1,918 research outputs found

    Inter-individual variation of the human epigenome & applications

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    Undergraduate Catalog of Studies, 2023-2024

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    Graduate Catalog of Studies, 2023-2024

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    Undergraduate Catalog of Studies, 2023-2024

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    UMSL Bulletin 2023-2024

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    The 2023-2024 Bulletin and Course Catalog for the University of Missouri St. Louis.https://irl.umsl.edu/bulletin/1088/thumbnail.jp

    Graduate Catalog of Studies, 2023-2024

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    Inter-individual variation of the human epigenome & applications

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    Genome-wide association studies (GWAS) have led to the discovery of genetic variants influencing human phenotypes in health and disease. However, almost two decades later, most human traits can still not be accurately predicted from common genetic variants. Moreover, genetic variants discovered via GWAS mostly map to the non-coding genome and have historically resisted interpretation via mechanistic models. Alternatively, the epigenome lies in the cross-roads between genetics and the environment. Thus, there is great excitement towards the mapping of epigenetic inter-individual variation since its study may link environmental factors to human traits that remain unexplained by genetic variants. For instance, the environmental component of the epigenome may serve as a source of biomarkers for accurate, robust and interpretable phenotypic prediction on low-heritability traits that cannot be attained by classical genetic-based models. Additionally, its research may provide mechanisms of action for genetic associations at non-coding regions that mediate their effect via the epigenome. The aim of this thesis was to explore epigenetic inter-individual variation and to mitigate some of the methodological limitations faced towards its future valorisation.Chapter 1 is dedicated to the scope and aims of the thesis. It begins by describing historical milestones and basic concepts in human genetics, statistical genetics, the heritability problem and polygenic risk scores. It then moves towards epigenetics, covering the several dimensions it encompasses. It subsequently focuses on DNA methylation with topics like mitotic stability, epigenetic reprogramming, X-inactivation or imprinting. This is followed by concepts from epigenetic epidemiology such as epigenome-wide association studies (EWAS), epigenetic clocks, Mendelian randomization, methylation risk scores and methylation quantitative trait loci (mQTL). The chapter ends by introducing the aims of the thesis.Chapter 2 focuses on stochastic epigenetic inter-individual variation resulting from processes occurring post-twinning, during embryonic development and early life. Specifically, it describes the discovery and characterisation of hundreds of variably methylated CpGs in the blood of healthy adolescent monozygotic (MZ) twins showing equivalent variation among co-twins and unrelated individuals (evCpGs) that could not be explained only by measurement error on the DNA methylation microarray. DNA methylation levels at evCpGs were shown to be stable short-term but susceptible to aging and epigenetic drift in the long-term. The identified sites were significantly enriched at the clustered protocadherin loci, known for stochastic methylation in neurons in the context of embryonic neurodevelopment. Critically, evCpGs were capable of clustering technical and longitudinal replicates while differentiating young MZ twins. Thus, discovered evCpGs can be considered as a first prototype towards universal epigenetic fingerprint, relevant in the discrimination of MZ twins for forensic purposes, currently impossible with standard DNA profiling. Besides, DNA methylation microarrays are the preferred technology for EWAS and mQTL mapping studies. However, their probe design inherently assumes that the assayed genomic DNA is identical to the reference genome, leading to genetic artifacts whenever this assumption is not fulfilled. Building upon the previous experience analysing microarray data, Chapter 3 covers the development and benchmarking of UMtools, an R-package for the quantification and qualification of genetic artifacts on DNA methylation microarrays based on the unprocessed fluorescence intensity signals. These tools were used to assemble an atlas on genetic artifacts encountered on DNA methylation microarrays, including interactions between artifacts or with X-inactivation, imprinting and tissue-specific regulation. Additionally, to distinguish artifacts from genuine epigenetic variation, a co-methylation-based approach was proposed. Overall, this study revealed that genetic artifacts continue to filter through into the reported literature since current methodologies to address them have overlooked this challenge.Furthermore, EWAS, mQTL and allele-specific methylation (ASM) mapping studies have all been