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Characterization and Engineering of Basidiomycota-derived Lipases: Structural Insights and Optimization of hydrolytic Selectivity and Stability
(2026) Henrich, Lea
In the production of piquant cheese varieties, such as Feta and Provolone, pregastric esterases (PGEs) are commonly used to generate characteristic piquant flavor profiles. These enzymes hydrolyze triglycerides present in milk fat during cheese ripening, releasing volatile free fatty acids that contribute to the distinctive aroma of these cheeses. These enzymes are traditionally derived from animal origin, and their use conflicts with the requirements for foods labeled as vegetarian, kosher, or halal. This interference raises the demand for enzymes with similar activity but of different origin. A lipase from the basidiomycete Pleurotus citrinopileatus (PCI_Lip) has been identified as a promising fungal substitute. To further adapt its catalytic properties to those of PGEs, a semi-rational protein engineering approach was applied to modify the chain length specificity for cheese production. Key residues influencing substrate specificity were identified in silico based on an AlphaFold3 model of PCI_Lip. In three rounds of mutagenesis, a small but smart library was generated. The library was screened for mutants with increased release of short to medium chain fatty acids and reduced release of long chain fatty acids. Alteration of substrate specificity was proven by changes in kinetic values compared to the wild type (WT) enzyme. Mutants with improved hydrolysis profiles were used for cheese production. The analysis of volatile free fatty acids and sensory evaluation showed that some mutants are suitable substitutes for PGEs. For a comprehensive characterization, stability, effects of immobilization, and regioselectivity of PCI_Lip were examined. The lipase was found to be highly thermostable with the potential to further increase its stability through suitable buffers and additives, as well as by immobilization. In the hydrolysis of triglycerides, the enzyme prefers the outer ester positions, therefore being characterized as an sn 1,3-selective lipase. Investigations on further Basidiomycota-derived lipases gave deeper insights into the structure and function of these enzymes. The characterization of a Pleurotus sapidus lipase (PSA_Lip) showed its thermostability and alkali tolerance. Besides activity on fatty acid esters, PSA_Lip was also active on ferulic acid esters. Sequence alignments with other hydrolases revealed that it does not belong to the big group of the α/β-hydrolases but displays a unique SGNH-like motif in the active site of the enzyme. The comparison of PCI_Lip with a lipase from Phlebia centrifuga (PCE_Lip) emphasized the role of the lid domain. A lack of this domain could be connected to reduced solubility and activity. The present work provides a comprehensive characterization of PCI_Lip and demonstrates strategies to optimize its catalytic properties and stability for various industrial applications. Through a combination of structural analysis, protein engineering, and immobilization techniques, the enzyme can be adapted to different parameters such as substrate selectivity and thermostability, making it applicable in various biotechnological applications. Comparative results with other Basidiomycota-derived lipases contribute to a better understanding of fungal hydrolases.
QCD Interactions in Hadrons
(2026) Hagel, Stephan
In this work, we use functional methods, namely the formalism of Dyson-Schwinger and Bethe-Salpeter equations (DSEs and BSEs) and the n-particle irreducible effective action formalism, to describe properties of quarks and mesons as bound states of quarks and antiquarks. We explore how truncations of these equations relate to the phenomenon of dynamical chiral symmetry breaking in quantum chromodynamics (QCD). Within the rainbow-ladder truncation, we explore, how different effective running couplings give rise to interaction potentials and how they relate to Regge behaviour in the resulting meson spectra. In particular, we explore spectra of light mesons, kaons, ss-states, charmonia and bottomonia with total angular momenta up to J ≤ 5 within this truncation. Furthermore, we use a universal kernel-first truncation to construct a quark self-energy from a quark-antiquark scattering kernel, such that the axialvector Ward-Takahashi identity and thus the effects of dynamical chiral symmetry breaking are conserved. In this truncation we solve the equations of motion for the quark propagator and quark-photon vertex to investigate, whether the vector Ward- Takahashi identity is fulfilled. Starting from a three particle irreducible effective action, we use this method to extract exploratory spectra of light mesons, kaons and ss-states with total angular momentum up to J ≤ 4 and sketch a way to expand this framework to heavier quarkonia and heavy-light mesons.
