens as soon as they translocate into our bloodstream. Preventing this is the task of the intestinal barrier, which is influenced by the brain-gut axis. The aim of this dissertation was to investigate whether there is a significant association between bacterial translocation and sepsis and whether enterocytes or tight junction components are suitable biomarkers for translocation. Translocation was detected by PCR from patient blood of two exemplary commensals with facultative pathogenic properties: Bacteroides spp. and Enterococcus faecalis. Furthermore, serial determination of iFABP and Claudin 3 as permeability markers of the intestinal barrier, laboratory parameters for determining the SOFA score for the diagnosis of sepsis, and the common parameters of inflammation, kidney function, and gastrointestinal function were performed. Although translocation was detected in some patients, no correlation with elevated permeability markers could be observed. A slightly elevated translocation rate in septic patients was not statistically significant.2026-03-2
Proinflammatorische Zytokine und vitale Erschöpfung bei Patienten mit kardiovaskulären Risikofaktoren
No full textDie Entwicklung einer Herzinsuffizienz wird nicht mehr ausschließlich als Erkrankung des Herzens betrachtet. Neben klassischen Risikofaktoren wie Alter, Geschlecht, Tabakkonsum, arterieller Hypertonie, Hyperlipidämie, Diabetes mellitus, Adipositas und Bewegungsmangel werden auch
biologische Risikofaktoren, darunter erhöhte Spiegel inflammatorischer Biomarker wie die Zytokine Interleukin-1β, Interleukin-6 und Tumornekrosefaktor-α und psychosoziale Risikofaktoren beschrieben. Zu letzteren zählen unter anderem psychologischer Stress (akut, episodisch oder chronisch), Persönlichkeitsmerkmale wie die Typ-D-Persönlichkeit, negative Affekte, ein niedriger sozioökonomischer Status sowie mangelnde soziale Unterstützung. Unter den psychosozialen Risikofaktoren stellt die vitale Erschöpfung einen signifikanten unabhängigen Prädiktor der
koronaren Herzkrankheit und einen relevanten Risikofaktor für kardiovaskuläre Ereignisse dar.
Ziel der vorliegenden Arbeit war es, Zusammenhänge zwischen vitaler Erschöpfung und ausgewählten pro- und antiinflammatorischen Zytokinen bei Patienten mit kardiovaskulären Risikofaktoren zu untersuchen.
Die vorliegende Arbeit ist ein Teilprojekt der MedViP-Studie (Medizinische Versorgung in der Praxis), einer prospektiven Beobachtungsstudie an der Universitätsmedizin Göttingen. In die Analysen wurden 356 allgemeinärztliche Patienten mit Risikofaktoren für die Entwicklung einer Herzinsuffizienz eingeschlossen, die umfassend kardiologisch (einschließlich Echokardiografie) und psychometrisch untersucht wurden. Die Typ-D-Persönlichkeit wurde mithilfe der DS14, die vitale Erschöpfung anhand des Maastricht-Fragebogens erfasst. Anhand der Summenscores des Maastricht-Fragebogens wurden Patienten mit hoher und niedriger Ausprägung der vitalen Erschöpfung unterschieden. Bei vital erschöpften und nicht vital erschöpften Patienten wurden die Serumkonzentrationen der proinflammatorischen Zytokine Interleukin-1β, Interleukin-6 und Tumornekrosefaktor-α und des antiinflammatorischen Zytokins Interleukin-10 bestimmt. Die Auswertung erfolgte auf Grundlage der Baseline-Daten sowie der Follow-up-Daten nach einem Jahr.
In der untersuchten Stichprobe zeigten sich Zusammenhänge zwischen vitaler Erschöpfung und ausgewählten Zytokinen, während sich für andere Zytokine keine entsprechenden Assoziationen ergaben. Darüber hinaus bestanden Zusammenhänge zwischen vitaler Erschöpfung sowie
psychosozialen und klinischen Merkmalen. In multivariaten Analysen erwiesen sich neben dem Geschlecht und dem Body-Mass-Index insbesondere psychosoziale Faktoren als relevante Prädiktoren der vitalen Erschöpfung. In der Längsschnittbetrachtung zeigten sich Veränderungen des
Erschöpfungsgrades über den Zeitraum von einem Jahr, die im Zusammenhang mit biologischen und klinischen Parametern analysiert wurden.
