@phdthesis{Bakirci2024,
  author    = {Bakirci, Ezgi},
  title     = {Development of \(In\) \(vitro\) Models for Tissue Engineering Applications Using a High-Resolution 3D Printing Technology},
  doi       = {10.25972/OPUS-25164},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-251645},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {In vitro models mimic the tissue-specific anatomy and play essential roles in personalized medicine and disease treatments. As a sophisticated manufacturing technology, 3D printing overcomes the limitations of traditional technologies and provides an excellent potential for developing in vitro models to mimic native tissue. This thesis aims to investigate the potential of a high-resolution 3D printing technology, melt electrowriting (MEW), for fabricating in vitro models. MEW has a distinct capacity for depositing micron size fibers with a defined design. In this thesis, three approaches were used, including 1) extending the MEW polymer library for different biomedical applications, 2) developing in vitro models for evaluation of cell growth and migration toward the different matrices, and 3) studying the effect of scaffold designs and biochemical cues of microenvironments on cells. First, we introduce the MEW processability of (AB)n and (ABAC)n segmented copolymers, which have thermally reversible network formulation based on physical crosslinks. Bisurea segments are combined with hydrophobic poly(dimethylsiloxane) (PDMS) or hydrophilic poly(propylene oxide)-poly(ethylene oxide)-poly(propylene oxide) (PPO-PEG-PPO) segments to form the (AB)n segmented copolymers. (ABAC)n segmented copolymers contain all three segments: in addition to bisurea, both hydrophobic and hydrophilic segments are available in the same polymer chain, resulting in tunable mechanical and biological behaviors. MEW copolymers either support cells attachment or dissolve without cytotoxic side effects when in contact with the polymers at lower concentrations, indicating that this copolymer class has potential in biological applications. The unique biological and surface properties, transparency, adjustable hydrophilicity of these copolymers could be beneficial in several in vitro models. The second manuscript addresses the design and development of a melt electrowritten competitive 3D radial migration device. The approach differs from most of the previous literature, as MEW is not used here to produce cell invasive scaffolds but to fabricate an in vitro device. The device is utilized to systematically determine the matrix which promotes cell migration and growth of glioblastoma cells. The glioblastoma cell migration is tested on four different Matrigel concentrations using a melt electrowritten radial device. The glioblastoma U87 cell growth and migration increase at Matrigel concentrations 6 and 8 mg mL-1 In the development of this radial device, the accuracy, and precision of melt electrowritten circular shapes were investigated. The results show that the printing speed and design diameter are essential parameters for the accuracy of printed constructs. It is the first instance where MEW is used for the production of in vitro devices. The influence of biochemical cues and scaffold designs on astrocytes and glioblastoma is investigated in the last manuscript. A fiber comprising the box and triangle-shaped pores within MEW scaffolds are modified with biochemical cues, including RGD and IKVAV peptides using a reactive NCO-sP(EO-stat-PO) macromer. The results show that astrocytes and glioblastoma cells exhibit different phenotypes on scaffold designs and peptide-coated scaffolds.},
  subject      = {3D-Druck},
  language  = {en}
}
@phdthesis{Blahetek2024,
  author    = {Blahetek, Gina},
  title     = {The role of alternative intronic polyadenylation on microRNA biogenesis in melanoma},
  doi       = {10.25972/OPUS-25474},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-254743},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {mRNA is co- or post-transcriptionally processed from a precursor mRNA to a mature mRNA. In addition to 5'capping and splicing, these modifications also include polyadenylation, the addition of a polyA tail to the 3'end of the mRNA. In recent years, alternative polyadenylation in particular has increasingly been taken into account as a mechanism for regulating gene expression. It is assumed that approximately 70-75 \% of human protein coding genes contain alternative polyadenylation signals, which are often located within intronic sequences of protein-coding genes. The use of such polyadenylation signals leads to shortened mRNA transcripts and thus to the generation of C-terminal shortened protein isoforms. Interestingly, the majority of microRNAs, small non-coding RNAs that play an essential role in post-transcriptional gene regulation, are also encoded in intronic sequences of protein-coding genes and are co-transcriptionally expressed with their host genes. The biogenesis of microRNA has been well studied and is well known, but mechanisms that may influence the expression regulation of mature microRNAs are just poorly understood. In the presented work, I aimed to investigate the influence of alternative intronic polyadenylation on the biogenesis of microRNAs. The human ion channel TRPM1 could already be associated with melanoma pathogenesis and truncated isoforms of this protein have already been described in literature. In addition, TRPM1 harbors a microRNA, miR211, in its sixth intron, which is assumed to act as a tumor suppressor. Since both, TRPM1 and miR211 have already been associated with melanoma pathogenesis, the shift towards truncated transcripts during the development of various cancers is already known and it has been shown that certain microRNAs play a crucial role in the development and progression of melanoma, melanoma cell lines were used as an in vitro model for these investigations.},
  subject      = {Polyadenylierung},
  language  = {en}
}
@phdthesis{Behne2024,
  author    = {Behne, Robert Stefan Friedrich},
  title     = {Development Of A Human iPSC-Derived Cortical Neuron Model Of Adaptor- Protein-Complex-4-Deficiency},
  doi       = {10.25972/OPUS-35139},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-351390},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Adaptor-protein-4-deficiency (AP-4-deficiency) is an autosomal-recessive childhood- onset form of complicated hereditary spastic paraplegia (HSP) caused by bi-allelic loss- of-function mutations in one of the four subunits of the AP-4-complex. These four conditions are named SPG47 (AP4B1, OMIM \#614066), SPG50 (AP4M1, OMIM \#612936), SPG51 (AP4E1, OMIM \#613744) and SPG52 (AP4S1, OMIM \#614067), respectively and all present with global developmental delay, progressive spasticity and seizures. Imaging features include a thinning of the corpus callosum, ventriculomegaly and white matter changes. AP-4 is a highly conserved heterotetrameric complex, which is responsible for polarized sorting of transmembrane cargo including the autophagy- related protein 9 A (ATG9A). Loss of any of the four subunits leads to an instable complex and defective sorting of AP-4-cargo. ATG9A is implicated in autophagosome formation and neurite outgrowth. It is missorted in AP-4-deficient cells and CNS-specific knockout of Atg9a in mice results in a phenotype reminiscent of AP-4-deficiency. However, the AP-4-related cellular phenotypes including ATG9A missorting have not been investigated in human neurons. Thus, the aim of this study is to provide the first human induced pluripotent stem cell- derived (iPSC) cortical neuron model of AP-4-deficiency to explore AP-4-related phenotypes in preparation for a high-content screening. Under the hypothesis that AP-4- deficiency leads to ATG9A missorting, elevated ATG9A levels, impaired autophagy and neurite outgrowth in human iPSC-derived cortical neurons, in vitro biochemical and imaging assays including automated high-content imaging and analysis were applied. First, these phenotypes were investigated in fibroblasts from three patients with compound heterozygous mutations in the AP4B1 gene and their sex-matched parental controls. The same cell lines were used to generate iPSCs and differentiate them into human excitatory cortical neurons. This work shows that ATG9A is accumulating in the trans-Golgi-network in AP-4- deficient human fibroblasts and that ATG9A levels are increased compared to parental controls and wild type cells suggesting a compensatory mechanism. Protein levels of the AP4E1-subunit were used as a surrogate marker for the AP-4-complex and were decreased in AP-4-deficient fibroblasts with co-immunoprecipitation confirming the instability of the complex. Lentiviral re-expression of the AP4B1-subunit rescues this corroborating the fact that a stable AP-4-complex is needed for ATG9A trafficking. Surprisingly, autophagic flux was present in AP-4-deficient fibroblasts under nutrient- rich and starvation conditions. These phenotypic markers were evaluated in iPSC-derived cortical neurons and here, a robust accumulation of ATG9A in the juxtanuclear area was seen together with elevated ATG9A protein levels. Strikingly, assessment of autophagy markers under nutrient-rich conditions showed alterations in AP-4-deficient iPSC- derived cortical neurons indicating dysfunctional autophagosome formation. These findings point towards a neuron-specific impairment of autophagy and need further investigation. Adding to the range of AP-4-related phenotypes, neurite outgrowth and branching are impaired in AP-4-deficient iPSC-derived cortical neurons as early as 24h after plating and together with recent studies point towards a distinct role of ATG9A in neurodevelopment independent of autophagy. Together, this work provides the first patient-derived neuron model of AP-4-deficiency and shows that ATG9A is sorted in an AP-4-dependent manner. It establishes ATG9A- related phenotypes and impaired neurite outgrowth as robust markers for a high-content screening. This disease model holds the promise of providing a platform to further study AP-4-deficiency and to search for novel therapeutic targets.},
  subject      = {Adaptorproteine},
  language  = {en}
}
@phdthesis{Reissland2024,
  author    = {Reissland, Michaela},
  title     = {USP10 is a \(de\) \(novo\) tumour-specific regulator of β-Catenin and contributes to cancer stem cell maintenance and tumour progression},
