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This compilation focuses on adolescent mental disorders and their prevention. It comprises three distinct studies, each contributing to a deeper understanding of this critical topic. This work addresses a critical gap in the understanding of, and approach to, adolescent mental health, and as a result reveals a critically important and urgently needed policy implication for action. The thematic structure of these studies begins with an examination of the epidemiology of child and adolescent mental disorders. Baseline data were collected from N = 877 adolescents with a mean age of 12.43 years (SD = 0.65). Mental health problems, such as depressive symptoms, non-suicidal self-injury, suicidal ideation, symptoms of eating disorders, and gender differences, are thoroughly examined. Results revealed a significant portion of our sample displaying mental health problems as early as the 6th and 7th grades, with girls generally being more affected than boys. The findings underscore the importance of early adolescence in the emergence of mental health problems and thereby emphasize the need for preventive measures. Moving beyond prevalence estimates, the compilation delves into the etiology of these disorders, exploring their potential correlation with a COVID-19 infection. Understanding the early signs and risk factors is crucial for timely support. While numerous studies have investigated potential risk and protective factors during the pandemic, our focus shifts to adolescents’ coping when an infection with the virus was involved (N = 2,154, M = 12.31, SD = 0.67). We hypothesized that students infected or with close family members infected, would exhibit an increased psychopathology and a decreased functioning of protective factors such as self-efficacy or self-esteem. We found no connection between infection and the mental health status within our sample, but protective factors and mental well-being were positively associated. Thus, universal primary prevention appears to be the preferred approach for promoting mental health. Lastly, the compilation introduces LessStress, a noteworthy contribution to more evidence-based prevention programs. This universal approach is designed to reduce stress in schools, accompanied by a cluster-randomized trial to evaluate its effectiveness (estimated sample size N = 1,894). Existing studies have demonstrated the effectiveness of stress prevention, leading us to introduce a short and easy-to-implement prevention program. There is positive evidence for one-lesson interventions in schools for promoting well-being and health behaviors among adolescents. LessStress is designed based on a life skills approach that not only imparts psychoeducational content but also teaches skills relevant to everyday life and directly applicable. Throughout these studies, a common thread emerges: the pressing need to address mental disorders during childhood and adolescence. These formative years play a pivotal role in the development of mental health problems. These formative years play a crucial role in the development of mental health problems. They highlight the importance of epidemiological data collection and analysis based on the latest models to develop prevention interventions that are not only effective but also reach young people on a global level.
This decade saw the development of new high-end light microscopy approaches. These technologies are increasingly used to expand our understanding of cellular function and the molecular mechanisms of life and disease. The precision of state-of-the-art super resolution microscopy is limited by the properties of the applied fluorescent label. Here I describe the synthesis and evaluation of new functional fluorescent probes that specifically stain gephyrin, universal marker of the neuronal inhibitory post-synapse. Selected probe precursor peptides were synthesised using solid phase peptide synthesis and conjugated with selected super resolution capable fluorescent dyes. Identity and purity were defined using chromatography and mass spectrometric methods. To probe the target specificity of the resulting probe variants in cellular context, a high-throughput assay was established. The established semi-automated and parallel workflow was used for the evaluation of three selected probes by defining their co-localization with the expressed fluorescent target protein. My work provided NN1Dc and established the probe as a visualisation tool for essentially background-free visualisation of the synaptic marker protein gephyrin in a cellular context. Furthermore, NN1DA became part of a toolbox for studying the inhibitory synapse ultrastructure and brain connectivity and turned out useful for the development of a label-free, high-throughput protein interaction quantification assay.
The mammalian central clock, located in the suprachiasmatic nucleus (SCN) of the anterior hypothalamus, controls circadian rhythms in behaviour such as the sleep-wake cycle. It is made up of approximately 20,000 heterogeneous neurons that can be classified by their expression of neuropeptides. There are three major populations: AVP neurons (arginine vasopressin), VIP neurons (vasoactive intestinal peptide), and GRP neurons (gastrin releasing peptide). How these neuronal clusters form functional units to govern various aspects of rhythmic behavior is poorly understood. At a molecular level, biological clocks are represented by transcriptional-posttranslational feedback loops that induce circadian oscillations in the electrical activity of the SCN and hence correlate with behavioral circadian rhythms. In mammals, the sleep wake cycle can be accurately predicted by measuring electrical muscle and brain activity. To investigate the link between the electrical activity of heterogeneous neurons of the SCN and the sleep wake cycle, we optogenetically manipulated AVP neurons in vivo with SSFO (stabilized step function opsin) and simultaneously recorded an electroencephalogram (EEG) and electromyogram (EMG) in freely moving mice. SSFO-mediated stimulation of AVP positive neurons in the anterior hypothalamus increased the total amount of wakefulness during the hour of stimulation. Interestingly, this effect led to a rebound in sleep in the hour after stimulation. Markov chain sleep-stage transition analysis showed that the depolarization of AVP neurons through SSFO promotes the transition from all states to wakefulness. After the end of stimulation, a compensatory increase in transitions to NREM sleep was observed. Ex vivo, SSFO activation in AVP neurons causes depolarization and modifies the activity of AVP neurons. Therefore, the results of this thesis project suggest an essential role of AVP neurons as mediators between circadian rhythmicity and sleep-wake behaviour.
