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Autism spectrum disorder (ASD) is a common neurodevelopmental disorder with a multifarious clinical presentation. Even though many genetic risk factors have been identified and studied in mouse models, the neurophysiological mechanisms underlying the autistic phenotype are still unclear. Based on the high rates of comorbidity with epilepsy, it was hypothesized that the balance between excitation and inhibition in neural circuits may be disrupted in autistic individuals.
In this dissertation, synaptic and network activity was measured in three different genetically modified mouse models that exhibit the characteristic behavioral abnormalities of the disorder: the Neurobeachin (Nbea) haploinsufficient mouse, the Neuroligin-3 (Nlgn3) knockout (KO) mouse, and the Neuroligin-4 (Nlgn4) KO mouse. Each of the affected proteins is involved in the formation and/or function of synapses in the central nervous system. Therefore, it was posited that the reduction or deletion of these proteins might alter the balance of excitatory to inhibitory synaptic transmission in individual neurons and in neural circuits. Extracellular recordings in the hippocampal dentate gyrus of anesthetized mice revealed that the excitation-inhibition (E-I) balance was reduced in Nbea haploinsufficient and Nlgn4 KO mice, but unchanged in Nlgn3 KO mice despite a reduction in excitatory synaptic transmission to dentate granule cells. Unexpectedly, the intrinsic excitability of dentate granule cells was altered in all three mouse models. These results imply that a homeostatic increase in the intrinsic excitability is able to compensate for the decreased excitatory transmission in Nlgn3 KO mice, whereas the decreased intrinsic excitability in the Nbea haploinsufficient and Nlgn4 KO mice leads to a reduction in the E-I balance. Taken together, these findings suggest that the influence of genetic factors on the E-I balance might be a potential common mechanism underlying the development of ASD.
The epitranscriptome embodies many new and largely unexplored functions of RNA. A major roadblock in the epitranscriptomics field is the lack of transcriptome-wide methods to detect more than a single RNA modification type at a time, identify RNA modifications in individual molecules, and estimate modification stoichiometry accurately. We address these issues with CHEUI (CH3 (methylation) Estimation Using Ionic current), a new method that concurrently detects N6-methyladenosine (m6A) and 5-methylcytidine (m5C) in individual RNA molecules from the same sample, as well as differential methylation between any two conditions, using signals from nanopore direct RNA sequencing. CHEUI processes observed and expected signals with convolutional neural networks to achieve high single-molecule accuracy and outperform other methods in detecting m6A and m5C sites and quantifying their stoichiometry. CHEUI’s unique capability to identify two modification types in the same sample reveals a non-random co-occurrence of m6A and m5C in mRNA transcripts in cell lines and tissues. CHEUI unlocks an unprecedented potential to study RNA modification configurations and discover new epitranscriptome functions.
The ability of wild animals to navigate and survive in complex and dynamic environments depends on their ability to store relevant information and place it in a spatial context. Despite the centrality of spatial memory, and given our increasing ability to observe animal movements in the wild, it is perhaps surprising how difficult it is to demonstrate spatial memory empirically. We present a cognitive analysis of movements of several wolves (Canis lupus) in Finland during a summer period of intensive hunting and den-centered pup-rearing. We tracked several wolves in the field by visiting nearly all GPS locations outside the den, allowing us to identify the species, location and timing of nearly all prey killed. We then developed a model that assigns a spatially explicit value based on memory of predation success and territorial marking. The framework allows for estimation of multiple cognitive parameters, including temporal and spatial scales of memory. For most wolves, fitted memory-based models outperformed null models by 20 to 50% at predicting locations where wolves chose to forage. However, there was a high amount of individual variability among wolves in strength and even direction of responses to experiences. Some wolves tended to return to locations with recent predation success—following a strategy of foraging site fidelity—while others appeared to prefer a site switching strategy. These differences are possibly explained by variability in pack sizes, numbers of pups, and features of the territories. Our analysis points toward concrete strategies for incorporating spatial memory in the study of animal movements while providing nuanced insights into the behavioral strategies of individual predators.
Bird-mediated seed dispersal is crucial for the regeneration and viability of ecosystems, often resulting in complex mutualistic species networks. Yet, how this mutualism drives the evolution of seed dispersing birds is still poorly understood. In the present study we combine whole genome re-sequencing analyses and morphometric data to assess the evolutionary processes that shaped the diversification of the Eurasian nutcracker (Nucifraga), a seed disperser known for its mutualism with pines (Pinus). Our results show that the divergence and phylogeographic patterns of nutcrackers resemble those of other non-mutualistic passerine birds and suggest that their early diversification was shaped by similar biogeographic and climatic processes. The limited variation in foraging traits indicates that local adaptation to pines likely played a minor role. Our study shows that close mutualistic relationships between bird and plant species might not necessarily act as a primary driver of evolution and diversification in resource-specialized birds.
