Refine
Has Fulltext
- yes (101)
Is part of the Bibliography
- yes (101) (remove)
Year of publication
- 2022 (101) (remove)
Document Type
- Doctoral Thesis (101) (remove)
Keywords
- Angststörung (3)
- DNA damage (3)
- GPCR (3)
- Jugend (3)
- Kind (3)
- T-Lymphozyt (3)
- Tissue Engineering (3)
- Transcription (3)
- 3D-Druck (2)
- Angst (2)
Institute
- Graduate School of Life Sciences (101) (remove)
Sonstige beteiligte Institutionen
- Helmholtz Institute for RNA-based Infection Research (HIRI) (2)
- Department of Cellular Therapies, University of Navarra, Pamplona, Spain (1)
- Department of X-ray Microscopy, University of Würzburg, Würzburg, Germany (1)
- Interdisziplinäre Zentrum für Klinische Forschung (IZKF) (1)
- Politecnico di Milano (1)
- Translationale soziale Neurowissenschaften (1)
- Universitätsklinikum Würzburg (UKW) (1)
- Zentrum für Interdisziplinäre Medizin der Uni Würzburg (1)
In this work, accelerated non-Cartesian Magnetic Resonance Imaging (MRI) methods were established and applied to cardiovascular imaging (CMR) at different magnetic field strengths (3T and 7T).
To enable rapid data acquisition, highly efficient spiral k-space trajectories were created. In addition, hybrid sampling patterns such as the twisting radial lines (TWIRL) k-space trajectory were studied.
Imperfections of the dynamic gradient system of a MR scanner result in k-space sampling errors. Ultimately, these errors can lead to image artifacts in non-Cartesian acquisitions.
Among other reasons such as an increased reconstruction complexity, they cause the lack of spiral sequences in clinical routine compared to standard Cartesian imaging.
Therefore, the Gradient System Transfer Functions (GSTFs) of both scanners were determined and used for k-space trajectory correction in post-correction as well as in terms of a pre-emphasis.
The GSTF pre-emphasis was implemented as a fully automatic procedure, which enabled a precise correction of arbitrary gradient waveforms for double-oblique slice orientations.
Consequently, artifacts due to trajectory errors could be mitigated, which resulted in high image quality in non-Cartesian MRI.
Additionally, the GSTF correction was validated by measuring pre-emphasized spiral gradient outputs, which showed high agreement with the theoretical gradient waveforms.
Furthermore, it could be demonstrated that the performance of the GSTF correction is superior to a simple delay compensation approach.
The developed pulse sequences were applied to gated as well as real-time CMR. Special focus lied on the implementation of a spiral imaging protocol to resolve the beating heart of animals and humans in real time and free breathing.
In order to achieve real-time CMR with high spatiotemporal resolution, k-space undersampling was performed. For this reason, efficient sampling strategies were developed with the aim to facilitate compressed sensing (CS) during image reconstruction.
The applied CS approach successfully removed aliasing artifacts and yielded high-resolution cardiac image series. Image reconstruction was performed offline in all cases such that the images were not available immediately after acquisition at the scanner.
Spiral real-time CMR could be performed in free breathing, which led to an acquisition time of less than 1 minute for a whole short-axis stack.
At 3T, the results were compared to the gold standard of electrocardiogram-gated Cartesian CMR in breath hold, which revealed similar values for important cardiovascular functional and volumetric parameters.
This paves the way to an application of the developed framework in clinical routine of CMR.
In addition, the spiral real-time protocol was transferred to swallowing and speech imaging at 3T, and first images were presented.
The results were of high quality and confirm the straightforward utilization of the spiral sequence in other fields of MRI.
In general, the GSTF correction yielded high-quality images at both field strengths, 3T and 7T.
Off-resonance related blurring was mitigated by applying non-Cartesian readout gradients of short duration. At 7T, however, B1-inhomogeneity led to image artifacts in some cases.
All in all, this work demonstrated great advances in accelerating the MRI process by combining efficient, undersampled non-Cartesian k-space coverage with CS reconstruction.
Trajectory correction using the GSTF can be implemented at any scanner model and enables non-Cartesian imaging with high image quality.
Especially MRI of dynamic processes greatly benefits from the presented rapid imaging approaches.
