@phdthesis{Eckert2023,
  author    = {Eckert, Ina-Nathalie},
  title     = {Molecular markers of myeloid-derived suppressor cells and their functional role for homing and in disease models in mice},
  doi       = {10.25972/OPUS-31997},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-319974},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {MDSCs are suppressive immune cells with a high relevance in various pathologies including cancer, autoimmunity, and chronic infections. Surface marker expression of MDSCs resembles monocytes and neutrophils which have immunostimulatory functions instead of suppressing T cells. Therefore, finding specific surface markers for MDSCs is important for MDSC research and therapeutic MDSC manipulation. In this study, we analyzed if the integrin VLA-1 has the potential as a novel MDSC marker. VLA-1 was expressed by M-MDSCs but not by G-MDSCs as well as by Teff cells. VLA-1 deficiency did not impact iNOS expression, the distribution of M-MDSC and G-MDSC subsets, and the suppressive capacity of MDSCs towards na{\"i}ve and Teff cells in vitro. In mice, VLA-1 had no effect on the homing capability of MDSCs to the spleen, which is a major reservoir for MDSCs. Since the splenic red pulp contains collagen IV and VLA-1 binds collagen IV with a high affinity, we found MDSCs and Teff cells in this area as expected. We showed that T cell suppression in the spleen, indicated by reduced T cell recovery and proliferation as well as increased apoptosis and cell death, partially depended on VLA-1 expression by the MDSCs. In a mouse model of multiple sclerosis, MDSC injection prior to disease onset led to a decrease of the disease score, and this effect was significantly reduced when MDSCs were VLA-1 deficient. The expression of Sema7A by Teff cells, a ligand for VLA-1 which is implicated in negative T cell regulation, resulted in a slightly stronger Teff cell suppression by MDSCs compared to Sema7A deficient T cells. Live cell imaging and intravital 2-photon microscopy showed that the interaction time of MDSCs and Teff cells was shorter when MDSCs lacked VLA 1 expression, however VLA-1 expression had no impact on MDSC mobility. Therefore, the VLA-1-dependent interaction of MDSC and Teff cells on collagen IV in the splenic red pulp is implicated MDSC-mediated Teff cell suppression.},
  subject      = {Immunologie},
  language  = {en}
}
@article{LibreSeisslerGuerreroetal.2021,
  author    = {Libre, Camille and Seissler, Tanja and Guerrero, Santiago and Batisse, Julien and Verriez, C{\´e}dric and Stupfler, Benjamin and Gilmer, Orian and Cabrera-Rodriguez, Romina and Weber, Melanie M. and Valenzuela-Fernandez, Agustin and Cimarelli, Andrea and Etienne, Lucie and Marquet, Roland and Paillart, Jean-Christophe},
  title     = {A conserved uORF regulates APOBEC3G translation and is targeted by HIV-1 Vif protein to repress the antiviral factor},
  series = {Biomedicines},
  volume    = {10},
  journal   = {Biomedicines},
  number    = {1},
  issn      = {2227-9059},
  doi       = {10.3390/biomedicines10010013},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-252147},
  year      = {2021},
  abstract  = {The HIV-1 Vif protein is essential for viral fitness and pathogenicity. Vif decreases expression of cellular restriction factors APOBEC3G (A3G), A3F, A3D and A3H, which inhibit HIV-1 replication by inducing hypermutation during reverse transcription. Vif counteracts A3G at several levels (transcription, translation, and protein degradation) that altogether reduce the levels of A3G in cells and prevent its incorporation into viral particles. How Vif affects A3G translation remains unclear. Here, we uncovered the importance of a short conserved uORF (upstream ORF) located within two critical stem-loop structures of the 5′ untranslated region (5′-UTR) of A3G mRNA for this process. A3G translation occurs through a combination of leaky scanning and translation re-initiation and the presence of an intact uORF decreases the extent of global A3G translation under normal conditions. Interestingly, the uORF is also absolutely required for Vif-mediated translation inhibition and redirection of A3G mRNA into stress granules. Overall, we discovered that A3G translation is regulated by a small uORF conserved in the human population and that Vif uses this specific feature to repress its translation.},
  language  = {en}
}
@phdthesis{Somody2023,
  author    = {Somody, Joseph Christian Campbell},
  title     = {Leveraging deep learning for identification and structural determination of novel protein complexes from \(in\) \(situ\) electron cryotomography of \(Mycoplasma\) \(pneumoniae\)},
  doi       = {10.25972/OPUS-31344},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-313447},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {The holy grail of structural biology is to study a protein in situ, and this goal has been fast approaching since the resolution revolution and the achievement of atomic resolution. A cell's interior is not a dilute environment, and proteins have evolved to fold and function as needed in that environment; as such, an investigation of a cellular component should ideally include the full complexity of the cellular environment. Imaging whole cells in three dimensions using electron cryotomography is the best method to accomplish this goal, but it comes with a limitation on sample thickness and produces noisy data unamenable to direct analysis. This thesis establishes a novel workflow to systematically analyse whole-cell electron cryotomography data in three dimensions and to find and identify instances of protein complexes in the data to set up a determination of their structure and identity for success. Mycoplasma pneumoniae is a very small parasitic bacterium with fewer than 700 protein-coding genes, is thin enough and small enough to be imaged in large quantities by electron cryotomography, and can grow directly on the grids used for imaging, making it ideal for exploratory studies in structural proteomics. As part of the workflow, a methodology for training deep-learning-based particle-picking models is established. As a proof of principle, a dataset of whole-cell Mycoplasma pneumoniae tomograms is used with this workflow to characterize a novel membrane-associated complex observed in the data. Ultimately, 25431 such particles are picked from 353 tomograms and refined to a density map with a resolution of 11 {\AA}. Making good use of orthogonal datasets to filter search space and verify results, structures were predicted for candidate proteins and checked for suitable fit in the density map. In the end, with this approach, nine proteins were found to be part of the complex, which appears to be associated with chaperone activity and interact with translocon machinery. Visual proteomics refers to the ultimate potential of in situ electron cryotomography: the comprehensive interpretation of tomograms. The workflow presented here is demonstrated to help in reaching that potential.},
  subject      = {Kryoelektronenmikroskopie},
  language  = {en}
}
@phdthesis{Lueffe2023,
  author    = {L{\"u}ffe, Teresa Magdalena},
  title     = {Behavioral and pharmacological validation of genetic zebrafish models for ADHD},
  doi       = {10.25972/OPUS-25716},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-257168},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2023},