employed to map epigenetic variation but require matching phenotypic/genotypic data and can only map specific components of epigenetic inter-individual variation. Inspired by the previously proposed co-methylation strategy, Chapter 4 describes a novel method to simultaneously map inter-haplotype, inter-cell and inter-individual variation without these requirements. Specifically, binomial likelihood function-based bootstrap hypothesis test for co-methylation within reads (Binokulars) is a randomization test that can identify jointly regulated CpGs (JRCs) from pooled whole genome bisulfite sequencing (WGBS) data by solely relying on joint DNA methylation information available in reads spanning multiple CpGs. Binokulars was tested on pooled WGBS data in whole blood, sperm and combined, and benchmarked against EWAS and ASM. Our comparisons revealed that Binokulars can integrate a wide range of epigenetic phenomena under the same umbrella since it simultaneously discovered regions associated with imprinting, cell type- and tissue-specific regulation, mQTL, ageing or even unknown epigenetic processes. Finally, we verified examples of mQTL and polymorphic imprinting by employing another novel tool, JRC_sorter, to classify regions based on epigenotype models and non-pooled WGBS data in cord blood. In the future, we envision how this cost-effective approach can be applied on larger pools to simultaneously highlight regions of interest in the methylome, a highly relevant task in the light of the post-GWAS era.Moving towards future applications of epigenetic inter-individual variation, Chapters 5 and 6 are dedicated to solving some of methodological issues faced in translational epigenomics.Firstly, due to its simplicity and well-known properties, linear regression is the starting point methodology when performing prediction of a continuous outcome given a set of predictors. However, linear regression is incompatible with missing data, a common phenomenon and a huge threat to the integrity of data analysis in empirical sciences, including (epi)genomics. Chapter 5 describes the development of combinatorial linear models (cmb-lm), an imputation-free, CPU/RAM-efficient and privacy-preserving statistical method for linear regression prediction on datasets with missing values. Cmb-lm provide prediction errors that take into account the pattern of missing values in the incomplete data, even at extreme missingness. As a proof-of-concept, we tested cmb-lm in the context of epigenetic ageing clocks, one of the most popular applications of epigenetic inter-individual variation. Overall, cmb-lm offer a simple and flexible methodology with a wide range of applications that can provide a smooth transition towards the valorisation of linear models in the real world, where missing data is almost inevitable. Beyond microarrays, due to its high accuracy, reliability and sample multiplexing capabilities, massively parallel sequencing (MPS) is currently the preferred methodology of choice to translate prediction models for traits of interests into practice. At the same time, tobacco smoking is a frequent habit sustained by more than 1.3 billion people in 2020 and a leading (and preventable) health risk factor in the modern world. Predicting smoking habits from a persistent biomarker, such as DNA methylation, is not only relevant to account for self-reporting bias in public health and personalized medicine studies, but may also allow broadening forensic DNA phenotyping. Previously, a model to predict whether someone is a current, former, or never smoker had been published based on solely 13 CpGs from the hundreds of thousands included in the DNA methylation microarray. However, a matching lab tool with lower marker throughput, and higher accuracy and sensitivity was missing towards translating the model in practice. Chapter 6 describes the development of an MPS assay and data analysis pipeline to quantify DNA methylation on these 13 smoking-associated biomarkers for the prediction of smoking status. Though our systematic evaluation on DNA standards of known methylation levels revealed marker-specific amplification bias, our novel tool was still able to provide highly accurate and reproducible DNA methylation quantification and smoking habit prediction. Overall, our MPS assay allows the technological transfer of DNA methylation microarray findings and models to practical settings, one step closer towards future applications.Finally, Chapter 7 provides a general discussion on the results and topics discussed across Chapters 2-6. It begins by summarizing the main findings across the thesis, including proposals for follow-up studies. It then covers technical limitations pertaining bisulfite conversion and DNA methylation microarrays, but also more general considerations such as restricted data access. This chapter ends by covering the outlook of this PhD thesis, including topics such as bisulfite-free methods, third-generation sequencing, single-cell methylomics, multi-omics and systems biology.<br/

    LIPIcs, Volume 251, ITCS 2023, Complete Volume

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    LIPIcs, Volume 251, ITCS 2023, Complete Volum

