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A novel USP11-TCEAL1-mediated mechanism protects transcriptional elongation by RNA Polymerase II
(2024)
Deregulated expression of MYC oncoproteins is a driving event in many human cancers. Therefore, understanding and targeting MYC protein-driven mechanisms in tumor biology remain a major challenge.
Oncogenic transcription in MYCN-amplified neuroblastoma leads to the formation of the MYCN-BRCA1-USP11 complex that terminates transcription by evicting stalling RNAPII from chromatin. This reduces cellular stress and allows reinitiation of new rounds of transcription. Basically, tumors with amplified MYC genes have a high demand on well orchestration of transcriptional processes-dependent and independent from MYC proteins functions in gene regulation. To date, the cooperation between promoter-proximal termination and transcriptional elongation in cancer cells remains still incomplete in its understanding.
In this study the putative role of the dubiquitinase Ubiquitin Specific Protease 11 (USP11) in transcription regulation was further investigated. First, several USP11 interaction partners involved in transcriptional regulation in neuroblastoma cancer cells were identified. In particular, the transcription elongation factor A like 1 (TCEAL1) protein, which assists USP11 to engage protein-protein interactions in a MYCN-dependent manner, was characterized. The data clearly show that TCEAL1 acts as a pro-transcriptional factor for RNA polymerase II (RNAPII)-medi- ated transcription. In detail, TCEAL1 controls the transcription factor S-II (TFIIS), a factor that assists RNAPII to escape from paused sites. The findings claim that TCEAL1 outcompetes the transcription elongation factor TFIIS in a non-catalytic manner on chromatin of highly expressed genes. This is reasoned by the need regulating TFIIS function in transcription. TCEAL1 equili- brates excessive backtracking and premature termination of transcription caused by TFIIS.
Collectively, the work shed light on the stoichiometric control of TFIIS demand in transcriptional regulation via the USP11-TCEAL1-USP7 complex. This complex protects RNAPII from TFIIS-mediated termination helping to regulate productive transcription of highly active genes in neuroblastoma.
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.
Human prosociality, encompassing generosity, cooperation, and volunteering, holds a vital role in our daily lives. Over the last decades, the question of whether prosociality undergoes changes over the adult lifespan has gained increased research attention. Earlier studies suggested increased prosociality in older compared to younger individuals. However, recent meta-analyses revealed that this age effect might be heterogeneous and modest. Moreover, the contributing factors and mechanisms behind these age-related variations remain to be identified. To unravel age-related differences in prosociality, the first study of this dissertation employed a meta-analytical approach to summarize existing findings and provide insight into their heterogeneity by exploring linear and quadratic age effects on self-reported and behavioral prosociality. Additionally, two empirical research studies investigated whether these age-related differences in prosociality were observed in real life, assessed through ecological momentary assessment (Study 2), and in a controlled laboratory setting by applying a modified dictator game (Study 3). Throughout these three studies, potential underlying behavioral and computational mechanisms were explored. The outcome of the meta-analysis (Study 1) revealed small linear age effects on prosociality and significant age group differences between younger and older adults, with higher levels of prosociality in older adults. Explorative evidence emerged in favor of a quadratic age effect on behavioral prosociality, indicating the highest levels in midlife. Additionally, heightened prosocial behavior among middle-aged adults was observed compared to younger adults, whereas no significant differences in prosocial behavior were noted between middle-aged and older adults. Situational and contextual features, such as the setting of the study and specific paradigm characteristics, moderated the age-prosociality relationship, highlighting the importance of the (social) context when studying prosociality. For Study 2, no significant age effect on real-life prosocial behavior was observed. However, evidence for a significant linear and quadratic age effect on experiencing empathy in real life emerged, indicating a midlife peak. Additionally, across all age groups, the link between an opportunity to empathize and age significantly predicted real-life prosocial behavior. This effect, indicating higher levels of prosocial behavior when there was a situation possibly evoking empathy, was most pronounced in midlife. Study 3 presented age differences in how older and younger adults integrate values related to monetary gains for self and others to make a potential prosocial decision. Younger individuals effectively combined both values in a multiplicative fashion, enhancing decision-making efficiency. Older adults showed an additive effect of values for self and other and displayed increased decision-making efficiency when considering the values separately. However, among older adults, individuals with better inhibitory control were better able to integrate information about both values in their decisions. Taken together, the findings of this dissertation offer new insights into the multi-faceted nature of prosociality across adulthood and the mechanisms that help explain these age-related disparities. While this dissertation observed increasing prosociality across the adult lifespan, it also questions the assumption that older adults are inherently more prosocial. The studies highlight midlife as a potential peak period in social development but also emphasize the importance of the (social) context and that different operationalizations might capture distinct facets of prosociality. This underpins the need for a comprehensive framework to understand age effects of prosociality better and guide potential interventions.
