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Malaria and HIV are among the most important global health problems of our time and together are responsible for approximately 3 million deaths annually. These two diseases overlap in many regions of the world including sub-Saharan Africa, Southeast Asia and South America, leading to a higher risk of co-infection. In this study, we generated and characterized hybrid molecules to target P. falciparum and HIV simultaneously for a potential HIV/malaria combination therapy. Hybrid molecules were synthesized by covalent fusion between azidothymidine (AZT) and dihydroartemisinin (DHA), tetraoxane or chloroquine (CQ); and a small library was generated and tested for antiviral and antimalarial activity. Our data suggest that dihyate is the most potent molecule in vitro, with antiplasmodial activity comparable to that of DHA (IC50 = 26 nM, SI > 3000), a moderate activity against HIV (IC50 = 2.9 µM; SI > 35) and safe to HeLa cells at concentrations used in the assay (CC50 > 100 µM). Pharmacokinetic studies further revealed that dihyate is metabolically unstable and is cleaved following an O-dealkylation once in contact with cytochrome P450 enzymes. The later further explains the uneffectiveness of dihyate against the CQ-sensitive P. berghei N strain in mice when administered by oral route at 20 mg/kg. Here, we report on a first approach to develop antimalarial/anti-HIV hybrid molecules and future optimization efforts will aim at producing second generation hybrid molecules to improve activity against HIV as well as compound bioavailability. With the emergence of resistant parasites against all the counterpart drugs of artemisinin derivatives used in artemisinin based combination therapies (ACTs), the introduction of antibiotics in the treatment of malaria has renewed interest on the identification of antibiotics with potent antimalarial properties. In this study we also investigated the antiplasmodial potential of thiostrepton and derivatives, synthesized using combinations of tail truncation, oxidation, and addition of lipophilic thiols to the terminal dehydroamino acid. We showed that derivatives SS231 and SS234 exhibit a better antiplasmodial activity (IC50 = 1 µM SI > 59 and SI > 77 respectively) than thiostrepton (IC50 = 8.95 µM, SI = 1.7). The antiplasmodial activity of these derivatives was observed at concentrations which are not hemolytic and non-toxic to human cell lines. Thiostrepton and derivatives appeared to exhibit transmission blocking properties when administered at their IC50 or IC90 concentrations and our data also showed that they attenuate proteasome activity of Plasmodium, which resulted in an accumulation of ubiquitinated proteins after incubation with their IC80 concentrations. Our results indicate that the parasite’s proteasome could be an attractive target for therapeutic intervention. In this regard, thiostrepton derivatives are promising candidates by dually acting on two independent targets, the proteasome and the apicoplast, with the capacity to eliminate both intraerythrocytic asexual and transmission stages of the parasite. To further support our findings, we evaluated the activity of a new class of antimalarial and proteasome inhibitors namely peptidyl sulfonyl fluorides on gametocyte maturation and analogues AJ34 and AJ38 were able to completely suppress gametocytogenesis at IC50 concentrations (0.23 µM and 0.17 µM respectively) suggesting a strong transmission blocking potential. The proteasome, a major proteolytic complex, responsible for the degradation and re-cycling of non-functional proteins has been studied only indirectly in P. falciparum. In addition, an apparent proteasome-like protein with similarity to bacterial ClpQ/hslV threonine-peptidases was predicted in the parasite. Antibodies were generated against the proteasome subunits alpha type 5 (α5-SU), beta type 5 (β5-SU) and pfhslV in mice and we showed that the proteasome is expressed in both sexual and asexual blood stages of P. falciparum, where they localize in the nucleus and in the cytoplasm. However, expression of PfhslV was only observed in trophozoites and shizonts. The trafficking of the studied proteasome subunits was further investigated by generating parasites expressing GFP tagged proteins. The expression of α5-SU-GFP in transgenic parasite appeared to localize abundantly in the cytoplasm of all blood stages, and no additional information was obtained from this parasite line. In conclusion, our data highlight two new tools towards combination therapy. Hybrid molecules represent promising tools for the cure of co-infected individuals, while very potent antibiotics with a wide scope of activities could be useful in ACTs by eliminating resistant parasites and limiting transmission of both, resistances and disease.
The interaction between circadian clocks and metabolism is of increasing interest, since clock dysfunction often correlates with metabolic pathologies. Many research articles have been published analysing the impact of factors such as circadian clock, light, feeding time and diet-type on energy homeostasis in various tissues/organs of organisms with most of the findings done in mammals. Little is known about the impact of circadian clock and the above-mentioned factors on circulating lipids, especially the transport form of lipids - diacylglycerol (DG) and membrane lipids such as phosphatidylethanolamine (PE) and phosphatidylcholine (PC) in the Drosophila hemolymph. The fruit fly Drosophila is a prime model organism in circadian, behaviour and metabolism research.
To study the role of circadian clock and behaviour in metabolism, we performed an extensive comparative hemolymph lipid (diacylglycerol: DG, phosphatidylethanolamine: PE, phosphatidylcholine: PC) analysis using ultra performance liquid chromatography coupled to time-of-flight mass spectrometry (UPLC-MS) between wild-type flies (WTCS) and clock disrupted mutants (per01). In addition, clock controlled food intake– feeding behaviour was investigated. Time-dependent variation of transport (DG) and membrane lipids (PE and PC) were not rhythmic in WTCS under constant darkness and in per01 under LD, suggesting an impact of light and clock genes on daily lipid oscillations. Day-time and night-time restriction of food led to comparable lipid profiles, suggesting that lipid oscillations are not exclusively entrained by feeding but rather are endogenously regulated. Ultradian oscillations in lipid levels in WTCS under LD were masked by digested fatty acids since lipid levels peaked more robustly at the beginning and end of light phase when flies were fed a lipid- and protein-free diet. These results suggest that metabolite (DG, PE and PC) oscillation is influenced by complex interactions between nutrient-type, photic conditions, circadian clock and feeding time.
In conclusion, the results of this thesis suggest that circadian clocks determine transport and membrane lipid oscillation in Drosophila hemolymph in complex interactions between nutrient-type, photic conditions and feeding behaviour.
Infectious diseases caused by pathogenic microorganisms are one of the largest socioeconomic burdens today. Although infectious diseases have been studied for decades, in numerous cases, the precise mechanisms involved in the multifaceted interaction between pathogen and host continue to be elusive. Thus, it still remains a challenge for researchers worldwide to develop novel strategies to investigate the molecular context of infectious diseases in order to devise preventive or at least anti-infective measures. One of the major drawbacks in trying to obtain in-depth knowledge of how bacterial pathogens elicit disease is the lack of suitable infection models to authentically mimic the disease progression in humans. Numerous studies rely on animal models to emulate the complex temporal interactions between host and pathogen occurring in humans. While they have greatly contributed to shed light on these interactions, they require high maintenance costs, are afflicted with ethical drawbacks, and are not always predictive for the infection outcome in human patients. Alternatively, in-vitro two-dimensional (2D) cell culture systems have served for decades as representatives of human host environments to study infectious diseases. These cell line-based models have been essential in uncovering virulence-determining factors of diverse pathogens as well as host defense mechanisms upon infection. However, they lack the morphological and cellular complexity of intact human tissues, limiting the insights than can be gained from studying host-pathogen interactions in these systems.
The focus of this thesis was to establish and innovate intestinal human cell culture models to obtain in-vitro reconstructed three-dimensional (3D) tissue that can faithfully mimic pathogenesis-determining processes of the zoonotic bacterium Campylobacter jejuni (C. jejuni). Generally employed for reconstructive medicine, the field of tissue engineering provides excellent tools to generate organ-specific cell culture models in vitro, realistically recapitulating the distinctive architecture of human tissues. The models employed in this thesis are based on decellularized extracellular matrix (ECM) scaffolds of porcine intestinal origin. Reseeded with intestinal human cells, application of dynamic culture conditions promoted the formation of a highly polarized mucosal epithelium maintained by functional tight and adherens junctions. While most other in-vitro infection systems are limited to a flat monolayer, the tissue models developed in this thesis can display the characteristic 3D villi and crypt structure of human small intestine.