  doi       = {10.25972/OPUS-31957},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-319579},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Colorectal Cancer (CRC) is the third most common cancer in the US. The majority of CRC cases are due to deregulated WNT-signalling pathway. These alterations are mainly caused by mutations in the tumour suppressor gene APC or in CTNNB1, encoding the key effector protein of this pathway, β-Catenin. In canonical WNT-signalling, β-Catenin activates the transcription of several target genes, encoding for proteins involved in proliferation, such as MYC, JUN and NOTCH. Being such a critical regulator of these proto-oncogenes, the stability of β-Catenin is tightly regulated by the Ubiquitin-Proteasome System. Several E3 ligases that ubiquitylate and degrade β-Catenin have been described in the past, but the antagonists, the deubiquitylases, are still unknown. By performing an unbiased siRNA screen, the deubiquitylase USP10 was identified as a de novo positive regulator of β-Catenin stability in CRC derived cells. USP10 has previously been shown in the literature to regulate both mutant and wild type TP53 stability, to deubiquitylate NOTCH1 in endothelial cells and to be involved in the regulation of AMPKα signalling. Overall, however, its role in colorectal tumorigenesis remains controversial. By analysing publicly available protein and gene expression data from colorectal cancer patients, we have shown that USP10 is strongly upregulated or amplified upon transformation and that its expression correlates positively with CTNNB1 expression. In contrast, basal USP10 levels were found in non-transformed tissues, but surprisingly USP10 is upregulated in intestinal stem cells. Endogenous interaction studies in CRC-derived cell lines, with different extend of APCtruncation, revealed an APC-dependent mode of action for both proteins. Furthermore, by utilising CRISPR/Cas9, shRNA-mediated knock-down and overexpression of USP10, we could demonstrate a regulation of β-Catenin stability by USP10 in CRC cell lines. It is widely excepted that 2D cell culture systems do not reflect complexity, architecture and heterogeneity and are therefore not suitable to answer complex biological questions. To overcome this, we established the isolation, cultivation and genetically modification of murine intestinal organoids and utilised this system to study Usp10s role ex vivo. By performing RNA sequencing, dependent on different Usp10 levels, we were able to recapitulate the previous findings and demonstrated Usp10 as important regulator of β-dependent regulation of stem cell homeostasis. Since genetic depletion of USP10 resulted in down-regulation of β-Catenin-dependent transcription, therapeutic intervention of USP10 in colorectal cancer was also investigated. Commercial and newly developed inhibitors were tested for their efficacy against USP10, but failed to significantly inhibit USP10 activity in colorectal cancer cells. To validate the findings from this work also in vivo, development of a novel mouse model for colorectal cancer has begun. By combining CRISPR/Cas9 and classical genetic engineering with viral injection strategies, WT and genetically modified mice could be transformed and, at least in some animals, intestinal lesions were detectable at the microscopic level. The inhibition of USP10, which we could describe as a de novo tumour-specific regulator of β-Catenin, could become a new therapeutic strategy for colorectal cancer patients.},
  subject      = {Biomedizin},
  language  = {en}
}
@phdthesis{Gaballa2024,
  author    = {Gaballa, Abdallah Hatem Hassan Hosny Ahmed},
  title     = {PAF1c drives MYC-mediated immune evasion in pancreatic ductal adenocarcinoma},
  doi       = {10.25972/OPUS-36045},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-360459},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {The expression of the MYC proto-oncogene is elevated in a large proportion of patients with pancreatic ductal adenocarcinoma (PDAC). Previous findings in PDAC have shown that this increased MYC expression mediates immune evasion and promotes S-phase progression. How these functions are mediated and whether a downstream factor of MYC mediates these functions has remained elusive. Recent studies identifying the MYC interactome revealed a complex network of interaction partners, highlighting the need to identify the oncogenic pathway of MYC in an unbiased manner. In this work, we have shown that MYC ensures genomic stability during S-phase and prevents transcription-replication conflicts. Depletion of MYC and inhibition of ATR kinase showed a synergistic effect to induce DNA damage. A targeted siRNA screen targeting downstream factors of MYC revealed that PAF1c is required for DNA repair and S-phase progression. Recruitment of PAF1c to RNAPII was shown to be MYC dependent. PAF1c was shown to be largely dispensable for cell proliferation and regulation of MYC target genes. Depletion of CTR9, a subunit of PAF1c, caused strong tumor regression in a pancreatic ductal adenocarcinoma model, with long-term survival in a subset of mice. This effect was not due to induction of DNA damage, but to restoration of tumor immune surveillance. Depletion of PAF1c resulted in the release of RNAPII with transcription elongation factors, including SPT6, from the bodies of long genes, promoting full-length transcription of short genes. This resulted in the downregulation of long DNA repair genes and the concomitant upregulation of short genes, including MHC class I genes. These data demonstrate that a balance between long and short gene transcription is essential for tumor progression and that interference with PAF1c levels shifts this balance toward a tumor-suppressive transcriptional program. It also directly links MYC-mediated S-phase progression to immune evasion. Unlike MYC, PAF1c has a stable, known folded structure; therefore, the development of a small molecule targeting PAF1c may disrupt the immune evasive function of MYC while sparing its physiological functions in cellular growth.},
  subject      = {Myc},
  language  = {en}
}
@phdthesis{CruzdeCasas2024,
  author    = {Cruz de Casas, Paulina},
  title     = {Sphingolipids as modulators of T cell function},
  doi       = {10.25972/OPUS-35969},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-359698},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {The immune system is responsible for the preservation of homeostasis whenever a given organism is exposed to distinct kinds of perturbations. Given the complexity of certain organisms like mammals, and the diverse types of challenges that they encounter (e.g. infection or disease), the immune system evolved to harbor a great variety of distinct immune cell populations with specialized functions. For instance, the family of T cells is sub-divided into conventional (Tconv) and unconventional T cells (UTCs). Tconv form part of the adaptive arm of the immune system and are comprised of αβ CD4+ or CD8+ cells that differentiate from na{\"i}ve to effector and memory populations upon activation and are essential during infection and cancer. Furthermore, UTCs, which include γδ T cells, NKT and MAIT, are involved in innate and adaptive immune responses, due to their dual mode of activation, through cytokines (innate-like) or TCR (adaptive), and function. Despite our understanding of the basic functions of T cells in several contexts, a great number of open questions related to their basic biology remain. For instance, the mechanism behind the differentiation of na{\"i}ve CD4+ and CD8+ T cells into effector and memory populations is not fully understood. Moreover, the exact function and relevance of distinct UTC subpopulations in a physiological context have not been fully clarified. Here, we investigated the factors mediating na{\"i}ve CD8+ T cell differentiation into effector and memory cells. By using flow cytometry, mass spectrometry, enzymatic assays, and transgenic mouse models, we found that the membrane bound enzyme sphingomyelin-phosphodiesterase acid-like 3b (Smpdl3b) is crucial for the maintenance of memory CD8+ T cells. Our data show that the absence of Smpdl3b leads to diminished CD8+ T cell memory, and a loss of stem-like memory populations due to an aggravated contraction. Our scRNA-seq data suggest that Smpdl3b could be involved in clathrinmediated endocytosis through modulation of Huntingtin interacting protein 1 (Hip1) levels, likely regulating TCR-independent signaling events. Furthermore, in this study we explored the role of UTCs in lymph node-specific immune responses. By using transgenic mouse models for photolabeling, lymph node transplantation models, infection models and flow cytometry, we demonstrate that S1P regulates the migration of tissue-derived UTC from tissues to draining lymph nodes, resulting in heterogeneous immune responses mounted by lymph nodes draining different tissues. Moreover, our unbiased scRNAseq and single lineage-deficient mouse models analysis revealed that all UTC lineages (γδ T cells, NKT and MAIT) are organized in functional units, based on transcriptional homogeneity, shared microanatomical location and migratory behavior, and numerical and functional redundancy. Taken together, our studies describe additional cell intrinsic (Smpdl3b) and extrinsic (S1Pmediated migration) functions of sphingolipid metabolism modulating T cell biology. We propose the S1P/S1PR1/5 signaling axis as the potential survival pathway for Smpdl3b+ memory CD8+ T cells and UTCs, mainly in lymph nodes. Possibly, Smpdl3b regulates S1P/S1PR signaling by balancing ligandreceptor endocytosis, while UTCs migrate to lymph nodes during homeostasis to be exposed to specific levels of S1P that assure their maintenance. Our results are clinically relevant, since several drugs modulating the S1P/S1PR signaling axis or the levels of Smpdl3b are currently used to treat human diseases, such as multiple sclerosis and B cell-mediated diseases. We hope that our discoveries will inspire future studies focusing on sphingolipid metabolism in immune cell biology.},
  subject      = {T-Lymphozyt},
  language  = {en}
}
@phdthesis{Amini2024,
  author    = {Amini, Emad},
  title     = {How central and peripheral clocks and the neuroendocrine system interact to time eclosion behavior in \(Drosophila\) \(melanogaster\)},
  doi       = {10.25972/OPUS-36130},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-361309},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {To grow larger, insects must shed their old rigid exoskeleton and replace it with a new one. This process is called molting and the motor behavior that sheds the old cuticle is called ecdysis. Holometabolic insects have pupal stages in between their larval and adult forms, during which they perform metamorphosis. The pupal stage ends with eclosion, i.e., the emergence of the adult from the pupal shell. Insects typically eclose at a specific time during the day, likely when abiotic conditions are at their optimum. A newly eclosed insect is fragile and needs time to harden its exoskeleton. Hence, eclosion is regulated by sophisticated developmental and circadian timing mechanisms. In Drosophila melanogaster, eclosion is limited to a daily time window in the morning, regarded as the "eclosion gate". In a population of laboratory flies entrained by light/dark cycles, most of the flies eclose around lights on. This rhythmic eclosion pattern is controlled by the circadian clock and persists even under constant conditions. Developmental timing is under the control of complex hormonal signaling, including the steroid ecdysone, insulin-like peptides, and prothoracicotropic hormone (PTTH). The interactions of the central circadian clock in the brain and a peripheral clock in the prothoracic gland (PG) that produces ecdysone are important for the circadian timing of eclosion. These two clocks are connected by a bilateral pair of peptidergic PTTH neurons (PTTHn) that project to the PG. Before each molt, the ecdysone level rises and then falls shortly before ecdysis. The falling ecdysone level must fall below a certain threshold value for the eclosion gate to open. The activity of PTTHn is inhibited by short neuropeptide F (sNPF) from the small ventrolateral neurons (sLNvs) and inhibition is thought to lead to a decrease in ecdysone production. The general aim of this thesis is to further the understanding of how the circadian clock and neuroendocrinal pathways are coordinated to drive eclosion rhythmicity and to identify when these endocrinal signaling pathways are active. In Chapter I, a series of conditional PTTHn silencing-based behavioral assays, combined with neuronal activity imaging techniques such as non-invasive ARG-Luc show that PTTH signaling is active and required shortly before eclosion and may serve to phase-adjust the activity of the PG at the end of pupal development. Trans-synaptic anatomical stainings identified the sLNvs, dorsal neurons 1 (DN1), dorsal neurons 2 (DN2), and lateral posterior neurons (LPNs) clock neurons as directly upstream of the PTTHn. Eclosion motor behavior is initiated by Ecdysis triggering hormone (ETH) which activates a pair of ventromedial (Vm) neurons to release eclosion hormone (EH) which positively feeds back to the source of ETH, the endocrine Inka cells. In Chapter II trans-synaptic tracing showed that most clock neurons provide input to the Vm and non-canonical EH neurons. Hence, clock can potentially influence the ETH/EH feedback loop. The activity profile of the Inka cells and Vm neurons before eclosion is described. Vm and Inka cells are active around seven hours before eclosion. Interestingly, all EH neurons appear to be exclusively peptidergic. In Chapter III, using chemoconnectomics, PTTHns were found to express receptors for sNPF, allatostatin A (AstA), allatostatin C (AstC), and myosuppressin (Ms), while EH neurons expressed only Ms and AstA receptors. Eclosion assays of flies with impaired AstA, AstC, or Ms signaling do not show arrhythmicity under constant conditions. However, optogenetic activation of the AstA neurons strongly suppresses eclosion. Chapter IV focuses on peripheral ventral' Tracheal dendrite (v'Td) and class IV dendritic arborization (C4da) neurons. The C4da neurons mediate larval light avoidance through endocrine PTTH signaling. The v'Td neurons mainly receive O2/CO2 input from the trachea and are upstream of Vm neurons but are not required for eclosion rhythmicity. Conditional ablation of the C4da neurons or torso (receptor of PTTH) knock-out in the C4da neurons impaired eclosion rhythmicity. Six to seven hours before eclosion, PTTHn, C4da, and Vm neurons are active based on ARG-Luc imaging. Thus, C4da neurons may indirectly connect the PTTHn to the Vm neurons. In summary, this thesis advances our knowledge of the temporal activity and role of PTTH signaling during pupal development and rhythmic eclosion. It further provides a comprehensive characterization of the synaptic and peptidergic inputs from clock neurons to PTTHn and EH neurons. AstA, AstC, and Ms are identified as potential modulators of eclosion circuits and suggest an indirect effect of PTTH signaling on EH signaling via the peripheral sensory C4da neurons.},