Within this thesis, three main approaches for the assessment and investigation of altered hemodynamics like wall shear stress, oscillatory shear index and the arterial pulse wave velocity in atherosclerosis development and progression were conducted:
1. The establishment of a fast method for the simultaneous assessment of 3D WSS and PWV in the complete murine aortic arch via high-resolution 4D-flow MRI
2. The utilization of serial in vivo measurements in atherosclerotic mouse models using high-resolution 4D-flow MRI, which were divided into studies describing altered hemodynamics in late and early atherosclerosis
3. The development of tissue-engineered artery models for the controllable application and variation of hemodynamic and biologic parameters, divided in native artery models and biofabricated artery models, aiming for the investigation of the relationship between atherogenesis and hemodynamics
Chapter 2 describes the establishment of a method for the simultaneous measurement of 3D WSS and PWV in the murine aortic arch at, using ultra high-field MRI at 17.6T [16], based on the previously published method for fast, self-navigated wall shear stress measurements in the murine aortic arch using radial 4D-phase contrast MRI at 17.6 T [4]. This work is based on the collective work of Dr. Patrick Winter, who developed the method and the author of this thesis, Kristina Andelovic, who performed the experiments and statistical analyses. As the method described in this chapter is basis for the following in vivo studies and undividable into the sub-parts of the contributors without losing important information, this chapter was not split into the single parts to provide fundamental information about the measurement and analysis methods and therefore better understandability for the following studies. The main challenge in this chapter was to overcome the issue of the need for a high spatial resolution to determine the velocity gradients at the vascular wall for the WSS quantification and a high temporal resolution for the assessment of the PWV without prolonging the acquisition time due to the need for two separate measurements. Moreover, for a full coverage of the hemodynamics in the murine aortic arch, a 3D measurement is needed, which was achieved by utilization of retrospective navigation and radial trajectories, enabling a highly flexible reconstruction framework to either reconstruct images at lower spatial resolution and higher frame rates for the acquisition of the PWV or higher spatial resolution and lower frame rates for the acquisition of the 3D WSS in a reasonable measurement time of only 35 minutes. This enabled the in vivo assessment of all relevant hemodynamic parameters related to atherosclerosis development and progression in one experimental session. This method was validated in healthy wild type and atherosclerotic Apoe-/- mice, indicating no differences in robustness between pathological and healthy mice.
The heterogeneous distribution of plaque development and arterial stiffening in atherosclerosis [10, 12], however, points out the importance of local PWV measurements. Therefore, future studies should focus on the 3D acquisition of the local PWV in the murine aortic arch based on the presented method, in order to enable spatially resolved correlations of local arterial stiffness with other hemodynamic parameters and plaque composition.
In Chapter 3, the previously established methods were used for the investigation of changing aortic hemodynamics during ageing and atherosclerosis in healthy wild type and atherosclerotic Apoe-/- mice using the previously established methods [4, 16] based on high-resolution 4D-flow MRI. In this work, serial measurements of healthy and atherosclerotic mice were conducted to track all changes in hemodynamics in the complete aortic arch over time. Moreover, spatially resolved 2D projection maps of WSS and OSI of the complete aortic arch were generated. This important feature allowed for the pixel-wise statistical analysis of inter- and intragroup hemodynamic changes over time and most importantly – at a glance. The study revealed converse differences of local hemodynamic profiles in healthy WT and atherosclerotic Apoe−/− mice, with decreasing longWSS and increasing OSI, while showing constant PWV in healthy mice and increasing longWSS and decreasing OSI, while showing increased PWV in diseased mice. Moreover, spatially resolved correlations between WSS, PWV, plaque and vessel wall characteristics were enabled, giving detailed insights into coherences between hemodynamics and plaque composition. Here, the circWSS was identified as a potential marker of plaque size and composition in advanced atherosclerosis. Moreover, correlations with PWV values identified the maximum radStrain could serve as a potential marker for vascular elasticity. This study demonstrated the feasibility and utility of high-resolution 4D flow MRI to spatially resolve, visualize and analyze statistical differences in all relevant hemodynamic parameters over time and between healthy and diseased mice, which could significantly improve our understanding of plaque progression towards vulnerability. In future studies the relation of vascular elasticity and radial strain should be further investigated and validated with local PWV measurements and CFD.
Moreover, the 2D histological datasets were not reflecting the 3D properties and regional characteristics of the atherosclerotic plaques. Therefore, future studies will include 3D plaque volume and composition analysis like morphological measurements with MRI or light-sheet microscopy to further improve the analysis of the relationship between hemodynamics and atherosclerosis.
Chapter 4 aimed at the description and investigation of hemodynamics in early stages of atherosclerosis. Moreover, this study included measurements of hemodynamics at baseline levels in healthy WT and atherosclerotic mouse models. Due to the lack of hemodynamic-related studies in Ldlr-/- mice, which are the most used mouse models in atherosclerosis research together with the Apoe-/- mouse model, this model was included in this study to describe changing hemodynamics in the aortic arch at baseline levels and during early atherosclerosis development and progression for the first time. In this study, distinct differences in aortic geometries of these mouse models at baseline levels were described for the first time, which result in significantly different flow- and WSS profiles in the Ldlr-/- mouse model. Further basal characterization of different parameters revealed only characteristic differences in lipid profiles, proving that the geometry is highly influencing the local WSS in these models. Most interestingly, calculation of the atherogenic index of plasma revealed a significantly higher risk in Ldlr-/- mice with ongoing atherosclerosis development, but significantly greater plaque areas in the aortic arch of Apoe-/- mice. Due to the given basal WSS and OSI profile in these two mouse models – two parameters highly influencing plaque development and progression – there is evidence that the regional plaque development differs between these mouse models during very early atherogenesis.