G-protein-coupled receptors (GPCRs) are key biological switches that transmit both internal and external stimuli into the cell interior. Among the GPCRs, the “light receptor” rhodopsin has been shown to activate with a re-arrangement of the transmembrane helix bundle within ≈1 ms, while all other receptors are thought to become activated in subsecond range at saturating concentrations. Here we investigate activation kinetics of a dimeric GPCR, the metabotropic glutamate receptor-1 (mGluR1), and several class A GPCRs, as muscarinic receptor 3 (M3R), adrenergic (α2aAR and β1R) and opioid (µOR) receptors. We first used UV-light-triggered uncaging of glutamate in intact cells. Sub-millisecond Förster resonance energy transfer recordings between labels at intracellular receptor sites were used to record conformational changes in the mGluR1. At millimolar ligand concentrations the initial rearrangement between the mGluR1 subunits occurs at a speed of τ1≈1-2 ms. These rapid changes were followed by significantly slower conformational changes in the transmembrane domain (τ2≈20 ms). We further characterized novel photoswitchable negative allosteric modulators for mGluR1, which bind to its transmembrane core and block the conformational change as well as the downstream signaling. Effects of the compounds were quantified in pharmacological cell assays in the dark and using UV and green light illumination. We finally develop a framework for image-based kinetic analysis of GPCRs which allowed us to measure activation kinetics of several prototypical class A GPCRs and to discover membrane heterogeneities of GPCR activation. It appears that GPCR activation signal is not only dependent on the amount of activated receptors, but also has some level of correlation with the local density of activated receptors.
Transcription describes the process of converting the information contained in DNA into RNA. Although, tremendous progress has been made in recent decades to uncover this complex mechanism, it is still not fully understood. Given the advances and reduction in cost of high-throughput sequencing experiments, more and more data have been generated to help elucidating this complex process. Importantly, these sequencing experiments produce massive amounts of data that are incomprehensible in their raw form for humans. Further, sequencing techniques are not always 100% accurate and are subject to a certain degree of variability and, in special cases, they might introduce technical artifacts. Thus, computational and statistical methods are indispensable to uncover the information buried in these datasets.
In this thesis, I worked with multiple high throughput datasets from herpes simplex virus 1 (HSV-1) and human cytomegalovirus (HCMV) infections. During the last decade, it has became clear that a gene might not have a single, but multiple sites at which transcription initiates. These multiple transcription start sites (TiSS) demonstrated to have regulatory effects on the gene itself depending on which TiSS is used. Specialized experimental approaches were developed to help identify TiSS (TiSS-profiling). In order to facilitate the identification of all potential TiSS that are used for cell type- and condition-specific transcription, I developed the tool iTiSS. By using a new general enrichment-based approach to predict TiSS, iTiSS proved to be applicable in integrated studies and made it less prone to false positives compared to other TiSS-calling tools. Another improvement in recent years was made in metabolic labeling experiments such as SLAM-seq. Here, they removed the time consuming and laborious step of physically separating new from old RNA in the samples. This was achieved by inducing specific nucleotide conversions in newly synthesized RNA that are later visible in the data. Consequently, the separation of new and old RNA is now done computationally and, hence, tools are needed that accurately quantify these fold-changes. My second tool that I developed, called GRAND-SLAM proved to be capable to accomplish this task and outperform competing programs. As both of my tools, iTiSS and GRAND-SLAM are not specifically tailored to my own goals, but could also facilitate the research of other groups in this field, I made them publicly available on GitHub.
I applied my tools to datasets generated in our lab as well as to publicly available data sets from HSV-1 and HCMV, respectively. For HSV-1, I was able to predict and validate TiSS with nucleotide precision using iTiSS. This has lead to the most comprehensive annotation for HSV-1 to date, which now serves as the fundamental basis of any future transcriptomic research on HSV-1. By combining both my tools, I was further able to uncover parts of the highly complex gene kinetics in HCMV and to resolve the limitations caused by the densely packed genome of HCMV.
With the ever-increasing advances in sequencing techniques and their decrease in cost, the amounts of data produced will continue to rise massively in the future. Additionally, more and more specialized omics approaches are appearing, calling for new tools to leverage their full information potential. Consequently, it has become apparent that specialized computational tools such as iTiSS and GRAND-SLAM are needed and will become an essential and indispensable part of the analysis.