Bedeutung von BACE1 und UCP2 für den Metabolismus des Skelettmuskels
(2026) Richter, Clara Christiane
Sowohl eine gestörte Glukose-Homöostase als auch eine Insulinresistenz fördern die Entstehung von Typ-2-Diabetes und gelten zudem als Risikofaktor von Alzheimer Erkrankungen. Forschungsergebnisse haben gezeigt, dass BACE1 (β-site of APP cleaving enzyme 1) nicht nur an der Entstehung neurodegenerativer Erkrankungen wie Alzheimer beteiligt ist, sondern ebenfalls einen wichtigen Regulator in der Glukose-Homöostase des Skelettmuskels darstellt. (Meakin et al. 2012) UCP2 ist ein mitochondrialer Entkoppler und könnte als molekularer Schalter des Energiestoffwechsels fungieren (Kutsche et al. 2020), wobei die Interaktion zwischen BACE1 und UCP2 bislang unzureichend verstanden ist. Ziel dieser Promotionsarbeit war es daher, die Hypothese zu überprüfen, dass eine Verbesserung der skelettmuskulären mitochondriellen Effizienz mittels UCP2-Defizienz die Expression von BACE1 senkt und dadurch zur Glukosehomöostase beiträgt. Hierzu wurden Expressionsanalysen von UCP2 und BACE1 in metabolisch unterschiedlichen Skelettmuskeln (M. gastrocnemius, M. soleus) durchgeführt. Ergänzend erfolgten Proteinanalysen (Western Blot), Bestimmungen des oxidativen Stresses mittels DHE-Färbungen sowie histologische Untersuchungen der Skelettmuskel-Proben von UCP2-defizienten und hypertensiven Ratten. Zusätzlich kamen siRNA-transfizierte AC16-Zellen für in-vitro-Analysen UCP2-abhängiger Effekte zum Einsatz. Die Ergebnisse zeigten, dass chronisch hypertensive SHR-Tiere eine verminderte Expression von UCP2 sowie antioxidativen und metabolischen Enzymen bei gleichzeitig erhöhtem oxidativem Stress aufwiesen, was auf eine maladaptive Regulation der redox- und metabolischen Homöostase unter chronisch hypertensiven Bedingungen hindeutet. Im Gegensatz dazu führte eine Deletion von UCP2 zu muskelfasertypspezifischen Veränderungen im Energiestoffwechsel und der ROS-Homöostase. Besonders ausgeprägt waren diese Effekte im überwiegend glykolytisch arbeitenden M. gastrocnemius (Schmalbruch 1967), was mit seiner unter physiologischen Bedingungen höheren basalen UCP2-Expression im Einklang steht. Zudem deuten die Ergebnisse darauf hin, dass UCP2 zur metabolischen Spezifizierung von Muskelfasertypen beiträgt, da es infolge der UCP2-Deletion zur metabolischen Annäherung beider untersuchter Muskeln kam. Unter Bedingungen einer zusätzlichen L-Name induzierten Hypertonie zeigte sich eine muskelfaserspezifische Reduktion der BACE1-Proteinexpression, was auf posttranskriptionelle Regulationsmechanismen hinweist, die im Sinne eines vaskulär metabolischen „Double Hit“ interpretiert werden können. Die in-vitro Analysen konnten diese Befunde insofern bestätigen, dass eine Suppression der UCP2-Translation in AC16-Zellen mit einer Herabregulation von BACE1 auf mRNA- und Proteinebene assoziiert war. Die Übereinstimmung der Ergebnisse über normo- und hypertensive Tiermodelle sowie über in-vivo und in-vitro Ansätze hinweg spricht für eine UCP2-abhängige Modulation redoxabhängiger und metabolischer Regulationsmechanismen. Dabei deuten die Befunde auf eine indirekte Einbindung von UCP2 in die ROS- und NO-abhängigen Signalachsen hin. Die zugrundeliegenden molekularen Mechanismen konnten im Rahmen dieser Arbeit jedoch nicht abschließend geklärt werden. Zudem ist zu berücksichtigen, dass die vorliegenden Ergebnisse überwiegend auf strukturellen Analysen beruhen, sodass sich keine direkten Rückschlüsse auf funktionelle Parameter wie der Insulinsensitivität, der Glukoseaufnahme oder der mitochondriellen Effizienz ziehen lassen. Zusammenfassend unterstreichen die Ergebnisse dieser Promotionsarbeit die Relevanz von UCP2 für die Regulation der Energie- und ROS-Homöostase des Skelettmuskels. Die Arbeitshypothese, dass eine UCP2-Defizienz mit einer Reduktion von BACE1 korreliert und dadurch zur Glukosehomöostase beiträgt, konnte teilweise bestätigt werden und erwies sich als muskelfaser- und kontextabhängig. Die identifizierte muskelfasertypspezifische UCP2-BACE1-Korrelation und deren Abhängigkeit von vaskulären und redoxabhängigen Faktoren könnten zukünftig für das Verständnis von metabolischen und degenerativen Erkrankungen von klinischer und therapeutischer Relevanz sein. Weitere Studien sind erforderlich, um die zugrunde liegenden molekularen Signalwege zu charakterisieren und deren medizinisches Potential zu evaluieren.