Therefore, future studies should focus on the spatiotemporal evaluation of plaque development and composition in the three defined aortic regions using morphological measurements with MRI or 3D histological analyses like LSFM. Moreover, this study offers an excellent basis for future studies incorporating CFD simulations, analyzing the different measured parameter combinations (e.g., aortic geometry of the Ldlr-/- mouse with the lipid profile of the Apoe-/- mouse), simulating the resulting plaque development and composition. This could help to understand the complex interplay between altered hemodynamics, serum lipids and atherosclerosis and significantly improve our basic understanding of key factors initiating atherosclerosis development.
Chapter 5 describes the establishment of a tissue-engineered artery model, which is based on native, decellularized porcine carotid artery scaffolds, cultured in a MRI-suitable bioreactor-system [23] for the investigation of hemodynamic-related atherosclerosis development in a controllable manner, using the previously established methods for WSS and PWV assessment [4, 16]. This in vitro artery model aimed for the reduction of animal experiments, while simultaneously offering a simplified, but completely controllable physical and biological environment. For this, a very fast and gentle decellularization protocol was established in a first step, which resulted in porcine carotid artery scaffolds showing complete acellularity while maintaining the extracellular matrix composition, overall ultrastructure and mechanical strength of native arteries. Moreover, a good cellular adhesion and proliferation was achieved, which was evaluated with isolated human blood outgrowth endothelial cells. Most importantly, an MRI-suitable artery chamber was designed for the simultaneous cultivation and assessment of high-resolution 4D hemodynamics in the described artery models. Using high-resolution 4D-flow MRI, the bioreactor system was proven to be suitable to quantify the volume flow, the two components of the WSS and the radStrain as well as the PWV in artery models, with obtained values being comparable to values found in literature for in vivo measurements. Moreover, the identification of first atherosclerotic processes like intimal thickening is achievable by three-dimensional assessment of the vessel wall morphology in the in vitro models. However, one limitation is the lack of a medial smooth muscle cell layer due to the dense ECM. Here, the utilization of the laser-cutting technology for the generation of holes and / or pits on a microscale, eventually enabling seeding of the media with SMCs showed promising results in a first try and should be further investigated in future studies. Therefore, the proposed artery model possesses all relevant components for the extension to an atherosclerosis model which may pave the way towards a significant improvement of our understanding of the key mechanisms in atherogenesis.
Chapter 6 describes the development of an easy-to-prepare, low cost and fully customizable artery model based on biomaterials. Here, thermoresponsive sacrificial scaffolds, processed with the technique of MEW were used for the creation of variable, biomimetic shapes to mimic the geometric properties of the aortic arch, consisting of both, bifurcations and curvatures. After embedding the sacrificial scaffold into a gelatin-hydrogel containing SMCs, it was crosslinked with bacterial transglutaminase before dissolution and flushing of the sacrificial scaffold. The hereby generated channel was subsequently seeded with ECs, resulting in an easy-to-prepare, fast and low-cost artery model. In contrast to the native artery model, this model is therefore more variable in size and shape and offers the possibility to include smooth muscle cells from the beginning. Moreover, a custom-built and highly adaptable perfusion chamber was designed specifically for the scaffold structure, which enabled a one-step creation and simultaneously offering the possibility for dynamic cultivation of the artery models, making it an excellent basis for the development of in vitro disease test systems for e.g., flow-related atherosclerosis research. Due to time constraints, the extension to an atherosclerosis model could not be achieved within the scope of this thesis. Therefore, future studies will focus on the development and validation of an in vitro atherosclerosis model based on the proposed bi- and three-layered artery models.
In conclusion, this thesis paved the way for a fast acquisition and detailed analyses of changing hemodynamics during atherosclerosis development and progression, including spatially resolved analyses of all relevant hemodynamic parameters over time and in between different groups. Moreover, to reduce animal experiments, while gaining control over various parameters influencing atherosclerosis development, promising artery models were established, which have the potential to serve as a new platform for basic atherosclerosis research.
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.
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.
Adoptive immunotherapy using chimeric antigen receptor (CAR)-modified T cells is an effective treatment for hematological malignancies that are refractory to conventional chemotherapy. To address a wider variety of cancer entities, there is a need to identify and characterize additional target antigens for CAR-T cell therapy. The two members of the receptor tyrosine kinase-like orphan receptor family, ROR1 and ROR2, have been found to be overexpressed on cancer cells and to correlate with aggressive cancer phenotypes. Recently, ROR1-specific CAR-T cells have entered testing in phase I clinical trials, encouraging us to assess the suitability of ROR2 as a novel target for CAR-T cell therapy. To study the therapeutic potential of targeting ROR2 in solid and hematological malignancies, we selected two representative cancer entities with high unmet medical need: renal cell carcinoma and multiple myeloma.