  subject      = {Neuroendokrines System},
  language  = {en}
}
@phdthesis{Glueck2024,
  author    = {Gl{\"u}ck, Valentina},
  title     = {Habitual avoidance in trait anxiety and anxiety disorders},
  doi       = {10.25972/OPUS-36022},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-360227},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Maladaptive avoidance behaviors can contribute to the maintenance of fear, anxiety, and anxiety disorders. It has been proposed that, throughout anxiety disorder progression, extensively repeated avoidance may become a habit (i.e., habitual avoidance) instead of being controlled by internal threat-related goals (i.e., goal-directed avoidance). However, the process of the acquisition of habitual avoidance in anxiety disorders is not yet well understood. Accordingly, the current thesis aimed to investigate experimentally whether trait anxiety and anxiety disorders are associated with an increased shift from goal-directed to habitual avoidance. The aim of Study 1 was to develop an experimental operationalization of maladaptive habitual avoidance. To this end, we adapted a commonly used action control task, the outcome devaluation paradigm. In this task, habitual avoidance was operationalized as persistent responses after extensive training to avoid an unpleasant stimulus when the aversive outcome was devalued, i.e., when individuals knew the aversive outcome could not occur anymore. We included indicators for costly and low-cost habitual avoidance, whereby habitual avoidance was associated with a monetary cost, while low-cost habitual avoidance was not associated with monetary costs. In Experiment 1 of Study 1, a pronounced costly and non-costly outcome devaluation effect was observed. However, this result may have partly resulted from trial-and-error learning or a better-safe-than-sorry strategy since not instructions about the stimulus-response-outcome contingencies after the outcome devaluation procedure had been provided to the participants. In Experiment 2 of Study 1, instructions on these stimulus-response-outcome contingencies were included to prevent the potential confounders. As a result, we observed no indicators for costly habitual avoidance, but evidence for low-cost habitual avoidance, potentially because competing goal-directed responses could easily be implemented and inhibited costly habitual avoidance tendencies. In Study 2, the strength of habitual avoidance acquisition was compared between participants with and without anxiety disorders, using the experimental task of Experiment 1 in Study 1. The results indicated that costly and low-cost habitual avoidance was not more pronounced in participants with anxiety disorders than in the healthy control group. However, in an exploratory subgroup comparison, panic disorder predicted more substantial habitual avoidance acquisition than social anxiety disorder. In Study 3, we investigated whether trait anxiety as a risk factor for anxiety disorders is associated with a specific increased shift from goal-directed to habitual avoidance and approach. The task from the Experiment 1 of Study 1 was adapted to include parallel versions for operationalizing habitual avoidance and habitual approach responses. Using a within-subjects design, the individuals - pre-screened for high and low trait anxiety - took part in the approach and the avoidance outcome devaluation task version. The results suggested stronger non-costly habitual responses in more highly trait-anxious individuals independent of the task version, and suggested a tendency towards an impact of trait anxiety on costly habitual approach rather than on costly habitual avoidance. In summary, individuals with high trait anxiety or anxiety disorders did not develop habitual avoidance more readily than individuals with low trait anxiety or without anxiety disorders. Therefore, this thesis does not support the assumption that an increased tendency to acquire habitual avoidance contributes to persistent maladaptive avoidance in anxiety disorders. The thesis also contributes to the discourse on the validity of outcome devaluation studies in general by highlighting the impact of task features, such as the instructions after the outcome devaluation procedure or the task difficulty in the test phase, on the experimental results. Such validity issues may partly explain the heterogeneity of findings in research with the outcome devaluation paradigm. We suggest ways towards more valid operationalizations of habitual avoidance in future studies.},
  subject      = {Gewohnheit},
  language  = {en}
}
@phdthesis{Nirchal2024,
  author    = {Nirchal, Naveen Kumar},
  title     = {Mechanistische Regulierung des gastro{\"o}sophagealen {\"U}bergangs und die Rolle der Retins{\"a}ure bei der Entwicklung des Barrett-{\"O}sophagus},
  doi       = {10.25972/OPUS-31155},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-311556},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Der gastro{\"o}sophageale {\"U}bergang (GEJ), der die Region abgrenzt, in der der distale {\"O}sophagus auf die proximale Magenregion trifft, ist bekannt f{\"u}r die Entwicklung pathologischer Zust{\"a}nde, wie Metaplasie und Adenokarzinom des {\"O}sophagus (EAC). Es ist wichtig, die Mechanismen der Entwicklungsstadien zu verstehen, die zu EAC f{\"u}hren, da die Inzidenzrate von EAC in den letzten 4 Jahrzehnten um das 7-fache gestiegen ist und die Gesamt{\"u}berlebensrate von 5 Jahren 18,4 \% betr{\"a}gt. In den meisten F{\"a}llenwird die Diagnose im fortgeschrittenen Stadium ohne vorherige Symptome erstellt. Der Hauptvorl{\"a}ufer f{\"u}r die Entwicklung von EAC ist eine pr{\"a}maligne Vorstufe namens Barrett-{\"O}sophagus (BE). BE ist der metaplastische Zustand, bei dem das mehrschichtige Plattenepithel des nativen {\"O}sophagus durch ein spezialisiertes einschichtiges S{\"a}ulenepithel ersetzt wird, das die molekularen Eigenschaften des Magen- sowie des Darmepithels aufweist. Zu den wichtigsten Risikofaktoren f{\"u}r die Entwicklung von BE geh{\"o}ren die chronische gastro{\"o}sophageale Refluxkrankheit (GERD), eine ver{\"a}nderte Mikrobiota und ver{\"a}nderte Retins{\"a}ure-Signalwege (RA). Es ist unklar, welche Zelle der Ursprung f{\"u}r BE ist, da es keine eindeutigen Beweisen f{\"u}r den Prozess der BE-Initiation gibt. In dieser Arbeit habe ich untersucht, wie die GEJ-Hom{\"o}ostase in gesundem Gewebe durch stammzellregulatorische Morphogene aufrechterhalten wird, welche Rolle der Vitamin-A (RA-Signal{\"u}bertragung) spieltund wie ihre Ver{\"a}nderung zur BE-Entwicklung beitr{\"a}gt. Im ersten Teil meiner Dissertation habe ich anhand von Einzelmolek{\"u}l-RNA in situ-Hybridisierung und Immunhistochemie eindeutig das Vorhandensein von zwei Arten von Epithelzellen nachweisen k{\"o}nnen, dem Plattenepithel in der Speiser{\"o}hre und dem S{\"a}ulenepithel imMagenbereich des GEJ. Mittels Abstammungsanalysen im Mausmodell konnte ich zeigen, dass die Epithelzellen des {\"O}sophagus und des Magens von zwei verschiedenen epithelialen Stammzelllinien imGEJ abstammen. Die Grenze zwischen Plattenepithel und S{\"a}ulenepithelzellen im SCJ des GEJ wirddurch gegens{\"a}tzliche Wnt-Mikroumgebungen streng reguliert. Plattenepithelstammzellen des {\"O}sophagus werden durch das Wnt-hemmende Mikroumgebungssignal aufrechterhalten, w{\"a}hrend Magens{\"a}ulenepithelzellen durch das Wnt-aktivierende Signal aus dem Stromakompartiment erhalten werden. Ich habe die in vivo Erhaltung der Epithelstammzellen des GEJ mit Hilfe eines in vitro Epithel-3D-Organoidkulturmodells rekonstruiert. Das Wachstum und die Vermehrung von Magens{\"a}ulenepithel-Organoiden h{\"a}ngen von Wnt-Wachstumsfaktoren ab, w{\"a}hrend das Wachstum von Plattenepithel-Organoiden von Wnt-defizienten Kulturbedingungen abh{\"a}ngt. Dar{\"u}ber hinaus zeigte die Einzelzell-RNA-Sequenzanalyse (scRNA-seq) der aus Organoiden gewonnenenEpithelzellen, dass der nicht-kanonische Wnt/ planar cell polarity (PCP) Signalweg an der Regulierung der Plattenepithelzellen beteiligt ist. Im Gegensatz dazu werden s{\"a}ulenf{\"o}rmige Magenepithelzellen durch den kanonischen Wnt/beta-Catenin- und den nicht-kanonischen Wnt/Ca2+-Weg reguliert. Meine Daten zeigen, dass die SCJ-Epithelzellen, die am GEJ verschmelzen, durch entgegengesetzte stromale Wnt-Faktoren und unterschiedliche Wnt-Weg-Signalee in den Epithelzellen reguliert werden. Im zweiten Teil der Dissertation untersuchte ich die Rolle der bioaktiven Vitamin A Verbindung RA auf {\"O}sophagus- und Magenepithelstammzellen. Die In-vitro-Behandlung von epithelialen Organoiden der Speiser{\"o}hre und des Magens mitRA oder seinem pharmakologischen Inhibitors BMS 493 zeigte, dass jeder Zelltyp unterschiedlich reguliert wurde. Ich beobachtete, dass eine verst{\"a}rkte RA die Differenzierung von Stammzellen und den Verlust der Schichtung f{\"o}rderte, w{\"a}hrend die RA-Hemmung zu einer verst{\"a}rkten Stammzellbildung und Regeneration im mehrschichtigen Epithel der Speiser{\"o}hre f{\"u}hrte. Im Gegensatz zur Speiser{\"o}hre ist der RA-Signalweg in Magen-Organoiden aktiv, und die Hemmung von RA hat ein reduziertes Wachstum von Magen-Organoiden. Globale transkriptomische Daten und scRNA-seq-Daten zeigten, dass derRA-Signalweg einen Ruheph{\"a}notyp in den {\"O}sophaguszellen induziert. Dagegen f{\"u}hrt das Fehlen von RA in Magenepithelzellen zur Expression von Genen, die mit BE assoziiert sind. Daher isteine r{\"a}umlich definierte Regulation der Wnt- und Retins{\"a}ure-Signalgebung amGEJ entscheidend f{\"u}r eine gesunde Hom{\"o}ostase, und ihre St{\"o}rung f{\"u}hrt zur Entwicklung von Krankheiten.},
  subject      = {Retinoes{\"a}ure},
  language  = {en}
}
@phdthesis{Scheiner2024,
  author    = {Scheiner, Christin},
  title     = {Vulnerability in adolescence: prevalence, pandemic impact and prevention},