Parkinson’s disease (PD), which is the most common motor neurodegenerative disorder has attracted a tremendous amount of research advancement amid the challenges of the lack of an appropriate model that summate all the features of the human disease. Nevertheless, an aspect of the disease that is yet to be fully elucidated is the role of the immune system particularly the adaptive arm in the pathogenesis of PD. The focus of this study therefore was to characterize the contribution of lymphocytes in PD using the AAV1/2-A53T-α-synuclein mouse model of the disease that encodes for human mutated A53T-α-synuclein. This model was suitable for this research because it reflects more faithfully the molecular pathology underlying the human disease by exhibition of insoluble α-synuclein containing Lewy-like protein aggregates as compared to the more classical toxin models used in PD research. The outcome of this study showed that stereotaxic delivery of pathogenic α-synuclein via a viral vector into the substantia nigra engender the invasion of activated CD4+ and CD8+ T lymphocytes in the brain. The invasion of activated T cells in the brain especially in the substantia nigra then results in enhanced microglial activation and the disintegration of dopaminergic neurons. In addition, it was also discovered that CD4+ T cells augmented dopaminergic cell death to a greater extent than CD8+ T cells although; axonal degeneration occurred relatively independent from T cells contribution. The ex vivo and in vitro, experiments also indicated that the T cells were not only activated but they were specific to the mutated human α-synuclein antigen. As a result, they demonstrated selectivity in inducing more cell death to primary hippocampal neurons transduced with AAV1/2-A53T-α-synuclein vector than neurons with empty viral vector infection. The mechanism of T cell induced neuronal cell loss could not be attributed to the presence of cytokines neither was it mediated through MHC I and II. On the whole, this research has established that the presence of pathogenic α-synuclein in the substantia nigra has the potential to trigger immune responses that involve the transmigration of adaptive immune cells into the brain. The infiltration of the T cells consequently has a detrimental effect on the survival of dopaminergic neurons and the progression of the disease
Estrogens, namely 17β-estradiol (E2) and estrone (E1) are considered to play an important role in the initiation and promotion of breast cancer (summarized in Raftogianis et al., 2000), a malignancy responsible for around 500,000 deaths per year (summarized in Ghislain et al., 2016). Two major mechanisms have been postulated to explain the carcinogenic effects of estrogens: (1) the estrogen receptor-mediated stimulation of breast cell proliferation with a concomitant enhanced rate of mutations and (2) the metabolism of hydroxylated estrogens to quinone derivatives which can react with the DNA (Russo and Russo, 2006, summarized in Yager and Davidson, 2006). Nevertheless, as a detoxifying mechanism, E1, E2, and their hydroxylated and methoxylated metabolites are reversibly conjugated into sulfates and glucuronides devoid of biological activity (summarized in Guillemette et al., 2004). Yet, despite the key detoxifying function of these conjugates, the study of their circulating levels face some significant problems: (1) analysis by techniques such as radioimmunoassay lack specificity and accuracy and requires enzymatic/chemical hydrolysis before analysis, being unable to differentiate between sulfates and glucuronides (summarized in Stanczyk et al., 2007, summarized in Wang et al., 2016), (2) very little knowledge in healthy women, which has been identified as a barrier to advance in breast cancer research (summarized in Liu, 2000), and (3) far fewer studies in pre- than in postmenopausal women (summarized in Samavat and Kurzer, 2015). Therefore, to get more insights into the research of breast cancer etiology and prevention, the analysis of circulating levels of estrogens (including metabolites and conjugates) in women without breast cancer through reliable analytical techniques, is required.