Die Ergebnisse der vorliegenden Arbeit beschreiben Zusammenhänge zwischen vitaler Erschöpfung sowie psychosozialen Faktoren und ausgewählten pro- und antiinflammatorischen Zytokinen bei Patienten mit kardiovaskulären Risikofaktoren.The development of heart failure is no longer regarded exclusively as a disease of the heart. In addition to classical risk factors such as age, sex, smoking, arterial hypertension, hyperlipidemia, diabetes mellitus, obesity, and physical inactivity, biological risk factors—including elevated levels of inflammatory biomarkers such as the cytokines interleukin-1β, interleukin-6, and tumor necrosis factor-α—as well as psychosocial risk factors have been described. The latter include psychological stress (acute, episodic, or chronic), personality traits such as type D personality, negative affective states, low socioeconomic status, and lack of social support. Among psychosocial risk factors, vital exhaustion represents a significant independent predictor of coronary heart disease and a relevant risk factor for cardiovascular events.
The aim of the present study was to investigate associations between vital exhaustion and selected pro- and anti-inflammatory cytokines in patients with cardiovascular risk factors.
This study represents a subproject of the MedViP study (Medical Care in General Practice), a prospective observational study conducted at the University Medical Center Göttingen. A total of 356 primary care patients with risk factors for the development of heart failure were included in the analyses and underwent comprehensive cardiological (including echocardiography) and psychometric assessment. Type D personality was assessed using the DS14 questionnaire, and vital exhaustion was assessed using the Maastricht Questionnaire. Based on the distribution of the Maastricht Questionnaire sum scores, patients with high and low levels of vital exhaustion were distinguished. In patients with and without vital exhaustion,
serum concentrations of the pro-inflammatory cytokines interleukin-1β, interleukin-6, and tumor necrosis factor-α and the anti-inflammatory cytokine interleukin-10 were determined. Analyses were based on baseline data and follow-up data obtained after one year.
In the study population, associations between vital exhaustion and selected cytokines were observed, whereas no corresponding associations were found for other cytokines. In addition, vital exhaustion was associated with psychosocial and clinical characteristics. In multivariate analyses, psychosocial factors emerged as relevant predictors of vital exhaustion, alongside sex and body mass index. Longitudinal analyses demonstrated changes in the degree of vital exhaustion over the one-year follow-up period, which were examined in relation to biological and clinical parameters.
The results of the present study describe associations between vital exhaustion, psychosocial factors, and selected pro- and anti-inflammatory cytokines in patients with cardiovascular risk factors.2026-03-2
Essays on the Economics of Behavioral and Structural Determinants of Health
No full textDiese Dissertation untersucht gesundheitsbezogene Entscheidungsprozesse im Kontext von Infektionskrankheiten. In vier empirischen Kapiteln wird aus einer mikro- und verhaltensökonomischen Perspektive Gesundheitsverhalten sowie dessen Determinanten entlang des Krankheitsverlaufs analysiert. Die Arbeit nutzt experimentelle und quasi-experimentelle Methoden und stützt sich auf eine Kombination aus Primär- und Sekundärdaten.
Kapitel 1 evaluiert die Wirksamkeit eines routinemäßigen staatlichen Programms in Togo, welches darauf abzielt, Wissen über Lepra zu erhöhen und Stigmatisierung zu reduzieren. Auf Grundlage einer randomisierten Zuweisung der Programmdurchführung auf 52 Gemeinden finden wir keinen Effekt auf das Wissen und, entgegen den Erwartungen, einen Anstieg stigmatisierender Einstellungen infolge der Kampagne. Eine Ergänzung der Intervention durch audiobasierte Geräte zur Verbesserung der Informationsvermittlung mildert diese negativen Effekte teilweise.
Kapitel 2 untersucht, ob Krankenversicherung die Inanspruchnahme von HIV-Tests erhöht. Unter Verwendung national repräsentativer Querschnittsdaten aus Ghana aus den Jahren 2014 und 2022 zeigen wir, dass Versicherungsschutz positiv mit HIV-Testen assoziiert ist. Die Ergebnisse legen nahe, dass dieser Zusammenhang über eine erhöhte Nutzung von Gesundheitseinrichtungen und durch anbieterseitig initiierte Tests vermittelt wird.