First, experimental conditions were established for infection of a previously developed, statically cultivated intestinal tissue model with C. jejuni. This included successful isolation of bacterial colony forming units (CFUs), measurement of epithelial barrier function, as well as immunohistochemical and histological staining techniques. In this way, it became possible to follow the number of viable bacteria during the infection process as well as their translocation over the polarized epithelium of the tissue model. Upon infection with C. jejuni, disruption of tight and adherens junctions could be observed via confocal microscopy and permeability measurements of the epithelial barrier. Moreover, C. jejuni wildtype-specific colonization and barrier disruption became apparent in addition to niche-dependent bacterial localization within the 3D microarchitecture of the tissue model. Pathogenesis-related phenotypes of C. jejuni mutant strains in the 3D host environment deviated from those obtained with conventional in-vitro 2D monolayers but mimicked observations made in vivo. Furthermore, a genome-wide screen of a C. jejuni mutant library revealed significant differences for bacterial factors required or dispensable for interactions with unpolarized host cells or the highly prismatic epithelium provided by the intestinal tissue model. Elucidating the role of several previously uncharacterized factors specifically important for efficient colonization of a 3D human environment, promises to be an intriguing task for future research.
At the frontline of the defense against invading pathogens is the protective, viscoelastic mucus layer overlying mucosal surfaces along the human gastrointestinal tract (GIT). The development of a mucus-producing 3D tissue model in this thesis was a vital step towards gaining a deeper understanding of the interdependency between bacterial pathogens and host-site specific mucins. The presence of a mucus layer conferred C. jejuni wildtype-specific protection against epithelial barrier disruption by the pathogen and prevented a high bacterial burden during the course of infection. Moreover, results obtained in this thesis provide evidence in vitro that the characteristic corkscrew morphology of C. jejuni indeed grants a distinct advantage in colonizing mucous surfaces.
Overall, the results obtained within this thesis highlight the strength of the tissue models to combine crucial features of native human intestine into accessible in-vitro infection models. Translation of these systems into infection research demonstrated their ability to expose in-vivo like infection outcomes. While displaying complex organotypic architecture and highly prismatic cellular morphology, these tissue models still represent an imperfect reflection of human tissue. Future advancements towards inclusion of human primary and immune cells will strive for even more comprehensive model systems exhibiting intricate multicellular networks of in-vivo tissue. Nevertheless, the work presented in this thesis emphasizes the necessity to investigate host-pathogen interactions in infection models authentically mimicking the natural host environment, as they remain among the most vital parts in understanding and counteracting infectious diseases.
The superfamily of G protein-coupled receptors (GPCRs) comprises more than 800 members, which are divided into five families based on phylogenetic analyses (GRAFS classification): Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2 and Secretin. The adhesion G protein-coupled receptor (aGPCR) family forms with 33 homologs in Mammalia the second largest and least investigated family of GPCRs. The general architecture of an aGPCR comprises the GPCR characteristics of an extracellular region (ECR), a seven transmembrane (7TM) domain and an intracellular region (ICR). A special feature of aGPCRs is the extraordinary size of the ECR through which they interact with cellular and matricellular ligands via adhesion motif folds. In addition, the ECR contains a so-called GPCR autoproteolysis-inducing (GAIN) domain, which catalyzes autoproteolytic cleavage of the protein during maturation. This cleavage leads to the formation of an N-terminal (NTF) and a C-terminal fragment (CTF), which build a unit by means of hydrophobic interactions and therefore appear as a heterodimeric receptor at the cell surface. In the past, it has been shown that the first few amino acids of the CTF act as a tethered agonist (TA) that mediates the activation of the receptor through the interaction with the 7TM domain. However, the molecular mechanism promoting the TA-7TM domain interaction remains elusive. This work reveals a novel molecular mechanism that does not require the dissociation of the NTF-CTF complex to promote release of the TA and thus activation of the aGPCR. The introduction of bioorthogonal labels into receptorsignaling- relevant regions of the TA of various aGPCRs demonstrated that the TA is freely accessible within the intact GAIN domain. This suggests a structural flexibility of the GAIN domain, which allows a receptor activation independent of the NTF-CTF dissociation, as found in cleavage-deficient aGPCR variants. Furthermore, the present study shows that the cellular localization and the conformation of the 7TM domain depends on the activity state of the aGPCR, which in turn indicates that the TA mediates conformational changes through the interaction with the 7TM domain, which ultimately regulates the receptor activity. In addition, biochemical analyses showed that the GAIN domain-mediated autoproteolysis of the human aGPCR CD97 (ADGRE5/E5) promotes further cleavage events within the receptor. This suggests that aGPCRs undergo cleavage cascades, which are initialized by the autoproteolytic reaction of the GAIN domain. Thus, it can be assumed that aGPCRs are subject to additional proteolytic events. Finally, the constitutive internalization of the NTF and the CTF of E5 was demonstrated by various labeling methods. It was possible to label both fragments independently and to follow their subcellular location in vitro. In summary, these obtained results contribute to a better understanding about the molecular mechanisms of activity and signaling of aGPCRs.
This work developed during the first funding period of the subproject B05 in the framework of the interdisciplinary research consortium TRR 225 ‘From the Fundamentals of Biofabrication toward functional Tissue Models’ and was part of a cooperation between the Orthopedic Department represented by Prof. Dr. Regina Ebert and the Institute of Organic Chemistry represented by Prof. Dr. Jürgen Seibel.
This project dealed with cellular behavior during the bioprinting process and how to influence it by modifying the cell glycocalyx with functional target molecules. The focus was on the impact of potential shear stress, that cells experience when they get processed in thermoresponsive bioinks, and a way to increase the cell stiffness via metabolic glycoengineering to attenuate shear forces. For the characterization of the metabolic glycoengineering, four different peracetylated and four non-acetylated modified monosaccharides (two mannose and two sialic acid sugars) were tested in primary human mesenchymal stromal cells (hMSC) and telomerase-immortalized hMSC (hMSC-TERT). Viability results demonstrated a dose-dependent correlation for all sugars, at which hMSC-TERT seemed to be more susceptible leading to lower viability rates. The assessment of the incorporation efficiencies was performed by click chemistry using fluorescent dyes and revealed also a dose-dependent correlation for all mannose and sialic acid sugars, while glucose and galactose variants were not detected in the glycocalyx. However, incorporation efficiencies were highest when using mannose sugars in the primary hMSC. A subsequent analysis of the temporal retention of the incorporated monosaccharides showed a constant declining fluorescence signal up to 6 d for azido mannose in hMSC-TERT, whereas no signal could be detected for alkyne mannose after 2 d. Investigation of the differentiation potential and expression of different target genes revealed no impairment after incubation with mannose sugars, indicating a normal phenotype for hMSC-TERT. Following the successful establishment of the method, either a coumarin derivative or an artificial galectin 1 ligand were incorporated into the cell glycocalyx of hMSC-TERT as functional target molecule. The biophysical analysis via shear flow deformation cytometry revealed a slightly increased cell stiffness and lowered fluidity for both molecules. A further part of this project aimed to control lectin-mediated cell adhesion by artificial galectin 1 ligands. As that hypothesis was settled in the work group of Prof. Dr. Jürgen Seibel, this work supported with an initial characterization of galectin 1 as part of the hMSC biology. A stable galectin 1 expression at gene and protein level in both hMSC and hMSC-TERT could be confirmed, at which immunocytochemical stainings could detect the protein only in the glycocalyx. The treatment of hMSC-TERT with a galectin 1 ligand in different concentrations did not show an altered gene expression of galectin 1. However, these first data in addition to the investigation of stiffness confirmed the applicability of specific and artificial
IV
galectin 1 ligands in biofabrication approaches to alter cell properties of hMSC. To conclude, metabolic glycoengineering has been successfully implemented in hMSC and hMSC-TERT to introduce glycocalyx modifications which reside there for several days. A proof of concept was carried out by the increase of cell stiffness and fluidity by the incorporation of a coumarin derivative or an artificial galectin 1 ligand.