  abstract  = {Attention-deficit/hyperactivity disorder (ADHD) is the most prevalent neurodevelopmental disorder described in psychiatry today. ADHD arises during early childhood and is characterized by an age-inappropriate level of inattention, hyperactivity, impulsivity, and partially emotional dysregulation. Besides, substantial psychiatric comorbidity further broadens the symptomatic spectrum. Despite advances in ADHD research by genetic- and imaging studies, the etiopathogenesis of ADHD remains largely unclear. Twin studies suggest a heritability of 70-80 \% that, based on genome-wide investigations, is assumed to be polygenic and a mixed composite of small and large, common and rare genetic variants. In recent years the number of genetic risk candidates is continuously increased. However, for most, a biological link to neuropathology and symptomatology of the patient is still missing. Uncovering this link is vital for a better understanding of the disorder, the identification of new treatment targets, and therefore the development of a more targeted and possibly personalized therapy. The present thesis addresses the issue for the ADHD risk candidates GRM8, FOXP2, and GAD1. By establishing loss of function zebrafish models, using CRISPR/Cas9 derived mutagenesis and antisense oligonucleotides, and studying them for morphological, functional, and behavioral alterations, it provides novel insights into the candidate's contribution to neuropathology and ADHD associated phenotypes. Using locomotor activity as behavioral read-out, the present work identified a genetic and functional implication of Grm8a, Grm8b, Foxp2, and Gad1b in ADHD associated hyperactivity. Further, it provides substantial evidence that the function of Grm8a, Grm8b, Foxp2, and Gad1b in activity regulation involves GABAergic signaling. Preliminary indications suggest that the three candidates interfere with GABAergic signaling in the ventral forebrain/striatum. However, according to present and previous data, via different biological mechanisms such as GABA synthesis, transmitter release regulation, synapse formation and/or transcriptional regulation of synaptic components. Intriguingly, this work further demonstrates that the activity regulating circuit, affected upon Foxp2 and Gad1b loss of function, is involved in the therapeutic effect mechanism of methylphenidate. Altogether, the present thesis identified altered GABAergic signaling in activity regulating circuits in, presumably, the ventral forebrain as neuropathological underpinning of ADHD associated hyperactivity. Further, it demonstrates altered GABAergic signaling as mechanistic link between the genetic disruption of Grm8a, Grm8b, Foxp2, and Gad1b and ADHD symptomatology like hyperactivity. Thus, this thesis highlights GABAergic signaling in activity regulating circuits and, in this context, Grm8a, Grm8b, Foxp2, and Gad1b as exciting targets for future investigations on ADHD etiopathogenesis and the development of novel therapeutic interventions for ADHD related hyperactivity. Additionally, thigmotaxis measurements suggest Grm8a, Grm8b, and Gad1b as interesting candidates for prospective studies on comorbid anxiety in ADHD. Furthermore, expression analysis in foxp2 mutants demonstrates Foxp2 as regulator of ADHD associated gene sets and neurodevelopmental disorder (NDD) overarching genetic and functional networks with possible implications for ADHD polygenicity and comorbidity. Finally, with the characterization of gene expression patterns and the generation and validation of genetic zebrafish models for Grm8a, Grm8b, Foxp2, and Gad1b, the present thesis laid the groundwork for future research efforts, for instance, the identification of the functional circuit(s) and biological mechanism(s) by which Grm8a, Grm8b, Foxp2, and Gad1b loss of function interfere with GABAergic signaling and ultimately induce hyperactivity.},
  language  = {en}
}
@phdthesis{Ferretti2022,
  author    = {Ferretti, Pamela},
  title     = {\(Clostridioides\) \(difficile\) beyond the disease-centred perspective: Beneficial properties in healthy infants and over-diagnosis in diseased adults identified by species- and SNV-based metagenomic analysis},
  doi       = {10.25972/OPUS-25417},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-254170},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Clostridioides difficile is a bacterial species well known for its ability to cause C. difficile infection (also known as CDI). The investigation of the role of this species in the human gut has been so far dominated by a disease-centred perspective, focused on studying C. difficile in relation to its associated disease. In this context, the first aim of this thesis was to combine publicly available metagenomic data to analyse the microbial composition of stool samples from patients diagnosed with CDI, with a particular focus on identifying a CDI-specific microbial signature. However, similarly to many other bacterial species inhabiting the human gut, C. difficile association with disease is not valid in absolute terms, as C. difficile can be found also among healthy subjects. Further aims of this thesis were to 1) identify potential C. difficile reservoirs by screening a wide range of habitats, hosts, body sites and age groups, and characterize the biotic context associated with C. difficile presence, and 2) investigate C. difficile within-species diversity and its toxigenic potential across different age groups. The first part of the thesis starts with the description of the concepts and definitions used to identify bacterial species and within-species diversity, and then proceeds to provide an overview of the bacterial species at the centre of my investigation, C. difficile. The first Chapter includes a detailed description of the discovery, biology and physiology of this clinically relevant species, followed by an overview of the diagnostic protocols used in the clinical setting to diagnose CDI. The second part of the thesis describes the methodology used to investigate the questions mentioned above, while the third part presents the results of such investigative effort. I first show that C. difficile could be found in only a fraction of the CDI samples and that simultaneous colonization of multiple enteropathogenic species able to cause CDI-like clinical manifestations is more common than previously thought, raising concerns about CDI overdiagnosis. I then show that the CDIassociated gut microbiome is characterized by a specific microbial signature, distinguishable from the community composition associated with non-CDI diarrhea. Beyond the nosocomial and CDI context, I show that while rarely found in adults, C. difficile is a common member of the infant gut microbiome, where its presence is associated with multiple indicators typical of a desirable healthy microbiome development. In addition, I describe C. difficile extensive carriage among asymptomatic subjects, of all age groups and a potentially novel clade of C. difficile identified exclusively among infants. Finally, I discuss the limitations, challenges and future perspectives of my investigation.},
  language  = {en}
}
@phdthesis{Amelingmeier2022,
  author    = {Amelingmeier, Florian},
  title     = {Identifizierung und Untersuchung TOP-mRNA - bindender Faktoren},
  doi       = {10.25972/OPUS-28923},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-289231},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Im Zellkern eukaryotischer Zellen werden Gene in mRNAs transkribiert, welche umfangreich prozessiert und aus dem Zellkern exportiert werden. Im Zytoplasma erfolgt die Translation der mRNAs in Proteine, ein Prozess, welcher viel Energie ben{\"o}tigt und daher mittels vielf{\"a}ltiger Mechanismen streng reguliert wird. Ein Beispiel hierf{\"u}r stellt die Klasse der TOP-mRNAs dar, eine RNA-Spezies, welche haupts{\"a}chlich Transkripte von Genen umfasst, die selbst in die Translation involviert sind. Die prominentesten Vertreter dieser Klasse sind die Proteine der kleinen und großen ribosomalen Untereinheiten. TOP-mRNAs zeichnen sich durch ein gemeinsames Sequenz-Motiv am Anfang Ihrer 5'-UTR aus, welches aus einem Pyrimidinstrang besteht und unmittelbar nach dem Cap mit einem Cytosin beginnt. Dieses allen TOP-RNAs gemeinsame Motiv erm{\"o}glicht die zeitgleiche Translationskontrolle dieser RNA-Klasse. So kann die Translation der TOP-mRNAs unter Stressbedingungen wie z.B. N{\"a}hrstoffmangel koordiniert inhibiert werden, wodurch Energie eingespart wird. Bereits lange wird nach einem Regulator gesucht, der an dieses TOP-Motiv bindet und die koordinierte Regulation erm{\"o}glicht. Man kann sich hier einen Inhibitor oder auch einen Aktivator vorstellen. Verschiedene Proteine wurden bereits in Erw{\"a}gung gezogen. In dieser Arbeit wurde das Protein TIAR mittels Massenspektrometrie als TOP-interagierender Faktor identifiziert und dessen Bindungseigenschaften mit dem TOP-Motiv durch Shift Assays untersucht. Hierbei konnten Minimalkonstrukte verschiedener Organismen sowie RNA-TOP - Sequenzen identifiziert werden, welche sich f{\"u}r Strukturanalysen eignen w{\"u}rden. Als weiterer TOP-interagierender Faktor wurde {\"u}ber verschiedene sequenzielle Reinigungsschritte das Protein 14-3-3ε identifiziert. Weiterhin wurden die TOP-Motiv-bindenden Proteine LARP1 und LARP7 auf Ihre Bindungseigenschaften mit Ihren Zielsequenzen untersucht. W{\"a}hrend gezeigt werden konnte, dass LARP1 einen inhibierenden Einfluss auf TOP-RNAs hat, wurde in weiteren Shift-Assays die Bindungseigenschaften von LARP7 mit 7SK untersucht, wobei ebenfalls ein minimales LARP7-Konstrukt sowie 7SK-Konstrukte f{\"u}r Strukturanalysen identifiziert werden konnten. Weiterhin konnte gezeigt werden, dass verschiedene Substanzen wie tRNA und Arginin einen starken Einfluss auf die LARP7-7SK - Interaktion aus{\"u}ben, welcher in weiteren Studien ber{\"u}cksichtigt werden sollte.},