  doi       = {10.25972/OPUS-35164},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-351644},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {This compilation focuses on adolescent mental disorders and their prevention. It comprises three distinct studies, each contributing to a deeper understanding of this critical topic. This work addresses a critical gap in the understanding of, and approach to, adolescent mental health, and as a result reveals a critically important and urgently needed policy implication for action. The thematic structure of these studies begins with an examination of the epidemiology of child and adolescent mental disorders. Baseline data were collected from N = 877 adolescents with a mean age of 12.43 years (SD = 0.65). Mental health problems, such as depressive symptoms, non-suicidal self-injury, suicidal ideation, symptoms of eating disorders, and gender differences, are thoroughly examined. Results revealed a significant portion of our sample displaying mental health problems as early as the 6th and 7th grades, with girls generally being more affected than boys. The findings underscore the importance of early adolescence in the emergence of mental health problems and thereby emphasize the need for preventive measures. Moving beyond prevalence estimates, the compilation delves into the etiology of these disorders, exploring their potential correlation with a COVID-19 infection. Understanding the early signs and risk factors is crucial for timely support. While numerous studies have investigated potential risk and protective factors during the pandemic, our focus shifts to adolescents' coping when an infection with the virus was involved (N = 2,154, M = 12.31, SD = 0.67). We hypothesized that students infected or with close family members infected, would exhibit an increased psychopathology and a decreased functioning of protective factors such as self-efficacy or self-esteem. We found no connection between infection and the mental health status within our sample, but protective factors and mental well-being were positively associated. Thus, universal primary prevention appears to be the preferred approach for promoting mental health. Lastly, the compilation introduces LessStress, a noteworthy contribution to more evidence-based prevention programs. This universal approach is designed to reduce stress in schools, accompanied by a cluster-randomized trial to evaluate its effectiveness (estimated sample size N = 1,894). Existing studies have demonstrated the effectiveness of stress prevention, leading us to introduce a short and easy-to-implement prevention program. There is positive evidence for one-lesson interventions in schools for promoting well-being and health behaviors among adolescents. LessStress is designed based on a life skills approach that not only imparts psychoeducational content but also teaches skills relevant to everyday life and directly applicable. Throughout these studies, a common thread emerges: the pressing need to address mental disorders during childhood and adolescence. These formative years play a pivotal role in the development of mental health problems. These formative years play a crucial role in the development of mental health problems. They highlight the importance of epidemiological data collection and analysis based on the latest models to develop prevention interventions that are not only effective but also reach young people on a global level.},
  subject      = {Jugend},
  language  = {en}
}
@phdthesis{Knorr2024,
  author    = {Knorr, Susanne},
  title     = {Pathophysiology of early-onset isolated dystonia in a DYT-TOR1A rat model with trauma-induced dystonia-like movements},
  doi       = {10.25972/OPUS-20609},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-206096},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Early-onset torsion dystonia (DYT-TOR1A, DYT1) is an inherited hyperkinetic movement disorder caused by a mutation of the TOR1A gene encoding the torsinA protein. DYT-TOR1A is characterized as a network disorder of the central nervous system (CNS), including predominantly the cortico-basal ganglia-thalamo-cortical loop resulting in a severe generalized dystonic phenotype. The pathophysiology of DYTTOR1A is not fully understood. Molecular levels up to large-scale network levels of the CNS are suggested to be affected in the pathophysiology of DYT-TOR1A. The reduced penetrance of 30\% - 40\% indicates a gene-environmental interaction, hypothesized as "second hit". The lack of appropriate and phenotypic DYT-TOR1A animal models encouraged us to verify the "second hit" hypothesis through a unilateral peripheral nerve trauma of the sciatic nerve in a transgenic asymptomatic DYT-TOR1A rat model (∆ETorA), overexpressing the human mutated torsinA protein. In a multiscale approach, this animal model was characterized phenotypically and pathophysiologically. Nerve-injured ∆ETorA rats revealed dystonia-like movements (DLM) with a partially generalized phenotype. A physiomarker of human dystonia, describing increased theta oscillation in the globus pallidus internus (GPi), was found in the entopeduncular nucleus (EP), the rodent equivalent to the human GPi, of nerve-injured ∆ETorA rats. Altered oscillation patterns were also observed in the primary motor cortex. Highfrequency stimulation (HFS) of the EP reduced DLM and modulated altered oscillatory activity in the EP and primary motor cortex in nerve-injured ∆ETorA rats. Moreover, the dopaminergic system in ∆ETorA rats demonstrated a significant increased striatal dopamine release and dopamine turnover. Whole transcriptome analysis revealed differentially expressed genes of the circadian clock and the energy metabolism, thereby pointing towards novel, putative pathways in the pathophysiology of DYTTOR1A dystonia. In summary, peripheral nerve trauma can trigger DLM in genetically predisposed asymptomatic ΔETorA rats leading to neurobiological alteration in the central motor network on multiple levels and thereby supporting the "second hit" hypothesis. This novel symptomatic DYT-TOR1A rat model, based on a DYT-TOR1A genetic background, may prove as a valuable chance for DYT-TOR1A dystonia, to further investigate the pathomechanism in more detail and to establish new treatment strategies.},
  subject      = {Dystonie},
  language  = {en}
}
@phdthesis{PenaMosca2024,
  author    = {Pe{\~n}a Mosca, Mar{\´i}a Josefina},
  title     = {Local regulation of T-cell immunity in the intestinal mucosa},
  doi       = {10.25972/OPUS-35266},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-352665},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {After priming in Peyer's patches (PPs) and mesenteric lymph nodes (mLN) T- cells infiltrate the intestine through lymphatic draining and homing through the bloodstream. However, we found that in mouse models of acute graft-versus-host disease (GvHD), a subset of alloreactive T-cells directly migrates from PPs to the adjacent intestinal lamina propria (LP), bypassing the normal lymphatic drainage and vascular trafficking routes. Notably, this direct migration occurred in irradiated and unirradiated GvHD models, indicating that irradiation is not a prerequisite for this observed behavior. Next, we established a method termed serial intravascular staining (SIVS) in mouse models to systematically investigate the trafficking and migration of donor T- cells in the early stages of acute GvHD initiation. We found that the direct migration of T-cells from PPs to LP resulted in faster recruitment of cells after allogeneic hematopoietic cell transplantation (allo-HCT). These directly migrating T-cells were found to be in an activated and proliferative state, exhibiting a TH1/TH17-like phenotype and producing cytokines such as IFN-γ and TNF-α. Furthermore, we observed that the directly migrating alloreactive T-cells expressed specific integrins (α4+, αE+) and chemokine receptors (CxCR3+, CCR5+, and CCR9+). Surprisingly, blocking these integrins and chemokine-coupled receptors did not hinder the direct migration of T- cells from PPs to LP, suggesting the involvement of alternative mechanisms. Previous experiments ruled out the involvement of S1PR1 and topographical features of macrophages, leading us to hypothesize that mediators of cytoskeleton reorganization, such as Coro1a, Dock2, or Cdc42, may play a role in this unique migration process. Additionally, we observed that directly migrating T-cells created a local inflammatory microenvironment, which attracts circulating T-cells. Histological analysis confirmed that alloreactive PPs-derived T-cells and bloodborne T-cells colocalized. We employed two experimental approaches, including either photoconversion of T-cells in PPs or direct transfer of activated T-cells into the vasculature, to demonstrate this colocalization. We hypothesize that cytokines released by migrating T-cells, such as IFN-γ and TNF-α, may play a role in recruiting T-cells from the vasculature, as inhibiting chemokine-coupled receptors did not impair recruitment.},
  subject      = {T-Lymphozyt},
  language  = {en}
}
@phdthesis{Neagoe2024,
  author    = {Neagoe, Raluca Alexandra Iulia},
  title     = {Development of techniques for studying the platelet glycoprotein receptors GPVI and GPIb localisation and signalling},
  doi       = {10.25972/OPUS-31306},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-313064},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Platelets play an important role in haemostasis by mediating blood clotting at sites of blood vessel damage. Platelets, also participate in pathological conditions including thrombosis and inflammation. Upon vessel damage, two glycoprotein receptors, the GPIb-IX-V complex and GPVI, play important roles in platelet capture and activation. GPIb-IX-V binds to von Willebrand factor and GPVI to collagen. This initiates a signalling cascade resulting in platelet shape change and spreading, which is dependent on the actin cytoskeleton. This thesis aimed to develop and implement different super-resolution microscopy techniques to gain a deeper understanding of the conformation and location of these receptors in the platelet plasma membrane, and to provide insights into their signalling pathways. We suggest direct stochastic optical reconstruction microscopy (dSTORM) and structured illumination microscopy (SIM) as the best candidates for imaging single platelets, whereas expansion microscopy (ExM) is ideal for imaging platelets aggregates. Furthermore, we highlighted the role of the actin cytoskeleton, through Rac in GPVI signalling pathway. Inhibition of Rac, with EHT1864 in human platelets induced GPVI and GPV, but not GPIbα shedding. Furthermore, EHT1864 treatment did not change GPVI dimerisation or clustering, however, it decreased phospholipase Cγ2 phosphorylation levels, in human, but not murine platelets, highlighting interspecies differences. In summary, this PhD thesis demonstrates that; 1) Rac alters GPVI signalling pathway in human but not mouse platelets; 2) our newly developed ExM protocol can be used to image platelet aggregates labelled with F(ab') fragments},
  subject      = {Platelet-Membranglykoprotein p62},
  language  = {en}
}
@phdthesis{Weisert2024,
  author    = {Weisert, Nadine},
  title     = {Characterization of telomere-associated proteins in \(Trypanosoma\) \(brucei\)},
