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Eine randomisierte placebo-kontrollierte Doppelblindstudie zum Nachweis des Einflusses der transkraniellen Gleichstromstimulation (tDCS) auf die Raucherentwöhnung
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Dissecting the nutrient-driven role of Creb3L transcription factor family to coordinate ER function
Get PDFDie Bedeutung des Thioredoxins Aspf3 für Wachstum und Virulenz des opportunistisch humanpathogenen Schimmelpilzes Aspergillus fumigatus
Get PDFAspergillus fumigatus is an important fungal pathogen that causes allergic reactions but also life-threatening infections. One of the most abundant A. fumigatus proteins is Asp f3. This peroxiredoxin is a major fungal allergen and known for its role as a virulence factor, vaccine candidate, and scavenger of reactive oxygen species. Based on the hypothesis that Asp f3 protects A. fumigatus against killing by immune cells, we investigated the susceptibility of a conditional aspf3 mutant by employing a novel assay. Surprisingly, Asp f3-depleted hyphae were killed as efficiently as the wild type by human granulocytes. However, we identified an unexpected growth defect of mutants that lack Asp f3 under low-iron conditions, which explains the avirulence of the Δaspf3 deletion mutant in a murine infection model. A. fumigatus encodes two Asp f3 homologues which we named Af3l (Asp f3-like) 1 and Af3l2. Inactivation of Af3l1, but not of Af3l2, exacerbated the growth defect of the conditional aspf3 mutant under iron limitation, which ultimately led to death of the double mutant. Inactivation of the iron acquisition repressor SreA partially compensated for loss of Asp f3 and Af3l1. However, Asp f3 was not required for maintaining iron homeostasis or siderophore biosynthesis. Instead, we show that it compensates for a loss of iron-dependent antioxidant enzymes. Iron supplementation restored the virulence of the Δaspf3 deletion mutant in a murine infection model. Our results unveil the crucial importance of Asp f3 to overcome nutritional immunity and reveal a new biological role of peroxiredoxins in adaptation to iron limitation
Das atypische Hämolytisch-Urämische Syndrom in der kardiovaskulären Chirurgie
Get PDFThe aim of this work is the research of the postoperative acute kidney injury (AKI) requiring renal replacement therapy (RRT), with emphasis on the prevalence of atypical haemolytic uremic syndrome (aHUS) in patients treated on the cardiothoracic ICU of the LMU University Hospital after cardiac surgery.
Except for some few case reports, the correlation between cardiac surgery requiring cardio-pulmonary-bypass (CPB) and the manifestation of aHUS has not been described so far in the field literature.
As the treatment of the AKI caused by aHUS widely varies from the management of the classical CSA-AKI (cardiac-surgery associated AKI) and because the early initiation of the treatment is essential for a positive outcome, the detailed analysis of this pathology is fully justified and imperative.
The underlying hypothesis of this work is that in certain high-risk patients, there is a correlation between the intraoperative use of CPB, which leads to an activation of the complement system and the clinical manifestation of aHUS, causing AKI.
In the beginning, there were analyzed all the patients who developed AKI requiring RRT in the early postoperative course after cardiac surgery. The target was to find out the significance of aHUS as a potential cause of postoperative AKI. Special attention was attached to the identification of the risk factors for developing aHUS. Demographic parameters, comorbidities, the surgical diagnosis and the unfolding of the surgical procedures were thoroughly analyzed.
Because of the highly significant statistical correlation between the manifestation of aHUS and procedures performed on the thoracic aorta, the work was expanded by a detailed analysis of this certain subgroup.
In this cohort too the primary endpoint was to identify potential risk factors for the postoperative manifestation of aHUS. The focus in this case went to the surgical procedure itself, as well as to the intraoperative course: the applied quantity of cardioplegic solution, the use of a (hypothermic) circulatory arrest and the exact time on the CPB.
Among the secondary endpoints mention should be made of the correlation between postoperative aHUS and other complications (such as bleedings, need for a durable pacemaker, a.o.) and the outcome of aHUS patients after adequate treatment. The outcome-analysis was based on data regarding survival, persistent need of RRT and relevant laboratory parameters at discharge and 6 months after surgery
Combination of deep behavioral phenotyping with brain region and cell type specific manipulations of FKBP51
Get PDFThe increasing prevalence of stress-related disorders, such as major depressive disorder (MDD) has become a significant global concern, with devastating effects on individuals' personal lives and societal well-being. The exposure to severe and chronic stressors is a major risk factor for the development of such disorders, and recent traumatic events have further exacerbated this mental health crisis. The susceptibility to MDD is determined by a complex interplay of genetic, epigenetic, and environmental factors. One specific gene of significance in this context is FKBP5 (Fkbp5 in rodents), encoding the co-chaperone FK506 binding protein 51 (FKBP51). The interplay between severe stress exposure and genetic risk variants of FKBP5 has been associated with an increased vulnerability to psychopathology.