Every year, stroke affects over 100 million people worldwide and the number of cases continues to grow. Ischemic stroke is the most prevalent form of stroke and rapid restoration of blood flow is the primary therapeutic aim. However, recanalization might fail or reperfusion itself induces detrimental processes leading to infarct progression. Previous studies identified platelets and immune cells as drivers of this so-called ischemia/reperfusion (I/R) injury, establishing the concept of ischemic stroke as thrombo-inflammatory disease. Reduced cerebral blood flow despite recanalization promoted the hypothesis that thrombus formation within the cerebral microcirculation induces further tissue damage. The results presented in this thesis refute this: using complementary methodologies, it was shown that infarct growth precedes the occurrence of thrombi excluding them as I/R injury-underlying cause. Blood brain barrier disruption is one of the hallmarks of ischemic stroke pathology and was confirmed as early event during reperfusion injury in the second part of this study. Abolished platelet α-granule release protects mice from vascular leakage in the early reperfusion phase resulting in smaller infarcts. Using in vitro assays, platelet α-granule-derived PDGF-AB was identified as one factor contributing to blood-brain barrier disruption.
In vivo visualization of platelet activation would provide important insights in the spatio-temporal context of platelet activation in stroke pathology. As platelet signaling results in elevated intracellular Ca2+ levels, this is an ideal readout. To overcome the limitations of chemical calcium indicators, a mouse line expressing an endogenous calcium reporter specifically in platelets and megakaryocytes was generated. Presence of the reporter did not interfere with platelet function, consequently these mice were characterized in in vivo and ex vivo models.
Upon ischemic stroke, neutrophils are among the first cells that are recruited to the brain. Since for neutrophils both, beneficial and detrimental effects are described, their role was investigated within this thesis. Neither neutrophil depletion nor absence of NADPH-dependent ROS production (Ncf-/- mice) affected stroke outcome. In contrast, abolished NET-formation in Pad4-/- mice resulted in reduced infarct sizes, revealing detrimental effects of NETosis in the context of ischemic stroke, which might become a potential therapeutic target.
Cerebral venous (sinus) thrombosis, CV(S)T is a rare type of stroke with mainly idiopathic onset. Whereas for arterial thrombosis a critical contribution of platelets is known and widely accepted, for venous thrombosis this is less clear but considered more and more. In the last part of this thesis, it was shown that fab-fragments of the anti-CLEC-2 antibody INU1 trigger pathological platelet activation in vivo, resulting in foudroyant CVT accompanied by heavy neurological symptoms. Using this novel animal model for CVT, cooperative signaling of the two platelet receptors CLEC-2 and GPIIb/IIIa was revealed as major trigger of CVT and potential target for treatment.
Pancreatic ductal adenocarcinoma (PDAC) is predominantly driven by mutations in KRAS and TP53. However, PDAC tumors display deregulated levels of MYC and are a paradigm example for MYC-driven and -addicted tumors. For many years MYC was described as a transcription factor that regulates a pleiotropic number of genes to drive proliferation. Recent work sheds a different light on MYC biology. First, changes in gene expression that come along with the activation of MYC are mild and MYC seems to act more as a factor that reduces stress and increases resilience towards challenges during transcription. Second, MYC is a strong driver of immune evasion in different entities. In this study we depleted MYC in murine PDAC cells and revealed the immune dependent regression of tumors in an orthotope transplant model, as well as the activation of the innate immune system using global expression analysis, immunoblotting and fCLIP.
These experiments revealed that endogenous double-stranded RNA is binding as a viral mimicry to Toll-like receptor 3, causing activation of TBK1 and downstream activation of a proimmunogenic transcription program. The regression of tumors upon depletion of MYC is dependent on this pathway since the knockout of TBK1 prevents regression of tumors after depletion of MYC.
We can summarize this study in three main findings: First, the dominant and most important function of MYC in tumors is not to drive proliferation but to promote immune evasion and prevent immune-dependent regression of tumors. Second, cells monitor defects or delay in splicing and RNA processing and activate the immune system to clear cells that face problems with co-transcriptional processing. Third, MYC suppresses the activation of the cell-intrinsic innate immune system and shields highly proliferating cells from the recognition by the immune system.