With 5-10 newly diagnosed patients per 100,000 people every year, glioblastoma is the most common malignant primary brain tumor. Despite extensive research activity in the last decades, clinical effectiveness of the currently available therapy standard of surgery, radiochemotherapy and tumor-treating fields is still limited and mean survival rates in unselected collectives are only about one year. Accordingly, there is an urgent need to explore new therapeutic options. The current standard of care includes surgery followed by radiation therapy in combination with the alkylating chemotherapeutic agent Temozolomide. Even with successful initial therapy, tumor recurrence is still inevitable. Currently, there are no defined recommendations for clinical management of the disease in the event of tumor recurrence. Only 20-30% of patients qualify for a second surgical resection, while other options include retreatment with Temozolomide, CCNU (Lomustine) or Regorafenib and enrollment in a clinical trial.
The development of immunotherapies for glioblastoma, in particular, has been the focus of intense preclinical and clinical efforts. However, low numbers of mutations and a highly immunosuppressive tumor microenvironment result in glioblastoma being considered an immunologically “cold” tumor. Strategies successfully established in mutagen-induced tumors with antibodies directed against the PD-1, PD-L1 or CTLA-A4 immune checkpoints have therefore failed in glioblastoma.
Cellular immunotherapies based on chimeric antigen receptor (CAR)-technology have emerged as an alternative powerful option to tackle immunologically “cold” tumors. Several CAR-T cell products targeting glioma antigens have been developed and some evidence of clinical activity has been demonstrated. Natural killer (NK) cells as carriers of CAR constructs have several advantages over T cells, including a much lower risk of neurotoxicity and better interaction with immune cells in the microenvironment. Based on the human NK cell line NK-92, a clinical-grade product, suitable as an off-the-shelf therapeutic, has been developed. The NK-92/5.28.z clone (CAR-NK) expresses a CAR based on the HER2-specific antibody FRP5 in addition to signal-enhancing CD28 and CD3ζ domains. Similar to several other tumor entities, overexpression of the growth factor receptor HER2 is often found in glioblastoma patients. Because of its substantial role in the regulation of cell proliferation, survival, differentiation, angiogenesis and invasion, this receptor is classified as an oncogene. HER2 overexpression plays a major role in the malignant transformation of cells and its oncogenic potential has been studied in detail in breast cancer. However, HER2 expression was also found in up to 80% of glioblastomas, which correlates with an impaired probability of survival. Under physiological conditions, HER2 is not expressed in the adult central nervous system, making it a promising target antigen for glioblastoma immunotherapy.
In previous projects, it has already been shown that these CAR-NK cells exhibit a high and specific lytic activity towards HER2+ glioblastoma cells. While repetitive intratumoral injections of CAR-NK cells already significantly extended symptom-free survival in murine orthotopic xenograft models, CAR-NK cell therapy in immunocompetent mice promotes an endogenous anti-tumor immune response which improves tumor control and provides persisting anti-tumor immunity after therapy of early-stage tumors. However, in more advanced tumor models, efficacy is limited and induction of the checkpoint-molecule PD-L1 in response to CAR-NK-cell therapy was identified as a key mechanism of therapy resistance.
Immunotherapy employing the intravenous administration of checkpoint inhibitors has already revolutionized the treatment of various malignant diseases such as melanoma or lung cancer. In particular, the approach of cancer immunotherapy has focused on the systemic administration of antibodies directed against immune checkpoints such as PD-1, PD-L1 and CTLA-4. In glioblastoma, both tumor cells and microglia, the brain-resident macrophages, express PD-L1, which hinders the activation of CD8+ and CD4+ T cells. Therefore, immunotherapy directed against the PD-1/PD-L1 axis represents a promising approach for the treatment of glioblastoma. One problem, however, is the severe toxicity caused by the systemic effects of checkpoint inhibitors, since the immune response is stimulated not only in tumor tissue but also in healthy organs. Serious side effects such as colitis, hepatitis, pancreatitis or hypophysitis, including numerous deaths, have been reported.
This study aimed to improve the efficacy of CAR-NK cell therapy by combining it with adeno-associated virus (AAV)-mediated transfer of anti-PD-1 antibodies as a strategy to enable local combination therapy to control intracranial tumors.
AAVs carrying a payload coding for an anti-PD-1 immunoadhesin (aPD-1) retargeted to HER2-expressing cells by fusion of so-called Designed Ankyrin Repeat Proteins (DARPins) with a viral capsid protein were employed for this to focus checkpoint inhibitor therapy to the tumor area, resulting in high intratumoral and low systemic drug concentrations. ...