For the characterization of shear stress impact on cells after printing in thermoresponsive bioinks, the processing of hMSC-TERT (mixing or additionally printing) with Pluronic F127 or Polyoxazoline-Polyoxazine (POx-POzi) polymer solution was investigated. While there were no changes in viability when using POx-POzi bioink, processing with Pluronic F127 indicated slightly lower viability and increased apoptosis activity. Assessment of cellular responses to potential shear stress showed no reorganization of the cytoskeleton independent of the bioink, but highly increased expression of the mechanoresponsive proto-oncogene c Fos which was more pronounced when using Pluronic F127 and just mixed with the bioinks. Interestingly, processing of the mechanoresponsive reporter cell line hMSC-TERT-AP1 revealed slightly elevated mechanotransduction activity when using POx-POzi polymer and just mixed with the bioinks as well. In conclusion, hMSC-TERT embedded in thermoresponsive bioinks might shortly experience shear stress during the printing process, but that did not lead to remarkable cell damage likely due to the rheological properties of the bioinks. Furthermore, the printing experiments also suggested that cells do not sense more shear stress when additionally printed.
In this work we wanted to investigate the role of NFATc1 in lymphocyte physiology and in pathological conditions (eg. psoriasis). NFATc1 is part of the signal transduction
pathways that regulates B cells activation and function. NFATc1 has different isoforms that are due to different promoters (P1 and P2), polyadenylation and alternative splicing. Moreover, we tried to elucidate the points of interactions between the NFAT and the NF-κB pathways in
activated B-cell fate. NFAT and NF-κB factors share several properties, such as a similar mode of induction and architecture in their DNA binding domain. We used mice which over-express a constitutive active version of NFATc1/α in their B cells with -or without- an ablated IRF4. IRF4 inhibits cell cycle progression of germinal center B cell-derived Burkitt’s lymphoma cells and
induces terminal differentiation toward plasma cells. Our experiments showed that a ‘double hit’ in factors affecting B cell activation (NFATc1 in this case) and late B cell Differentiation (IRF4 in this case) alter the development of the B cells, lead to increase in their numbers and increase in stimulation induced proliferation. Therefore, the overall picture indicates a link between these 2 genes and probable carcinogenic alterations that may occur in B cells.
We also show that in splenic B cells, c-Rel (of the NF-κB canonical pathway) Support the induction of NFATc1/αA through BCR signals. We also found evidence that the lack of NFATc1 affects the expression of Rel-B (of the NF-κB non-canonical pathway). These data suggest a tight interplay between NFATc1 and NF-κB in B cells, influencing the competence of B cells and their functions in peripheral tissues.
We also used IMQ-induced psoriasis-like inflammation on mice which either lack NFATc1 from B cell. Psoriasis is a systemic chronic immunological disease characterized
primarily by abnormal accelerated proliferation of the skin keratinocytes. In psoriasis, the precipitating event leads to immune cell activation. Our experiments showed that NFATc1 is needed for the development of psoriasis. It also showed that IL-10 is the link that enables NFAT
from altering the B cell compartment (eg Bregs) in order to affect inflammation. The important role of B cell in psoriasis is supported by the flared up psoriasis-like inflammation in mice that lack B cells. Bregs is a special type of B cells that regulate other B cells and T cells; tuning the immunological response through immunomodulatory cytokines.
Frizzled (FZD) are highly conserved receptors that belong to class F of the G protein-coupled receptor (GPCR) superfamily. They are involved in a great variety of processes during embryonic development, organogenesis, and adult tissue homeostasis. In particular, FZD5 is an important therapeutic target due to its involvement in several pathologies, such as tumorigenesis. Nevertheless, little is known regarding the activation of FZD receptors and the signal initiation, and their GPCR nature has been debated. In order to investigate the activation mechanism of these receptors, FRET (Förster Resonance Energy Transfer)-based biosensors for FZD5 have been developed and characterized. A cyan fluorescent protein (CFP) was fused to the C-terminus of the receptor and the specific FlAsH-binding sequence (CCPGCC) was inserted within the 2nd or the 3rd intracellular loop. Single-cell FRET experiments performed using one of these sensors, V5-mFZD5-FlAsH436-CFP, reported structural rearrangements in FZD5 upon stimulation with the endogenous ligand WNT-5A. These movements are similar to those observed in other GPCRs using the same technique, which suggests an activation mechanism for FZD reminiscent of GPCRs. Furthermore, stimulation of the FZD5 FRET-based sensor with various recombinant WNT proteins in a microplate FRET reader allowed to obtain concentration-response curves for several ligands, being possible to distinguish between full and partial agonists. This technology allowed to address the selectivity between WNTs and FZD5 using a full-length receptor in living cells. In addition, G protein FRET-based sensors revealed that WNT-5A specifically induced Gαq activation mediated by FZD5, but not Gαi activation. Other WNT proteins were also able to induce Gαq activation, but with lower efficacy than WNT-5A. In addition, a dual DAG/calcium sensor further showed that WNT-5A stimulation led to the activation of the Gαq-dependent signaling pathway mediated by FZD5, which outcome was the activation of Protein Kinase C (PKC) and the release of intracellular calcium. Altogether, these data provide evidence that the activation process of FZD5 resembles the general characteristics of class A and B GPCR activation, and this receptor also mediates the activation of the heterotrimeric Gαq protein and its downstream signaling pathway. In addition, the FZD5 receptor FRET-based sensor provides a valuable tool to characterize the pharmacological properties of WNTs and other potential ligands for this receptor.
Introduction.
Mobile health (mHealth) integrates mobile devices into healthcare, enabling remote monitoring, data collection, and personalized interventions. Machine Learning (ML), a subfield of Artificial Intelligence (AI), can use mHealth data to confirm or extend domain knowledge by finding associations within the data, i.e., with the goal of improving healthcare decisions. In this work, two data collection techniques were used for mHealth data fed into ML systems: Mobile Crowdsensing (MCS), which is a collaborative data gathering approach, and Ecological Momentary Assessments (EMA), which capture real-time individual experiences within the individual’s common environments using questionnaires and sensors. We collected EMA and MCS data on tinnitus and COVID-19. About 15 % of the world’s population suffers from tinnitus.
Materials & Methods.
This thesis investigates the challenges of ML systems when using MCS and EMA data. It asks: How can ML confirm or broad domain knowledge? Domain knowledge refers to expertise and understanding in a specific field, gained through experience and education. Are ML systems always superior to simple heuristics and if yes, how can one reach explainable AI (XAI) in the presence of mHealth data? An XAI method enables a human to understand why a model makes certain predictions. Finally, which guidelines can be beneficial for the use of ML within the mHealth domain? In tinnitus research, ML discerns gender, temperature, and season-related variations among patients. In the realm of COVID-19, we collaboratively designed a COVID-19 check app for public education, incorporating EMA data to offer informative feedback on COVID-19-related matters. This thesis uses seven EMA datasets with more than 250,000 assessments. Our analyses revealed a set of challenges: App user over-representation, time gaps, identity ambiguity, and operating system specific rounding errors, among others. Our systematic review of 450 medical studies assessed prior utilization of XAI methods.
Results.
ML models predict gender and tinnitus perception, validating gender-linked tinnitus disparities. Using season and temperature to predict tinnitus shows the association of these variables with tinnitus. Multiple assessments of one app user can constitute a group. Neglecting these groups in data sets leads to model overfitting. In select instances, heuristics outperform ML models, highlighting the need for domain expert consultation to unveil hidden groups or find simple heuristics.