The epithelial layer of the gastrointestinal (GI) tract provides a barrier between the environment and the body. Dysfunction of the epithelium, including changes of the innate immune response facilitated by pattern recognition receptors (PRRs), plays a major role in the development of GI disorders. However, the organization of innate immune sensing, the expression and activity of PRRs and the factors contri¬buting to such possible organization along the GI tract are unclear. In recent years, stem cell-derived organoids gained increasing attention as promising tissue models. Here, a biobank of human and murine organoids comprising three lines from each GI segment; corpus, pylorus, duodenum, jejunum, ileum, colon was generated. RNA sequencing of 42 lines confirmed the preservation of tissue identity and revealed an extensive organization of innate immune signaling components along the cephalocaudal axis, giving each segment a specific innate immune profile. Comple-menting the region-specific expression analysis, several PRRs in human and murine organoids showed region- and species-specific function. To investigate the factors contributing to the patterning of innate immunity in the GI tract, the impact of microbial components was analyzed using murine embryo-derived, never colonized gastric and proximal intestinal organoids. Transcriptional profiling of embryo-derived organoids showed that while expression of some PRRs may depend on environmental cues as expected, an unexpectedly large part of segment-specific expression of PRR signaling components is independent of prior contact with microbial products. Further, analysis of published RNA-seq data as well as in vitro experiments using directed differentiation of organoids into specific cell types showed that expression of innate immune gene also depended on cellular differentiation along the crypt-villus axis. This underlined the importance of cellular differentiation rather than contact to microbial compounds for expression of PRRs. Lastly, analysis of published datasets of RNA-seq and ATAC-seq after knockout of the intestinal transcription factor Cdx2 demonstrated that Cdx2 is likely important for the expression of Nlrp6 and Naip1 in the murine intestine. Future experiments have to support these preliminary findings. Taken together, the expression of a large part of epithelial innate immunity is develop¬mentally defined and conserved in tissue-resident stem cells. The identification of mechanisms governing expression of genes related to immunity will provide further insights into the mechanisms that play a role in the progress of inflammatory diseases.
Wilms tumor (WT) is the most common renal tumor in childhood. Among others, MYCN copy number gain and MYCN P44L and MAX R60Q mutations have been identified in WT. The proto-oncogene MYCN encodes a transcription factor that requires dimerization with MAX to activate transcription of numerous target genes. MYCN gain has been associated with adverse prognosis. The MYCN P44L and MAX R60Q mutations, located in either the transactivating or basic helix-loop-helix domain, respectively, are predicted to be damaging by different pathogenicity prediction tools. These mutations have been reported in several other cancers and remain to be functionally characterized.
In order to further describe these events in WT, we screened both mutations in a large cohort of unselected WT patients, to check for an association of the mutation status with certain histological or clinical features. MYCN P44L and MAX R60Q revealed frequencies of 3 % and 0.9 % and also were significantly associated to higher risk of relapse and metastasis, respectively. Furthermore, to get a better understanding of the MAX mutational landscape in WT, over 100 WT cases were analyzed by Sanger sequencing to identify other eventual MAX alterations in its coding sequence. R60Q remained the only MAX CDS alteration described in WT to date.
To analyze the potential functional consequences of these mutations, we used a doxycycline-inducible system to overexpress each mutant in HEK293 cells. This biochemical characterization identified a reduced transcriptional activation potential for MAX R60Q, while the MYCN P44L mutation did not change activation potential or protein stability. The protein interactome of N-MYC-P44L was likewise not altered as shown by mass spectrometric analyses of purified N-MYC complexes. However, we could identify a number of novel N-MYC partner proteins, several of these known for their oncogenic potential. Their correlated expression in WT samples suggested a role in WT oncogenesis and they expand the range of potential biomarkers for WT stratification and targeting, especially for high-risk WT.
Bacteria thrive and survive in many different environments, and as a result, they have developed robust mechanisms to adapt rapidly to alterations in their surroundings. The protection against osmotic forces is provided by mechanosensitive channels: their primary function is to maintain the integrity of the cell upon a hypoosmotic shock. The mechanosensitive channel of small conductance (MscS) is not only the smallest common structural unit of a diverse family that allows for a tailored response in osmoregulation; it is also the most intensively studied homologue. Mechanosensitive channels directly sense elevated membrane tension levels generated by increased pressure within the cell and open transiently. Escherichia coli has six paralogues that differ in their gating properties and the number of additional transmembrane (TM) helices. These TM helices, termed sensor paddles, are essential for sensing, as they directly contact the surrounding membrane; however, the role of the additional TM helices is still unclear. Furthermore, lipids occupy hydrophobic pockets far away from the membrane plane. A recent gating model for MscS states that increased membrane tension triggers the expulsion of lipids out of those pockets, modulating different conformational states of MscS. This model focuses on bound lipids, but it is still unclear to what extent the direct interaction with the membrane influences sensing and how relevant it is for the larger paralogues.