To translate this into a therapeutically approach, we replaced the shRNA mediated depletion of MYC by treatment with cardiac glycosides. Upon treatment with cardiac glycosides tumor cells reduce uptake of nutrients, causing a downregulation of MYC translation, inhibition of proliferation, glycolysis and lactate secretion. Lactate is a major reason for immune evasion in solid tumors since it dampens, amongst others, cytotoxic T cells and promotes regulatory T cells.
Treatment of mice with cardiac glycosides causes a complete and immune-dependent remission of PDAC tumors in vivo, pointing out that cardiac glycosides have strong proimmunogenic, anti-cancer effects. More detailed analyses will be needed to dissect the full mechanism how cardiac glycosides act on MYC translation and immune evasion in PDAC tumors.
Bulk RNA sequencing technologies have provided invaluable insights into host and bacterial gene expression and associated regulatory networks. Nevertheless, the majority of these approaches report average expression across cell populations, hiding the true underlying expression patterns that are often heterogeneous in nature. Due to technical advances, single-cell transcriptomics in bacteria has recently become reality, allowing exploration of these heterogeneous populations, which are often the result of environmental changes and stressors. In this work, we have improved our previously published bacterial single-cell RNA sequencing (scRNA-seq) protocol that is based on multiple annealing and deoxycytidine (dC) tailing-based quantitative scRNA-seq (MATQ-seq), achieving a higher throughput through the integration of automation. We also selected a more efficient reverse transcriptase, which led to reduced cell loss and higher workflow robustness. Moreover, we successfully implemented a Cas9-based rRNA depletion protocol into the MATQ-seq workflow. Applying our improved protocol on a large set of single Salmonella cells sampled over different growth conditions revealed improved gene coverage and a higher gene detection limit compared to our original protocol and allowed us to detect the expression of small regulatory RNAs, such as GcvB or CsrB at a single-cell level. In addition, we confirmed previously described phenotypic heterogeneity in Salmonella in regard to expression of pathogenicity-associated genes. Overall, the low percentage of cell loss and high gene detection limit makes the improved MATQ-seq protocol particularly well suited for studies with limited input material, such as analysis of small bacterial populations in host niches or intracellular bacteria.
IMPORTANCE: Gene expression heterogeneity among isogenic bacteria is linked to clinically relevant scenarios, like biofilm formation and antibiotic tolerance. The recent development of bacterial single-cell RNA sequencing (scRNA-seq) enables the study of cell-to-cell variability in bacterial populations and the mechanisms underlying these phenomena. Here, we report a scRNA-seq workflow based on MATQ-seq with increased robustness, reduced cell loss, and improved transcript capture rate and gene coverage. Use of a more efficient reverse transcriptase and the integration of an rRNA depletion step, which can be adapted to other bacterial single-cell workflows, was instrumental for these improvements. Applying the protocol to the foodborne pathogen Salmonella, we confirmed transcriptional heterogeneity across and within different growth phases and demonstrated that our workflow captures small regulatory RNAs at a single-cell level. Due to low cell loss and high transcript capture rates, this protocol is uniquely suited for experimental settings in which the starting material is limited, such as infected tissues.
Chemical synapses are a physically and functionally varied type of cell-cell contact specialized in conducting communication between neurons. They are the smallest "computational" unit of the brain and are often classified as electrical and chemical, and they can be distinguished based on their transmission mechanism. These categories could be further broken into many kinds, each having a specific structure-function repertoire that is hypothesized to provide neural networks with distinct computational capabilities. Heterogeneity refers to the variety of structures and functions present in a particular category of synapses. Contributing factors for this heterogeneity may be the synaptic vesicles, the active zone (AZ), the synaptic cleft, the postsynaptic density, and the glial processes associated with the synaptic contacts. Each of these five structural modules has its own set of functions, and their combination determines the spectrum of functional heterogeneity at mammalian excitatory synapses. This work focused on the changes in AZ protein expression after chemical induction of plasticity with forskolin in synaptic contacts of the hippocampal mossy fibers. With the nanoscopic resolution provided by dSTORM, along with the multicolor SIM imaging capabilities, changes in expression of key presynaptic AZ components were analyzed. Using SIM imaging along with a standardized stimulation protocol in acute brain slices from male 16-week old Thy1-mEGFP (Lsi1) mice, the changes of the key AZ proteins Bassoon, Munc 13-1 and Tomosyn were investigated 30 min after stimulation with forskolin (50 μM for 30 min). Forskolin induced changes in these proteins largely in small synaptic contacts whereas no clear changes were detected in large mossy fiber boutons. However, due to the high variability it cannot be ruled out that forskolin may differentially modify AZ protein composition depending on experimental circumstances such as age and gender of mice or the time point and duration of forskolin stimulation. The dSTORM data demonstrated feasibility to perform single molecule 3D imaging of hippocampal presynaptic AZs and allowed quantitative mapping of molecular changes in AZ proteins after induction of plasticity. The findings suggest high heterogeneity in mossy fiber synaptic contacts that may have an impact on the function of neural networks. These imaging approaches may now be used to identify potential differences in functional molecular rearrangements of synaptic proteins in healthy and diseased brain (e.g. after induction of traumatic brain injury).