A significant symptom observed in individuals with MDD is social dysfunction, characterized by the avoidance of social interactions and the display of maladaptive behaviors, such as aggression or irritability. However, traditional preclinical assessment methods for stress-induced behavioral symptoms, such as social aversion, have faced criticism due to their reductionistic nature, often failing to capture ethologically relevant behavioral constructs. Advancements in high-throughput pose estimation tools have provided opportunities for comprehensive behavioral analysis through automatically annotated behavioral assessments. This thesis explores various tools for automatically annotated behavioral assessment in preclinical psychiatry research, employing both supervised classification and unsupervised clustering strategies.
Applying the newly established ad validated deep phenotyping methods, the thesis further investigates the brain region and cell type specific role of FKBP51 across different stress models and uncovers the underlying neurobiological mechanisms and behavioral profiles using automatically annotated behavioral assessment. The effectiveness of both supervised classification and unsupervised clustering strategies is demonstrated in characterizing individual and social behavioral profiles in mice subjected to various stress conditions. Moreover, the thesis highlights the distinct sex-specific effects of different stress paradigms on the regulation of the hypothalamic-pituitary-adrenal (HPA) axis, including the expression of Fkbp5 in several stress-related brain regions, in particular the Locus Coeruleus (LC).
Taken together, the current thesis emphasizes the importance of brain region and cell type specific regulation of Fkbp5 and underscores the benefits of automatically annotated behavioral assessment tools. This is put into perspective with future research prospects, advocating for the integration of diverse data modalities, such as in vivo measurements of stress mediators and neuronal activity recordings. This integrated approach aims to enhance our understanding of complex behaviors and the underlying molecular mechanisms. Ultimately, this can contribute to a better comprehension of the behavioral phenotypes and associated neurobiological alterations in stress-related disorders. These insights hold potential to facilitate the development of novel treatments for psychiatric disorders
Mechanistic investigation of eukaryotic quality control factors RQT and Mbf1
Get PDFTranslation is a central cellular process and thus tightly regulated by quality control mechanisms. Monitoring the ribosome during translation is an elegant way to track the progress and to catch a variety of errors before they lead to detrimental effects in the cell. Recently, ribosomal collisions have emerged as a trigger for such quality control pathways, and various collision sensors have been identified. One of those pathways called ribosome associated quality control (RQC) deals with ribosomes stuck on an open reading frame (e.g. due to stable mRNA structures or inhibitory codons). Here, ubiquitination of ribosomal proteins serves as a signal for dissociation of the stuck ribosome by the RQC-trigger (RQT) complex. Subsequently the aberrant mRNA and the truncated nascent peptide are degraded and intact components such as ribosomal subunits or tRNAs can be recycled. Although this pathway has been studied in detail over the last years, the exact mechanism by which RQT leads to dissociation of stalled ribosomes remains unclear.
This thesis aimed to elucidate the RQT-mediated dissociation mechanism by setting up an in vitro splitting system and subsequent cryo-EM analysis of the splitting reactions. To generate suitable substrates for the dissociation process, collisions were generated using known ribosome stalling sequences in a cell free in vitro translation system. Splitting assays showed that an in vitro ubiquitination step for collided ribosomes is crucial for splitting. Moreover, such assays revealed that efficient splitting is dependent on ATPase activity of the N-terminal helicase cassette of RQT component Slh1, on the presence of a neighboring ribosome and on availability of a 3’ mRNA overhang. Structural analysis of the ribosome-bound RQT complex divulged stable positioning of RQT on the lead ribosome of a collided ‘disome’ unit, as well as on 80S and 40S. The 80S-RQT complex was observed in two different states located in close proximity to the entry of the mRNA channel. Together with the observed requirement of available 3’mRNA and helicase activity of Slh1, this suggests that Slh1 can pull on the mRNA, leading to an initial model for ribosome dissociation.
Ribosome stalling and subsequent collisions increase the probability of frameshifting and thus translation of an aberrant protein. Structural analysis of three collided ribosomes, so called trisomes, revealed the presence of multiprotein bridging factor (Mbf1), previously identified as a frameshift inhibitor. This small protein was found on the second and third colliding ribosomes, positioned between beak and body of the 40S subunit. Comparison with the human homolog EDF1, which was found to be recruited to emetine induced collisions, showed that those proteins bind in the exact same fashion. The position on the 40S subunit of the collided ribosomes suggests that both proteins interact directly with the mRNA to prevent frameshifting, probably in combination with preventing conformational changes required for translocation of the ribosome.
In conclusion, high resolution cryo-EM structures of both RQT and Mbf1 on ribosomes enabled detailed insights into the intricate quality control network targeting collisions in the cell. From this, molecular models for both a helicase driven dissociation mechanism by RQT and the frameshifting inhibition by Mbf1 could be derived. These results, together with the developed optimization strategies, provide the basis for future works, leading to a detailed understanding of these pathways