The brains of black 6 mice (Mus musculus) and Seba’s short-tailed bats (Carollia perspicillata) weigh roughly the same and share the mammalian neocortical laminar architecture. Bats have highly developed sonar calls and social communication and are an excellent neuroethological animal model for auditory research. Mice are olfactory and somatosensory specialists and are used frequently in auditory neuroscience, particularly for their advantage of standardization and genetic tools. Investigating their potentially different general auditory processing principles would advance our understanding of how the ecological needs of a species shape the development and function of the mammalian nervous system. We compared two existing datasets, recorded with linear multichannel electrodes down the depth of the primary auditory cortex (A1) while awake, across both species while presenting repetitive stimulus trains with different frequencies (∼5 and ∼40 Hz). We found that while there are similarities between cortical response profiles in bats and mice, there was a better signal to noise ratio in bats under these conditions, which allowed for a clearer following response to stimuli trains. This was most evident at higher frequency trains, where bats had stronger response amplitude suppression to consecutive stimuli. Phase coherence was far stronger in bats during stimulus response, indicating less phase variability in bats across individual trials. These results show that although both species share cortical laminar organization, there are structural differences in relative depth of layers. Better signal to noise ratio in bats could represent specialization for faster temporal processing shaped by their individual ecological niches.
The opportunistic human pathogen Acinetobacter baumannii can grow with carnitine but its metabolism, regulation and role in virulence remained elusive. Recently, we identified a carnitine transporter encoded by a gene closely associated with potential carnitine degradation genes. Among those is a gene coding for a putative d-malate dehydrogenase (Mdh). Deletion of the mdh gene led to a loss of growth with carnitine but not l-malate; growth with d-malate was strongly reduced. Therefore, it is hypothesized that d-malate is formed during carnitine oxidation and further oxidized to CO2 and pyruvate and, that not, as previously suggested, l-malate is the product and funnelled directly into the TCA cycle. Mutant analyses revealed that the hydrolase in this cluster funnels acetylcarnitine into the degradation pathway by deacetylation. A transcriptional regulator CarR bound in a concentration-dependent manner to the intergenic region between the mdh gene, the first gene of the carnitine catabolic operon and the carR gene in the presence and absence of carnitine. Both carnitine and d-malate induced CarR-dependent expression of the carnitine operon. Infection studies with Galleria mellonella larvae demonstrated a strong increase in virulence by addition of carnitine indicating that carnitine degradation plays a pivotal role in virulence of A. baumannii.
Establishing management programs to preserve the benthic communities along the NW Pacific and the Arctic Ocean (AO) requires a deep understanding of the composition of communities and their responses to environmental stressors. In this study, we thus examine patterns of benthic community composition and patterns of species richness along the NW Pacific and Arctic Seas and investigate the most important environmental drivers of those patterns. Overall we found a trend of decreasing species richness toward higher latitudes and deeper waters, peaking in coastal waters of the eastern Philippines. The most dominant taxa along the entire study area were Arthropoda, Mollusca, Cnidaria, Echinodermata, and Annelida. We found that depth, not temperature, was the main driver of community composition along the NW Pacific and neighboring Arctic Seas. Depth has been previously suggested as a factor driving species distribution in benthic fauna. Following depth, the most influential environmental drivers of community composition along the NW Pacific and the Arctic Ocean were silicate, light, and currents. For example, silicate in Hexactinellida, Holothuroidea, and Ophiuroidea; and light in Cephalopoda and Gymnolaemata had the highest correlations with community composition. In this study, based on a combination of new samples and open-access data, we show that different benthic communities might respond differently to future climatic changes based on their taxon-specific biological, physiological, and ecological characteristics. International conservation efforts and habitat preservation should take an adaptive approach and apply measures that take the differences among benthic communities in responding to future climate change into account. This facilitates implementing appropriate conservation management strategies and sustainable utilization of the NW Pacific and Arctic marine ecosystems.
Besides transcription, RNA decay accounts for a large proportion of regulated gene expression and is paramount for cellular functions. Classical RNA surveillance pathways, like nonsense-mediated decay (NMD), are also implicated in the turnover of non-mutant transcripts. Whereas numerous protein factors have been assigned to distinct RNA decay pathways, the contribution of long non-coding RNAs (lncRNAs) to RNA turnover remains unknown. Here we identify the lncRNA CALA as a potent regulator of RNA turnover in endothelial cells. We demonstrate that CALA forms cytoplasmic ribonucleoprotein complexes with G3BP1 and regulates endothelial cell functions. A detailed characterization of these G3BP1-positive complexes by mass spectrometry identifies UPF1 and numerous other NMD factors having cytoplasmic G3BP1-association that is CALA-dependent. Importantly, CALA silencing impairs degradation of NMD target transcripts, establishing CALA as a non-coding regulator of RNA steady-state levels in the endothelium.
To fight the global problems of humanity, the United Nations has adopted 17 Sustainable Development Goals (SDGs). To achieve these goals, it is necessary that future decision-makers and stakeholders in society consider these goals to be important. Therefore, in this study, we examined how important students in 41 countries directly related to the environmental sector rated each of the 17 SDGs. Based on the analysis of these ratings, it was possible to categorize the SDGs into three higher-level factors that reflect the three pillars of sustainability (social, economic, environmental). These three pillars are considered to be of varying importance in different countries. We also correlated the ratings of these higher-level factors with country-specific indicators, such as the Human Development Index. The correlations between the indicators and the higher-level factors revealed that in countries with higher indices, the SDGs are rated as less important compared to in countries with lower indices. These results provide stakeholders with important guidance on how the SDGs should be promoted in their country.