    UMSL Bulletin 2022-2023

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    The 2022-2023 Bulletin and Course Catalog for the University of Missouri St. Louis.https://irl.umsl.edu/bulletin/1087/thumbnail.jp

    Signaling Mechanisms Behind the Benefits of Sleep

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    Hintergrund: Schlaf ist ein streng regulierter Zustand körperlicher Ruhe und reduzierten Bewusstseins, der evolutionär im ganzen Tierreich konserviert ist. Schlafmangel ist in der modernen Gesellschaft weit verbreitet und betrifft 10 – 30 % der Erwachsenen. Dies stellt ein ernstes gesundheitliches Problem dar, da Schlafmangel mit vielen Krankheiten assoziiert ist, darunter Depressionen, Krebs und Herz-Kreislauf-Erkrankungen. Umgekehrt beeinflussen auch Krankheiten und das Immunsystem das Schlafverhalten. Trotz der fundamentalen Rolle dieser Wechselbeziehung sind grundlegende molekulare Mechanismen, die Funktionen des Immunsystems und Schlafkontrolle verbinden, bisher kaum verstanden. Da die Schlafregulation in Säugetieren sehr komplex ist, ist es sinnvoll konservierte Mechanismen zuerst in einfacheren Modellorganismen zu untersuchen. Der Rundwurm C. elegans ist ein solcher etablierter, simpler und vielseitiger Modellorganismus für die Schlafforschung. Er schläft sowohl im Rhythmus seiner Larvenentwicklung immer jeweils während des Lethargus kurz vor der Häutung, als auch nach besonderem Stress, wie zum Beispiel Hunger oder Hitze. C. elegans besitzt ein invariantes Nervensystem, in dem eine rapide Depolarisation des einzelnen RIS-Interneurons genügt, um Schlaf zu induzieren. Eine Mutation des AP2 Transkriptionsfaktors APTF-1 verhindert die Expression von FLP-11, dem schlafinduzierenden Neuropeptid von RIS. Dies führt praktisch zu völliger Schlaflosigkeit, die in C. elegans in der Regel nicht tödlich ist, und deshalb ein nützliches Modell für genetisch-chronischen Schlafmangel darstellt. Unser Labor fand heraus, dass eine Gain-of-function-Mutation in der Kollagenase NAS-38 über Signalwege der angeborenen Immunität und RIS-Aktivierung zu vermehrtem Schlaf während des Lethargus führt. Gleichzeitig wird dabei die Expression einer ganzen Familie antimikrobieller Peptide (AMP) hochreguliert. Derselbe Signalweg, einschließlich der AMP, sowie das Schlafverhalten werden auch durch Verletzungen induziert. Interessanterweise sterben nicht-schlafende Würmer nach einer Verletzung häufiger. Insgesamt deutet dies darauf hin, dass AMP als Signalmoleküle fungieren könnten, die Schlaf als Teil einer globalen Schutzreaktion vom peripheren Gewebe zum Nervensystem signalisieren. Für diese Hypothese fehlten bisher jedoch die Beweise. Fragestellungen und Hypothesen: Mein Ziel war es, den molekularen Mechanismus zu entschlüsseln, durch den verschiedene Reize der angeborenen Immunität, das heißt NAS-38 sowie epidermale Verletzungen, Schlaf induzieren. Zwei Fragen habe ich hierbei im Speziellen adressiert: Welche Domänen des NAS 38-Proteins sind an der Schlafregulation beteiligt? Da die Astacin-Domäne als aktive Proteasedomäne von NAS-38 angesehen wird, erwartete ich eine Schlüsselrolle dieser Domäne auch in der Schlafinduktion. Zweitens, welche Rolle spielen AMP bei der Signalisierung von immunitätsinduziertem Schlaf? Da gezeigt wurde, dass AMP während des NAS-38 Schlafes und auch nach Verwundung hochreguliert sind, erwartete ich, dass AMP an der Signalisierung von Schlaf von der Epidermis zum Nervensystem beteiligt sind. In einem zweiten Schritt untersuchte ich die molekularen Mechanismen, die den Vorteilen von Schlaf