Our data show that ROR2 is commonly expressed on primary samples and cell lines of clear cell renal cell carcinoma and multiple myeloma. To study the efficacy of ROR2-specific CAR T cell therapy, we designed two CAR constructs with 10-fold binding affinity differences for the same epitope of ROR2. We found both cell products to exhibit antigen-specific anti-tumor reactivity in vitro, including tumor cell lysis, secretion of the effector cytokines interleukin-2 (IL-2) and interferon-gamma (IFNγ), and T cell proliferation. In vivo studies revealed ROR2 specific CAR-T cells to confer durable responses, significant survival benefits and long-term persistence of CAR-expressing T cells. Overall, there was a trend towards more potent anti-tumor efficacy upon treatment with T cells that expressed the CAR with higher affinity for ROR2, both in vitro and in vivo.
We performed a preclinical safety and toxicology assessment comprising analyses of ROR2 expression in healthy human and murine tissues, cross-reactivity, and adoptive T cell transfer in immunodeficient mice. We found ROR2 expression to be conserved in mice, and low-level expression was detectable in the male and female reproductive system as well as parts of the gastrointestinal tract. CAR-T cells targeting human ROR2 were found to elicit similarly potent reactivity upon recognition of murine ROR2. In vivo analyses showed transient tissue-specific enrichment and activation of ROR2-specific CAR-T cells in organs with high blood circulation, such as lung, liver, or spleen, without evidence for clinical toxicity or tissue damage as determined by histological analyses.
Furthermore, we humanized the CAR binding domain of ROR2-specific CAR-T cells to mitigate the risk of adverse immune reactions and concomitant CAR-T cell rejection. Functional analyses confirmed that humanized CARs retained their specificity and functionality against ROR2-positive tumor cells in vitro.
In summary, we show that ROR2 is a prevalent target in RCC and MM, which can be addressed effectively with ROR2-specific CAR-T cells in preclinical models. Our preliminary toxicity studies suggest a favorable safety profile for ROR2-specific CAR-T cells. These findings support the potential to develop ROR2-specific CAR-T cells clinically to obtain cell products with broad utility.
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.
Development Of A Human iPSC-Derived Cortical Neuron Model Of Adaptor- Protein-Complex-4-Deficiency
(2024)
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.
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.
Gold nanoparticles of diameter ca. 60 nm have been synthesized based on Turkevich and Frens protocols. We have demonstrated that the carboxyl-modified gold nanoparticles can be coupled covalently with antibodies (Ab) of interest using the EDC/NHS coupling procedure. Binding studies with Ab-grafted AuNPs and GpL fusion proteins proved that conjugation of AuNPs with antibodies enables immobilization of antibodies with preservation of a significant antigen binding capacity. More importantly, our findings showed that the conjugation of types of anti-TNF receptors antibodies such as anti-Fn14 antibodies (PDL192 and 5B6) (Aido et al., 2021), anti-CD40, anti-4-1BB and anti-TNFR2 with gold nanoparticles confers them with potent agonism. Thus, our results suggest that AuNPs can be utilized as a platform to immobilize anti-TNFR antibodies which, on the one hand, helps to enhance their agonistic activity in comparison to “free” inactive antibodies by mimicking the effect of cell-anchored antibodies or membrane-bound TNF ligands and, on the other hand, allows to develop new generations of drug delivery systems. These constructs are characterized with their biocompatibility and their tunable synthesis process.
In a further work part, we combined the benefits of the established system of Ab-AuNPs with materials used widely in the modern biofabrication approaches such as the photo-crosslinked hydrogels, methacrylate-modified gelatin (GelMA), combined with embedded variants of human cell lines. The acquired results demonstrated clearly that the attaching of proteins like antibodies to gold nanoparticles might reduce their release rate from the crosslinked hydrogels upon the very low diffusion of gold nanoparticles from the solid constructs to the surrounding medium yielding long-term local functioning proteins-attached particles. Moreover, our finding suggests that hydrogel-embedded AuNP-immobilized antibodies, e.g. anti-TNFα-AuNPs or anti-IL1-AuNPs enable local inhibitory functions,
To sum up, our results demonstrate that AuNPs can act as a platform to attach anti-TNFR antibodies to enhance their agonistic activity by resembling the output of cell-anchoring or membrane bounding. Gold nanoparticles are considered, thus, as promising tool to develop the next generation of drug delivery systems, which may contribute to cancer therapy. On top of that, the embedding of anti-inflammatory-AuNPs in the biofabricated hydrogel presents new innovative strategy of the treatment of autoinflammatory diseases.
In this study, we developed an innovative nanoparticle formulation to facilitate the delivery of antitumor antibodies to tumor sites. The study commenced with the utilization of 13 bispecific antibody fusion proteins, which targeted the Fn14 receptor, thereby validating the pivotal role of crosslinking in Fn14 receptor activation. Subsequently, gold nanoparticles were activated using COOH-PEG-SH in combination with EDC/NHS, and subsequently conjugated with two Fn14-targeting antibodies, PDL192 and 5B6. Following this, a pH-sensitive shell was generated on the outer layer of the antibody-coupled gold nanoparticles through the application of chemically modified polylysine. The resultant complexes, termed MPL-antibody-AuNP, demonstrated a release profile reminiscent of the tumor microenvironment (TME). Notably, these complexes released antibody-AuNPs only in slightly acidic conditions while remaining intact in neutral or basic environments. Functionality analysis further affirmed the pH-sensitive property of MPL-antibody-AuNPs, demonstrating that the antibodies only initiated potent Fn14 activation in slightly acidic environments. This formulation holds potential for applicability to antibodies or ligands targeting the 80 TNFRSF family, given that gold nanoparticles successfully served as platforms for antibody crosslinking, thereby transforming these antibodies into potent agonists. Moreover, the TME disintegration profile of MPL mitigates the potential cytotoxic effects of antibodies, thereby circumventing associated adverse side effects. This study not only showcases the potential of nanoparticle formulations in targeted therapy, but also provides a solid foundation for further investigations on their clinical application in the context of targeting category II TNFRSF receptors with antibodies or ligands.