  doi       = {10.25972/OPUS-35273},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-352732},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {The unicellular pathogen Trypanosoma brucei is the causative agent of African trypanosomiasis, an endemic disease prevalent in sub-Saharan Africa. Trypanosoma brucei alternates between a mammalian host and the tsetse fly vector. The extracellular parasite survives in the mammalian bloodstream by periodically exchanging their ˈvariant surface glycoproteinˈ (VSG) coat to evade the host immune response. This antigenic variation is achieved through monoallelic expression of one VSG variant from subtelomeric ˈbloodstream form expression sitesˈ (BES) at a given timepoint. During the differentiation from the bloodstream form (BSF) to the procyclic form (PCF) in the tsetse fly midgut, the stage specific surface protein is transcriptionally silenced and replaced by procyclins. Due to their subtelomeric localization on the chromosomes, VSG transcription and silencing is partly regulated by homologues of the mammalian telomere complex such as TbTRF, TbTIF2 and TbRAP1 as well as by ˈtelomere-associated proteinsˈ (TelAPs) like TelAP1. To gain more insights into transcription regulation of VSG genes, the identification and characterization of other TelAPs is critical and has not yet been achieved. In a previous study, two biochemical approaches were used to identify other novel TelAPs. By using ˈco-immunoprecipitationˈ (co-IP) to enrich possible interaction partners of TbTRF and by affinity chromatography using telomeric repeat oligonucleotides, a listing of TelAP candidates has been conducted. With this approach TelAP1 was identified as a novel component of the telomere complex, involved in the kinetics of transcriptional BES silencing during BSF to PCF differentiation. To gain further insights into the telomere complex composition, other previously enriched proteins were characterized through a screening process using RNA interference to deplete potential candidates. VSG expression profile changes and overall proteomic changes after depletion were analyzed by mass spectrometry. With this method, one can gain insights into the functions of the proteins and their involvement in VSG expression site regulation. To validate the interaction of proteins enriched by co-IP with TbTRF and TelAP1 and to identify novel interaction proteins, I performed reciprocal affinity purifications of the four most promising candidates (TelAP2, TelAP3, PPL2 and PolIE) and additionally confirmed colocalization of two candidates with TbTRF via immunofluorescence (TelAP2, TelAP3). TelAP3 colocalizes with TbTRF and potentially interacts with TbTRF, TbTIF2, TelAP1 and TelAP2, as well as with two translesion polymerases PPL2 and PolIE in BSF. PPL2 and PolIE seem to be in close contact to each other at the telomeric ends and fulfill different roles as only PolIE is involved in VSG regulation while PPL2 is not. TelAP2 was previously characterized to be associated with telomeres by partially colocalizing with TbTRF and cells show a VSG derepression phenotype when the protein was depleted. Here I show that TelAP2 interacts with the telomere-binding proteins TbTRF and TbTIF2 as well as with the telomere-associated protein TelAP1 in BSF and that TelAP2 depletion results in a loss of TelAP1 colocalization with TbTRF in BSF. In conclusion, this study demonstrates that characterizing potential TelAPs is effective in gaining insights into the telomeric complex's composition and its role in VSG regulation in Trypanosoma brucei. Understanding these interactions could potentially lead to new therapeutic targets for combatting African trypanosomiasis.},
  subject      = {Telomer <Molekulargenetik>},
  language  = {en}
}
@phdthesis{Choi2024,
  author    = {Choi, Jihyoung},
  title     = {Development of an Add-On Electrode for Non-Invasive Monitoring in Bioreactor Cultures and Medical Devices},
  doi       = {10.25972/OPUS-35823},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-358232},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Electrochemical impedance spectroscopy (EIS) is a valuable technique analyzing electrochemical behavior of biological systems such as electrical characterization of cells and biomolecules, drug screening, and biomaterials in biomedical field. In EIS, an alternating current (AC) power signal is applied to the biological system, and the impedance of the system is measured over a range of frequencies. In vitro culture models of endothelial or epithelial barrier tissue can be achieved by culturing barrier tissue on scaffolds made with synthetic or biological materials that provide separate compartments (apical and basal sides), allowing for further studies on drug transport. EIS is a great candidate for non-invasive and real-time monitoring of the electrical properties that correlate with barrier integrity during the tissue modeling. Although commercially available transendothelial/transepithelial electrical resistance (TEER) measurement devices are widely used, their use is particularly common in static transwell culture. EIS is considered more suitable than TEER measurement devices in bioreactor cultures that involve dynamic fluid flow to obtain accurate and reliable measurements. Furthermore, while TEER measurement devices can only assess resistance at a single frequency, EIS measurements can capture both resistance and capacitance properties of cells, providing additional information about the cellular barrier's characteristics across various frequencies. Incorporating EIS into a bioreactor system requires the careful optimization of electrode integration within the bioreactor setup and measurement parameters to ensure accurate EIS measurements. Since bioreactors vary in size and design depending on the purpose of the study, most studies have reported using an electrode system specifically designed for a particular bioreactor. The aim of this work was to produce multi-applicable electrodes and established methods for automated non-invasive and real-time monitoring using the EIS technique in bioreactor cultures. Key to the electrode material, titanium nitride (TiN) coating was fabricated on different substrates (materials and shape) using physical vapor deposition (PVD) and housed in a polydimethylsiloxane (PDMS) structure to allow the electrodes to function as independent units. Various electrode designs were evaluated for double-layer capacitance and morphology using EIS and scanning electron microscopy (SEM), respectively. The TiN-coated tube electrode was identified as the optimal choice. Furthermore, EIS measurements were performed to examine the impact of influential parameters related to culture conditions on the TiN-coated electrode system. In order to demonstrate the versatility of the electrodes, these electrodes were then integrated into in different types of perfusion bioreactors for monitoring barrier cells. Blood-brain barrier (BBB) cells were cultured in the newly developed dynamic flow bioreactor, while human umblical vascular endothelial cells (HUVECs) and Caco-2 cells were cultured in the miniature hollow fiber bioreactor (HFBR). As a result, the TiN-coated tube electrode system enabled investigation of BBB barrier integrity in long-term bioreactor culture. While EIS measurement could not detect HUVECs electrical properties in miniature HFBR culture, there was the possibility of measuring the barrier integrity of Caco-2 cells, indicating potential usefulness for evaluating their barrier function. Following the bioreactor cultures, the application of the TiN-coated tube electrode was expanded to hemofiltration, based on the hypothesis that the EIS system may be used to monitor clotting or clogging phenomena in hemofiltration. The findings suggest that the EIS monitoring system can track changes in ion concentration of blood before and after hemofiltration in real-time, which may serve as an indicator of clogging of filter membranes. Overall, our research demonstrates the potential of TiN-coated tube electrodes for sensitive and versatile non-invasive monitoring in bioreactor cultures and medical devices.},
  subject      = {Monitoring},
  language  = {en}
}
@phdthesis{Kutschka2024,
  author    = {Kutschka, Ilona},
  title     = {Activation of the integrated stress response induces remodeling of cardiac metabolism in Barth Syndrome},
  doi       = {10.25972/OPUS-35818},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-358186},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Barth Syndrome (BTHS) is an inherited X-chromosomal linked disorder, characterized by early development of cardiomyopathy, immune system defects, skeletal muscle myopathy and growth retardation. The disease displays a wide variety of symptoms including heart failure, exercise intolerance and fatigue due to the muscle weakness. The cause of the disease are mutations in the gene encoding for the mitochondrial transacylase Tafazzin (TAZ), which is important for remodeling of the phospholipid cardiolipin (CL). All mutations result in a pronounced decrease of the functional enzyme leading to an increase of monolysocardiolipin (MLCL), the precursor of mature CL, and a decrease in mature CL itself. CL is a hallmark phospholipid of mitochondrial membranes, highly enriched in the inner mitochondrial membrane (IMM). It is not only important for the formation of the cristae structures, but also for the function of different protein complexes associated with the mitochondrial membrane. Reduced levels of mature CL cause remodeling of the respiratory chain supercomplexes, impaired respiration, defects in the Krebs cycle and a loss of mitochondrial calcium uniporter (MCU) protein. The defective Ca2+ handling causes impaired redox homeostasis and energy metabolism resulting in cellular arrhythmias and defective electrical conduction. In an uncompensated situation, blunting mitochondrial Ca2+ uptake provokes increased mitochondrial emission of H2O2 during workload transitions, related to oxidation of NADPH, which is required to regenerate anti-oxidative enzymes. However, in the hearts and cardiac myocytes of mice with a global knock-down of the Taz gene (Taz-KD), no increase in mitochondrial ROS was observed, suggesting that other metabolic pathways may have compensated for reduced Krebs cycle activation. The healthy heart produces most of its energy by consuming fatty acids. In this study, the fatty acid uptake into mitochondria and their further degradation was investigated, which showed a switch of the metabolism in general in the Taz-KD mouse model. In vivo studies revealed an increase of glucose uptake into the heart and decreased fatty acid uptake and oxidation. Disturbed energy conversion resulted in activation of retrograde signaling pathways, implicating overall changes in the cell metabolism. Upregulated integrated stress response (ISR) was confirmed by increased levels of the downstream target, i.e., the activating transcription factor 4 (ATF4). A Tafazzin knockout mouse embryonal fibroblast cell model (TazKO) was used to inhibit the ISR using siRNA transfection or pharmaceutical inhibition. This verified the central role of II the ISR in regulating the metabolism in BTHS. Moreover, an increased metabolic flux into glutathione biosynthesis was observed, which supports redox homeostasis. In vivo PET-CT scans depicted elevated activity of the xCT system in the BTHS mouse heart, which transports essential amino acids for the biosynthesis of glutathione precursors. Furthermore, the stress induced signaling pathway also affected the glutamate metabolism, which fuels into the Krebs cycle via -ketoglutarate and therefore supports energy converting pathways. In summary, this thesis provides novel insights into the energy metabolism and redox homeostasis in Barth syndrome cardiomyopathy and its regulation by the integrated stress response, which plays a central role in the metabolic alterations. The aim of the thesis was to improve the understanding of these metabolic changes and to identify novel targets, which can provide new possibilities for therapeutic intervention in Barth syndrome.},
  subject      = {Herzmuskelkrankheit},
  language  = {en}
}
@phdthesis{Kuehnemundt2024,
  author    = {K{\"u}hnemundt, Johanna},
  title     = {Defined microphysiologic 3D tumour models with aspects from the tumour microenvironment for the evaluation of cellular immunotherapies},