  subject      = {Proteinbiosynthese},
  language  = {de}
}
@phdthesis{JimenezMartin2022,
  author    = {Jim{\´e}nez Mart{\´i}n, Ovidio Manuel},
  title     = {Analysis of MYCN and MAX alterations in Wilms Tumor},
  doi       = {10.25972/OPUS-24291},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-242919},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Wilms tumor (WT) is the most common renal tumor in childhood. Among others, MYCN copy number gain and MYCN P44L and MAX R60Q mutations have been identified in WT. The proto-oncogene MYCN encodes a transcription factor that requires dimerization with MAX to activate transcription of numerous target genes. MYCN gain has been associated with adverse prognosis. The MYCN P44L and MAX R60Q mutations, located in either the transactivating or basic helix-loop-helix domain, respectively, are predicted to be damaging by different pathogenicity prediction tools. These mutations have been reported in several other cancers and remain to be functionally characterized. In order to further describe these events in WT, we screened both mutations in a large cohort of unselected WT patients, to check for an association of the mutation status with certain histological or clinical features. MYCN P44L and MAX R60Q revealed frequencies of 3 \% and 0.9 \% and also were significantly associated to higher risk of relapse and metastasis, respectively. Furthermore, to get a better understanding of the MAX mutational landscape in WT, over 100 WT cases were analyzed by Sanger sequencing to identify other eventual MAX alterations in its coding sequence. R60Q remained the only MAX CDS alteration described in WT to date. To analyze the potential functional consequences of these mutations, we used a doxycycline-inducible system to overexpress each mutant in HEK293 cells. This biochemical characterization identified a reduced transcriptional activation potential for MAX R60Q, while the MYCN P44L mutation did not change activation potential or protein stability. The protein interactome of N-MYC-P44L was likewise not altered as shown by mass spectrometric analyses of purified N-MYC complexes. However, we could identify a number of novel N-MYC partner proteins, several of these known for their oncogenic potential. Their correlated expression in WT samples suggested a role in WT oncogenesis and they expand the range of potential biomarkers for WT stratification and targeting, especially for high-risk WT.},
  subject      = {Nephroblastom},
  language  = {en}
}
@phdthesis{Christmann2022,
  author    = {Christmann, Gabriel},
  title     = {USP 10 als Deubiquitinase f{\"u}r β-Catenin},
  doi       = {10.25972/OPUS-27299},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-272998},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {Der WNT-Signalweg ist ein hochkonservierter Signalweg, dessen zentraler intrazellul{\"a}rer Regulationsschritt die Proteinstabilit{\"a}t des Proteins β-Catenin ist. Deregulierende Mutationen in diesem sind fr{\"u}he Ereignisse bei der Entstehung von Darmtumoren. Ist der Abbau von β-Catenin gest{\"o}rt, so ist unabh{\"a}ngig von {\"a}ußerer Kontrolle der Signalweg konstitutiv aktiviert und liefert ein Wachstumssignal. Untersuchungen haben aber gezeigt, dass beim Vorliegen solcher Mutationen immer noch eine - unzureichende - Ubiquitinylierung und ein Abbau von β-Catenin stattfindet. Ziel dieser Studie war Deubiquitinasen (DUBs) zu finden, die durch ihre Aktivit{\"a}t den Abbau von β-Catenin verhindern. Mithilfe eines siRNA Screens in der Vorarbeit konnten DUBs als Kandidaten f{\"u}r einen CRISPR Ansatz ausgew{\"a}hlt werden. APC Wildtyp HEK293T Zellen und Darmkrebszellen wurden mit lentiviralen CRISPR/Cas9 Vektoren infiziert, in welche sgRNAs gegen exonische Sequenzen von DUBs geklont waren. Einzelne Zellklone von USP10 CRISPR Zellen wurden weiter untersucht. In Western Blots und Immunofluoreszenz zeigte sich bei den USP10 CRISPR Zellen eine verminderte Expression von USP10 und damit einhergehend β-Catenin. Proteinstabilit{\"a}tsversuche mit MG132 und Cycloheximid zeigten einen erh{\"o}hten Abbau von β-Catenin in HEK293T USP10 CRISPR Zellen, vor allem nach Stimulierung des WNT-Signalwegs durch LiCl. In Aktivierungsassays (Luciferase und TOP-GFP FACS) des WNT-Signalwegs zeigte sich in HEK293T Zellen nach Behandlung mit LiCl eine geringere Aktivierung in den USP10 CRISPR Zellen. In einem Wachstumsassay zeigten HT29 USP10 CRISPR ein geringeres Wachstum als Kontrollzellen. W{\"a}hrend in einer histologischen F{\"a}rbung von Mausgewebe eine erh{\"o}hte Expression von USP10 nachweisbar war, zeigten sich in einer TMA F{\"a}rbung kein eindeutiger Unterschied zwischen gesundem Gewebe und Tumorgewebe. Die Studie identifiziert USP10 als eine m{\"o}gliche DUB f{\"u}r β-Catenin und potenzielles Ziel f{\"u}r eine Beeinflussung des mutierten WNT-Signalwegs in Darmkrebszellen.},
  subject      = {Darmkrebs},
  language  = {de}
}
@phdthesis{Amatobi2022,
  author    = {Amatobi, Kelechi Michael},
  title     = {Circadian clocks determine transport and membrane lipid oscillation in \(Drosophila\) hemolymph in complex interactions between nutrient-type, photic conditions and feeding behaviour},
  doi       = {10.25972/OPUS-24446},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-244462},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {The interaction between circadian clocks and metabolism is of increasing interest, since clock dysfunction often correlates with metabolic pathologies. Many research articles have been published analysing the impact of factors such as circadian clock, light, feeding time and diet-type on energy homeostasis in various tissues/organs of organisms with most of the findings done in mammals. Little is known about the impact of circadian clock and the above-mentioned factors on circulating lipids, especially the transport form of lipids - diacylglycerol (DG) and membrane lipids such as phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in the Drosophila hemolymph. The fruit fly Drosophila is a prime model organism in circadian, behaviour and metabolism research. To study the role of circadian clock and behaviour in metabolism, we performed an extensive comparative hemolymph lipid (diacylglycerol: DG, phosphatidylethanolamine: PE, phosphatidylcholine: PC) analysis using ultra performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC-MS) between wild-type flies (WTCS) and clock disrupted mutants (per01). In addition, clock controlled food intake- feeding behaviour was investigated. Time-dependent variation of transport (DG) and membrane lipids (PE and PC) were not rhythmic in WTCS under constant darkness and in per01 under LD, suggesting an impact of light and clock genes on daily lipid oscillations. Day-time and night-time restriction of food led to comparable lipid profiles, suggesting that lipid oscillations are not exclusively entrained by feeding but rather are endogenously regulated. Ultradian oscillations in lipid levels in WTCS under LD were masked by digested fatty acids since lipid levels peaked more robustly at the beginning and end of light phase when flies were fed a lipid- and protein-free diet. These results suggest that metabolite (DG, PE and PC) oscillation is influenced by complex interactions between nutrient-type, photic conditions, circadian clock and feeding time. In conclusion, the results of this thesis suggest that circadian clocks determine transport and membrane lipid oscillation in Drosophila hemolymph in complex interactions between nutrient-type, photic conditions and feeding behaviour.},