Neisseria meningitidis (the meningococcus) is one of the major causes of bacterial meningitis, a life-threatening inflammation of the meninges. Traversal of the meningeal blood-cerebrospinal fluid barrier (mBCSFB), which is composed of highly specialized brain endothelial cells (BECs), and subsequent interaction with leptomeningeal cells (LMCs) are critical for disease progression. Due to the human-exclusive tropism of N. meningitidis, research on this complex host-pathogen interaction is mostly limited to in vitro studies. Previous studies have primarily used peripheral or immortalized BECs alone, which do not retain relevant barrier phenotypes in culture. To study meningococcal interaction with the mBCSFB in a physiologically more accurate context, BEC-LMC co-culture models were developed in this project using BEC-like cells derived from induced pluripotent stem cells (iBECs) or hCMEC/D3 cells in combination with LMCs derived from tumor biopsies.
Distinct BEC and LMC layers as well as characteristic expression of cellular markers were observed using transmission electron microscopy (TEM) and immunofluorescence staining. Clear junctional expression of brain endothelial tight and adherens junction proteins was detected in the iBEC layer. LMC co-culture increased iBEC barrier tightness and stability over a period of seven days, as determined by sodium fluorescein (NaF) permeability and transendothelial electrical resistance (TEER). Infection experiments demonstrated comparable meningococcal adhesion and invasion of the BEC layer in all models tested, consistent with previously published data. While only few bacteria crossed the iBEC-LMC barrier initially, transmigration rates increased substantially over 24 hours, despite constant high TEER. After 24 hours of infection, deterioration of the barrier properties was observed including loss of TEER and altered expression of tight and adherens junction components. Reduced mRNA levels of ZO-1, claudin-5, and VE-cadherin were detected in BECs from all models. qPCR and siRNA knockdown data suggested that transcriptional downregulation of these genes was potentially but not solely mediated by Snail1. Immunofluorescence staining showed reduced junctional coverage of occludin, indicating N. meningitidis-induced post-transcriptional modulation of this protein, as previous studies have suggested. Together, these results suggest a potential combination of transcellular and paracellular meningococcal traversal of the mBCSFB, with the more accessible paracellular route becoming available upon barrier disruption after prolonged N. meningitidis infection. Finally, N. meningitidis induced cellular expression of pro-inflammatory cytokines and chemokines such as IL-8 in all mBCSFB models. Overall, the work described in this thesis highlights the usefulness of advanced in vitro models of the mBCSFB that mimic native physiology and exhibit relevant barrier properties to study infection with meningeal pathogens such as N. meningitidis.
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
Different effects of conditional Knock-Out of Stat3 on the sensory epithelium of the Organ of Corti
(2024)
The mammalian cochlea detects sound in response to vibration at frequency-dependent positions along the cochlea duct. The sensory outer hair cells, which are surrounded by supporting cells, act as a signal amplifier by changing their cell length. This is called electromotility. To ensure correct electrical transmission during mechanical forces, a certain resistance of the sensory epithelium is a prerequisite for correct transduction of auditory information. This resistance is managed by microtubules and its posttranslational modification in the supporting cells of the sensory epithelium of the cochlea. Stat3 is a transcription factor, with its different phosphorylation sites, is involved in many cellular processes like differentiation, inflammation, cell survival and microtubule dynamics, depending on cell type and activated pathway. While Stat3 has a wide range of intracellular roles, the question arose, how and if Stat3 is involved in cells of the organ of Corti to ensure a correct hearing.
To test this, Cre/loxp system were used to perform conditional Knock-Out (cKO) of Stat3 in outer hair cells or supporting cells either before hearing onset or after hearing onset. Hearing performances included DPOAE and ABR measurements, while molecular were performed by sequencing. Additionally, morphological examination was used by immunohistochemistry and electron microscopy.
A cKO of Stat3 before and after hearing onset in outer hair cells leads to hearing impairments, whereas synapses, nerve fibers and mitochondria were not affected. Bulk sequencing analyzation of outer hair cells out of cKO mice before hearing onset resulted in a disturbance of cellular homeostasis and extracellular signals. A cKO of Stat3 in the outer hair cells after hearing onset resulted in inflammatory signaling pathway with increased cytokine production and upregulation of NF-kb pathway. In supporting cells, cKO of Stat3 only after hearing onset resulted in a hearing impairment. However, synapses, nerve soma and fibers were not affected of a cKO of Stat3 in supporting cells. Nevertheless, detyronisated modification of microtubules were altered, which can lead to an instability of supporting cells during hearing.