In the herein described work, structural studies on two larger paralogues, the medium-sized channel YnaI and the large channel YbiO were realised using electron cryomicroscopy (cryo-EM). Lipids were identified in YnaI in the pockets in a similar position and orientation as in MscS, suggesting a conserved sensing mechanism. Moreover, the copolymer diisobutylene/maleic acid (DIBMA) allowed the extraction of artificially activated YnaI from plasma membranes, leading to an open-like form of this channel. This novel conformation indicated that the pore helices bend at a GGxGG motif during gating, which is unique among the Escherichia coli paralogues, concomitant with a structural reorganisation of the sensor paddles. Thus, despite a high similarity of their closed states, the gating mechanisms of MscS and YnaI are surprisingly different. Furthermore, the comparison of MscS, YnaI, and YbiO accentuates variations and similarities between the differently sized family members, implying fine-tuning of channel properties in the pore regions and the cytosolic lateral entry sides into the channel. Structural analyses of MscS reconstituted into different systems showed the advantages and disadvantages of certain polymers and detergents. The novel DIBMA copolymer and the more conventional amphiphilic polymers, so-called Amphipols, perturb contacting transmembrane helices or lead to their denaturation. Due to this observation, the obtained structures of YnaI must also be cautiously considered. The structures obtained in detergents resulted in unaffected channels; however, the applicability of detergents for MscS-like channels is limited by the increased required sample concentration.
The role of lipids for gating MscS in the absence of a membrane was examined by deliberately removing coordinated lipid molecules from MscS using different amounts and kinds of detergent. The effects on the channel were inspected by cryo-EM. These experiments showed that closed MscS adopts the open conformation when it is enough delipidated by incubation with the detergent n-dodecyl-β-D-maltoside, and adding lipids to the open channel reverses this process. The results agree with the state-of-the-art model that the amount of lipid molecules in the pockets and grooves is responsible for the conformational state of MscS. Furthermore, incubation with the detergent lauryl maltose neopentyl glycol, which has stabilising and delipidating characteristics, resulted in a high-resolution structure of open MscS exhibiting an intricate network of ligands. Based on this structure, an updated gating model is proposed, which states that upon opening, lipids from the pockets migrate into the cytosolic membrane leaflet, while lipids from the periplasmic leaflet enter the grooves that arise between the sensor paddles.
RNA molecules play diverse roles in biological systems. Post-transcriptional RNA modifications and dynamic structures enhance the functional diversity of RNA. A prerequisite for studying their biological significance is the availability of reliable methods for the detection of RNA modifications and structures. Several promising approaches have been developed in the last few decades; however, efficient, and versatile tools are still required to study the dynamic features of RNA. This thesis focuses on the development of nucleic acid catalysts as a tool to address the current needs in studying RNA. The major part of this thesis aimed at the development of deoxyribozymes as a tool for the detection of RNA modifications. Using in vitro selection from a random DNA library, we found deoxyribozymes that are sensitive to N 6 -isopentenyladenosine (i6A), a native tRNA modification and structural analogue of m6A. The in vitro evolution identified three classes of DNA enzymes: AA, AB08, and AC17 DNAzymes that showed distinct response to i6A modification and showed strong discrimination between structural analogues, i.e., m6A and i6A. In the continuation of the project, we attempted to develop RNA-cleaving deoxyribozymes that differentially respond to monomethylated cytidine isomers, 3-methylcytidine (m3C), N4 - methylcytidine (m4C), and 5-methylcytidine (m5C). Several deoxyribozymes were identified from in vitro selection, which are selective for a specific methylated cytidine isomer. The characterization of AL112, AM101, AN05, and AK104 catalysts confirmed the successful evolution of modification-specific and general deoxyribozymes that showed a broad substrate scope. In order to accelerate the DNAzymes discovery, a high throughput sequencing method (DZ-seq) was established that directly quantifies the RNA cleavage activity and cleavage site from deep sequencing data. The libraries contained information about cleavage status, cleavage site and sequence of deoxyribozymes and RNA substrate. The fraction cleaved (FC) data obtained from Dz-seq was validated for a subset of deoxyribozmes using conventional gel based kinetic assay and showed a good linear correlation (R2 = 0.91). Dz-seq possesses a great potential for the discovery of novel deoxyribozymes for the analysis of various RNA modifications in the future. The second objective of the current study was the development of structure-specific RNA labeling ribozymes. Here, we attempted to develop ribozymes that targets RNA of interest by structure-specific interaction rather than base-pairing and focused on a specific RNA G-quadruplex as the target. Two subsequent selection experiments led to the identification of the adenylyltransferase ribozymes AO10.2 and AR9. The partial characterization of these catalysts showed that A010.2 was unable to recognize intact BCL2 structure, but it turned out as the first reported trans-active ribozyme that efficiently labeled uridine in a defined substrate RNA hybridized to the ribozyme. The other ribozyme AR9 was shown to serve as a trans-active, self-labeling ribozyme that catalyzed adenylyl transferase reaction in the presence of the intact BCL2 sequence. Based on these preliminary findings, we envision that AR9 could potentially serve as a reporter RNA by self-labeling in the presence of an RNA G-quadruplex. However, both AO10.2 and AR9 still require more detailed characterization for their potential applications.