für das Überleben von Verletzungen zugrunde liegen. Auch hier habe ich speziell zwei Fragestellungen untersucht: Verändert genetischer Schlafentzug die transkriptionelle Reaktion auf epidermale Verletzungen? Da Schlaf für viele fundamentale Prozesse wichtig ist und Schlaflosigkeit die Sterblichkeit nach Verletzungen erhöht, vermutete ich, dass genetischer Schlafentzug die transkriptionelle Reaktion auf Verletzungen beeinträchtigt. Zweitens, ist Schlaf wichtig für die Entwicklung von Robustheit, um im Falle einer Verletzung weniger Schaden zu nehmen? Während der Larvenentwicklung fällt die Cuticula-Synthese mit Schlaf zeitlich zusammen. Daher stellte ich die Hypothese auf, dass Schlafentzug die korrekte Bildung einer Cuticula beeinträchtigt. Methoden: Zur Analyse der Signalmechanismen, durch die sowohl NAS-38 als auch Verletzungen Schlaf induzieren, filmte ich das Schlafverhalten von C. elegans mittels Langzeit-Bildgebung in Agarose-Mikrokammern. So führte ich eine Struktur-Funktions-Analyse mit verschiedenen nas-38 Mutanten durch, in denen jeweils eine andere NAS-38 Domäne deletiert war. Darüber hinaus testete ich verschiedene Suppressoren für immunvermittelten Schlaf, der durch NAS 38 oder Verletzungen induziert war. Die Redundanz des Suppressionseffektes der verschiedenen Mitglieder der AMP-Familie auf immunvermittelten Schlaf testete ich, indem ich den Suppressionsphänotyp einer CRISPR/Cas9-editierten Multi-Knockout-Mutante analysierte, in der insgesamt 19 AMP deletiert waren. Um Effektoren zu identifizieren, die den AMP nachgeschaltet sind, induzierte ich Schlaf durch Überexpression des AMP NLP 29 unter der Kontrolle eines Hitzeschock-Promotors und analysierte die Sschlafsuppression durch verschiedene Knockout-Mutanten. Im zweiten Projekt beschäftigte ich mich mit der Frage, wie genau Schlaf das Überleben nach Verletzungen unterstützt. Ich verglich die Expression von literaturbekannten Reportern für verschiedene Aspekte der Verwundungsreaktion mittels Langzeit-Fluoreszenzmikroskopie im Wildtyp sowie dem Modell für chronisch-genetischen Schlafmangel. Darüber hinaus habe ich die Transkriptome zwischen jeweils adulten verwundeten und unverwundeten Wildtypen und schlaflosen Mutanten verglichen. Um die Struktur der Cuticula des Wildtyps und der schlaflosen Mutante zu vergleichen, analysierte ich außerdem rasterelektronen-mikroskopische Aufnahmen. Ergebnisse: Im ersten Projekt konnte ich zeigen, dass NAS-38 Schlaf durch seine Astacin-Domäne verlängert. Dieser Prozess wird moderiert durch die TSP-1-Domäne. Weiterhin konnte ich zeigen, dass viele AMP redundant wirken um immunvermittelten Schlaf, verursacht durch NAS-38 oder Verletzungen, zu signalisieren. Ich konnte zeigen, dass das AMP NLP-29 über den Neuropeptidrezeptor NPR-12 wirkt. Dieser kann NLP-29-induzierten Schlaf vermitteln, wenn er in einem neuronalen Netzwerk exprimiert wird, welches nachweislich RIS aktiviert. Interessanterweise fand ich außerdem heraus, dass für NLP-29-vermittelten Schlaf der EGFR Signalweg notwendig ist. Im zweiten Projekt entdeckte ich, dass Schlaflosigkeit die transkriptionelle Reaktion auf Verletzungen nicht dramatisch verändert. Allerdings ist das Transkriptionsprofil bereits in der unverletzten schlaflosen Mutante verändert. Dies betraf unter anderem eine Gruppe oszillierender Gene, die Cuticula-assoziierte Proteine codieren, und deren Expression normalerweise ihren Höhepunkt gegen Ende des Lethargus erreicht. Da angenommen wird, dass der Zeitpunkt der Kollagenexpression entscheidend für eine fehlerfreie Cuticula-Bildung