Due to their sessile nature, plants are constantly exposed to an everchanging environment. When these changes exceed certain limits, they can significantly impact plant growth and development, which, in case of crop plants, has consequences on food security. Exposure to high temperatures causes heat stress (HS), one of the most devastating stresses that plants can face. The survival and recovery from HS are dependent on the activation of the HS response (HSR), a collection of molecular mechanisms conferring HS tolerance by maintaining the cellular homeostasis. Stress responses follow a strictly orchestrated network of signal perception and -transduction, ultimately resulting in an adaptive cellular output. Thereby, the massive reshaping of the transcriptome plays a major part, in which heat stress transcription factors (HSFs) play the key role by inducing the expression of HS-responsive genes, including heat shock proteins and other transcription factors. Additionally, alternative splicing (AS), the selective usage of splice sites, contributes to the rapid adjustment of the transcriptome landscape by producing different mRNA variants from a single gene. Consequently, this results in the reduction of translatable transcripts by nonsense-mediated mRNA-decay or nuclear retention, but also enhances the proteome diversity by allowing the synthesis of protein isoforms with distinct functions. AS thereby modulates the activity of important regulatory factors like HSFA2 in Solanum lycopersicum (tomato). HSFA2 is the key factor of acquired thermotolerance (ATT), which enables the ability to survive a potentially lethal HS through pre-exposure to a preceding mild HS. Temperature-dependent AS leads to the synthesis of two HSFA2 protein variants, whereby inhibition of splicing ensures the synthesis of the stable isoform HSFA2-I that is required for ATT.
Transcriptome analysis of several plant species exposed to HS has highlighted the strong impact of high temperatures on the regulation of pre-mRNA splicing. Despite its importance, little is known about the molecular basis of the AS regulation in plants. Particularly for an economically important crop like tomato, understanding the regulation of HS-sensitive AS will contribute to the description of such an important regulatory mechanism but also might offer new insights for increasing HS resilience. Serine/arginine-rich proteins (SR proteins) are central regulators of constitutive and AS by modulating the splice site selection by the spliceosome. This study describes two members of the RS2Z subfamily of SR proteins in tomato, namely RS2Z35 and RS2Z36, which act as core regulators of AS under HS and consequently as central factors for thermotolerance. This study investigates the interaction of the two RS2Z proteins with the HSFA2 pre-mRNA and provides evidence for their function as splicing repressors in this particular AS event. Thereby, RS2Z proteins play an important role in the HSR by modulating the AS of the key factor of the ATT. Furthermore, based on global transcriptome analysis of knockout mutants of single or both RS2Z genes, it is demonstrated that RS2Z proteins are involved in the splicing of pre-mRNAs of almost 2000 genes. Moreover, RS2Z proteins act as splicing regulators and take part in a large portion of HS-induced AS events, thus playing a broader role in AS regulation. Furthermore, the HS-induced RS2Z36 is involved in basal thermotolerance (BTT), highlighting its importance for the basic HS resilience capacity of tomato. In addition, RNA sequencing demonstrates that RS2Z proteins–especially RS2Z36–regulate the expression of proteins involved in plant immunity. The study thereby provides experimental evidence for the important and essential role of SR proteins for plant thermotolerance and suggests the existence of RS2Z-mediated crossroads of different stress responses.
Heart development is a dynamic process modulated by various extracellular and intracellular cues. Cardiac progenitors in vertebrates such as the zebrafish, migrate over to the midline after differentiation from the epiblast (Bakkers, 2011; Rosenthal & Harvey, 2010; Stainier et al., 1996; Trinh & Stainier, 2004). These progenitors form a cardiac disc at the midline which elongates into the linear heart tube. The differentiation and migration of cardiac precursors is modulated by signaling interactions between cardiac precursor cells and their extracellular environment known as the Extracellular Matrix (ECM). Studies have shown that Cell-ECM interactions play a crucial role in sculpting the heart during early morphogenic events (Davis CL, 1924; Männer & Yelbuz, 2019; Rosenthal & Harvey, 2010). One key factor to these processes is the presence of a specialized ECM known as the Basement Membrane (BM). Extracellular basement membrane proteins such as Fibronectin have been shown to modulate these very early migration processes of the cardiomyocyte progenitors (Trinh & Stainier, 2004). As the heart develops further, the linear heart tube is composed of myocardial cells with an inner endothelial cell lining separated by a layer of thick jelly like substance called the cardiac jelly (Barry A, 1948; Davis CL, 1924; Little et al., 1989). The cardiac jelly also called the cardiac basement membrane, has been shown to regulate distinct developmental events during cardiogenesis. This early CJ contains components of the basal lamina such as laminins, fibronectin, hyaluronan as well as non-fibrillar collagens such as Collagen IV (Little et al., 1989). In this study, I aimed to identify ECM molecules of the Basement Membrane in the heart and identify their role in the modulation of cardiac development and regeneration using the zebrafish as my model organism.