  subject      = {Pharmaceutische Biologie},
  language  = {en}
}
@article{HicklHeintzBuschartTrautweinSchultetal.2019,
  author    = {Hickl, Oskar and Heintz-Buschart, Anna and Trautwein-Schult, Anke and Hercog, Rajna and Bork, Peer and Wilmes, Paul and Becher, D{\"o}rte},
  title     = {Sample preservation and storage significantly impact taxonomic and functional profiles in metaproteomics studies of the human gut microbiome},
  series = {Microorganisms},
  volume    = {7},
  journal   = {Microorganisms},
  number    = {9},
  issn      = {2076-2607},
  doi       = {10.3390/microorganisms7090367},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-195976},
  year      = {2019},
  abstract  = {With the technological advances of the last decade, it is now feasible to analyze microbiome samples, such as human stool specimens, using multi-omic techniques. Given the inherent sample complexity, there exists a need for sample methods which preserve as much information as possible about the biological system at the time of sampling. Here, we analyzed human stool samples preserved and stored using different methods, applying metagenomics as well as metaproteomics. Our results demonstrate that sample preservation and storage have a significant effect on the taxonomic composition of identified proteins. The overall identification rates, as well as the proportion of proteins from Actinobacteria were much higher when samples were flash frozen. Preservation in RNAlater overall led to fewer protein identifications and a considerable increase in the share of Bacteroidetes, as well as Proteobacteria. Additionally, a decrease in the share of metabolism-related proteins and an increase of the relative amount of proteins involved in the processing of genetic information was observed for RNAlater-stored samples. This suggests that great care should be taken in choosing methods for the preservation and storage of microbiome samples, as well as in comparing the results of analyses using different sampling and storage methods. Flash freezing and subsequent storage at -80 °C should be chosen wherever possible.},
  language  = {en}
}
@phdthesis{Strack2022,
  author    = {Strack, Stefanie},
  title     = {Rolle der Polycomb Faktoren PCGF6 und E2F6 in undifferenzierten und differenzierenden embryonalen Stammzellen der Maus},
  doi       = {10.25972/OPUS-25560},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-255600},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2022},
  abstract  = {To investigate the role of PCGF6 and E2F6 in murine embryonic stem cells (mESCs) and at the beginning of differentiation, knockout cell lines of both proteins and in combination were generated by the CRISPR/Cas9n system. Characterization of these knockout cell lines (KO) was performed by growth analysis in mESCs and differentiating murine stem cells (EBs). It was found that Pcgf6 KO cells formed smaller EBs that also could not be maintained in culture for an extended period. To resolve this specific phenotype, further molecular analyses were performed by flow cytometry (FACS). Cells of the Pcgf6 KO exhibited an increased proportion of cells in G1 phase during differentiation as well as an increased apoptotic frequency. Supporting the assumption of a cell cycle defect, RNASeq data were analysed. It could be shown that cells of the Pcgf6 KO differentiated in a temporally uncontrolled manner. Evaluation of differentially expressed genes revealed that expression of E2f6, a regulator of the cell cycle and another component of the non-canonical PRC1.6, was downregulated in mESC and EB cultures, whereas cell cycle-specific targets of E2F6-dependent gene regulation were upregulated at day 2 of differentiation. These results indicated that deletion of Pcgf6 at the beginning of differentiation must have effects on E2F6-dependent cell cycle regulation. Due to mycoplasma contamination in the cell culture at this time point, the Pcgf6 KO cell line had to be re-established. In addition, KO cell lines of E2f6 in Wt and in Pcgf6 KO mESCs were established. The replication of cellular characterization of the phenotype revealed that EB cultures of the Pcgf6 KO and the double knockout of Pcgf6 and E2f6 (dKOPcgf6/E2f6) exhibited reduced cell numbers during differentiation. Molecular characterizations of the phenotype revealed that the increased proportion of cells in the G1 phase of the Pcgf6 KO, which was detected before mycoplasma contamination, could not be reproduced. However, an increased frequency of cells in the G2 phase of dKOPcgf6/E2f6 was detected in mESC and EB culture. Analysis of apoptotic frequency in all KO cell lines indicated an increase during differentiation. RNASeq data from two publications of PCGF6 and E2F6 were used to support the analyses performed to this point (Qui et al, 2021; Dahlet et al, 2021). Gene Ontology Enrichment analyses of these data revealed that germline genes were independently upregulated in both KO cell lines in mESCs. However, both KO cell lines also showed an overlap of commonly upregulated germline genes. Following these publications, gene expression analysis of individual germline genes revealed that loss of E2f6 leads to de-repression of genes that have a binding site for E2F6. In contrast, loss of Pcgf6 had no effect on expression of these targets. These results, as well as previously published data, support the assumption that there are distinct subcomplexes that regulate the expression of germline genes in mESC and EB cultures.},
  subject      = {Murine embryonale Stammzellen},
  language  = {de}
}
@misc{Gamaleldin2021,
  type      = {Master Thesis},
  author    = {Gamaleldin, Mariam},
  title     = {Non-canonical Signaling of μ-opioid Receptors},
  doi       = {10.25972/OPUS-24032},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-240327},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {According to the "canonical" paradigm of GPCR signaling, agonist-bound GPCRs only signal to the downstream adenylyl cyclase enzyme when they are seated at the plasma membrane. Upon prolonged binding of an agonist, receptor internalization usually takes place, leading to the termination of this downstream signaling pathway and activation of alternative ones. However, a set of recent studies have shown that at least some GPCRs (e.g. thyroid stimulating hormone receptor) continue signaling to adenylyl cyclase after internalization. In this study, I aimed to investigate canonical signaling by internalized μ opioid receptors (MORs), which are Gi-coupled receptors, using a fluorescence resonance energy transfer (FRET) sensor for cyclic AMP (cAMP) known as Epac1-camps. My results show that the cyclic AMP inhibition signal induced by the binding of DAMGO, a MOR agonist, persists after agonist washout. We hypothesized that this persistent signal might come from internalized DAMGO-bound receptors located in the endosomal compartment. To test this hypothesis, I used dynasore and Dyngo 4a, two dynamin inhibitors that are known to prevent clathrin-mediated endocytosis. Interestingly, dynasore but not Dyngo 4a pretreatment largely blunted the response to MOR activation as well as to adenylyl cyclase activation with Forskolin (FSK). In addition, DAMGO-induced cAMP signal remained persistent even in the presence of 30 M Dyngo 4a. These results might point to a complex interplay between clathrin-mediated internalization and MOR signaling. Further experiments are required to elucidate the mechanisms underlying the persistent MOR signaling and to fully clarify whether MORs are capable of Gi signaling in the endosomal compartment.},
  language  = {en}
}
@phdthesis{Bluemel2021,