In conclusion, Stat3 likely interact in a cell-specific and function-specific manner in cells of the organ of Corti. While a cKO in outer hair cells resulted in increased cytokine production, supporting cells altered its stability due to decreased detyronisated modification of microtubules. Together the results indicated that Stat3 is an important protein for hearing performances. However, additional investigations of the molecular mechanism are needed to understand the role of Stat3 in the cells of the organ of Corti.
Drug Discovery based on Oxidative Stress and HDAC6 for Treatment of Neurodegenerative Diseases
(2024)
Most antioxidants reported so far only achieved limited success in AD clinical trials. Growing evidences suggest that merely targeting oxidative stress will not be sufficient to fight AD. While multi-target directed ligands could synergistically modulate different steps in the neurodegenerative process, offering a promising potential for treatment of this complex disease.
Fifteen target compounds have been designed by merging melatonin and ferulic acid into the cap group of a tertiary amide HDAC6 inhibitor. Compound 10b was screened as the best hybrid molecule exhibit potent HDAC6 inhibition and potent antioxidant capacity. Compound 10b also alleviated LPS-induced microglia inflammation and led to a switch from neurotoxic M1 to the neuroprotective M2 microglial phenotype. Moreover, compound 10b show pronounced attenuation of spatial working memory and long-term memory damage in an in vivo AD mouse model. Compound 10b can be a potentially effective drug candidate for treatment of AD and its druggability worth to be further studied.
We have designed ten novel neuroprotectants by hybridizing with several common antioxidants, including ferulic acid, melatonin, lipoic acid, and trolox. The trolox hybrid compound exhibited the most potent neuroprotective effects in multiple neuroprotection assays. Besides, we identified the synergistic effects between trolox and vitamin K derivative, and our trolox hybrid compound showed comparable neuroprotection with the mixture of trolox and vitamin K derivative.
We have designed and synthesized 24 quinone derivatives based on five kinds of different quinones including ubiquinone, 2,3,5-trimethyl-1,4-benzoquinone, memoquin, thymoquinone, and anthraquinone. Trimethylbenzoquinone and thymoquinone derivatives showed more potent neuroprotection than other quinones in oxytosis assay. Therefore, trimethylbenzoquinone and thymoquinone derivatives can be used as lead compounds for further mechanism study and drug discovery for treatment of neurodegenerative disease.
We designed a series of photoswitchable HDAC inhibitors, which could be effective molecular tools due to the high spatial and temporal resolution. In total 23 target compounds were synthesized and photophysicochemically characterized. Azoquinoline-based compounds possess more thermally stable cis-isomers in buffer solution, which were further tested in enzyme-based HDAC inhibition assay. However, none of those tested compounds show significant differences in activities between trans-isomers and corresponding cis-isomers.
After myocardial infarction, an inflammatory response is induced characterized by a sterile inflammation, followed by a reparative phase in order to induce cardiac healing. Neutrophils are the first immune cells that enter the ischemic tissue. Neutrophils have various functions in the ischemic heart, such as phagocytosis, production of reactive oxygen species or release of granule components. These functions can not only directly damage cardiac tissue, but are also necessary for initiating reparative effects in post-ischemic healing, indicating a dual role of neutrophils in cardiac healing after infarction.
In recent years, evidence has been growing that neutrophils show phenotypic and functional differences in distinct homeostatic and pathogenic settings.
Preliminary data of my working group using single-cell RNA-sequencing revealed the time- dependent heterogeneity of neutrophils, with different populations showing distinct gene expression profiles in ischemic hearts of mice, including the time-dependent appearance of a SiglecFhigh neutrophil population. To better understand the dynamics of neutrophil heterogeneity in the ischemic heart, my work aimed to validate previous findings at the protein level, as well as to investigate whether the distinct neutrophil populations show functional differences. Furthermore, in vivo depletion experiments were performed in order to modulate circulating neutrophil levels.
Hearts, blood, bone marrow and spleens were processed and analyzed from mice after 1 day and 3 days after the onset of cardiac ischemia and analyzed using flow cytometry.
Results showed that the majority of cardiac neutrophils isolated at day 3 after myocardial infarction were SiglecFhigh, whereas nearly no SiglecFhigh neutrophils could be isolated from ischemic hearts at day 1 after myocardial infarction.
No SiglecFhigh neutrophils could be found in the blood, spleen and bone marrow either after 1 day or 3 days after myocardial infarction, indicating that the SiglecFhigh state of neutrophils is unique to the ischemic cardiac tissue.
When I compared SiglecFhigh and SiglecFlow neutrophils regarding their phagocytosis activity and ROS production, SiglecFhigh neutrophils showed a higher phagocytosis ability than their SiglecFlow counterparts, as well as higher ROS production capacity.
In vivo depletion experiments could not achieve successful and efficient depletion of cardiac neutrophils either 1 day or 3 days after myocardial infarction, but led to a shift of a higher percentage of SiglecFhigh expressing neutrophils in the depletion group. Bone marrow neutrophil levels only showed partial depletion at day 3 after MI. Regarding blood neutrophils, depletion efficiently reduced circulating neutrophils at both time points, 1 and 3 days after MI. To summarize, this work showed the time-dependent presence of different neutrophil states in the ischemic heart. The main population of neutrophils isolated 3 days after MI showed a high expression of SiglecF, a unique state that could not be detected at different time points or other organs. These SiglecFhigh neutrophils showed functional differences regarding their phagocytosis ability and ROS production. Further investigation is needed to reveal what role these SiglecFhigh neutrophils could play within the ischemic heart.