Conclusion.
This thesis suggests guidelines for mHealth related data analyses and improves estimates for ML performance. Close communication with medical domain experts to identify latent user subsets and incremental benefits of ML is essential.
Pro-migratory signals mediated by the tumor microenvironment contribute to the cancer progression cascade, including invasion, metastasis and resistance to therapy. Derived from in vitro studies, isolated molecular steps of cancer invasion programs have been identified but their integration into the tumor microenvironment and suitability as molecular targets remain elusive. The purpose of the study was to visualize central aspects of tumor progression, including proliferation, survival and invasion by real-time intravital microscopy. The specific aims were to monitor the kinetics, mode, adhesion and chemoattraction mechanisms of tumor cell invasion, the involved guidance structures, and the response of invasion zones to anti-cancer therapy. To reach deeper tumor regions by optical imaging with subcellular resolution, near-infrared and infrared excited multiphoton microscopy was combined with a modified dorsal skinfold chamber model. Implanted HT-1080 fibrosarcoma and B16/F10 and MV3 melanoma tumors developed zones of invasive growth consisting of collective invasion strands that retained cell-cell contacts and high mitotic activity while invading at velocities of up to 200 μm per day. Collective invasion occurred predominantly along preexisting tissue structures, including blood and lymph vessels, collagen fibers and muscle strands of the deep dermis, and was thereby insensitive to RNAi based knockdown and/or antibody-based treatment against β1 and β3 integrins, chemokine (SDF-1/CXCL12) and growth factor (EGF) signaling. Therapeutic hypofractionated irradiation induced partial to complete regression of the tumor main mass, yet failed to eradicate the collective invasion strands, suggesting a microenvironmentally privileged niche. Whereas no radiosensitization was achieved by interference with EGFR or doxorubicin, the simultaneous inhibition of β1 and β3 integrins impaired cell proliferation and survival in spontaneously growing tumors and strongly enhanced the radiation response up to complete eradication of both main tumor and invasion strands. In conclusion, collective invasion in vivo is a robust process which follows preexisting tissue structures and is mainly independent of established adhesion and chemoattractant signaling. Due to its altered biological response to irradiation, collective invasion strands represent a microenvironmentally controlled and clinically relevant resistance niche to therapy. Therefore supportive regimens, such as anoikisinduction by anti-integrin therapy, may serve to enhance radio- and chemoefficacy and complement classical treatment regimens.
Melanoma arises from the malignant transformation of melanocytes and is one of the most aggressive forms of human cancer. In fish of the genus Xiphophorus, melanoma development, although very rarely, happens spontaneously in nature and can be induced by interspecific crossing. The oncogenic receptor tyrosine kinase, Xmrk, is responsible for melanoma formation in these fishes. Since Xiphophorus are live-bearing fishes and therefore not compatible with embryonic manipulation and transgenesis, the Xmrk melanoma model was brought to the medaka (Oryzias latipes) system. Xmrk expression under the control of the pigment cell specific mitf promoter leads to melanoma formation with 100% penetrance in medaka. Xmrk is an orthologue of the human epidermal growth factor receptor (EGFR) and activates several downstream signaling pathways. Examples of these pathways are the direct phosphorylation of BRAF and Stat5, as well as the enhanced transcription of C-myc. BRAF is a serine-threonine kinase which is found mutated at high frequencies in malignant melanomas. Stat5 is a transcription factor known to be constitutively activated in fish melanoma. C-myc is a transcription factor that is thought to regulate the expression of approximately 15% of all human genes and is involved in cancer progression of a large number of different tumors. To gain new in vivo information on candidate factors known to be involved in melanoma progression, I identified and analysed BRAF, Stat5 and C-myc in the laboratory fish model system medaka. BRAF protein motifs are highly conserved among vertebrates and the results of this work indicate that its function in the MAPK signaling is maintained in medaka. Transgenic medaka lines carrying a constitutive active version of BRAF (V614E) showed more pigmented skin when compared to wild type. Also, some transiently expressing BRAF V614E fishes showed a disrupted eye phenotype. In addition, I was able to identify two Stat5 copies in medaka, named Stat5ab/a and Stat5ab/b. Sequence analysis revealed a higher similarity between both Stat5 sequences when compared to either human Stat5a or Stat5b. This suggests that the two Stat5 copies in medaka arose by an independent duplication processes. I cloned these two Stat5 present in medaka, produced constitutive active and dominant negative gene versions and successfully established transgenic lines carrying each version under the control of the MITF promoter. These lines will help to elucidate questions that are still remaining in Stat5 biology and its function in melanoma progression, like the role of Stat5 phosphorylation on tumor invasiveness. In a third project during my PhD work, I analysed medaka C-myc function and indentified two copies of this gene in medaka, named c-myc17 and c-myc20, according to the chromosome where they are located. I produced conditional transgenic medaka lines carrying the c-myc17 gene coupled to the hormone binding domain of the estrogen receptor to enable specific transgene activation at a given time point. Comparable to human C-myc, medaka C-myc17 is able to induce proliferation and apoptosis in vivo after induction. Besides that, C-myc17 long-term activation led to liver hyperplasia. In summary, the medaka models generated in this work will be important to bring new in vivo information on genes involved in cancer development. Also, the generated transgenic lines can be easily crossed to the melanoma developing Xmrk medaka lines, thereby opening up the possibility to investigate their function in melanoma progression. Besides that, the generated medaka fishes make it possible to follow the whole development of melanocytes, since the embryos are transparent and can be used for high throughput chemical screens.
Arrhythmogene Kardiomyopathie (ACM) ist eine genetische Herzerkrankung, die durch Herzinsuffizienz, ventrikuläre Arrhythmien und plötzlichen Herztod gekennzeichnet ist. Mutationen in desmosomalen Proteinen der Zelladhäsion, wie Plakophilin 2 (PKP2) und Plakoglobin (PG), sind die häufigste Ursache der familiären ACM. Wie gestörte Zelladhäsion zum ACM-Phänotyp führt, ist jedoch nur teilweise geklärt. Potentielle Mechanismen sind eine gestörte Kalzium-(Ca2+)-Homöostase, mitochondrialer oxidativer Stress und metabolische Störungen. Ziel dieser Studie ist es, die mitochondriale Energetik und die Ca2+ -Homöostase in kardio-restriktiven PKP2-Knockout-Mäusen (KO) im Alter von 4, 8 und 12 Wochen sowie in PG-Knockout- Mäusen im Alter von 6 Wochen zu untersuchen. Vier Wochen alte PKP2-KO-Mäuse zeigten frühe Anzeichen von ACM, während alle anderen Altersgruppen typische Kennzeichen von ACM rekapitulierten. Kontraktilität, die damit verbundenen Ca2+ - Transienten, der Redoxstatus und das mitochondriale Membranpotenzial (ΔΨm) isolierter Kardiomyozyten wurden mit einem IonOptix-System bei elektrischer und β- adrenerger Stimulation untersucht. Alle desmosomalen KO-Kardiomyozyten zeigten eine verringerte diastolische Sarkomerlänge, was auf eine diastolische Dysfunktion hinwies. In allen PKP2 KO Kardiomyozyten lag außerdem ein erhöhter intrazellulärer Ca2+ -Spiegel vor, während in den PG KO-Kardiomyozyten das intrazellulärer Ca2+ unverändert war. PKP2 KO- und PG KO-Kardiomyozyten wiesen keine Ca2+ - Sensibilisierung der Myofilamente auf. Zur weiteren Bewertung der mitochondrialen Funktion wurde eine hochauflösende Respirometrie in isolierten Herzmitochondrien bei gleichzeitiger Überwachung von ΔΨm in PKP2 KO und PG KO Mäusen durchgeführt, welche in allen Versuchs- und Kontrollgruppen vergleichbar war. Im Verlauf der Versuche blieb der Redoxstatus stabil und es konnte kein Exzess reaktiver Sauerstoffspezies (ROS) festgestellt werden. Daraus konnte gefolgert werden, dass weder PKP2 KO noch PG KO-Mäuse eine beeinträchtigte mitochondriale Atmung aufwiesen. Diese Studie zeigt, dass isolierte PKP2 KO- oder PG KO-Kardiomyozyten EC-Kopplungsdefekte ohne mitochondriale Dysfunktion aufwiesen. Eine mitochondriale Dysfunktion konnte als treibender Faktor für die Progression des ACM- Phänotyps in den vorgestellten Mausmodellen ausgeschlossen werden. Weitere Studien sind erforderlich, um die mitochondriale Funktion im Zusammenhang mit ACM zu entschlüsseln.