  doi       = {10.25972/OPUS-27667},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-276674},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Adoptive cellular immunotherapy with chimeric antigen receptor (CAR) T cells is highly effective in haematological malignancies. This success, however, has not been achieved in solid tumours so far. In contrast to hematologic malignancies, solid tumours include a hostile tumour microenvironment (TME), that poses additional challenges for curative effects and consistent therapeutic outcome. These challenges manifest in physical and immunological barriers that dampen efficacy of the CAR T cells. Preclinical testing of novel cellular immunotherapies is performed mainly in 2D cell culture and animal experiments. While 2D cell culture is an easy technique for efficacy analysis, animal studies reveal information about toxicity in vivo. However, 2D cell culture cannot fully reflect the complexity observed in vivo, because cells are cultured without anchorage to a matrix and only short-term periods are feasible. Animal studies provide a more complex tissue environment, but xenografts often lack human stroma and tumour inoculation occurs mostly ectopically. This emphasises the need for standardisable and scalable tumour models with incorporated TME-aspects, which enable preclinical testing with enhanced predictive value for the clinical outcome of immunotherapies. Therefore, microphysiologic 3D tumour models based on the biological SISmuc (Small Intestinal mucosa and Submucosa) matrix with preserved basement membrane were engaged and improved in this work to serve as a modular and versatile tumour model for efficacy testing of CAR T cells. In order to reflect a variety of cancer entities, TME-aspects, long-term stability and to enhance the read-out options they were further adapted to achieve scalable and standardisable defined microphysiologic 3D tumour models. In this work, novel culture modalities (semi-static, sandwich-culture) were characterised and established that led to an increased and organised tissue generation and long-term stability. Application of the SISmuc matrix was extended to sarcoma and melanoma models and serial bioluminescence intensity (BLI)-based in vivo imaging analysis was established in the microphysiologic 3D tumour models, which represents a time-efficient read-out method for quality evaluation of the models and treatment efficacy analysis, that is independent of the cell phenotype. Isolation of cancer-associated-fibroblasts (CAFs) from lung (tumour) tissue was demonstrated and CAF-implementation further led to stromal-enriched microphysiologic 3D tumour models with in vivo-comparable tissue-like architecture. Presence of CAFs was confirmed by CAF-associated markers (FAP, α-SMA, MMP-2/-9) and cytokines correlated with CAF phenotype, angiogenesis, invasion and immunomodulation. Additionally, an endothelial cell barrier was implemented for static and dynamic culture in a novel bioreactor set-up, which is of particular interest for the analysis of immune cell diapedesis. Studies in microphysiologic 3D Ewing's sarcoma models indicated that sarcoma cells could be sensitised for GD2-targeting CAR T cells. After enhancing the scale of assessment of the microphysiologic 3D tumour models and improving them for CAR T cell testing, the tumour models were used to analyse their sensitivity towards differently designed receptor tyrosine kinase-like orphan receptor 1 (ROR1) CAR T cells and to study the effects of the incorporated TME-aspects on the CAR T cell treatment respectively. ROR1 has been described as a suitable target for several malignancies including triple negative breast cancer (TNBC), as well as lung cancer. Therefore, microphysiologic 3D TNBC and lung cancer models were established. Analysis of ROR1 CAR T cells that differed in costimulation, spacer length and targeting domain, revealed, that the microphysiologic 3D tumour models are highly sensitive and can distinguish optimal from sub-optimal CAR design. Here, higher affinity of the targeting domain induced stronger anti-tumour efficacy and anti-tumour function depended on spacer length, respectively. Long-term treatment for 14 days with ROR1 CAR T cells was demonstrated in dynamic microphysiologic 3D lung tumour models, which did not result in complete tumour cell removal, whereas direct injection of CAR T cells into TNBC and lung tumour models represented an alternative route of application in addition to administration via the medium flow, as it induced strong anti-tumour response. Influence of the incorporated TME-aspects on ROR1 CAR T cell therapy represented by CAF-incorporation and/or TGF-β supplementation was analysed. Presence of TGF-β revealed that the specific TGF-β receptor inhibitor SD-208 improves ROR1 CAR T cell function, because it effectively abrogated immunosuppressive effects of TGF-β in TNBC models. Implementation of CAFs should provide a physical and immunological barrier towards ROR1 CAR T cells, which, however, was not confirmed, as ROR1 CAR T cell function was retained in the presence of CAFs in stromal-enriched microphysiologic 3D lung tumour models. The absence of an effect of CAF enrichment on CAR T cell efficacy suggests a missing component for the development of an immunosuppressive TME, even though immunomodulatory cytokines were detected in co-culture models. Finally, improved gene-edited ROR1 CAR T cells lacking exhaustion-associated genes (PD-1, TGF-β-receptor or both) were challenged by the combination of CAF-enrichment and TGF-β in microphysiologic 3D TNBC models. Results indicated that the absence of PD-1 and TGF-β receptor leads to improved CAR T cells, that induce strong tumour cell lysis, and are protected against the hostile TME. Collectively, the microphysiologic 3D tumour models presented in this work reflect aspects of the hostile TME of solid tumours, engage BLI-based analysis and provide long-term tissue homeostasis. Therefore, they present a defined, scalable, reproducible, standardisable and exportable model for translational research with enhanced predictive value for efficacy testing and candidate selection of cellular immunotherapy, as exemplified by ROR1 CAR T cells.},
  subject      = {Immuntherapie},
  language  = {en}
}
@phdthesis{Yu2024,
  author    = {Yu, Yanying},
  title     = {Applied machine learning for the analysis of CRISPR-Cas systems},
  doi       = {10.25972/OPUS-32021},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-320219},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Among the defense strategies developed in microbes over millions of years, the innate adaptive CRISPR-Cas immune systems have spread across most of bacteria and archaea. The flexibility, simplicity, and specificity of CRISPR-Cas systems have laid the foundation for CRISPR-based genetic tools. Yet, the efficient administration of CRISPR-based tools demands rational designs to maximize the on-target efficiency and off-target specificity. Specifically, the selection of guide RNAs (gRNAs), which play a crucial role in the target recognition of CRISPR-Cas systems, is non-trivial. Despite the fact that the emerging machine learning techniques provide a solution to aid in gRNA design with prediction algorithms, design rules for many CRISPR-Cas systems are ill-defined, hindering their broader applications. CRISPR interference (CRISPRi), an alternative gene silencing technique using a catalytically dead Cas protein to interfere with transcription, is a leading technique in bacteria for functional interrogation, pathway manipulation, and genome-wide screens. Although the application is promising, it also is hindered by under-investigated design rules. Therefore, in this work, I develop a state-of-art predictive machine learning model for guide silencing efficiency in bacteria leveraging the advantages of feature engineering, data integration, interpretable AI, and automated machine learning. I first systematically investigate the influential factors that attribute to the extent of depletion in multiple CRISPRi genome-wide essentiality screens in Escherichia coli and demonstrate the surprising dominant contribution of gene-specific effects, such as gene expression level. These observations allowed me to segregate the confounding gene-specific effects using a mixed-effect random forest (MERF) model to provide a better estimate of guide efficiency, together with the improvement led by integrating multiple screens. The MERF model outperformed existing tools in an independent high-throughput saturating screen. I next interpret the predictive model to extract the design rules for robust gene silencing, such as the preference for cytosine and disfavoring for guanine and thymine within and around the protospacer adjacent motif (PAM) sequence. I further incorporated the MERF model in a web-based tool that is freely accessible at www.ciao.helmholtz-hiri.de. When comparing the MERF model with existing tools, the performance of the alternative gRNA design tool optimized for CRISPRi in eukaryotes when applied to bacteria was far from satisfying, questioning the robustness of prediction algorithms across organisms. In addition, the CRISPR-Cas systems exhibit diverse mechanisms albeit with some similarities. The captured predictive patterns from one dataset thereby are at risk of poor generalization when applied across organisms and CRISPR-Cas techniques. To fill the gap, the machine learning approach I present here for CRISPRi could serve as a blueprint for the effective development of prediction algorithms for specific organisms or CRISPR-Cas systems of interest. The explicit workflow includes three principle steps: 1) accommodating the feature set for the CRISPR-Cas system or technique; 2) optimizing a machine learning model using automated machine learning; 3) explaining the model using interpretable AI. To illustrate the applicability of the workflow and diversity of results when applied across different bacteria and CRISPR-Cas systems, I have applied this workflow to analyze three distinct CRISPR-Cas genome-wide screens. From the CRISPR base editor essentiality screen in E. coli, I have determined the PAM preference and sequence context in the editing window for efficient editing, such as A at the 2nd position of PAM, A/TT/TG downstream of PAM, and TC at the 4th to 5th position of gRNAs. From the CRISPR-Cas13a screen in E. coli, in addition to the strong correlation with the guide depletion, the target expression level is the strongest predictor in the model, supporting it as a main determinant of the activation of Cas13-induced immunity and better characterizing the CRISPR-Cas13 system. From the CRISPR-Cas12a screen in Klebsiella pneumoniae, I have extracted the design rules for robust antimicrobial activity across K. pneumoniae strains and provided a predictive algorithm for gRNA design, facilitating CRISPR-Cas12a as an alternative technique to tackle antibiotic resistance. Overall, this thesis presents an accurate prediction algorithm for CRISPRi guide efficiency in bacteria, providing insights into the determinants of efficient silencing and guide designs. The systematic exploration has led to a robust machine learning approach for effective model development in other bacteria and CRISPR-Cas systems. Applying the approach in the analysis of independent CRISPR-Cas screens not only sheds light on the design rules but also the mechanisms of the CRISPR-Cas systems. Together, I demonstrate that applied machine learning paves the way to a deeper understanding and a broader application of CRISPR-Cas systems.},
  subject      = {Maschinelles Lernen},
  language  = {en}
}
@phdthesis{Ramirez2024,
  author    = {Ramirez, Yesid A.},
  title     = {Structural basis of ubiquitin recognition and rational design of novel covalent inhibitors targeting Cdu1 from \(Chlamydia\) \(Trachomatis\)},