To better target neutrophil depletion in vivo, more efficient or different anti-neutrophil strategies are needed.
Ecophysiological adaptations of the cuticular water permeability within the Solanaceae family
(2024)
The cuticle, a complex lipidic layer synthesized by epidermal cells, covers and protects primary organs of all land plants. Its main function is to avoid plant desiccation by limiting non-stomatal water loss. The cuticular properties vary widely among plant species. So far, most of the cuticle-related studies have focused on a limited number of species, and studies addressing phylogenetically related plant species are rare. Moreover, comparative studies among organs from the same plant species are still scarce.
Thus, this study focus on organ-specificities of the cuticle within and between plant species of the Solanaceae family. Twenty-seven plant species of ten genera, including cultivated and non- cultivated species, were investigated to identify potential cuticular similarities. Structural, chemical and functional traits of fully expanded leaves, inflated fruiting calyces, and ripe fruits were analyzed.
The surface morphology was investigated by scanning electron microscopy. Leaves were mainly amphistomatic and covered by an epicuticular wax film. The diversity and distribution of trichomes varied among species. Only the leaves of S. grandiflora were glabrous. Plant species of the Leptostemonum subgenus had numerous prickles and non-glandular stellate trichomes. Fruits were stomata-free, except for S. muricatum, and a wax film covered their surface. Last, lenticel- like structures and remaining scars of broken trichomes were found on the surface of some Solanum fruits.
Cuticular water permeability was used as indicators of the cuticular transpiration barrier efficiency. The water permeability differed among plant species, organs and fruit types with values ranging up to one hundred-fold. The minimum leaf conductance ranged from 0.35 × 10-5 m s-1 in S. grandiflora to 31.54 × 10-5 m s-1 in S. muricatum. Cuticular permeability of fruits ranged from 0.64 × 10-5 m s-1 in S. dulcamara (fleshy berry) to 34.98 × 10-5 m s-1 in N. tabacum (capsule). Generally, the cuticular water loss of dry fruits was about to 5-fold higher than that of fleshy fruits.
Interestingly, comparisons between cultivated and non-cultivated species showed that wild species have the most efficient cuticular transpiration barrier in leaves and fruits. The average permeability of leaves and fruits of wild plant species was up to three-fold lower in comparison to the cultivated ones. Moreover, ripe fruits of P. ixocarpa and P. peruviana showed two-times lower cuticular transpiration when enclosed by the inflated fruiting calyx.
The cuticular chemical composition was examined using gas chromatography. Very-long-chain aliphatic compounds primarily composed the cuticular waxes, being mostly dominated by n- alkanes (up to 80% of the total wax load). Primary alkanols, alkanoic acids, alkyl esters and branched iso- and anteiso-alkanes were also frequently found. Although in minor amounts, sterols, pentacyclic triterpenoids, phenylmethyl esters, coumaric acid esters, and tocopherols were identified in the cuticular waxes. Cuticular wax coverages highly varied in solanaceous (62- fold variation). The cuticular wax load of fruits ranged from 0.55 μg cm−2 (Nicandra physalodes) to 33.99 μg cm−2 (S. pennellii), whereas the wax amount of leaves varied from 0.90 μg cm−2 (N. physalodes) to 28.42 μg cm−2 (S. burchellii). Finally, the wax load of inflated fruiting calyces ranged from 0.56 μg cm−2 in P. peruviana to 2.00 μg cm−2 in N. physalodes.
For the first time, a comparative study on the efficiency of the cuticular transpiration barrier in different plant organs of closely related plant species was conducted. Altogether, the cuticular chemical variability found in solanaceous species highlight species-, and organ-specific wax biosynthesis. These chemical variabilities might relate to the waterproofing properties of the plant cuticle, thereby influencing leaf and fruit performances. Additionally, the high cuticular water permeabilities of cultivated plant species suggest a potential existence of a trade-off between fruit organoleptic properties and the efficiency of the cuticular transpiration barrier. Last, the high cuticular water loss of the solanaceous dry fruits might be a physiological adaptation favouring seed dispersion.
Biofabrication technologies must address numerous parameters and conditions to reconstruct tissue complexity in vitro. A critical challenge is vascularization, especially for large constructs exceeding diffusion limits. This requires the creation of artificial vascular structures, a task demanding the convergence and integration of multiple engineering approaches. This doctoral dissertation aims to achieve two primary objectives: firstly, to implement and refine engineering methods for creating artificial microvascular structures using Melt Electrowriting (MEW)-assisted sacrificial templating, and secondly, to deepen the understanding of the critical factors influencing the printability of bioink formulations in 3D extrusion bioprinting.
In the first part of this dissertation, two innovative sacrificial templating techniques using MEW are explored. Utilizing a carbohydrate glass as a fugitive material, a pioneering advancement in the processing of sugars with MEW with a resolution under 100 microns was made. Furthermore, by introducing the “print-and-fuse” strategy as a groundbreaking method, biomimetic branching microchannels embedded in hydrogel matrices were fabricated, which can then be endothelialized to mirror in vivo vascular conditions.