  author    = {Bl{\"u}mel, Rabea},
  title     = {Der Zebrab{\"a}rbling (Danio rerio) als in vivo Modell zur Untersuchung der Entstehung von Kraniosynostosen},
  doi       = {10.25972/OPUS-20743},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-207436},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Die Entwicklung des Sch{\"a}deldachs beginnt beim Menschen bereits in der fr{\"u}hen Embryogenese und ist erst im Erwachsenenalter abgeschlossen. Das Wachstum der Sch{\"a}delknochen muss sich w{\"a}hrend der Entwicklung fortw{\"a}hrend dem Gehirnwachstum anpassen. An den Stellen, wo zwei Sch{\"a}delknochen aufeinandertreffen, formen sich Sch{\"a}deln{\"a}hte, die aus mesenchymalem Bindegewebe bestehen und als Wachstumsfugen des Sch{\"a}dels dienen. Tritt eine fr{\"u}hzeitige Verkn{\"o}cherung innerhalb einer oder mehrerer Sch{\"a}deln{\"a}hte auf, spricht man von einer Kraniosynostose. Als Konsequenz wird ein weiteres Knochenwachstum verhindert, sodass sich das Neurokranium in dieser Region nicht dem expansiven Wachstum des Gehirns anpassen kann. Dies geht in der Regel mit einem kompensatorischen Wachstum des Sch{\"a}dels und infolgedessen mit kraniofazialen Dysmorphien und einem erh{\"o}hten intrakraniellen Druck einher. Klinische Studien und Forschungen an Modellorganismen konnten bereits eine Vielzahl an Genen mit der Entstehung von Kraniosynostosen assoziieren, darunter die Transkriptionsfaktoren TCF12 und TWIST1. Beim Menschen sind heterozygote Mutationen in TCF12 und TWIST1 mit Kraniosynostosen der Koronarnaht assoziiert. Bei M{\"a}usen hingegen f{\"u}hrt eine heterozygote Tcf12 Mutation nur in Kombination mit einer heterozygoten Twist1 Mutation zu Fusionen der Koronarnaht. Der Zebrab{\"a}rbling (Danio rerio, {\"u}berwiegend auch Zebrafisch genannt) weist eine bemerkenswerte {\"A}hnlichkeit bez{\"u}glich der Anatomie und Morphologie des Sch{\"a}deldachs zum Menschen auf. Um die genaue Funktion von TCF12 bei der Ausbildung der Sch{\"a}deln{\"a}hte zu untersuchen, wurde im Rahmen dieser Arbeit der Zebrafisch als in vivo Modell f{\"u}r die Entstehung tcf12-induzierter Kraniosynostosen etabliert. Zu Beginn der Arbeit wurde das Expressionsmuster von tcf12 {\"u}ber die Entwicklung hinweg analysiert. Ein besonderer Fokus lag dabei auf einem Expressionsnachweis w{\"a}hrend der Entwicklung der Sch{\"a}delplatten und der Sch{\"a}deln{\"a}hte. Ein erster Expressionsnachweis von tcf12 mittels PCR-Analysen und Whole-mount RNA in-situ Hybridisierungen zeigte eine breite Expression von tcf12 ab dem 1-3 Somiten Stadium an. F{\"u}r tiefergehende in vivo Analysen wurden im Zuge dieser Arbeit tcf12:EGFP Reportergenlinien generiert. Mit diesen gelang ein Nachweis der tcf12 Expression entlang der Wachstumsfronten der Sch{\"a}delplatten, innerhalb der Sch{\"a}deln{\"a}hte sowie im Periost und der Dura mater. Mit den tcf12:EGFP Fischen als Referenz wurde in weiterf{\"u}hrenden Experimenten die Aktivit{\"a}t drei hochkonservierter CNEs (engl. conserved non-coding elements) in vivo im Zebrafisch untersucht. Zwei der CNEs konnten als tcf12 Enhancer verifiziert werden, die eine Genexpression w{\"a}hrend der Neurogenese des zentralen Nervensystems (ZNS) steuern. Die beiden Enhancer-Elemente zeichnen sich durch eine hohe Konservierung vom Menschen bis hin zum Zebrafisch aus. Aufgrund der unterschiedlichen Sensitivit{\"a}t gegen{\"u}ber einem Funktionsverlust von TCF12 und TWIST1 in Mensch und Maus sollte die Auswirkung eines Knockouts der orthologen Gene auf die Entwicklung der Sch{\"a}deln{\"a}hte des Zebrafisches untersucht werden. Mittels CRISPR/Cas9 wurden verschiedene Knockout-Linien f{\"u}r die Gene tcf12, twist1a und twist1b generiert. Analysen der Knockoutmutanten zeigten, dass ein heterozygoter Verlust von tcf12 und twist1b in seltenen F{\"a}llen zu partiellen Fusionen der Koronarn{\"a}hte im Zebrafisch f{\"u}hrt. Des Weiteren konnte bei tcf12 und twist1b Einzel- und Doppelmutanten ein abnormes Wachstum der Sch{\"a}delplatten im Bereich der Suturen beobachtet werden. Die Expressionsstudien und die Analysen der Knockoutmutanten deuten auf eine Regulation von TCF12 bei der Differenzierung der Stammzellen sowie der Proliferation der Osteoblasten innerhalb der Sch{\"a}deln{\"a}hte hin. Um die Auswirkung von TCF12 Mutationen auf funktioneller Ebene zu untersuchen wurden im Verlauf dieser Arbeit Luciferase-Reporter Assays durchgef{\"u}hrt. Anhand dieser konnte nachgewiesen werden, dass Mutationen, die die basic helix-loop-helix (bHLH)-Dom{\"a}ne beeintr{\"a}chtigen, die Transaktivierungsf{\"a}higkeit von TCF12 aufheben. Co-Transfektions-Experimente mit TWIST1 offenbarten eine Regulation der Transaktivierung von TCF12 durch TWIST1, sowohl im Menschen, als auch im Zebrafisch. Im Rahmen dieser Arbeit konnten die genauen Expressionsorte von TCF12 w{\"a}hrend der Morphogenese des Sch{\"a}deldachs nachgwiesen und die Funktion von TCF12 und seinem Interaktionspartner TWIST1 bei der Entstehung von Kraniosynostosen weiter aufgekl{\"a}rt werden.},
  subject      = {Kraniosynostose},
  language  = {de}
}
@phdthesis{Yang2021,
  author    = {Yang, Tao},
  title     = {Functional insights into the role of a bacterial virulence factor and a host factor in Neisseria gonorrhoeae infection},
  doi       = {10.25972/OPUS-20895},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-208959},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {Neisseria gonorrhoeae (GC) is a human specific pathogenic bacterium. Currently, N. gonorrhoeae developed resistance to virtually all the available antibiotics used for treatment. N. gonorrhoeae starts infection by colonizing the cell surface, followed by invasion of the host cell, intracellular persistence, transcytosis and exit into the subepithelial space. Subepithelial bacteria can reach the bloodstream and disseminate to other tissues causing systemic infections, which leads to serious conditions such as arthritis and pneumonia. A number of studies have well established the host-pathogen interactions during the initial adherence and invasion steps. However, the mechanism of intracellular survival and traversal is poorly understood so far. Hence, identification of novel bacterial virulence factors and host factors involved in the host-pathogen interaction is a crucial step in understanding disease development and uncovering novel therapeutic approaches. Besides, most of the previous studies about N. gonorrhoeae were performed in the conventional cell culture. Although they have provided insights into host-pathogen interactions, much information about the native infection microenvironment, such as cell polarization and barrier function, is still missing. This work focused on determining the function of novel bacterial virulence factor NGFG_01605 and host factor (FLCN) in gonococcal infection. NGFG_01605 was identified by Tn5 transposon library screening. It is a putative U32 protease. Unlike other proteins in this family, it is not secreted and has no ex vivo protease activity. NGFG_01605 knockout decreases gonococcal survival in the epithelial cell. 3D models based on T84 cell was developed for the bacterial transmigration assay. NGFG_01605 knockout does not affect gonococcal transmigration. The novel host factor FLCN was identified by shRNA library screening in search for factors that affected gonococcal adherence and/or internalization. We discovered that FLCN did not affect N. gonorrhoeae adherence and invasion but was essential for bacterial survival. Since programmed cell death is a host defence mechanism against intracellular pathogens, we further explored apoptosis and autophagy upon gonococcal infection and determined that FLCN did not affect apoptosis but inhibited autophagy. Moreover, we found that FLCN inhibited the expression of E-cadherin. Knockdown of E- cadherin decreased the autophagy flux and supported N. gonorrhoeae survival. Both non-polarized and polarized cells are present in the cervix, and additionally, E-cadherin represents different polarization properties on these different cells. Therefore, we established 3-D models to better understand the functions of FLCN. We discovered that FLCN was critical for N. gonorrhoeae survival in the 3-D environment as well, but not through inhibiting autophagy. Furthermore, FLCN inhibits the E-cadherin expression and disturbs its polarization in the 3-D models. Since N. gonorrhoeae can cross the epithelial cell barriers through both cell-cell junctions and transcellular migration, we further explored the roles FLCN and E-cadherin played in transmigration. FLCN delayed N. gonorrhoeae transmigration, whereas the knockdown of E-cadherin increased N. gonorrhoeae transmigration. In summary, we revealed roles of the NGFG_01605 and FLCN-E-cadherin axis play in N. gonorrhoeae infection, particularly in relation to intracellular survival and transmigration. This is also the first study that connects FLCN and human-specific pathogen infection.},