I identified genes belonging to the Zebrafish Matrisome expressed during cardiac developmental and regeneration and performed CRISPR/Cas9 sgRNA mediated mutagenesis. I also developed overexpression tools for these genes.
Agrinp168 mutants exhibited no obvious gross morphology defects during cardiac development and were adult viable. Adult mutants exhibited reduced cardiomyocyte proliferation, but no significant difference in cardiomyocyte dedifferentiation post cardiac cryoinjury.
Decorin overexpression through mRNA injections led to increased myocardial wall thickness and DN dcn overexpression through mRNA injections led to loss of cardiac looping during early development.
Mutants for Small Leucine Rich Proteoglycan (SLRP) prelp generated using CRISPR/Cas9 mutagenesis exhibited cardiovascular defects. Close observation of prelp mutant hearts revealed a reduced heart rate and impaired fractional shortening of the ventricle. prelp mutants exhibited an enlarged atrium at 48 hpf and 72 hpf as well as a reduced ventricle size at 72 hpf. Chamber size in the mutant hearts were enlarged irrespective of contractility of the heart. Mutants showed an increased number of Atrial cardiomyocytes, but no change in cell size. On the molecular level, extracellular Laminin localization was disrupted in prelp mutants along with an increase in thickness and volume of the cardiac HA in the CJ suggesting a potential compensatory role, or retention of immaturity of the cardiac jelly in the prelp mutants. Transcriptomics analysis on the prelp mutant hearts revealed downregulation of ECM organization and ECM-Receptor interaction processes in the mutants. Gene Ontology analysis on prelp mutants hearts transcriptome revealed increased MAPK signaling. Interestingly, genes related to degradation of cardiac HA and maturation of cardiac jelly were downregulated, and genes related to epithelial identity of cardiomyocytes were upregulated. Analysis of the mutant hearts at single cell resolution revealed increased number of mutants exhibiting rounded up cardiomyocytes and loss of apical Podocalyxin. Truncated forms of prelp were generated to identify domain specific roles for Prelp, and reintroduction of N-terminal truncated Prelp into the mutants rescued the basal lamina localization and cardiac jelly volume phenotypes. Myocardium specific re-establishment of prelp expression revealed a marked rescue of the mutant cardiovascular phenotype suggesting that tissue specific expression of prelp is not required so long as Prelp is secreted into the CJ. With these data, I’ve elucidated the role of ECM SLRPs in modulation of cardiac chamber morphogenesis process and regeneration of the heart.
Die Vorläuferform der eukaryotischen mRNA (prä-mRNA) durchläuft, eine Reihe von Prozessierungs-Schritte, die schließlich zu der Synthese einer „reifen“ und Exportkompetenten mRNA führt. prä-mRNA Spleißen ist ein essentieller Teilschritt dieser Reifung bei der intragene Sequenzen, sogenannte Introns, von der prä-mRNA entfernt werden, während Exons legiert werden. Das prä-mRNA Spleißen wird durch das Spleißosom katalysiert. Dieser Mega-Dalton Komplex, besteht aus fünf Sub-Komplexen, die sich wiederum aus katalytisch aktiven „kleinen nukleären Ribonukleinsäuren“ (snRNAs) und einer Vielzahl von proteinogenen Faktoren zusammensetzen. Diese Subkomplexe, bezeichnet als snRNPs (small nuclear Ribonucleoprotein Particles), binden die prä-mRNA an charakteristischen Sequenzen und richten die prä-mRNA durch eine Reihe von Konformations-Änderungen so aus, dass benachbarte Exons in Kontakt treten und über eine biochemische Ligations-Reaktion verbunden werden können.
Die Exon- bzw Intronerkennung der snRNPs wird durch zahlreiche Spleißfaktoren reguliert. Eine Proteinfamilie, die essentiell für die Regulierung des Spleißens ist, sind Serin/Arginin-reiche Proteine (SR-Proteine). Diese binden vorzugsweise an das 3‘ oder 5’ Ende von Exons, rekrutieren snRNPs und stimulieren dadurch die Exon-Inklusion. Durch diese Stimulierung können Spleiß-Events reguliert und gezielt spezifische Exons ausgeschlossen oder eingeschlossen werden. Dieser Prozess, der als alternatives Spleißen (AS) bezeichnet wird, tritt in 95% des menschlichen Transkriptoms auf und erweitert die Diversität eines Organismus, da verschiedene Transkripte von demselben Gen erzeugt werden können und folglich die Translation unterschiedlicher Proteine mit distinkten Funktionen ermöglicht wird.