Functional Characterization of RBPMS-Positive Ribonucleoprotein Granules in Cardiomyocytes
(2025) Eibach, Yvonne
RNA-Binding Protein with Multiple Splicing (RBPMS) is a member of the RNA-binding protein family and localizes to both the nucleus and cytoplasm. In addition to its function in transcriptional co-regulation, RBPMS may also play a role in the cytoplasm for regulation of mRNA stability, transport and recruitment into cytoplasmic granules. Although aberrant RNA processing and dysregulated ribonucleoprotein assemblies are known to contribute to the pathogenesis of neurodegenerative diseases, the composition of RNPs and their (patho)physiological roles in the cardiovascular system remain poorly understood. In this study, I investigated the role of RBPMS in regulating RNP assemblies and their dynamics in response to cardiovascular stress. I developed an optimized protocol for APEX2-mediated proximity labeling in murine HL-1 cardiomyocytes. The approach enabled high-resolution mapping of the cardiac stress granule proteome, revealing a previously uncharacterized complexity and tissue-specific adaptation in SG composition. Interestingly, proximity interactions between SG components in unstressed cells point to pre-assembly mechanisms facilitating rapid SG formation during stress. Interactome analysis of GFP-tagged RBPMSA and RBPMSB isoforms identified key partners involved in DNA damage response and phase separation. Additionally, RBPMSB was found to interact with mitochondrial proteins, potentially linking phase-separated compartments and cellular organelles in stress responses. Upon doxorubicin-induced DNA damage, RBPMS initially translocates to SGs in the cytoplasm and subsequently forms nuclear puncta, coinciding with chromatin reorganization. These observations suggest that RBPMS contributes to both acute and long-term stress adaptation, potentially modulating DDR and chromatin phase transitions. Importantly, doxorubicin-induced genotoxic stress alters the abundance and localization of RBPMS isoforms, disrupts splicing factor dynamics, and impairs mitochondrial biogenesis, collectively contributing to cardiotoxicity. The identification of age-dependent differences in alternative splicing regulation further highlights the complexity of the cardiac stress response. The interplay between RBPMS, splicing factors, and SGs emerged as a critical determinant of cardiomyocyte survival and function under stress. Taken together, my findings establish RBPMS as a regulator of RNP assembly and cellular stress responses in cardiovascular disease, highlighting its potential as a therapeutic target and providing a foundation for further investigation into its role in DNA damage response and stress-related cardiac pathologies.