  language  = {en}
}
@phdthesis{Figueiredo2021,
  author    = {Figueiredo, Ludmilla},
  title     = {Extinction debt of plants, insects and biotic interactions: interactive effects of habitat fragmentation and climate change},
  doi       = {10.25972/OPUS-23873},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-238738},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The importance of understanding species extinctions and its consequences for ecosystems and human life has been getting increasing public attention. Nonetheless, regardless of how pressing the current biodiversity loss is, with rare exceptions, extinctions are actually not immediate. Rather, they happen many generations after the disturbance that caused them. This means that, at any point in time after a given disturbance, there is a number of extinctions that are expected to happen. This number is the extinction debt. As long as all the extinctions triggered by the disturbance have not happened, there is a debt to be paid. This delay in extinctions can be interpreted as a window of opportunity, when conservation measures can be implemented. In this thesis, I investigated the relative importance of ecological and evolutionary processes unfolding after different disturbances scenarios, to understand how this knowledge can be used to improve conservation practices aiming at controlling extinctions. In the Introduction (chapter 1), I present the concept of extinction debts and the complicating factors behind its understanding. Namely, I start by presenting i) the theoretical basis behind the definition of extinction debts, and how each theory informed different methodologies of study, ii) the complexity of understanding and predicting eco-evolutionary dynamics, and iii) the challenges to studying extinctions under a regime of widespread and varied disturbance of natural habitats. I start the main body of the thesis (chapter 2) by summarizing the current state of empirical, theoretical, and methodological research on extinction debts. In the last 10 years, extinction debts were detected all over the globe, for a variety of ecosystems and taxonomic groups. When estimated - a rare occurrence, since quantifying debts requires often unavailable data - the sizes of these debts range from 9 to 90\\% of current species richness and they have been sustained for periods ranging from 5 to 570 yr. I identified two processes whose contributions to extinction debts have been studied more often, namely 1) life-history traits that prolong individual survival, and 2) population and metapopulation dynamics that maintain populations under deteriorated conditions. Less studied are the microevolutionary dynamics happening during the payment of a debt, the delayed conjoint extinctions of interaction partners, and the extinction dynamics under different regimes of disturbances (e.g. habitat loss vs. climate change). Based on these observations, I proposed a roadmap for future research to focus on these less studies aspects. In chapters 3 and 4, I started to follow this roadmap. In chapter 3, I used a genomically-explicit, individual-based model of a plant community to study the microevolutionary processes happening after habitat loss and climate change, and potentially contributing to the settlement of a debt. I showed that population demographic recovery through trait adaptation, i.e. evolutionary rescue, is possible. In these cases, rather than directional selection, trait change involved increase in trait variation, which I interpreted as a sign of disruptive selection. Moreover, I disentangled evolutionary rescue from demographic rescue and show that the two types of rescue were equally important for community resistance, indicating that community re-assembly plays an important role in maintaining diversity following disturbance. The results demonstrated the importance of accounting for eco-evolutionary processes at the community level to understand and predict biodiversity change. Furthermore, they indicate that evolutionary rescue has a limited potential to avoid extinctions under scenarios of habitat loss and climate change. In chapter 4, I analysed the effects of habitat loss and disruption of pollination function on the extinction dynamics of plant communities. To do it, I used an individual, trait-based eco-evolutionary model (Extinction Dynamics Model, EDM) parameterized according to real-world species of calcareous grasslands. Specifically, I compared the effects of these disturbances on the magnitude of extinction debts and species extinction times, as well as how species functional traits affect species survival. I showed that the loss of habitat area generates higher number of immediate extinctions, but the loss of pollination generates higher extinction debt, as species take longer to go extinct. Moreover, reproductive traits (clonal ability, absence of selfing and insect pollination) were the traits that most influenced the occurrence of species extinction as payment of the debt. Thus, the disruption of pollination functions arose as a major factor in the creation of extinction debts. Thus, restoration policies should aim at monitoring the status of this and other ecological processes and functions in undisturbed systems, to inform its re-establishment in disturbed areas. Finally, I discuss the implications of these findings to i) the theoretical understanding of extinction debts, notably via the niche, coexistence, and metabolic theories, ii) the planning conservation measures, including communicating the very notion of extinction debts to improve understanding of the dimension of the current biodiversity crisis, and iii) future research, which must improve the understanding of the interplay between extinction cascades and extinction debts.},
  subject      = {Aussterbedynamik},
  language  = {en}
}
@phdthesis{Kunz2021,
  author    = {Kunz, Tobias C.},
  title     = {Expansion Microscopy (ExM) as a tool to study organelles and intracellular pathogens},
  doi       = {10.25972/OPUS-22333},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-223330},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2021},