Kapitel 3 analysiert die Verhaltensauswirkungen des Lernens des eigenen HIV-Statuses auf sexuelles Risikoverhalten. Mithilfe eines Instrumentvariablenansatzes und fünfjähriger Paneldaten aus KwaZulu-Natal, Südafrika, zeigen wir, dass sexuelles Risikoverhalten bei Personen ohne feste Partnerschaft auf der intensiven Marge reduziert wird. Unabhängig davon, ob das Testergebnis positiv oder negativ ist, steigt die Kondomnutzung. Innerhalb von Partnerschaften zeigen sich Verhaltensanpassungen ausschließlich bei Personen mit einem positiven HIV-Testergebnis --- in Form erhöhter sexueller Abstinenz.
Kapitel 4 untersucht die Auswirkungen einer HIV-Behandlung auf Fehlzeiten am Arbeitsplatz unter HIV-positiven Bergarbeitern in Südafrika. Unter Verwendung eines Instrumentvariablenansatzes finden wir eine substanzielle Reduktion der Fehlzeiten - um etwa einen Tag pro Arbeiter und Monat. Darüber hinaus zeigen wir, dass die durch verringerte Fehlzeiten erzielten Kosteneinsparungen im Bergbausektor in mehreren Ländern Subsahara-Afrikas die Behandlungskosten übersteigen. In Niedriglohnsektoren sind die Einsparungen nicht ausreichend um die Behandlungskosten vollständig zu kompensieren.This dissertation examines health-related decision-making in the context of infectious diseases. Across four empirical chapters, it adopts a microeconomic and behavioral perspective to study health behavior and its determinants along the disease pathway. The dissertation employs experimental and quasi-experimental methods and draws on a combination of primary and secondary data sources.
Chapter 1 evaluates the effectiveness of a routine government program aimed at increasing knowledge and reducing stigma related to leprosy in Togo. Using randomized assignment of program implementation across 52 communities, we find no effect on knowledge and, counterintuitively, an increase in stigmatizing attitudes following the campaign. Supplementing the intervention with audio-based tools designed to enhance information retention partially mitigates these adverse effects.
Chapter 2 investigates whether health insurance coverage increases HIV testing rates. Using nationally representative cross-sectional data from Ghana in 2014 and 2022, we show that insurance coverage is positively associated with HIV testing. The results suggest that this relationship operates through increased contact with health facilities and provider-initiated testing.
Chapter 3 examines the behavioral effects of learning one’s HIV status on sexual risk behavior. Using an instrumental variable approach and five years of follow-up data from KwaZulu-Natal, South Africa, we find that sexual risk behavior decreases on the intensive margin among individuals who are not in relationships. Regardless of whether test results are positive or negative, individuals increase condom use. Within relationships, however, behavioral adjustments are observed only among individuals who receive a positive HIV result, primarily through increased sexual abstinence.
Chapter 4 assesses the labor market impact of HIV treatment initiation on worker absenteeism among South African miners living with HIV. Using an instrumental variable approach, we find a substantial reduction in absenteeism - approximately one day per worker per month. We further show that absenteeism-related cost savings exceed treatment costs in the mining sector in several sub-Saharan African countries, while such savings are insufficient to fully offset treatment costs in lower-wage sectors.2026-02-2
A retrospective analysis of 279 cases
No full textDie Anzahl an Vergiftungen durch Quetiapin ist in den letzten Jahren sowohl in Deutschland als auch in anderen Ländern gestiegen. Diese Arbeit soll einen Beitrag dazu leisten, die Bedeutung und Tragweite dieser Entwicklung besser einschätzen zu können und gegebenenfalls Empfehlungen für die Verschreibung in der Therapie und für die Behandlung von Vergiftungsfällen aussprechen zu können. Sie beruht auf einer retrospektiven Analyse mittelschwerer, schwerer und letaler Fälle mit Vergiftung durch Quetiapin, die in den Jahren 2020, 2021 und 2022 im Giftinformationszentrum-Nord (GIZ-Nord) beraten wurden. 