  doi       = {10.25972/OPUS-19168},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-191683},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {The WHO-designated neglected-disease pathogen Chlamydia trachomatis (CT) is a gram-negative bacterium responsible for the most frequently diagnosed sexually transmitted infection worldwide. CT infections can lead to infertility, blindness and reactive arthritis, among others. CT acts as an infectious agent by its ability to evade the immune response of its host, which includes the impairment of the NF-κB mediated inflammatory response and the Mcl1 pro-apoptotic pathway through its deubiquitylating, deneddylating and transacetylating enzyme ChlaDUB1 (Cdu1). Expression of Cdu1 is also connected to host cell Golgi apparatus fragmentation, a key process in CT infections. Cdu1 may this be an attractive drug target for the treatment of CT infections. However, a lead molecule for the development of novel potent inhibitors has been unknown so far. Sequence alignments and phylogenetic searches allocate Cdu1 in the CE clan of cysteine proteases. The adenovirus protease (adenain) also belongs to this clan and shares a high degree of structural similarity with Cdu1. Taking advantage of topological similarities between the active sites of Cdu1 and adenain, a target-hopping approach on a focused set of adenain inhibitors, developed at Novartis, has been pursued. The thereby identified cyano-pyrimidines represent the first active-site directed covalent reversible inhibitors for Cdu1. High-resolution crystal structures of Cdu1 in complex with the covalently bound cyano-pyrimidines as well as with its substrate ubiquitin have been elucidated. The structural data of this thesis, combined with enzymatic assays and covalent docking studies, provide valuable insights into Cdu1s activity, substrate recognition, active site pocket flexibility and potential hotspots for ligand interaction. Structure-informed drug design permitted the optimization of this cyano-pyrimidine based scaffold towards HJR108, the first molecule of its kind specifically designed to disrupt the function of Cdu1. The structures of potentially more potent and selective Cdu1 inhibitors are herein proposed. This thesis provides important insights towards our understanding of the structural basis of ubiquitin recognition by Cdu1, and the basis to design highly specific Cdu1 covalent inhibitors.},
  subject      = {Ubiquitin},
  language  = {en}
}
@phdthesis{HuttererneeHerzog2024,
  author    = {Hutterer, n{\´e}e Herzog, Katharina},
  title     = {Treatment-like use of discrimination training to reduce generalization of conditioned fear},
  doi       = {10.25972/OPUS-31728},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-317286},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Anxiety patients overgeneralize fear, also because of an inability to perceptually discriminate threat and safety signals. Therefore, some studies have developed discrimination training that successfully reduced the occurrence of fear generalization. The present work is the first to take a treatment-like approach by using discrimination training after generalization has occurred. Therefore, two studies were conducted with healthy participants using the same fear conditioning and generalization paradigm, with two faces as conditioned stimuli (CSs), and four facial morphs between CSs as generalization stimuli (GSs). Only one face (CS+) was followed by a loud scream (unconditioned stimulus, US). In Study 1, participants underwent either fear-relevant (discriminating faces) or fear-irrelevant discrimination training (discriminating width of lines) or a non-discriminative control training between the two generalization tests, each with or without feedback (n = 20 each). Generalization of US expectancy was reduced more effectively by fear-relevant compared to fear-irrelevant discrimination training. However, neither discrimination training was more effective than non-discriminative control training. Moreover, feedback reduced generalization of US expectancy only in discrimination training. Study 2 was designed to replicate the effects of the discrimination-training conditions in a large sample (N = 244) and examine their benefits in individuals at risk for anxiety disorders. Again, feedback reduced fear generalization particularly well for US expectancy. Fear relevance was not confirmed to be particularly fear-reducing in healthy participants, but may enhance training effects in individuals at risk of anxiety disorder. In summary, this work provides evidence that existing fear generalization can be reduced by discrimination training, likely involving several (higher-level) processes besides perceptual discrimination (e.g., motivational mechanisms in feedback conditions). Its use may be promising as part of individualized therapy for patients with difficulty discriminating similar stimuli.},
  subject      = {Furcht},
  language  = {en}
}
@phdthesis{KuklovskyformerFinke2024,
  author    = {Kuklovsky [former Finke], Valerie},
  title     = {Are some bees smarter than others? An examination of consistent individual differences in the cognitive abilities of honey bees},
  doi       = {10.25972/OPUS-32301},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-323012},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Cognition refers to the ability to of animals to acquire, process, store and use vital information from the environment. Cognitive processes are necessary to predict the future and reduce the uncertainty of the ever-changing environment. Classically, research on animal cognition focuses on decisive cognitive tests to determine the capacity of a species by the testing the ability of a few individuals. This approach views variability between these tested key individuals as unwanted noise and is thus often neglected. However, inter-individual variability provides important insights to behavioral plasticity, cognitive specialization and brain modularity. Honey bees Apis mellifera are a robust and traditional model for the study of learning, memory and cognition due to their impressive capabilities and rich behavioral repertoire. In this thesis I have applied a novel view on the learning abilities of honey bees by looking explicitly at individual differences in a variety of learning tasks. Are some individual bees consistently smarter than some of her sisters? If so, will a smart individual always perform good independent of the time, the context and the cognitive requirements or do bees show distinct isolated 'cognitive modules'? My thesis presents the first comprehensive investigation of consistent individual differences in the cognitive abilities of honey bees. To speak of an individual as behaving consistently, a crucial step is to test the individual multiple times to examine the repeatability of a behavior. I show that free-flying bees remain consistent in a visual discrimination task for three consecutive days. Successively, I explored individual consistency in cognitive proficiency across tasks involving different sensory modalities, contexts and cognitive requirements. I found that free-flying bees show a cognitive specialization between visual and olfactory learning but remained consistent across a simple discrimination task and a complex concept learning task. I wished to further explore individual consistency with respect to tasks of different cognitive complexity, a question that has never been tackled before in an insect. I thus performed a series of four experiments using either visual or olfactory stimuli and a different training context (free-flying and restrained) and tested bees in a discrimination task, reversal learning and negative patterning. Intriguingly, across all these experiments I evidenced the same results: The bees' performances were consistent across the discrimination task and reversal learning and negative patterning respectively. No association was evidenced between reversal learning and negative patterning. After establishing the existence of consistent individual differences in the cognitive proficiency of honey bees I wished to determine factors which could underlie these differences. Since genetic components are known to underlie inter-individual variability in learning abilities, I studied the effects of genetics on consistency in cognitive proficiency by contrasting bees originating from either from a hive with a single patriline (low genetic diversity) or with multiple patrilines (high genetic diversity). These two groups of bees showed differences in the patterns of individually correlated performances, indicating a genetic component accounts for consistent cognitive individuality. Another major factor underlying variability in learning performances is the individual responsiveness to sucrose solution and to visual stimuli, as evidenced by many studies on restrained bees showing a positive correlation between responsiveness to task relevant stimuli and learning performances. I thus tested whether these relationships between sucrose/visual responsiveness and learning performances are applicable for free-flying bees. Free-flying bees were again subjected to reversal learning and negative patterning and subsequently tested in the laboratory for their responsiveness to sucrose and to light. There was no evidence of a positive relationship between sucrose/visual responsiveness and neither performances of free-flying bees in an elemental discrimination, reversal learning and negative patterning. These findings indicate that relationships established between responsiveness to task relevant stimuli and learning proficiency established in the laboratory with restrained bees might not hold true for a completely different behavioral context i.e. for free-flying bees in their natural environment. These results show that the honey bee is an excellent insect model to study consistency in cognitive proficiency and to identify the underlying factors. I mainly discuss the results with respect to the question of brain modularity in insects and the adaptive significance of individuality in cognitive abilities for honey bee colonies. I also provide a proposition of research questions which tie in this theme of consistent cognitive proficiency and could provide fruitful areas for future research.},
  subject      = {Lernen},
  language  = {en}
}
@phdthesis{Beudert2024,
  author    = {Beudert, Matthias},
  title     = {Bioinspired Modification and Functionalization of Hydrogels for Applications in Biomedicine},
  doi       = {10.25972/OPUS-32288},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-322887},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Over the years, hydrogels have been developed and used for a huge variety of different applications ranging from drug delivery devices to medical products. In this thesis, a poly(2-methyl-2-oxazoline) (POx) / poly(2-n-propyl-2-oxazine) (POzi) bioink was modified and analyzed for the use in biofabrication and targeted drug delivery. In addition, the protein fibrinogen (Fbg) was genetically modified for an increased stability towards plasmin degradation for its use as wound sealant. In Chapter 1, a thermogelling, printable POx/POzi-based hydrogel was modified with furan and maleimide moieties in the hydrophilic polymer backbone facilitating post-printing maturation of the constructs via Diels-Alder chemistry. The modification enabled long-term stability of the hydrogel scaffolds in aqueous solutions which is necessary for applications in biofabrication or tissue engineering. Furthermore, we incorporated RGD-peptides into the hydrogel which led to cell adhesion and elongated morphology of fibroblast cells seeded on top of the scaffolds. Additional printing experiments demonstrate that the presented POx/POzi system is a promising platform for the use as a bioink in biofabrication. Chapter 2 highlights the versatility of the POx/POzi hydrogels by adapting the system to a use in targeted drug delivery. We used a bioinspired approach for a bioorthogonal conjugation of insulin-like growth factor I (IGF-I) to the polymer using an omega-chain-end dibenzocyclooctyne (DBCO) modification and a matrix metalloprotease-sensitive peptide linker. This approach enabled a bioresponsive release of IGF-I from hydrogels as well as spatial control over the protein distribution in 3D printed constructs which makes the system a candidate for the use in personalized medicine. Chapter 3 gives a general overview over the necessity of wound sealants and the current generations of fibrin sealants on the market including advantages and challenges. Furthermore, it highlights trends and potential new strategies to tackle current problems and broadens the toolbox for future generations of fibrin sealants. Chapter 4 applies the concepts of recombinant protein expression and molecular engineering to a novel generation of fibrin sealants. In a proof-of-concept study, we developed a new recombinant fibrinogen (rFbg) expression protocol and a Fbg mutant that is less susceptible to plasmin degradation. Targeted lysine of plasmin cleavage sites in Fbg were exchanged with alanine or histidine in different parts of the molecule. The protein was recombinantly produced and restricted plasmin digest was analyzed using high resolution mass spectrometry. In addition to that, we developed a novel time resolved screening protocol for the detection of new potential plasmin cleavage sites for further amino acid exchanges in the fibrin sealant.},