Darüber hinaus verfügt die Zelle durch das AS über eine weitere posttranskriptionale Genregulationsebene, die insbesondere unter zellulären Stressbedingungen zur Expression von alternativen Protein-Isoformen von der Zelle genutzt wird. Eine in medizinischer Hinsicht besonders relevante Stressbedingung ist die sogenannte Hypoxie, die eine Sauerstoff-Unterversorgung von Zellen oder Gewebebereichen beschreibt. Hypoxie bzw. hypoxische Bereiche finden sich in Krebszellen und treten in 90% aller soliden Tumoren auf. Als Teil der Hypoxie Stress-Antwort, verfügt die Zelle über einen Adaptations-Mechanismus, der durch Hypoxieinduzierbare Faktoren (HIF) vermittelt wird. Diese Faktoren induzieren die Transkription zahlreicher Gene und stimulieren die Expression von Stressfaktoren, die an der zellulären Adaption der Hypoxie beteiligt sind. Einer dieser Faktoren ist der vaskuläre endotheliale Wachstumsfaktor A (VEGFA), welcher unter hypoxischen Bedingungen sekretiert wird und dadurch die Proliferation von Endothelzellen, die Neubildung von Blutgefäßen und damit die Vaskularisation des hypoxischen Bereichs stimuliert.
Die zelluläre Anpassung ist jedoch nicht nur auf die transkriptionelle Regulation des HIF-vermittelten Hypoxie Signalwegs beschränkt, sondern wird auf multiplen Genexpressions-Ebenen reguliert. Obwohl bekannt ist, dass tausende Transkripte unter hypoxischen Bedingungen alternativ gespleißt werden, sind die Faktoren, die die zelluläre Stress-Antwort durch AS regulieren, sowie deren molekularer Mechanismus jedoch weitestgehend unbekannt.
Diese Arbeit umfasst die Identifizierung und Charakterisierung von AS Events, sowie den Einfluss und die Regulation von Spleißfaktoren auf AS unter hypoxischen Bedingungen. Hierzu führten wir globale Genexpressions- und AS-Analysen in HeLaKarzinomzelllinien unter Normoxie (21% O2) und Hypoxie (0.2% O2) durch und zeigen, dass 7962 Gene nach 24h Hypoxie unterschiedlich exprimiert werden. Über AS-Analysen konnten 4434 Transkripte identifiziert werden, die bei Hypoxie über AS reguliert sind. Dabei trat „Exon-Skipping“ als das am häufigsten auftretende AS-Events auf. Über PCR basierte Validierungs-Experimente konnten 5 regulierte Transkripte nachgewiesen werden. Dabei weisen Exon 3 und 4 in BORA, Exon 6 in MDM4 und Exon 4-5 in CSSP1 Exon-Skipping Events auf, während Exon-Inklusionen in CEP192 Exon 28 und in der 3’UTR von EIF4A2 validiert werden konnten.
Darüber hinaus wurde im Rahmen der AS-Analyse die Regulation des sogenannten „backsplicings“ bei Hypoxie untersucht. Im Gegensatz zum linearen Spleißens, wird beim backsplicing das 5’Ende und das 3’Ende von Exons verbunden, was die Bildung von sogenannten zirkulären RNAs (circRNAs) zufolge hat. Obwohl nur wenige Funktionen dieser RNA-Klasse bekannt sind, wurde die Regulation von circRNAs während der Zell-Differenzierung sowie in diversen Krebszellen beschrieben. Dabei können circRNAs als microRNA- oder Protein-Schwämme fungieren oder dienen als Protein-Interaktion Plattform und regulieren dabei die Genexpression.
Die akute myeloische Leukämie (AML) ist eine aggressive Erkrankung des Knochenmarks, welche die Hämatopoese beeinträchtigt und zu Knochenmarksversagen führt. Trotz des Fortschritts in der AML-Therapie bleibt die Prognose für die meisten Patienten schlecht, sodass neue Therapieansätze für die Behandlung dringend benötigt werden. Autophagie, ein kataboler Abbauprozess von zellulären Komponenten, ist nachweislich an der Entstehung von AML beteiligt. Als zentraler Regulator von Zellüberleben, Homöostase und Stoffwechsel, dient die Autophagie als Nährstoffquelle durch die Wiederverwertung von Makromolekülen während begrenzter Energieversorgung. AML-Zellen benötigen ein konstantes Nährstoff- und Energieniveau, um ihre Vermehrung aufrechtzuerhalten. Dies wird durch eine Umstellung von Stoffwechselwegen, insbesondere des mitochondrialen Stoffwechsels einschließlich der oxidativen Phosphorylierung (OXPHOS) und des Tricarbonsäurezyklus (TCA), erreicht.
Mehrere Studien haben die Hemmung der Autophagie für die Behandlung von Krebs als vielversprechenden Ansatz vorgestellt. Doch eine Monotherapie mit Autophagie-Inhibitoren erzielte nur eine geringfügige Wirksamkeit. Eine mögliche Erklärung hierfür ist die Entstehung von Kompensationsmechanismen, die zum Ausgleich der Autophagie-Hemmung in Krebszellen entstehen. Bis heute sind diese Kompensationsmechanismen kaum untersucht. Ziel dieser Arbeit ist es, ein geeignetes Autophagie-Gen zu identifizieren, mit dem sich die Rolle der Autophagie-Hemmung für das Überleben von AML-Zellen untersuchen lässt. Zusätzlich sollen die kompensatorischen Mechanismen, die durch die Autophagie-Hemmung in AML-Zellen entstehen können, untersucht werden, um neue metabolische Angriffspunkte zu identifizieren, die für Kombinationstherapien genutzt werden können.