279 Fälle entsprachen den Einschlusskriterien. Schwere und letale Verläufe kamen in 25,8 Prozent der Fälle vor und waren damit in einem deutlichen Anteil vertreten. Vergiftungen dieser Art sollten dementsprechend sehr ernst genommen und die Indikation zur Verschreibung gründlich geprüft werden. Risikokonstellationen für mittelschwere und schwere Verläufe ergaben sich aus dem durchschnittlichen Alter von 38 Jahren beziehungsweise der Altersgruppe zwischen 20 und 49 Jahren, dem weiblichen Geschlecht, der Medikamenteneinnahme zuhause, der oralen Aufnahme, suizidalen Absichten sowie psychischen Vorerkrankungen. In 64,5 Prozent der durchgeführten Ösophagogastroduodenoskopien wurden Tablettenreste geborgen, was auf einen maßgeblichen Stellenwert der Endoskopie in der Behandlung von Vergiftungsfällen hinweist und in diesem Kontext noch genauer untersucht werden sollte. Insbesondere hinsichtlich dessen, dass kein Antidot für Quetiapin bekannt ist, könnte die Endoskopie eine wesentliche Erweiterung der Therapieoptionen bei schwerwiegenden Vergiftungen durch Quetiapin darstellen.The number of cases of poisoning by quetiapine has risen in recent years, both in Germany as well as in other countries. This study aimed to contribute to a better understanding of this development and to evaluate whether there can be further recommendations to physicians prescribing quetiapine or to health services treating cases of poisoning. It is based on a retrospective analysis of moderate, severe and fatal cases of quetiapine poisoning in 2020, 2021 and 2022, documented and advised by the Giftinformationszentrum-Nord (GIZ-Nord). 279 cases met the inclusion criteria. Severe and fatal outcomes constituted 25,8% of all the cases. Risk factors for moderate and severe outcomes correlated with an age group between 20 and 49 years (average 38), with female gender, medication taken at home, oral intake, a history of suicidal intentions and a history of mental illnesses. The possibility of poisoning by quetiapine should therefore be taken seriously and the risk factors should be considered when prescribed. In 64,5% of cases where esophagogastroduodenoscopy (EGD) was used, tablet residues were recovered. This indicates the importance of endoscopy in the treatment of poisoning by quetiapine and should be investigated in more detail. Particularly in view of the fact that there is no known antidote for quetiapine, endoscopy could be a significant addition to the therapy of severe cases of poisoning by quetiapine.2026-03-2
Mechanism, regulation and structure of human pyruvate dehydrogenase complex
No full textThe human pyruvate dehydrogenase complex (hPDHc) catalyzes the irreversible conversion of pyruvate to acetyl-CoA, linking glycolysis to the citric acid cycle - a central hub of cellular metabolism. This reaction proceeds through three sequential steps catalyzed by the E1, E2, and E3 subunits, with E1 (a thiamine diphosphate-dependent enzyme) initiating the process through pyruvate decarboxylation and reductive acetylation. Despite extensive studies, the detailed reaction mechanism and regulatory control of E1 remain incompletely understood, partly due to a lack of structural evidence for key reaction intermediates. Additionally, the mechanism by which site 2 and site 3 phosphorylation inactivates E1 is not fully established.
In this thesis, we present the first crystal structures of human E1 (hE1) bound to pyruvate and a modified lipoyllysine, capturing key reaction intermediates in the wildtype enzyme. Our structural data reveal that the two active sites of hE1 exhibit asymmetry throughout catalysis, evident in both reaction intermediate distribution and loop conformations between the active sites. Furthermore, we propose that pyruvate decarboxylation is triggered in the presence of the lipoyl domain, as supported by intermediate distribution analysis and hE1 structures capturing a covalent adduct of lipoyllysine and ThDP. Combining structural and enzyme kinetics data, we suggest that H263 acts as the proton donor during reductive acetylation. MD simulations further strengthen the role of H263 as proton donor in hE1 Lipoyl domain complex. Structural and kinetic studies of site 2 and site 3 phosphomimic mutants show that site 2 phosphorylation disrupts catalytic competence, while site 3 phosphorylation reduces ThDP binding affinity, providing insights into PDHc regulation.