  subject      = {Hydrogel},
  language  = {en}
}
@phdthesis{Dietrich2024,
  author    = {Dietrich, Oliver},
  title     = {Integrating single-cell multi-omics to decipher host-pathogen interactions},
  doi       = {10.25972/OPUS-36013},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-360138},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Interactions between host and pathogen determine the development, progression and outcomes of disease. Medicine benefits from better descriptions of these interactions through increased precision of prevention, diagnosis and treatment of diseases. Single-cell genomics is a disruptive technology revolutionizing science by increasing the resolution with which we study diseases. Cell type specific changes in abundance or gene expression are now routinely investigated in diseases. Meanwhile, detecting cellular phenotypes across diseases can connect scientific fields and fuel discovery. Insights acquired through systematic analysis of high resolution data will soon be translated into clinical practice and improve decision making. Therefore, the continued use of single-cell technologies and their application towards clinical samples will improve molecular interpretation, patient stratification, and the prediction of outcomes. In the past years, I was fortunate to participate in interdisciplinary research groups bridging biology, clinical research and data science. I was able to contribute to diverse projects through computational analysis and biological interpretation of sequencing data. Together, we were able to discover cellular phenotypes that influence disease progression and outcomes as well as the response to treatment. Here, I will present four studies that I have conducted in my PhD. First, we performed a case study of relapse from cell-based immunotherapy in Multiple Myeloma. We identified genomic deletion of the epitope as mechanism of immune escape and implicate heterozygosity or monosomy of the genomic locus at baseline as a potential risk factor. Second, we investigated the pathomechanisms of severe COVID-19 at the earliest stage of the COVID- 19 pandemic in Germany in March 2020. We discovered that profibrotic macrophages and lung fibrosis can be caused by SARS-CoV-2 infection. Third, we used a mouse model of chronic infection with Staphylococcus aureus that causes Osteomyelitis similar to the human disease. We were able to identify dysregulated immunometabolism associated with the generation of myeloid-derived suppressor cells (MDSC). Fourth, we investigated Salmonella infection of the human small intestine in an in vitro model and describe features of pathogen invasion and host response. Overall, I have been able to successfully employ single-cell sequencing to discover important aspects of diseases ranging from development to treatment and outcome. I analyzed samples from the clinics, human donors, mouse models and organoid models to investigate different aspects of diseases and managed to integrate data across sample types, technologies and diseases. Based on successful studies, we increased our efforts to combine data from multiple sources to build comprehensive references for the integration of large collections of clinical samples. Our findings exemplify how single-cell sequencing can improve clinical research and highlights the potential of mechanistic discoveries to drive precision medicine.},
  subject      = {Einzelzellanalyse},
  language  = {en}
}
@phdthesis{Hartmann2024,
  author    = {Hartmann, Oliver},
  title     = {Development of somatic modified mouse models of Non-Small cell lung cancer},
  doi       = {10.25972/OPUS-36340},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-363401},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {In 2020, cancer was the leading cause of death worldwide, accounting for nearly 10 million deaths. Lung cancer was the most common cancer, with 2.21 million cases per year in both sexes. This non-homogeneous disease is further subdivided into small cell lung cancer (SCLC, 15\%) and non-small cell lung cancer (NSCLC, 85\%). By 2023, the American Cancer Society estimates that NSCLC will account for 13\% of all new cancer cases and 21\% of all estimated cancer deaths. In recent years, the treatment of patients with NSCLC has improved with the development of new therapeutic interventions and the advent of targeted and personalised therapies. However, these advances have only marginally improved the five-year survival rate, which remains alarmingly low for patients with NSCLC. This observation highlights the importance of having more appropriate experimental and preclinical models to recapitulate, identify and test novel susceptibilities in NSCLC. In recent years, the Trp53fl/fl KRaslsl-G12D/wt mouse model developed by Tuveson, Jacks and Berns has been the main in vivo model used to study NSCLC. This model mimics ADC and SCC to a certain extent. However, it is limited in its ability to reflect the genetic complexity of NSCLC. In this work, we use CRISPR/Cas9 genome editing with targeted mutagenesis and gene deletions to recapitulate the conditional model. By comparing the Trp53fl/fl KRaslsl- G12D/wt with the CRISPR-mediated Trp53mut KRasG12D, we demonstrated that both showed no differences in histopathological features, morphology, and marker expression. Furthermore, next-generation sequencing revealed a very high similarity in their transcriptional profile. Adeno-associated virus-mediated tumour induction and the modular design of the viral vector allow us to introduce additional mutations in a timely manner. CRISPR-mediated mutation of commonly mutated tumour suppressors in NSCLC reliably recapitulated the phenotypes described in patients in the animal model. Lastly, the dual viral approach could induce the formation of lung tumours not only in constitutive Cas9 expressing animals, but also in wildtype animals. Thus, the implementation of CRISPR genome editing can rapidly advance the repertoire of in vivo models for NSCLC research. Furthermore, it can reduce the necessity of extensive breeding.},
  subject      = {CRISPR/Cas-Methode},
  language  = {en}
}
@phdthesis{Hahn2024,
  author    = {Hahn, Sarah},
  title     = {Investigating non-canonical, 5' UTR-dependent translation of MYC and its impact on colorectal cancer development},
  doi       = {10.25972/OPUS-36420},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-364202},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Colorectal cancer (CRC) is the second most common tumour disease in Germany, with the sequential accumulation of certain mutations playing a decisive role in the transition from adenoma to carcinoma. In particular, deregulation of the Wnt signalling pathway and the associated deregulated expression of the MYC oncoprotein play a crucial role. Targeting MYC thus represents an important therapeutic approach in the treatment of tumours. Since direct inhibition of MYC is challenging, various approaches have been pursued to date to target MYC indirectly. The MYC 5' UTR contains an internal ribosomal entry site (IRES), which has a particular role in the initiation of MYC translation, especially in multiple myeloma. As basis for this work, it was hypothesised on the basis of previous data that translation of MYC potentially occurs via its IRES in CRC as well. Based on this, two IRES inhibitors were tested for their potential to regulate MYC expression in CRC cells. In addition, alternative, 5' UTR-dependent translation of MYC and interacting factors were investigated. EIF3D was identified as a MYC 5' UTR binding protein which has the potential to regulate MYC expression in CRC. The results of this work suggest that there is a link between eIF3D and MYC expression/translation, rendering eIF3D a potential therapeutic target for MYC-driven CRCs.},
  subject      = {Myc},
  language  = {en}
}
@phdthesis{Merscher2024,
  author    = {Merscher, Alma-Sophia},
  title     = {To Fear or not to Fear: Unraveling the (Oculo)motor and Autonomic Components of Defensive States in Humans},
  doi       = {10.25972/OPUS-32791},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-327913},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2024},
  abstract  = {Defensive behaviors in response to threats are key factors in maintaining mental and physical health, but their phenomenology remains poorly understood. Prior work reported an inhibition of oculomotor activity in response to avoidable threat in humans that reminded of freezing behaviors in rodents. This notion of a homology between defensive responding in rodents and humans was seconded by concomitant heart rate decrease and skin conductance increase. However, several aspects of this presumed defense state remained ambiguous. For example, it was unclear whether the observed oculomotor inhibition would 1) robustly occur during preparation for threat-avoidance irrespective of task demands, 2) reflect a threat-specific defensive state, 3) be related to an inhibition of somatomotor activity as both motion metrics have been discussed as indicators for freezing behaviors in humans, and 4) manifest in unconstrained settings. We thus embarked on a series of experiments to unravel the robustness, threat-specificity, and validity of previously observed (oculo)motor and autonomic dynamics upon avoidable threat in humans. We provided robust evidence for reduced gaze dispersion, significantly predicting the speed of subsequent motor reactions across a wide range of stimulus contexts. Along this gaze pattern, we found reductions in body movement and showed that the temporal profiles between gaze and body activity were positively related within individuals, suggesting that both metrics reflect the same construct. A simultaneous activation of the parasympathetic (i.e., heart rate deceleration) and sympathetic (i.e., increased skin conductance and pupil dilation) nervous system was present in both defensive and appetitive contexts, suggesting that these autonomic dynamics are not only sensitive to threat but reflecting a more general action-preparatory mechanism. We further gathered evidence for two previously proposed defensive states involving a decrease of (oculo)motor activity in a naturalistic, unconstrained virtual reality environment. Specifically, we observed a state consisting of a cessation of ongoing behaviors and orienting upon relatively distal, ambiguous threat (Attentive Immobility) while an entire immobilization and presumed allocation of attention to the threat stimulus became apparent upon approaching potential threat (Immobility under Attack). Taken together, we provided evidence for specific oculomotor and autonomic dynamics upon increasing levels of threat that may inspire future translational work in rodents and humans on shared mechanisms of threat processing, ultimately supporting the development of novel therapeutic approaches.},
  subject      = {Furcht},
  language  = {en}
}