Dual RNA-seq captures both host and pathogen transcriptomes at the site of infection, facilitating an exploration of processes that play an essential role in pathogenesis and the host defense. This work presents an application of this technique to explore processes occurring during the infection of the human endothelial cells with two clinical isolates of Orientia tsutsugamushi (Ot) — the causative agent of scrub typhus. Combining comparative genomics, transcriptomics, and proteomics, we investigated the transcriptional architecture of Ot and identified non-coding RNAs, operon structures, and widespread antisense transcription, that may have a role in regulation of repetitive genes that are abundant in the Ot genome. In addition, the comparative analysis of bacterial and eukaryotic transcriptomes allowed us to investigate factors that drive the difference in virulence between Karp and UT176 and the host response to these two Ot strains.
The host and pathogen transcriptional profiles in each dual RNA-seq study are obtained in‑silico by adopting tools developed for RNA-seq data analysis. The Dualrnaseq pipeline presented in the second part of this work is the first publicly available, highly reproducible, scalable, and user‑friendly workflow developed for processing dual RNA‑seq data of any eukaryotic and bacterial organisms with a reference genome and annotation. It provides three mapping and quantification strategies: (i) alignment-based mapping of reads onto the chimeric genome with STAR followed by counting of uniquely mapped reads with HTSeq; (ii) a fast transcriptome quantification method handling multi‑mapped reads (Salmon with Selective Alignment); (iii) and Salmon alignment-based mode which uses a STAR‑derived alignment combined with Salmon quantification. Performing an initial benchmark analysis of the employed methods we provided recommendations ensuring accurate estimation of host and pathogen transcript expression.
Purpose
The purpose of this study is to investigate changes over time in quality of life (QoL) in incurable lung cancer patients and the impact of determinants like molecular alterations (MA).
Methods
In a prospective, longitudinal, multicentric study, we assessed QoL, symptom burden, psychological distress, unmet needs, and prognostic understanding of patients diagnosed with incurable lung cancer at the time of the diagnosis (T0) and after 3 (T1), 6 (T2) and 12 months (T3) using validated questionnaires like FACT-L, National Comprehensive Cancer Network (NCCN) Distress Thermometer (DT), PHQ-4, SCNS-SF-34, and SEIQoL.
Results
Two hundred seventeen patients were enrolled, 22 (10%) with reported MA. QoL scores improved over time, with a significant trend for DT, PHQ-4, and SCNS-SF-34. Significant determinants for stable or improving scores over time were survival > 6 months, performance status at the time of diagnosis, and presence of MA. Patients with MA showed better QoL scores (FACT-L at T1 104.4 vs 86.3; at T2 107.5 vs 90.0; at T3 100.9 vs 92.8) and lower psychological distress (NCCN DT at T1 3.3 vs 5; at T2 2.7 vs 4.5; at T3 3.7 vs 4.5; PHQ-4 at T1 2.3 vs 4.1; at T2 1.7 vs 3.6; at T3 2.2 vs 3.6), but also a worsening of the scores at 1 year and a higher percentage of inaccurate prognostic understanding (27 vs 17%) compared to patients without MA.
Conclusion
Patients with tumors harboring MA are at risk of QoL deterioration during the course of the disease. Physicians should adapt their communication strategies in order to maintain or improve QoL.
Background
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. 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 in different childhood tumors including WT. 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, but the functional consequences remain to be characterized.
Methods
We screened a large cohort of unselected WTs for MYCN and MAX alterations. Wild-type and mutant protein function were characterized biochemically, and we analyzed the N-MYC protein interactome by mass spectrometric analysis of N-MYC containing protein complexes.
Results
Mutation screening revealed mutation frequencies of 3% for MYCN P44L and 0.9% for MAX R60Q that are associated with a higher risk of relapse. 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. Nevertheless, we could identify a number of novel N-MYC partner proteins, e.g. PEG10, YEATS2, FOXK1, CBLL1 and MCRS1, whose expression is correlated with MYCN in WT samples and several of these are known for their own oncogenic potential.
Conclusions
The strongly elevated risk of relapse associated with mutant MYCN and MAX or elevated MYCN expression corroborates their role in WT oncogenesis. Together with the newly identified co-expressed interactors they expand the range of potential biomarkers for WT stratification and targeting, especially for high-risk WT.