In the second part of this study, we present previously unrecognized regulatory mechanisms of hPDHc by the metabolites dephospho-coenzyme A and octanoyl-coenzyme A, supported by X-ray structures of the E2 and E3 subunits in complex with these molecules. For the first time, we provide structural evidence that dephospho-coenzyme A directly targets the NAD+ site of E3, while octanoyl-CoA binds within the coenzyme A channel of E2, revealing a novel mode of PDHc regulation.
Activation of hPDHc is crucial for treating metabolic disorders. In this study, we characterized the mechanism of activation of hPDHc by the synthetic molecule activators discovered in collaboration with Rutter’s lab. Our activity assays demonstrate that these effector molecules enhance hPDHc activity in both in vitro and in vivo conditions. Kinetic analyses indicate that these molecules facilitate E1-E2 binding and relieve feedback inhibition imposed by acetylCoA and octanoyl-CoA, thereby enhancing hPDHc activity. Cryo-EM structures of the E2 core
2
in complex with effector molecules reveal that these compounds bind at the lipoyl entry site, possibly influencing subunit dynamics and enzyme assembly.
These findings provide key mechanistic insights into hPDHc reaction mechanism and regulation, revealing potential therapeutic targets for metabolic disorders involving hPDHc dysfunction, such as hPDHc deficiency and lactic acidosis. By identifying small-molecule activators, this work lays the foundation for novel strategies to modulate hPDHc activity and restore metabolic balance in disease states.2026-04-2
Cytoskeletal networks in living cells under strain
No full textThe eukaryotic cytoskeleton consists of three types of filamentous proteins: actin filaments, microtubules and intermediate filaments (IFs). In contrast to actin and tubulin, IF proteins are expressed in a cell-type specific manner, and keratins are found in epithelial cells. In certain cell types, the keratins form a layer close to the membrane which may be referred to as an “IF-cortex”. It is hypothesized that this IF-cortex arranges with radial bundles in a “rim-and-spokes” structure in epithelia. Based on this hypothesis, IFs and actin filaments might add complementary mechanical properties to the cortex. It was previously shown that single IFs in vitro remain undamaged at high strains. We now ask the question of whether this unique force-extension behavior of single IFs is also relevant in the filament network within a cell. In this thesis, we combine cell stretching experiments on MDCK II wild-type and keratin-deficient cells with atomic force microscopy to measure the viscoelastic properties and determine the role of keratin for the cell cortex. Furthermore, we compare the influence of different stretching modes on the cell and nucleus shape to investigate the mechanical link from the periphery of the cell to the nucleus through the keratin network. Additionally, we stretch three-dimensional MDCK II cysts to high strains providing another point of view by enabling imaging of the cross section of the cells. Finally, we compare the structure of the actin and keratin networks in the cells under strain. We find that keratin-deficient cells compensate for the missing keratin, but are nevertheless very sensitive to external strain, whereas the intricate interplay between the actin and keratin cortices provides a protective mechanism to the cell by preserving the mechanical state and cell stability. Hence, this thesis provides intriguing insights on cells under strain from a mechanical to a structural level.2026-05-2
Improved genome and population genomics unveil the winter hardiness and breeding signatures in faba bean
No full textDie Ackerbohne (Vicia faba L.) ist eine global bedeutende Hülsenfrucht, die aufgrund ihres hohen Proteingehalts, ihrer stickstoffbindenden Fähigkeit und ihrer Anpassungsfähigkeit an diverse agroökologische Bedingungen geschätzt wird. Dennoch wird ihre breitere Nutzung durch zentrale Herausforderungen eingeschränkt, darunter ihre Anfälligkeit für Winterfrostschäden, begrenzte genomische Ressourcen und das Fehlen effizienter Züchtungswerkzeuge. Von besonderer Bedeutung ist die Verbesserung der Winterhärte der Ackerbohne, um ihren Nutzen in gemäßigten und mediterranen Regionen zu steigern, wo Winterkultivare deutliche Ertragsvorteile gegenüber Frühjahrstypen aufweisen.