Neurons are specialized cells dedicated to transmit the nerve impulses throughout the human body across specialized structures called synapses. At the synaptic terminals, a crosstalk between multiple macromolecules regulates the structure and function of the presynaptic nerve endings and the postsynaptic recipient sites.
Gephyrin is the central organizer at inhibitory postsynaptic specializations and plays a crucial role in the organization of these structures by anchoring GABAA receptors (GABAAR) and glycine receptors (GlyR) to the postsynaptic membrane. This 93 kDa protein features an N-terminal G domain and a C-terminal E domain and the latter interacts directly with the intracellular loop between transmembrane helices 3 and 4 of certain subunits of the GlyRs and GABAARs. Biochemical and structural analyses have already provided valuable insights into the gephyrin-GlyR interaction. Interestingly, biochemical studies on the gephyrin-GABAAR interaction demonstrated that the GABAARs also depend on the same binding site as the GlyRs for the interaction with the gephyrin, but the molecular basis for this receptor specific interaction of gephyrin was still unknown. Co-crystal structures of GephE-GABAAR α3- derived peptides with supporting biochemical data presented in this study deciphered the receptor-specific interactions of gephyrin in atomic detail.
In its moonlighting function, gephyrin also catalyzes the terminal step of the evolutionarily conserved molybdenum cofactor biosynthesis. Molybdenum, an essential transition element has to be complexed with a pterin-based cofactor resulting in the formation of the molybdenum cofactor (Moco). Moco is an essential component at the active site of all molybdenum-containing enzymes with the exception of nitrogenase. Mutations in enzymes involved in this pathway lead to a rare yet severe disease called Moco deficiency, which manifest itself in severe neurodevelopmental abnormalities and early childhood death. Moco biosynthesis follows a complex multistep pathway, where in the penultimate step, the N-terminal G domain of gephyrin activates the molybdopterin to form an adenylated molybdopterin intermediate. In the terminal step, this intermediate is then transferred to the C-terminal E domain of gephyrin, which catalyzes the metal insertion and deadenylation reaction to form active Moco. Previous biochemical and structural studies provided valuable insights into the penultimate step of the Moco biosynthesis but the terminal step remained elusive. Through the course of my dissertation, I crystallized the C-terminal E domain in the apo-form as well as in complex with ADP and AMP. These structures shed lightonto the deadenylation reaction and the formation of a ternary E-domain-ADP-Mo/W complex and thus provide structural insight into the metal insertion mechanism. Moreover, the structures also provided molecular insights into a mutation leading to Moco deficiency. Finally, ternary
complexes of GephE, ADP and receptor-derived peptides provided first clues regarding the integration of gephyrin’s dual functionality.
In summary, during the course of the dissertation I was able to derive high resolution structural insights into the interactions between gephyrin and GABAARs, which explain the receptor-specific interaction of gephyrin and, furthermore, these studies can be extended in the future to understand GABAAR subunit-specific interactions of gephyrin. Finally, the understanding of Moco biosynthesis shed light on the molecular basis of the fatal Moco deficiency.