Direct and time-lagged impact of heat stress on primary and functional traits in dairy and dual-purpose cattle at different “omics” levels
(2025) Halli, Kathrin
This habilitation thesis focuses on the impact of acute and maternal heat stress (HS) on production and functional traits and on metabolism of dairy and dual-purpose cattle and offspring considering the temperature humidity index (THI) as environmental descriptor. In this context, the comprehensive introduction, the discussion and the 10 original research papers address the different “omics” levels “phenomics” (phenotypic association studies), “genomics” (genetic parameters, genome wide association studies, gene annotations), “epigenomics” (DNA methylation profiles), “metabolomics” (metabolite concentrations) and “transcriptomics” (gene expression levels). The original research paper 1 focused on the impact of acute HS during the early life of dual-purpose calves on their weight development and of acute and time-lagged HS on fertility traits of dams under pasture-based conditions. Across-generation effects of maternal HS during late gestation are also content of this paper. Our results indicated acute and time-lagged HS effects and limited acclimatization processes to heat, especially during the cooler spring and autumn months. Similarly, the original research paper 2 focused on across-generation effects of maternal HS during late gestation on production and functional traits, however, in dairy cattle. We found an unfavorable impact of HS from the dry period on time-lagged performances in offspring on most primary and functional traits, even on longevity. Metabolic aspects of acute HS during late gestation on German dairy cows and of across-generation effects of maternal HS on female progeny are content of original research paper 3 and 4. The results showed altered concentrations of different plasma metabolites from different biochemical classes between animals suffering from direct or maternal HS or not with potential to serve as biomarkers for HS. The impact of time-lagged HS during late gestation and postpartum on genetic parameter estimates for birth weight and weight gain in dual-purpose calves is presented in original research paper 5. Additive genetic variances and heritabilities of all traits increased under challenging climatic conditions indicating a higher selection response in beef cattle traits under HS conditions. Genetic correlations substantially smaller than 0.80 indicated genotype by climate interactions, proven by substantial alterations of sire EBVs for production traits in dependency of time-lagged climatic alterations. In a similar context, maternal HS during late gestation was analyzed for its impact on production and functional traits in dairy cattle in the original research paper 6. Birth weights and birth weight genetic parameters of dual-purpose and dairy calves in context of time-lagged effects due to HS during late gestation and genotype by climate interactions for birth weight are also presented in this paper. The results showed detrimental effects of prenatal HS on birth weight in offspring. Direct and maternal heritabilities for calf birth weight were independent from prenatal HS effects. No genotype by climate interactions on calf birth weight were identified for maternal genetic effects. We concluded that the decline in birth weight is more likely caused by maternal permanent environmental effects instead of genetics, why we suggested to avoid HS during the dry period of dams. The original research paper 7 focused on direct and maternal genetic effects and the annotation of potential candidate genes for weight and meat quality traits of dual-purpose cattle, raised under outdoor climatic conditions. For intramuscular fat content (IMF), we estimated quite high heritabilities and we identified a quite strong maternal genetic component on weight traits as well as on IMF. This is probably due to the intensive calf-cow relationship in the outdoor suckler system. We suggest the application of models with maternal genetic effect in genetic evaluations, and we recommended to develop a breeding goal including IMF for the used dual-purpose cattle breed. Direct and maternal genetic effects, and maternal genetic sensitivity on prenatal HS for calf diseases and corresponding genomic loci in German Holsteins were analyzed in the original research paper 8. Genotype by climate interactions for calf diseases in dairy calves, suffering from prenatal HS were analyzed. Our results showed that HS, particularly during the last week of gestation, contributed to increased calf disease incidence. Maternal heritabilities for pneumonia increased with increasing heat. We identified 30 suggestive and 2 significant SNPs from the GWAS and 43 genes, annotated as potential candidate genes. Based on these genes, 3 biological processes were inferred, indicating association between the genetics of prenatal HS mechanisms and immune physiology and disease resistance mechanisms. Original research paper 9 addressed the estimation of genotype by time-lagged HS variance components for milk production traits of the offspring generation, and main and interaction SNP-marker effects for maternal HS during late pregnancy. The results indicated quite small effects of HS during late pregnancy on the test day and lactation milk production traits. Genotype by HS interaction variances varied, depending on the week with in-utero HS. Eight out of 31 suggestive SNPs affect cattle functionality, indicating a possible favorable correlation between functional traits and heat tolerance. Two biological processes contributing to immune response mechanisms, were inferred with suggestive interaction effects. In original research paper 10, we studied HS effects on 3 different “omics features” which were methylations, gene expressions and metabolic pattern from a direct perspective in pregnant cows and from an indirect time-lagged intergenerational perspective in offspring. Differences in DNA methylation between maternally heat stressed and not heat stressed calves were proven in this paper, supporting the hypothesis of an epigenetic percentage, explaining across generation effects of HS. With regard to the intergenerational perspective, transcriptomic and metabolic profiles showed obvious clustering between dams and calves. In the context of HS separation, gene expression and methylation data clearly separated HS and control groups. However, in the multi-omics analysis, methylation information was the least important omics tier to classify dams in terms of direct HS, when compared to the other omics datasets. Overall, the studies of this habilitation thesis present important findings for future research studies regarding heat stress mitigation strategies in cattle under German climatic conditions and breeding programs in context of heat stress resistance.