Diese Dissertation konzentriert sich auf die Identifizierung und Charakterisierung der genetischen Determinanten der Winterhärte bei Ackerbohnen. Unter Nutzung eines neu entwickelten hochwertigen chromosomenweiten Genomsequenzassemblies kombiniert diese Studie Genom-Resequenzierung, phänotypische Analysen und genomweite Assoziationsstudien (GWAS), um die genetische Grundlage der Frosttoleranz zu entschlüsseln. Ein Hauptlocus, der mit Winterhärte assoziiert ist, wurde identifiziert, wobei die höchstassoziierte Variante den größten Teil der phänotypischen Variation erklärt und eine klare Differenzierung zwischen Winter- und Sommerackerbohnen ermöglicht. Zusätzliche genetische Signale innerhalb des Winter-Genpools wurden aufgedeckt, die tiefere Einblicke in die Anpassung von Winterkultivaren bieten und wertvolle Ziele für Züchtungsprogramme darstellen.
Neben der Winterhärte trägt die vorliegende Studie zum übergeordneten Ziel bei, Züchtungswerkzeuge für Ackerbohnen weiterzuentwickeln. Um die effiziente Charakterisierung genetischer Diversität und Merkmalskartierung zu ermöglichen, wurde ein optimiertes Genotypisierung-durch-Sequenzierung (GBS)-Protokoll entwickelt. Die Kombination der Restriktionsenzyme ApeKI/MseI erwies sich als optimal für Ackerbohnen und lieferte hochwertige genomische Bibliotheken, die für genomische Regionen angereichert sind. Dieses Protokoll ermöglichte die Erstellung einer genetischen Karte mittels einer Poopulation rekombinanter Inzuchtlinien (RIL) und die Identifizierung wichtiger quantitativer Trait Loci (QTLs) für Hilum-Farbe, Pflanzenhöhe, Blütezeit und Hülsen-zahl. Die Ergebnisse unterstreichen den Nutzen von GBS zur Erschließung der ungenutzten genetischen Vielfalt der Ackerbohne für die Züchtung.
Zusammen bilden diese Fortschritte einen umfassenden Rahmen für die genetische Verbesserung der Ackerbohne. Die Identifizierung von Winterhärte-Loci adressiert eine kritische agronomische Herausforderung, während die optimierte Genotypisierungsplattform Forschung und Züchtung beschleunigt. Diese Dissertation legt den Grundstein für zukünftige Studien zur Verbesserung der Resilienz, Produktivität und Nachhaltigkeit der Ackerbohne als globale Proteinpflanze in diversen agroökologischen Systemen.Faba bean (Vicia faba L.) is a globally significant cool-season legume valued for its high protein content, nitrogen-fixing ability, and adaptability to diverse agroecological conditions. However, its broader adoption is constrained by key challenges, including its susceptibility to winter kill, limited genomic resources, and the lack of efficient breeding tools. Among these, improving the winter hardiness of faba bean is critical for enhancing its utility in temperate and Mediterranean regions, where winter cultivars exhibit substantial yield advantages over spring types.
This dissertation focuses on identifying and characterizing the genetic determinants of winter hardiness in faba bean. By leveraging a newly developed high-quality chromosome-scale genome assembly, this study combines genome resequencing, phenotypic analysis, and genome-wide association studies (GWAS) to dissect the genetic basis of frost tolerance. A major locus associated with winter hardiness was identified, with the most strongly associated variant explaining most of the phenotypic variation and clearly differentiating winter and spring faba beans. Additional genetic signals within the winter gene pool were uncovered, providing further insights into the adaptation of winter cultivars and offering valuable targets for breeding programs.
In addition to addressing winter hardiness, this research contributes to the broader goal of advancing faba bean breeding tools. To facilitate the efficient characterization of genetic diversity and trait mapping, an optimized genotyping-by-sequencing (GBS) protocol was developed. The combination of restriction enzymes ApeKI/MseI was identified as optimal for faba bean, yielding high-quality genomic libraries enriched for genomic space. This protocol enabled the construction of a genetic map using a recombinant inbred line (RIL) population and the identification of key quantitative trait loci (QTLs) for hilum color, plant height, flowering time, and pod number per plant. The findings demonstrate the utility of GBS in harnessing the untapped genetic diversity of faba bean for breeding purposes.
Together, these advancements establish a comprehensive framework for the genetic improvement of faba bean. The identification of winter hardiness loci addresses a critical agronomic challenge, while the optimized genotyping platform accelerates research and breeding efforts. This dissertation lays the groundwork for future studies aimed at improving the resilience, productivity, and sustainability of faba bean as a global protein crop in diverse agroecological systems.2026-07-0