Cardiovascular diseases are considered the leading cause of death worldwide according to the World Health Organization. Heart failure is the last stage of most of these diseases, where loss of myocardium leads to architectural and functional decline.
The definitive treatment option for patients with CVDs is organ or tissue transplantation, which relies on donor availability. Therefore, generating an autologous bioengineered myocardium or heart could overcome this limitation. In addition, generating cardiac patches will provide ventricular wall support and enable reparative stem cells delivery to damaged areas. Although many hurdles still exist, a good number of researches have attempted to create an engineered cardiac tissue which can induce endogenous cardiac repair by replacing damaged myocardium.
The present study provided cardiac patches in two models, one by a detergent coronary perfusion decellularization protocol that was optimized, and the other that resulted in a 3D cell-free extracellular matrix with intact architecture and preserved s-glycosaminoglycan and vasculature conduits. Perfusion with 1% Sodium dodecyle sulfate (SDS) under constant pressure resulted in cell-free porcine scaffold within two and cell-free rat scaffold in 7 days, whereas scaffold perfused with 4% sodium deoxycholate (SDO) was not able to remove cells completely. Re-reendothelialization of tissue vasculature was obtained by injecting human microvascular endothelial cell and human fibroblast in 2:1 ratio in a dynamic culture. One-week later, CD31 positive cells and endothelium markers were observed, indicating new blood lining. Moreover, functionality test of re-endothelialized tissue revealed improvement in clotting seen in decellularized tissues. When the tissue was ready to be repopulated, porcine induced pluripotent stem cells (PiPSc) were generated by transfected reprogramming of porcine skin fibroblast and then differentiated to cardiac cells following a robust protocol, for an autologous cardiac tissue model. However, due to the limitation in the PiPSc cell number, alternatively, human induced pluripotent stem cells generated cardiac cells were used.
For reseeding a coculture of human iPSc generated cardiac cells, human mesenchymal stem cells and human fibroblast in 2:1:1 ratio respectively were used in a dynamic culture for 6-8 weeks. Contractions at different areas of the tissue were recorded at an average beating rate of 67 beats/min. In addition, positive cardiac markers (Troponin T), Fibroblast (vemintin), and mesenchymal stem cells (CD90) were detected. Not only that, but by week 3, MSC started differentiating to cardiac cells progressively until few CD90 positive cells were very few by week 6 with increasing troponin t positive cells in parallel. Electrophysiological and drug studies were difficult to obtain due to tissue thickness and limited assessment sources. However, the same construct was established using small intestine submucosa (SISer) scaffold, which recorded a spontaneous beating rate between 0.88 and 1.2 Hz, a conduction velocity of 23.9 ± 0.74 cm s−1, and a maximal contraction force of 0.453 ± 0.015 mN. Moreover, electrophysiological studies demonstrated a drug-dependent response on beating rate; a higher adrenalin frequency was revealed in comparison to the untreated tissue and isoproterenol administration, whereas a decrease in beating rate was observed with propranolol and untreated tissue.
The present study demonstrated the establishment of vascularized cardiac tissue, which can be used for human clinical application.
In the recent years, translational studies comparing imaging data of animals and humans have gained increasing scientific interests with crucial findings stemming from both, human and animal work. In order to harmonize statistical analyses of data from different species and to optimize the transfer of knowledge between them, shared data acquisition protocols and combined statistical approaches have to be identified. Following this idea, methods of data analysis, which have until now mainly been used to model neural responses of electrophysiological recordings from rodent data, were applied on human hemodynamic responses (i.e. Blood-Oxygen-Level-Dependent BOLD signal) as measured via functional magnetic resonance imaging (fMRI).
At the example of two attention and impulsivity networks, timing dynamics and amplitude of the fMRI signal were determined (study 1). Study 2 described the same parameters frequency-specifically, and in study 3, the complexity of neural processing was quantified in terms of fractality. Determined parameters were compared with regard to the subjects’ task performance / impulsivity to validate findings with regard to reports of the current scientific debate.
In a general discussion, overlapping as well as additional information of methodological approaches were discussed with regard to its potential for biomarkers in the context of neuropsychiatric disorders.
In the recent years, translational studies comparing imaging data of animals and humans have gained increasing
scientific interests with crucial findings stemming from both, human and animal work. In order to harmonize
statistical analyses of data from different species and to optimize the transfer of knowledge between them, shared
data acquisition protocols and combined statistical approaches have to be identified. Following this idea, methods
of data analysis, which have until now mainly been used to model neural responses of electrophysiological
recordings from rodent data, were applied on human hemodynamic responses (i.e. Blood-Oxygen-Level-
Dependent BOLD signal) as measured via functional magnetic resonance imaging (fMRI).
At the example of two attention and impulsivity networks, timing dynamics and amplitude of the fMRI signal were
determined (study 1). Study 2 described the same parameters frequency-specifically, and in study 3, the
complexity of neural processing was quantified in terms of fractality. Determined parameters were compared with
regard to the subjects’ task performance / impulsivity to validate findings with regard to reports of the current
scientific debate.
In a general discussion, overlapping as well as additional information of methodological approaches were
discussed with regard to its potential for biomarkers in the context of neuropsychiatric disorders.
Platelets play an important role in the body, since they are part of the hemostasis
system, preventing and stopping blood loss. Nevertheless, when platelet or
coagulation system function are impaired, uncontrolled bleedings but also irreversible
vessel occlusion followed by ischemic tissue damage can occur. Therefore,
understanding platelet function and activation, mechanisms which are controlled by a
variety of platelet membrane receptors and other factors is important to advance out
knowledge of hemostasis and platelet malfunction. For a complete picture of platelet
function and their modulating behavior it is desired to be able to quantify receptor
distributions and interactions of these densely packed molecular ensembles in the
membrane. This challenges scientists for several reasons. Most importantly, platelets
are microscopically small objects, challenging the spatial resolution of conventional
light microscopy. Moreover, platelet receptors are highly abundant on the membrane
so even super-resolution microscopy struggles with quantitative receptor imaging on
platelets.
With Expansion microscopy (ExM), a new super-resolution technique was introduced,
allowing resolutions to achieve super-resolution without using a super-resolution
microscope, but by combining a conventional confocal microscopy with a highly
processed sample that has been expanded physically. In this doctoral thesis, I
evaluated the potential of this technique for super-resolution platelet imaging by
optimizing the sample preparation process and establishing an imaging and image
processing pipeline for dual-color 3D images of different membrane receptors. The
analysis of receptor colocalization using ExM demonstrated a clear superiority
compared to conventional microscopy. Furthermore, I identified a library of
fluorescently labeled antibodies against different platelet receptors compatible with
ExM and showed the possibility of staining membrane receptors and parts of the
cytoskeleton at the same time.
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.
The Role of Attentional Control and Fear Acquisition and Generalization in Social Anxiety Disorder
(2020)
Although Social Anxiety Disorder (SAD) is one of the most prevalent mental disorders, still little is known about its development and maintenance. Cognitive models assume that deviations in attentional as well as associative learning processes play a role in the etiology of SAD. Amongst others, deficits in inhibitory attentional control as well as aberrations during fear generalization, which have already been observed in other anxiety disorders, are two candidate mechanisms that might contribute to the onset and retention of SAD. However, a review of the literature shows that there is a lack of research relating to these topics. Thus, the aim of the present thesis was to examine in which way individuals with SAD differ from healthy controls regarding attentional control and generalization of acquired fear during the processing of social stimuli.