  abstract  = {The resolution of fluorescence light microscopy was long believed to be limited by the diffraction limit of light of around 200-250 nm described in 1873 by Ernst Abbe. Within the last decade, several approaches, such as structured illumination microscopy (SIM), stimulated emission depletion STED and (direct) stochastic optical reconstruction microscopy (d)STORM have been established to bypass the diffraction limit. However, such super-resolution techniques enabling a resolution <100 nm require specialized and expensive setups as well as expert knowledge in order to avoid artifacts. They are therefore limited to specialized laboratories. Recently, Boyden and colleagues introduced an alternate approach, termed expansion microscopy (ExM). The latter offers the possibility to perform superresolution microscopy on conventional confocal microscopes by embedding the sample into a swellable hydrogel that is isotropically expanded. Since its introduction in 2015, expansion microscopy has developed rapidly offering protocols for 4x, 10x and 20x expansion of proteins and RNA in cells, tissues and human clinical specimens. Mitochondria are double membrane-bound organelles and crucial to the cell by performing numerous tasks, from ATP production through oxidative phosphorylation, production of many important metabolites, cell signaling to the regulation of apoptosis. The inner mitochondrial membrane is strongly folded forming so-called cristae. Besides being the location of the oxidative phosphorylation and therefore energy conversion and ATP production, cristae have been of great interest because changes in morphology have been linked to a plethora of diseases from cancer, diabetes, neurodegenerative diseases, to aging and infection. However, cristae imaging remains challenging as the distance between two individual cristae is often below 100 nm. Within this work, we demonstrate that the mitochondrial creatine kinase MtCK linked to fluorescent protein GFP (MtCK-GFP) can be used as a cristae marker. Upon fourfold expansion, we illustrate that our novel marker enables visualization of cristae morphology and localization of mitochondrial proteins relative to cristae without the need for specialized setups. Furthermore, we show the applicability of expansion microscopy for several bacterial pathogens, such as Chlamydia trachomatis, Simkania negevensis, Neisseria gonorrhoeae and Staphylococcus aureus. Due to differences in bacterial cell walls, we reveal important aspects for the digestion of pathogens for isotropic expansion. We further show that expansion of the intracellular pathogens C. trachomatis and S. negevensis, enables the differentiation between the two distinct developmental forms, catabolic active reticulate bodies (RB) and infectious elementary bodies (EB), on a conventional confocal microscope. We demonstrate the possibility to precisely locate chlamydial effector proteins, such as CPAF or Cdu1, within and outside the chlamydial inclusion. Moreover, we show that expansion microscopy enables the investigation of bacteria, herein S. aureus, within LAMP1 and LC3-II vesicles. With the introduction of the unnatural α-NH2-ω-N3-C6-ceramide, we further present the first approach for the expansion of lipids that may also be suitable for far inaccessible molecule classes like carbohydrates. The efficient accumulation and high labeling density of our functionalized α-NH2-ω-N3-C6-ceramide in both cells and bacteria enables in combination with tenfold expansion nanoscale resolution (10-20 nm) of the interaction of proteins with the plasma membrane, membrane of organelles and bacteria. Ceramide is the central molecule of the sphingolipid metabolism, an important constituent of cellular membranes and regulates many important cellular processes such as differentiation, proliferation and apoptosis. Many studies report about the importance of sphingolipids during infection of various pathogens. While the transport of ceramide to Chlamydia has been reported earlier, one of the unanswered questions remaining was if ceramide forms parts of the outer or inner bacterial membrane. Expansion of α-NH2-ω-N3-C6-ceramide enabled the visualization of ceramide in the inner and outer membrane of C. trachomatis and their distance was determined to be 27.6 ± 7.7 nm.},
  subject      = {Fluoreszenzmikroskopie},
  language  = {en}
}
@phdthesis{Pickel2020,
  author    = {Pickel, Simone},
  title     = {Role of the β subunit of L-type calcium channels in cardiac hypertrophy},
  doi       = {10.25972/OPUS-19282},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-192829},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {L-type calcium channels (LTCCs) control crucial physiological processes in cardiomyocytes such as the duration and amplitude of action potentials, excitation-contraction coupling and gene expression, by regulating the entry of Ca2+ into the cells. Cardiac LTCCs consist of one pore-forming α1 subunit and the accessory subunits Cavβ, Cavα2δ and Cavγ. Of these auxiliary subunits, Cavβ is the most important regulator of the channel activity; however, it can also have LTCC-independent cellular regulatory functions. Therefore, changes in the expression of Cavβ can lead not only to a dysregulation of LTCC activity, but also to changes in other cellular functions. Cardiac hypertrophy is one of the most relevant risk factors for congestive heart failure and depends on the activation of calcium-dependent prohypertrophic signaling pathways. However, the role of LTCCs and especially Cavβ in this pathology is controversial and needs to be further elucidated. Of the four Cavβ isoforms, Cavβ2 is the predominant one in cardiomyocytes. Moreover, there are five different splice variants of Cavβ2 (Cavβ2a-e), differing only in the N-terminal region. We reported that Cavβ2b is the predominant variant expressed in the heart. We also revealed that a pool of Cavβ2 is targeted to the nucleus in cardiomyocytes. The expression of the nuclear Cavβ2 decreases during in vitro and in vivo induction of cardiomyocyte hypertrophy and overexpression of a nucleus-targeted Cavβ2 completely abolishes the in vitro induced hypertrophy. Additionally, we demonstrated by shRNA-mediated protein knockdown that downregulation of Cavβ2 enhances the hypertrophy induced by the α1-adrenergic agonist phenylephrine (PE) without involvement of LTCC activity. These results suggest that Cavβ2 can regulate cardiac hypertrophy through LTCC-independent pathways. To further validate the role of the nuclear Cavβ2, we performed quantitative proteome analyses of Cavβ2-deficient neonatal rat cardiomyocytes (NRCs). The results show that downregulation of Cavβ2 influences the expression of various proteins, including a decrease of calpastatin, an inhibitor of the calcium-dependent cysteine protease calpain. Moreover, downregulation of Cavβ2 during cardiomyocyte hypertrophy drastically increases calpain activity as compared to controls after treatment with PE. Finally, the inhibition of calpain by calpeptin abolishes the increase in PE-induced hypertrophy in Cavβ2-deficient cells. These results suggest that nuclear Cavβ2 has Ca2+- and LTCC-independent functions during the development of hypertrophy. Overall, our results indicate a new role for Cavβ2 in antihypertrophic signaling in cardiac hypertrophy.},
  subject      = {Herzhypertrophie},
  language  = {en}
}
@phdthesis{Sieger2020,
  author    = {Sieger, Charlotte Sophie},
  title     = {Potential evolutionary responses to landscape heterogeneity and systematic environmental trends},
  doi       = {10.25972/OPUS-21669},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-216690},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Over the course of the last century, humans have witnessed drastic levels of global environmental change that endangered both, the survival of single species as well as biodiversity itself. This includes climate change, in both environmental means and in variance and subsequently frequent extreme weather events, as well as land use change that species have to cope with. With increasing urbanization, increasing agricultural area and increasing intensification, natural habitat is not only lost, but also changes its shape and distribution in the landscape. Both aspects can heavily influence an individual's fitness and therefore act as a selective force promoting evolutionary change. This way climate change influences individuals' niches and dispersal. Local adaptation and dispersal are not independent of each other. Dispersal can have two opposite effects on local adaptation. It can oppose local adaptation, by promoting the immigration of maladapted indi- viduals or favor local adaptation by introducing better adapted genotypes. Which of those effects of dispersal on local adaptation emerges in a population depends on the dispersal strategies and the spatial structure of the landscape. In principle an adaptive response can include adjustment of the niche optimum as well as habitat tolerance (niche width) or (instead) ecological tracking of adequate conditions by dispersal and range shifting. So far, there has been no extensive modeling study of the evolution of the environmental niche optimum and tolerance along with dispersal probability in complex landscapes. Either only dispersal or (part of ) the environmental niche can evolve or the landscapes used are not realistic but rather a very abstract representation of spatial structures. I want to try and disentangle those different effects of both local adaptation and dispersal during global change, as well as their interaction, especially considering the separation between the effects of increasing mean and increasing variance. For this, I implemented an individual based model (IBM), with escalating complexity. I showed that both on a temporal as well as on a spatial scale, variation can be more influential then mean conditions. Indeed, the actual spatial configuration of this heterogeneity and the relationship between spatial and temporal heterogeneity affect the evolution of the niche and of dispersal probability more than temporal or spatial mean conditions. I could show that in isolated populations, an increase of an environmental attribute's mean or variance can lead to extinction, under certain conditions. In particular, increasing variance cannot be tracked forever, since increasing tolerance has distinct limits of feasibility. Increasing mean conditions can also occur too fast to be tracked, especially from generalist individuals. When expanding the model to the metapopulation level without a temporal environmental trend, the degree of spatial vs.temporal heterogeneity influenced the evolution of random dispersal heavily. With increasing spatial heterogeneity, individuals from extreme and rare patches evolve from being philopatric to dispersive, while individuals from average patches switch in the opposite direction. With the last expansion to a different set of landscapes with varying degrees of edge density, I could show that edge effects are strong in pseudo-agricultural landscapes, while in pseudo-natural habitats they were hardly found, regardless of emigration strategy. Sharp edges select against dispersal in the edge patches and could potentially further isolate populations in agricultural landscapes. The work I present here can also be expanded further and I present several suggestions on what to do next. These expansions could help the realism of the model and eventually shed light on its bearing on ecological global change predictions. For example species distribution models or extinction risk models would be more precise, if they included both spatial and temporal variation. The current modeling practices might not be suffcient to describe the possible outcomes of global change, because spatio-temporal heterogeneity and its influence on species' niches is too important to be ignored for longer.},
  language  = {en}
}
@phdthesis{Lekszas2020,
  author    = {Lekszas, Caroline},
  title     = {Erweiterung des genetischen Mutationsspektrums verschiedener Krankheitsbilder und Identifizierung neuer krankheitsrelevanter Gene im Menschen mittels Whole Exome Sequenzierung},
  doi       = {10.25972/OPUS-20880},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-208807},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Trotz der rasanten Entwicklung molekulargenetischer Analysemethoden sind die Ausl{\"o}ser vieler Erbrankheiten bislang ungekl{\"a}rt. Eine Identifikation der genetischen Ursache einer Erkrankung ist jedoch essenziell, um zus{\"a}tzliche invasive Tests vermeiden, ad{\"a}quate Therapiemaßnahmen in die Wege leiten, akkurate Prognosen stellen und eine entsprechende genetische Beratung anbieten zu k{\"o}nnen. Next Generation Sequencing (NGS)-basierte Techniken wie die Whole Exome Sequenzierung (WES) haben die humangenetische Forschung und Diagnostik in den letzten Jahren revolutioniert. Die WES erm{\"o}glicht die Sequenzierung der Exons aller proteincodierenden Gene von mehreren Individuen gleichzeitig und stellt ein hilfreiches Werkzeug bei der Suche nach neuen kranheitsrelevanten Genen im Menschen dar. Die vorliegende Arbeit besch{\"a}ftigt sich mit der Aufkl{\"a}rung genetischer Ursachen verschiedenster Erkrankungen in konsanguinen Familien aus dem nahen und mittleren Osten mittels WES. Insgesamt wurden 43 Patienten mit unterschiedlichen Krankheitsbildern untersucht, darunter viele mit Skelettdysplasien oder Neuropathien. In 22 F{\"a}llen (51\%) konnte die entsprechende krankheitsverursachende Mutation ausfindig gemacht werden. In 21\% der aufgekl{\"a}rten F{\"a}lle wurden Sequenzvarianten detektiert, die in der Literatur bereits als pathogen beschrieben wurden, w{\"a}hrend 63\% bisher noch unbekannte Mutationen in bereits als krankheitsrelevant beschriebenen Genen darstellten. Zudem konnten im Rahmen dieser Arbeit drei neue, f{\"u}r den Menschen krankheitsrelevante Gene identifiziert werden, solute carrier family 10 member 7 (SLC10A7), T-box 4 (TBX4) und MIA SH3 domain ER export factor 3 (MIA3). SLC10A7 codiert f{\"u}r einen Transporter aus der Familie der solute carrier, der in der Plasmamembran verankert ist. In dieser Arbeit geleistete Analyseergebnisse konnten zu der Erstbeschreibung von homozygoten pathogenen SLC10A7-Mutationen als Ursache f{\"u}r eine Skelettdysplasie mit Amelogenesis imperfecta beitragen. Bei TBX4 handelt es sich um einen hochkonservierten Transkriptionsfaktor, der w{\"a}hrend der embryonalen Entwicklung an der Ausbildung der unteren Extremit{\"a}ten beteiligt ist. Homozygote pathogene TBX4-Mutationen wurden im Kontext dieser Arbeit erstmalig mit einer posterioren Amelie mit Becken- und Lungenhypoplasie in Verbindung gebracht. MIA3 ist ein Transmembranprotein des endoplasmatischen Retikulums, das eine essenzielle Rolle bei der Proteinsekretion spielt. Die hier vorgestellten Patienten mit homozygoten pathogenen MIA3-Mutationen zeigen eine komplexe syndromale Erkrankung, die sich haupts{\"a}chlich in einer Kollagenopathie, Diabetes mellitus und milder mentaler Retardierung manifestiert und ein neues Krankheitsbild darstellt. Die im Rahmen dieser Arbeit erzielten Ergebnisse erweitern somit zum einen das Mutationsspektrum verschiedener bekannter Krankheitsbilder und offenbaren zum anderen neue krankheitsrelevante Gene im Menschen.},
  subject      = {Verwandtenehe},
  language  = {de}
}
@phdthesis{Bertolini2020,
  author    = {Bertolini, Enrico},
  title     = {Comparative analysis of insect circadian clocks: a behavioural, anatomical, and molecular study},
  doi       = {10.25972/OPUS-16465},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-164651},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2020},
  abstract  = {Biological clocks are endogenous oscillators that give organisms the sense of time. Insects, as the largest taxonomic group, offer fascinating models to study the evolution of clocks and their adaptation to various environments. Although the laboratory fruit fly, Drosophila melanogaster, led the role in the field of circadian biology as it provides a powerful genetic experimental tool, new model insect species need to be established to understand photoperiodic responses and to enable comparative studies. This work reports the behavioural, anatomical, and molecular characterization of the circadian clock of five insect species. The malt fly Chymomyza costata carries a D. melanogaster-like clock network, which supports circadian rhythms under rhythmic environment but cannot self-sustain when isolated from external time cues. The olive fly Bactrocera oleae is the major pest of olive plantations and the characterization of its circadian clock will improve future pest management strategies. The linden bug Pyrrhocoris apterus, a well suited model for investigating circadian and photoperiodic timing interactions, shows high degree of homology of the clock network with D. melanogaster. The scuttle flies Megaselia scalaris and Megaselia abdita represent new fascinating models to study how the clock network controls circadian behaviour. Overall, this work highlights high degree of homology between different circadian clock systems, but at the same time also dramatic differences in terms of circadian behaviour and neuro-anatomical expression of clock components. These have been mainly discussed in regards to the evolution of clocks in Diptera, and the adaptation of clocks to high latitudes.},
  language  = {en}
}