Zu Beginn der Arbeit wurde ein gezielter CRISPR/Cas9 Screen in zwei humanen AML-Zelllinien durchgeführt, um Autophagie-Gene zu identifizieren, deren Verlust eine Proliferationsstörung in AML-Zellen verursacht, welche überwunden werden kann. Validierungsexperimente zeigten, dass der Verlust von ATG3 das Zellwachstum signifikant verminderte. Außerdem zeigte die Messung des Autophagie-Fluxes, dass der Verlust von ATG3 die Autophagie stark beeinträchtigte. Dies wurde durch eine Western-Blot-Analyse, die eine beeinträchtigte LC3-Lipidierung zeigte, und durch eine Immunfluoreszenzanalyse der Autophagosomen-Bildung mittels konfokaler Mikroskopie, die eine geringere Anzahl von Autophagosomen in ATG3-defizienten Zellen ergab, bestätigt. Deshalb wurde der Knockdown von ATG3 in AML Zellen verwendet, um die Mechanismen, die zum Ausgleichen der Autophagie-Hemmung entstehen, zu untersuchen. Zuerst wurde die Zellproliferation in fünf verschiedenen AML Zelllinien über sieben Tage betrachtet. In allen Zellenlinien führte der Verlust von ATG3 mittels small hairpin RNA zu verminderter Zellproliferation. Diese Ergebnisse zeigen die wichtige Rolle von ATG3 in der Autophagie und dass Autophagie-Hemmung durch ATG3-Verlust das Wachstum von AML-Zellen beeinträchtigt.
Da der Verlust von ATG3 die Proliferation von AML-Zellen beeinträchtigte, wurde eine Zellzyklusanalyse durchgeführt. Eine reduzierte S-Phase bestätigte die verminderte Proliferation in ATG3-depletierten AML-Zellen, doch der Zellzyklus war grundsätzlich nicht gestoppt. Darüber hinaus ergab die Analyse der Apoptose, dass diese unter dem Verlust von ATG3 erhöht war, aber etwa 50% der Zellen blieben vital. Diese Beobachtungen deuten darauf hin, dass AML-Zellen trotz des Verlusts der ATG3-abhängigen Autophagie weiter proliferieren können.
Um die Mechanismen zur Kompensation der Autophagie-Hemmung zu untersuchen, wurden die Auswirkungen des ATG3-Verlusts auf die mitochondriale Homöostase untersucht. Die Mitophagie sowie das mitochondriale Membranpotenzial und die Masse unterschieden sich zwischen Kontroll- und ATG3-depletierten AML-Zellen nicht, was darauf hindeutet, dass die mitochondriale Homöostase durch den Verlust von ATG3 nicht beeinträchtigt ist. Als nächstes wurde die mitochondriale Funktion durch Messung des ATP-Spiegels und der OXPHOS untersucht. Die ATP-Level und die OXPHOS waren nach dem Verlust von ATG3 in AML-Zellen erhöht, was auf eine gesteigerte mitochondriale Aktivität bei Autophagie-Defizienz hinweist.
Ischemic heart disease caused by occlusion of coronary vessels leads to the death of downstream tissues, resulting in a fibrotic scar that cannot be resolved. In contrast to the adult mammalian heart, the adult zebrafish heart can regenerate following injury, enabling the study of the underlying cellular and molecular mechanisms. One of the earliest responses that take place after cardiac injury in adult zebrafish is coronary revascularization. Previous transcriptomic data from our lab show that vegfc, a well-known regulator of lymphatic development, is upregulated early after injury and peaks at 96 hours post cryoinjury, coinciding with the peak of coronary endothelial cell proliferation. To test the hypothesis that vegfc is involved in coronary revascularization, I examined its expression pattern and found that it is expressed by coronary endothelial cells after cardiac damage. Using a loss-of-function approach to block Vegfc signaling, I found that it is required for coronary revascularization during cardiac regeneration. Notably, blocking Vegfc signaling resulted in a significant reduction in cardiomyocyte regeneration. Using transcriptomic analysis, I identified the extracellular matrix component gene emilin2a and the chemokine gene cxcl8a as effectors of Vegfc signaling. During cardiac regeneration, cxcl8a is expressed in epicardium-derived cells, while the gene encoding its receptor cxcr1 is expressed on coronary endothelial cells. I found that overexpressing emilin2a increases coronary revascularization, and induces cxcl8a expression. Using loss-of-function approaches, I observed that both cxcl8a and cxcr1 are required for coronary revascularization after cardiac injury.
Altogether, my findings indicate that Vegfc acts as an angiocrine factor that plays an important role in regulating cardiac regeneration in zebrafish. Mechanistically, Vegfc promotes the expression of emilin2a, which promotes coronary proliferation, at least in part by enhancing Cxcl8a-Cxcr1 signaling. This study helps in understanding the mechanisms underlying coronary revascularization during cardiac regeneration, with promising therapeutic applications for human heart regeneration.