Study 1 tested whether impairment in the inhibitory control of attention is a feature of SAD, and how it might be influenced by emotional expression and gaze direction of an interactional partner. For this purpose, individuals with SAD and healthy controls (HC) participated in an antisaccade task with faces displaying different emotional expressions (angry, neutral and happy) and gaze directions (direct and averted) serving as target stimuli. While the participants performed either pro- or antisaccades in response to the peripherally presented faces, their gaze behavior was recorded via eye-tracking, and ratings of valence and arousal were obtained. Results revealed that both groups showed prolonged latencies and increased error rates in trials with correct anti- compared to prosaccades. However, there were no differences between groups with regard to response latency or error rates, indicating that SAD patients did not exhibit impairment on inhibitory attentional control in comparison to HC during eye-tracking. Possible explanations for this finding could be that reduced inhibitory attentional control in SAD only occurs under certain circumstances, for example, when these individuals currently run the risk of being negatively evaluated by others and not in the mere presence of phobic stimuli, or when the cognitive load of a task is so high that it cannot be unwound by compensatory strategies, such as putting more effort into a task.
As not only deviations in attentional, but also associative learning processes might be pathogenic markers of SAD, these mechanisms were further addressed in the following experiments. Study 2 is the first that attempted to investigate the generalization of conditioned fear in patients with SAD. To this end, patients with SAD and HC were conditioned to two neutral female faces serving as conditioned stimuli (CS+: reinforced; CS-: non-reinforced) and a fearful face paired with a loud scream serving as unconditioned stimulus (US). Fear generalization was tested by presenting morphs of the two faces (GS: generalization stimuli), which varied in their similarity to the original faces. During the whole experiment, self-report ratings, heart rate (HR) and skin conductance responses (SCR) were recorded. Results demonstrated that SAD patients rated all stimuli as less pleasant and more arousing, and overestimated the occurrence of the US compared to HC, indicating a general hyperarousal in individuals with SAD. In addition, ratings and SCR indicated that both groups generalized their acquired fear from the CS+ to intermediate GSs as a function of their similarity to the CS+. However, except for the HR data, which indicated that only SAD patients but not HC displayed a generalization response in this measure, most of the results did not support the hypothesis that SAD is characterized by overgeneralization. A plausible reason for this finding could be that overgeneralization is just a key characteristic of some anxiety disorders and SAD is not one of them. Still, other factors, such as comorbidities in the individuals with SAD, could also have had an influence on the results, which is why overgeneralization was further examined in study 3.
The aim of study 3 was to investigate fear generalization on a neuronal level. Hence, high (HSA) and low socially anxious participants (LSA) underwent a conditioning paradigm, which was an adaption of the experimental design used study 2 for EEG. During the experiment, steady-state visually evoked potentials (ssVEPs) and ratings of valence and arousal were recorded. Analyses revealed significant generalization gradients in all ratings with highest fear responses to the CS+ and a progressive decline of these reactions with increasing similarity to the CS-. In contrast, the generalization gradient on a neuronal level showed highest amplitudes for the CS+ and a reduction in amplitude to the most proximal, but not distal GSs in the ssVEP signal, which might be interpreted as lateral inhibition in the visual cortex. The observed dissociation among explicit and implicit measures points to different functions of behavioral and sensory cortical processes during fear generalization: While the ratings might reflect an individual’s consciously increased readiness to react to threat, the lateral inhibition pattern in the occipital cortex might serve to maximize the contrast among stimuli with and without affective value and thereby improve adaptive behavior. As no group differences could be observed, the finding of study 2 that overgeneralization does not seem to be a marker of SAD is further consolidated.
In sum, the conducted experiments suggest that individuals with SAD are characterized by a general hyperarousal during the exposition to disorder-relevant stimuli as indicated by enhanced arousal and reduced valence ratings of the stimuli compared to HC. However, the hypotheses that reduced inhibitory attentional control and overgeneralization of conditioned fear are markers of SAD were mostly not confirmed. Further research is required to elucidate whether they only occur under certain circumstances, such as high cognitive load (e.g. handling two tasks simultaneously) or social stress (e.g. before giving a speech), or whether they are not characteristics of SAD at all. With the help of these findings, new interventions for the treatment of SAD can be developed, such as attentional bias modification or discrimination learning.
Imprinted genes play important roles in brain development. As the neural developmental capabilities of human parthenogenetic embryonic stem cells (hpESCs) with only a maternal genome were not assessed in great detail, hence here the potential of hpESCs to differentiate into various neural subtypes was determined. In addition DNA methylation and expression of imprinted genes upon neural differentiation was also investigated. The results demonstrated that hpESC-derived neural stem cells (hpNSCs) showed expression of NSC markers Sox1, Nestin, Pax6, and Musashi1 (MS1), the silencing of pluripotency genes (Oct4, Nanog) and the absence of activation of neural crest (Snai2, FoxD3) and mesodermal (Acta1) markers. Moreover, confocal images of hpNSC cultures exhibited ubiquitous expression of NSC markers Nestin, Sox1, Sox2 and Vimentin. Differentiating hpNSCs for 28 days generated neural subtypes with neural cell type-specific morphology and expression of neuronal and glial markers, including Tuj1, NeuN, Map2, GFAP, O4, Tau, Synapsin1 and GABA. hpNSCs also responded to region-specific differentiation signals and differentiated into regional phenotypes such as midbrain dopaminergic- and motoneuron-type cells. hpESC-derived neurons showed typical neuronal Na+/K+ currents in voltage clamp mode, elicited multiple action potentials with a maximum frequency of 30 Hz. Cell depicted a typical neuron-like current pattern that responded to selective pharmacological blockers of sodium (tetrodotoxin) and potassium (tetraethylammonium) channels. Furthermore, in hpESCs and hpNSCs the majority of CpGs of the differentially methylated regions (DMRs) KvDMR1 were methylated whereas DMR1 (H19/Igf2 locus) showed partial or complete absence of CpG methylation, which is consistent with a parthenogenetic (PG) origin. Upon differentiation parent-of-origin-specific gene expression was maintained in hpESCs and hpNSCs as demonstrated by imprinted gene expression analyses. Together this shows that despite the lack of a paternal genome, hpNSCs are proficient in differentiating into glial- and neuron-type cells, which exhibit electrical activity similar to newly formed neurons. Moreover, maternal-specific gene expression and imprinting-specific DNA-methylation are largely maintained upon neural differentiation. hpESCs are a means to generate histocompatible and disease allele-free ESCs. Additionally, hpESCs are a unique model to study the influence of imprinting on neurogenesis.