The second part of the dissertation explores extrusion bioprinting. By introducing a simple binary bioink formulation, the correlation between physical properties and printability was showcased. In the next step, employing state-of-the-art machine-learning approaches revealed a deeper understanding of the correlations between bioink properties and printability in an extended library of hydrogel formulations.
This dissertation offers in-depth insights into two key biofabrication technologies. Future work could merge these into hybrid methods for the fabrication of vascularized constructs, combining MEW's precision with fine-tuned bioink properties in automated extrusion bioprinting.
According to the WHO, foodborne derived enteric infections are a global disease burden and often manifest in diseases that can potentially reach life threatening levels, especially in developing countries. These diseases are caused by a variety of enteric pathogens and affect the gastrointestinal tract, from the gastric to the intestinal to the rectal tissue. Although the complex mucosal structure of these organs is usually well prepared to defend the body against harmful agents, specialised pathogens such as Salmonella enterica can overcome the intestinal defence mechanism. After ingestion, Salmonella are capable of colonising the gut and establishing their proliferative niche, thereby leading to inflammatory processes and tissue damage of the host epithelium. In order to understand these processes, the scientific community in the last decades mostly used cell line based in vitro approaches or in vivo animal studies. Although these approaches provide fundamental insights into the interactions between bacteria and host cells, they have limited applicability to human pathology. Therefore, tissue engineered primary based approaches are important for modern infection research. They exhibit the human complexity better than traditional cell lines and can mimic human-obligate processes in contrast to animal studies.
Therefore, in this study a tissue engineered human primary model of the small intestinal epithelium was established for the application of enteric infection research with the exemplary pathogen Salmonella Typhimurium.
To this purpose, adult stem cell derived intestinal organoids were used as a primary human cell source to generate monolayers on biological or synthetic scaffolds in a Transwell®-like setting. These tissue models of the intestinal epithelium were examined for their comparability to the native tissue in terms of morphology, morphometry and barrier function. Further, the gene expression profiles of organotypical mucins, tight junction-associated proteins and claudins were investigated. Overall, the biological scaffold-based tissue models showed higher similarity to the native tissue - among others in morphometry and polarisation. Therefore, these models were further characterised on cellular and structural level. Ultrastructural analysis demonstrated the establishment of characteristic microvilli and tight-junction connections between individual epithelial cells. Furthermore, the expression pattern of typical intestinal epithelial protein was addressed and showed in vivo-like localisation. Interested in the cell type composition, single cell transcriptomic profiling revealed distinct cell types including proliferative cells and stem cells, progenitors, cellular entities of the absorptive lineage, Enterocytes and Microfold-like cells. Cells of the secretory lineage were also annotated, but without distinct canonical gene expression patterns. With the organotypical polarisation, protein expression, structural features and the heterogeneous cell composition including the rare Microfold-like cells, the biological scaffold-based tissue model of the intestinal epithelium demonstrates key requisites needed for infection studies with Salmonella.
In a second part of this study, a suitable infection protocol of the epithelial tissue model with Salmonella Typhimurium was established, followed by the examination of key features of the infection process. Salmonella adhered to the epithelial microvilli and induced typical membrane ruffling during invasion; interestingly the individual steps of invasion could be observed. After invasion, time course analysis showed that Salmonella resided and proliferated intracellularly, while simultaneously migrating from the apical to the basolateral side of the infected cell. Furthermore, the bacterial morphology changed to a filamentous phenotype; especially when the models have been analysed at late time points after infection. The epithelial cells on the other side released the cytokines Interleukin 8 and Tumour Necrosis Factor α upon bacterial infection in a time-dependent manner. Taken together, Salmonella infection of the intestinal epithelial tissue model recapitulates important steps of the infection process as described in the literature, and hence demonstrates a valid in vitro platform for the investigation of the Salmonella infection process in the human context.
During the infection process, intracellular Salmonella populations varied in their bacterial number, which could be attributed to increased intracellular proliferation and demonstrated thereby a heterogeneous behaviour of Salmonella in individual cells. Furthermore, by the application of single cell transcriptomic profiling, the upregulation of Olfactomedin-4 (OLFM4) gene expression was detected; OLFM4 is a protein involved in various functions including cell immunity as well as proliferating signalling pathways and is often used as intestinal stem cell marker. This OLFM4 upregulation was time-dependent, restricted to Salmonella infected cells and seemed to increase with bacterial mass. Investigating the OLFM4 regulatory mechanism, nuclear factor κB induced upregulation could be excluded, whereas inhibition of the Notch signalling led to a decrease of OLFM4 gene and protein expression. Furthermore, Notch inhibition resulted in decreased filamentous Salmonella formation. Taken together, by the use of the introduced primary epithelial tissue model, a heterogeneous intracellular bacterial behaviour was observed and a so far overlooked host cell response – the expression of OLFM4 by individual infected cells – could be identified; although Salmonella Typhimurium is one of the best-studied enteric pathogenic bacteria. This proves the applicability of the introduced tissue model in enteric infection research as well as the importance of new approaches in order to decipher host-pathogen interactions with higher relevance to the host.