Vascular integrity is essential for organ homeostasis to prevent edema formation and infiltration of inflammatory cells. Long non-coding RNAs (lncRNAs) are important regulators of gene expression and often expressed in a cell type-specific manner. By screening for endothelial-enriched lncRNAs, we identified the undescribed lncRNA NTRAS to control endothelial cell functions. Silencing of NTRAS induces endothelial cell dysfunction in vitro and increases vascular permeability and lethality in mice. Biochemical analysis revealed that NTRAS, through its CA-dinucleotide repeat motif, sequesters the splicing regulator hnRNPL to control alternative splicing of tight junction protein 1 (TJP1; also named zona occludens 1, ZO-1) pre-mRNA. Deletion of the hnRNPL binding motif in mice (Ntras∆CA/∆CA) significantly repressed TJP1 exon 20 usage, favoring expression of the TJP1α- isoform, which augments permeability of the endothelial monolayer. Ntras∆CA/∆CA mice further showed reduced retinal vessel growth and increased vascular permeability and myocarditis. In summary, this study demonstrates that NTRAS is an essential gatekeeper of vascular integrity.
Insects with aquatic life stages can transfer sediment and water pollutants to terrestrial ecosystems, which has been described for metals, polyaromatic hydrocarbons, and polychlorinated chemicals. However, knowledge of the transfer of aquatic micropollutants released by wastewater treatment plants is scarce despite some preliminary studies on their occurrence in riparian spiders. In our study, we address a major analytical gap focusing on the transfer of the micropollutant carbamazepine from the larvae to the adult midges of Chironomus riparius using an optimized QuEChERS extraction method and HPLC–MS/MS applicable to both life stages down to the level of about three individuals. We show that the uptake of carbamazepine by larvae is concentration-dependent and reduces the emergence rate. Importantly, the body burden remained constant in adult midges. Using this information, we estimated the daily exposure of insectivorous tree swallows as terrestrial predators to carbamazepine using the energy demand of the predator and the energy content of the prey. Assuming environmentally relevant water concentrations of about 1 μg/L, the daily dose per kilogram of body weight for tree swallows was estimated to be 0.5 μg/kg/day. At places of high water contamination of 10 μg/L, the exposure may reach 5 μg/kg/day for this micropollutant of medium polarity. Considering body burden changes upon metamorphosis, this study fills the missing link between aquatic contamination and exposure in terrestrial habitats showing that wastewater pollutants can impact birds’ life. Clearly, further analytical methods for biota analysis in both habitats are urgently required to improve risk assessment.
Acinetobacter baumannii can thrive on a broad range of substrates such as sugars, alcohols, lipids, amino acids and aromatic compounds. The latter three are abundant in the human host and are potential candidates as carbon sources for the metabolic adaptation of A. baumannii to the human host. In this study we determined the biodegradative activities of A. baumannii AYE with monocyclic aromatic compounds. Deletion of genes encoding the key enzymes of the ß-ketoadipate pathway, the protocatechuate-3,4-dioxygenase (ΔpcaHG) and the catechol-1,2-dioxygenase (ΔcatA), led to a complete loss of growth on benzoate and p-hydroxybenzoate, suggesting that these substrates are metabolized via the two distinct branches (pca and cat) of this pathway. Furthermore, we investigated the potential role of these gene products in host adaptation by analyzing the capability of the mutants to resist complement-mediated killing. These studies revealed that the mutants exhibit a decreased complement resistance, but a dramatic increase in survival in normal human serum in the presence of p-hydroxybenzoate or protocatechuate. These results indicate that the ß-ketoadipate pathway plays a role in adaptation of A. baumannii to the human host. Moreover, the single and double mutants exhibited increased antibiotic resistances indicating a link between the two dioxygenases and antibiotic resistance.
Tick-borne diseases are a major health problem worldwide and could become even more important in Europe in the future. Due to changing climatic conditions, ticks are assumed to be able to expand their ranges in Europe towards higher latitudes and altitudes, which could result in an increased occurrence of tick-borne diseases.
There is a great interest to identify potential (new) areas of distribution of vector species in order to assess the future infection risk with vector-borne diseases, improve surveillance, to develop more targeted monitoring program, and, if required, control measures.
Based on an ecological niche modelling approach we project the climatic suitability for the three tick species Ixodes ricinus, Dermacentor reticulatus and Dermacentor marginatus under current and future climatic conditions in Europe. These common tick species also feed on humans and livestock and are vector competent for a number of pathogens.
For niche modelling, we used a comprehensive occurrence data set based on several databases and publications and six bioclimatic variables in a maximum entropy approach. For projections, we used the most recent IPCC data on current and future climatic conditions including four different scenarios of socio-economic developments.
Our models clearly support the assumption that the three tick species will benefit from climate change with projected range expansions towards north-eastern Europe and wide areas in central Europe with projected potential co-occurrence.
A higher tick biodiversity and locally higher abundances might increase the risk of tick-borne diseases, although other factors such as pathogen prevalence and host abundances are also important.