Die Funktionalität β1- und β2-adrenerger Rezeptoren wird durch Polymorphismen in ihrer kodierenden Region moduliert. Wir haben uns die Technik des Fluoreszenz-Resonanz- Energie-Transfers (FRET) zu Nutze gemacht, um den Einfluss der am häufigsten vorkommenden Polymorphismen (Ser49Gly und Gly389Arg im β1AR, Arg16Gly und Gln27Glu im β2AR) auf die Rezeptorkonformation nach Aktivierung zu untersuchen. Dafür wurden FRET-Sensoren für die beiden βAR-Subtypen mit einem gelb-fluoreszierenden Protein (YFP) sowie einem cyan-fluoreszierenden Protein (CFP oder Cerulean) in der dritten intrazellulären Schleife bzw. am C-Terminus verwendet. Nach Stimulierung der βARSensoren konnte die Aktivierung der polymorphen Rezeptorvarianten in lebenden Zellen in Echtzeit untersucht werden. Dabei behielten die FRET-Sensoren sowohl die Bindungsaffinitäten der nativen Rezeptoren als auch eine intakte Funktionalität hinsichtlich der Bildung von sekundären Botenstoffen. Der Vergleich der Aktivierungskinetiken der verschieden polymorphen Varianten des β1AR und β2AR ergab keine signifikanten Unterschiede nach einer einmaligen Stimulation. Es zeigte sich jedoch, dass Rezeptorpolymorphismen die Aktivierungskinetik vorstimulierter βAR erheblich beeinflussen. So konnten wir im Vergleich zur ersten Aktivierung eine schnellere Aktivierung der Gly16-Varianten des β2AR sowie des Gly49Arg389-β1AR feststellen, während die Arg16-β2AR-Variante und der Ser49Gly389-β1AR dagegen bei einer wiederholten Stimulation langsamer aktiviert wurden. Diese Ergebnisse lassen auf ein "Rezeptorgedächtnis" schließen, das spezifisch für bestimmte polymorphe Rezeptorvarianten ist und eine βAR-Subtyp-spezische Ausprägung zeigt. Die Ausbildung der unterschiedlichen Aktivierungskinetiken hing von der Interaktion des Rezeptors mit löslichen intrazellulären Faktoren ab und bedurfte einer Phosphorylierung intrazellulärer Serin- und Threonin-Reste durch G-Protein-gekoppelte Rezeptorkinasen. Die Interaktion mit löslichen intrazellulären Faktoren scheint für den β1AR weniger stark ausgeprägt zu sein als für den β2AR. Die cAMP-Produktion war für die schneller werdenden, “hyperfunktionellen” Gly16-β2ARVarianten signifikant um mehr als 50% höher im Vergleich zur “hypofunktionellen” Arg16- Variante. Die unterschiedliche Funktionalität spiegelte sich im Therapieausgang bei Tokoysepatientinnen wider, dessen Erfolg mit dem Arg16Gly Polymorphismus verknüpft war. Die Daten implizieren eine intrinsische, polymorphismusabhängige Eigenschaft der βAR, die die Aktivierungskinetik der Rezeptoren bei wiederholten Stimulationen determiniert. Diese könnte auch für die zwischen Individuen variierende Ansprechbarkeit auf β-Agonisten und β-Blocker mitverantwortlich sein.
G protein-coupled receptor research looks out for new technologies to elucidate the complex
processes of receptor activation, function and downstream signaling with spatiotemporal
resolution, preferably in living cells and organisms. A thriving approach consists in making use
of the unsurpassed properties of light, including its high precision in space and time, noninvasiveness
and high degree of orthogonality regarding biological processes. This is realized
by the incorporation of molecular photoswitches, which are able to effectively respond to light,
such as azobenzene, into the structure of a ligand of a given receptor. The muscarinic
acetylcholine receptors belong to class A GPCRs and have received special attention in this
regard due to their role as a prototypic pharmacological system and their therapeutic potential.
They mediate the excitatory and inhibitory effects of the neurotransmitter acetylcholine and
thus regulate diverse important biological processes, especially many neurological functions in
our brain.
In this work, the application of photopharmacological tool compounds to muscarinic receptors
is presented, consisting of pharmacophores extended with azobenzene as light-responsive
motif. Making use of the dualsteric concept, such photochromic ligands can be designed to bind
concomitantly to the orthosteric and allosteric binding site of the receptor, which is
demonstrated for BQCAAI (M1) and PAI (M2) and may lead to subtype- and functionalselective
photoswitchable ligands, suitable for further ex vivo and in vivo studies.
Moreover, photoswitchable ligands based on the synthetic agonist iperoxo were investigated
extensively with regard to their photochemical behavior and pharmacological profile, outlining
the advantages and challenges of using red-shifted molecular photoswitches, such as tetraortho-
fluoro azobenzene. For the first time on a GPCR it was examined, which impact the
different substitution pattern has on both the binding and the activity on the M1 receptor. Results
show that substituted azobenzenes in photopharmacological compounds (F4-photoiperoxo and
F4-iper-azo-iper) not just represent analogs with other photophysical properties but can exhibit
a considerably different biological profile that has to be investigated carefully.
The achievements gained in this study can give important new insights into the binding mode
and time course of activation processes, enabling precise spatial and temporal resolution of the
complex signaling pathway of muscarinic receptors. Due to their role as exemplary model
system, these findings may be useful for the investigation into other therapeutically relevant
GPCRs.
The hallmark oncoprotein Myc is a major driver of tumorigenesis in various human cancer entities. However, Myc’s structural features make it challenging to develop small molecules against it. A promising strategy to indirectly inhibit the function of Myc is by targeting its interactors. Many Myc-interacting proteins have reported scaffolding functions which are difficult to target using conventional occupancy- driven inhibitors. Thus, in this thesis, the proteolysis targeting chimera (PROTAC) approach was used to target two oncoproteins interacting with Myc which promote the oncogenicity of Myc, Aurora-A and WDR5. PROTACs are bifunctional small molecules that bind to the target protein with one ligand and recruit a cellular E3- ligase with the other ligand to induce target degradation via the ubiquitin- proteasome system. So far, the most widely used E3-ligases for PROTAC development are Cereblon (CRBN) and von Hippel–Lindau tumor suppressor (VHL). Furthermore, there are cases of incompatibility between some E3-ligases and proteins to bring about degradation. Hence there is a need to explore new E3- ligases and a demand for a tool to predict degradative E3-ligases for the target protein in the PROTAC field.
In the first part, a highly specific mitotic kinase Aurora-A degrader, JB170, was developed. This compound utilized Aurora-A inhibitor alisertib as the target ligand and thalidomide as the E3-ligase CRBN harness. The specificity of JB170 and the ternary complex formation was supported by the interactions between Aurora-A and CRBN. The PROTAC-mediated degradation of Aurora-A induced a distinct S- phase defect rather than mitotic arrest, shown by its catalytic inhibition. The finding demonstrates that Aurora-A has a non-catalytic role in the S-phase. Furthermore, the degradation of Aurora-A led to apoptosis in various cancer cell lines.
In the second part, two different series of WDR5 PROTACs based on two protein- protein inhibitors of WDR5 were evaluated. The most efficient degraders from both series recruited VHL as a E3-ligase and showed partial degradation of WDR5. In addition, the degradation efficiency of the PROTACs was significantly affected by the linker nature and length, highlighting the importance of linker length and composition in PROTAC design. The degraders showed modest proliferation defects at best in cancer cell lines. However, overexpression of VHL increased the degradation efficiency and the antiproliferative effect of the PROTACs.
In the last part, a rapamycin-based assay was developed to predict the degradative E3-ligase for a target. The assay was validated using the WDR5/VHL and Aurora- A/CRBN pairs. The result that WDR5 is degraded by VHL but not CRBN and Aurora-A is degraded by CRBN, matches observations made with PROTACs. This technique will be used in the future to find effective tissue-specific and essential E3-ligases for targeted degradation of oncoproteins using PROTACs.
Collectively, the work presented here provides a strategy to improve PROTAC development and a starting point for developing Aurora-A and WDR5 PROTACs for cancer therapy.
The putative attachment protein G of pneumonia virus of mice (PVM), a member of the Pneumoviruses, is an important virulence factor with so far ambiguous function in a virus-cell as well as in virus-host context. The sequence of the corresponding G gene is characterized by significant heterogeneity between and even within strains, affecting the gene and possibly the protein structure. This accounts in particular for the PVM strain J3666 for which two differing G gene organizations have been described: a polymorphism in nucleotide 65 of the G gene results in the presence of an upstream open reading frame (uORF) that precedes the main ORF in frame (GJ366665A) or extension of the major G ORF for 18 codons (GJ366665U). Therefore, this study was designed to analyse the impact of the sequence variations in the respective G genes of PVM strains J3666 and the reference strain 15 on protein expression, replication and virulence.
First, the controversy regarding the consensus sequence of PVM J3666 was resolved. The analysis of 45 distinct cloned fragments showed that the strain separated into two distinct virus populations defined by the sequence and structure of the G gene. This division was further supported by nucleotide polymorphisms in the neighbouring M and SH genes. Sequential passage of this mixed strain in the cell line standardly used for propagation of virus stocks resulted in selection for the GJ366665A-containing population in one of two experiments pointing towards a moderate replicative advantage. The replacement of the G gene of the recombinant PVM 15 with GJ366665A or GJ366665U, respectively, using a reverse genetic approach indicated that the presence of uORF within the GJ366665A significantly reduced the expression of the main G ORF on translational level while the potential extension of the ORF in GJ366665U increased G protein expression. In comparison, the effect of the G gene-structure on virus replication was inconsistent and dependent on cell line and type. While the presence of uORF correlated with a replication advantage in the standardly used BHK-21 cells and primary murine embryonic fibroblasts, replication in the murine macrophage cell line RAW 264.7 did not. In comparison, the GJ366665U variant was not associated with any effect on replication in cultured cells at all. Nonetheless, in-vivo analysis of the recombinant viruses associated the GJ366665U gene variant, and hence an increased G expression, with higher virulence whereas the GJ366665A gene, and therefore an impaired G expression, conferred an attenuated phenotype to the virus.
To extend the study to other G gene organizations, a recombinant PVM expressing a G protein without the cytoplasmic domain and for comparison a G-deletion mutant, both known to be attenuated in vivo, were studied. Not noticed before, this structure of the G gene was associated with a 75% reduction in G protein expression and a significant attenuation of replication in macrophage-like cells. This attenuation was even more prominent for the virus lacking G. Taking into consideration the higher reduction in G protein levels compared to the GJ366665A variant indicates that a threshold amount of G is required for efficient replication in these cells.
In conclusion, the results gathered indicated that the expression levels of the G protein were modulated by the sequence of the 5’ untranslated region of the gene. At the same time the G protein levels modulated the virulence of PVM.
Disruptions in brain serotonin (5-hydroxytryptamine, 5-HT) signaling pathways have been associated with etiology and pathogenesis of various neuropsychiatric disorders, but specific neural mechanisms of 5-HT function are yet to be fully elucidated. Tryptophan hydroxylase 2 (TPH2) is the rate-limiting enzyme for brain 5-HT synthesis. Therefore, in this study a tamoxifen (Tam)-inducible cre-mediated conditional gene (Tph2) knockout in adult mouse brain (Tph2icKO) has been established to decipher the specific role of brain 5-HT in the regulation of behavior in adulthood.
Immunohistochemistry and high-performance liquid chromatography (HPLC) were used first to test the efficacy of Tam-inducible inactivation of Tph2 and consequential reduction of 5-HT in adult mouse brain. Tam treatment resulted in ≥90% reduction in the number of 5-HT immuno-reactive cells in the anterior raphe nuclei. HPLC revealed a significant reduction in concentration of 5-HT and its metabolite 5-hydroxyindole acetic acid (5-HIAA) in selected brain regions of Tph2icKO, indicating the effectiveness of the protocol used.
Second, standard behavioral tests were used to assess whether reduced brain 5-HT concentrations could alter anxiety-, fear- and depressive-like behavior in mice. No altered anxiety- and depressive-like behaviors were observed in Tph2icKO compared to control mice (Tph2CON) in all indices measured, but Tph2icKO mice exhibited intense and sustained freezing during context-dependent fear memory retrieval. Tph2icKO mice also exhibited locomotor hyperactivity in the aversive environments, such as the open field, and consumed more food and fluid than Tph2CON mice.
Lastly, the combined effect of maternal separation (MS) stress and adult brain 5-HT depletion on behavior was assessed in male and female mice. Here, MS stress, 5-HT depletion and their interaction elicited anxiety-like behavior in a sex-dependent manner. MS reduced exploratory behavior in both male and female mice. Reduced 5-HT enhanced anxiety in female, but not in male mice.
Furthermore, expression of genes related to the 5-HT system and emotionality (Tph2, Htr1a, Htr2a, Maoa and Avpr1a) was assessed by performing a quantitative real-time PCR. In Tph2icKO mice there was a reduction in expression of Tph2 in the raphe nuclei of both male and female mice. Interaction between MS stress and 5-HT deficiency was detected showing increased Htr2a and Maoa expression in raphe and hippocampus respectively of female mice. In male mice, MS stress and 5-HT depletion interaction effects reduced Avpr1a expression in raphe, while the expression of Htr1a, Htr2a and Maoa was differentially altered by 5-HT depletion and MS in various brain regions.
This thesis is divided into three parts with the main goal allocating novel antimicrobial compounds that could be used as future antibiotics. The first part aimed to evaluate the potential of plant suspension cultures for the production of antimicrobial proteins. The extracellular, intracellular and cell wall bound fractions of seven heterotrophic and photomixotrophic plant cell suspension cultures treated with nine different elicitors were tested for the elicitor dependent production of antimicrobial proteins. Bioactivities were tested against a selected panel of human isolates including Gram-positive and Gram-negative bacteria as well as fungi using the disc diffusion assay. The intracellular fractions of elicited cell cultures were more active than extracellular fractions while the cell wall bound fractions showed lowest activities. Among the 21 fractions tested, the intracellular fraction of Lavendula angustifolia elicited with DC3000 was most active against Candida maltosa. The second most active fraction was the intracellular fraction of Arabidopsis thaliana elicited with salicylic acid which was moreover active against all test strains. The antimicrobial activity of elicited Arabidopsis thaliana cell cultures was tested by bioautography to locate the antimicrobial proteins in the crude extract. The intracellular fraction of photomixotrophic Arabidopsis thaliana cells elicited with salicylic acid was selected for further gel filtration chromatography on S-200 column leading to the purification of one 19 kDa antimicrobially active protein, designated, AtAMP. Our findings suggest that elicited plant cell cultures may present a new promising alternative source of antimicrobial proteins. The second part comprises the isolation of actinomycetes associated with marine sponges and testing the bioactivities of new species for further investigations. Actinobacterial communities of eleven taxonomically different sponges that had been collected from offshore Ras Mohamed (Egypt) and from Rovinj (Croatia) were investigated by a culture-based approach using different standard media for isolation of actinomycetes and media enriched with aqueous sponge extract to target rare and new actinomycete species. Phylogenetic characterization of 52 representative isolates out of 90 based on almost complete sequences of genes encoding 16S rRNA supported their assignment to 18 different actinomycete genera. Altogether 14 putatively new species were identified based on sequence similarity values below 98.2% to other strains in the NCBI database. The use of M1 agar amended with aqueous sponge extract yielded a putative new genus related to Rubrobacter which highlighting the need for innovative cultivation protocols. Biological activity testing showed that five isolates were active against Gram-positives only, one isolate was active against Candida albicans only and one isolate showed activity against both groups of pathogens. Moreover, the antiparasistic activity was documented for four isolates. These results showed a high diversity of actinomycetes associated with marine sponges as well as highlighted their potential to produce anti-infective agents. The third part of the thesis focused on the isolation and structure elucidation of new bioactive compounds. Streptomyces strain RV15 recovered from sponge Dysidea tupha, was selected for further chemical analysis by virtue of the fact that it exhibited the greatest antimicrobial potential against Staphylococcus aureus as well as Candida albicans among the all tested strains. Moreover, members of the genus Streptomyces are well known as prolific producers of interesting pharmacologically active metabolites. Chemical analysis of the methanolic crude extract using different chromatographic tools yielded four new compounds. The structures of the new compounds were spectroscopically elucidated to be four new cyclic peptides, namely, cyclodysidins A-D. Their bioactivity was tested against different proteases, bacteria and Candida as well as tumor cell lines. The compounds did not show any significant activities at this point.