ist, analysierte ich die Cuticula der schlaflosen Mutante. Ich konnte zeigen, dass die Cuticula des adulten Tieres tatsächlich einen strukturellen Defekt aufweist. Dieser betrifft speziell Furchen in der Region nahe den Alae und könnte möglicherweise die Strapazierfähigkeit der Cuticula gegenüber bestimmten Belastungen verringern. Daher könnte Schlaf erforderlich sein, Robustheit in Form einer strukturierten Cuticula zu fördern. Schlussfolgerungen: In diesem Dissertationsprojekt vollendete ich die Charakterisierung eines neuentdeckten Mechanismus in C. elegans, durch den Verwundungen Schlaf als Teil der Immunantwort aus der Peripherie zum Nervensystem signalisieren. Ich konnte zeigen, dass AMP gewebeübergreifend Signale von der Epidermis an ein neuronales Netz vermitteln, welches wiederum RIS aktiviert und dadurch Schlaf induziert. Da Komponenten dieses Signalweges konserviert sind, könnten AMP auch in anderen Tieren, einschließlich des Menschen, Schlaf zur Genesung fördern. Darüber hinaus habe ich die Grundlagen für die Analyse molekularer Mechanismen geschaffen, die den essentiellen Funktionen des Schlafes für Heilung und Überleben zugrunde liegen. Obwohl Schlaflosigkeit die transkriptionelle Reaktion auf Verletzungen nicht drastisch zu verändern scheint, deuten meine Ergebnisse auf eine Rolle des Schlafes bei der richtigen Cuticula-Bildung und möglicherweise sogar auf eine vielfältigere Rolle bei der zeitlichen Regulierung der Genexpression hin.:Summary I Zusammenfassung IV Contents VII List of Figures XII List of Tables XIV Abbreviations XV 1. Introduction 1 1.1. Sleep is fascinating 1 1.1.1. The origin and basic features of sleep 1 1.1.2. Regulation of sleep in higher animals 3 1.1.2.1. Neuronal control of sleep 3 1.1.2.2. Molecular control of sleep 5 1.1.3. The functions of sleep 6 1.2. The immune system and its relationship to sleep 7 1.3. Wound healing and its relationship to sleep 10 1.4. Caenorhabditis elegans is a well-studied model organism 12 1.4.1. Sleep in C. elegans 15 1.4.2. The C. elegans cuticle 18 1.4.3. Immunity in C. elegans 19 1.4.4. Wound healing response in C. elegans 22 2. Previous results 25 2.1. A strong gain-of-function mutation in the astacin metallo-proteinase NAS 38 increases lethargus duration and movement quiescence in C. elegans 25 2.2. NAS-38 increases sleep mostly through the RIS neuron 25 2.3. NAS-38 is expressed in the epidermis and oscillates with the developmental rhythm 25 2.4. nas-38(ok3407) acts via innate immunity pathways to increase lethargus duration and AMP expression 27 2.5. Overexpression of AMPs induces RIS dependent quiescence 30 2.6. Epidermal wounding induces RIS-dependent sleep, which is beneficial for survival 31 3. Thesis Aims 34 3.1. Aim 1 – Characterizing the molecular mechanism through which NAS-38, innate immunity, and wounding induce sleep 34 3.2. Aim 2 – Analyzing how sleep promotes survival after wounding 35 4. Materials and Methods 36 4.1. C. elegans maintenance 36 4.2. C. elegans crossing and genotyping 41 4.3. Creation of transgenic animals 45 4.3.1. Creating the npr-12 rescue in nmr-1 expressing neurons 45 4.3.2. Microparticle bombardment 45 4.3.3. CRISPR/Cas9 system 46 4.4. Synchronizing worm cultures by hypochlorite treatment 48 4.5. Imaging 49 4.5.1. Imaging setups 49 4.5.2. DIC Imaging of worm development, lethargus, and sleep behavior 50 4.5.2.1. Imaging of heterozygous mutants 50 4.5.3. DIC imaging in the temperature control device 51 4.5.4. Fluorescent imaging experiments 51 4.5.4.1. nas-38p::d1GFP and nlp-29p::GFP during L1 development 51 4.5.4.2. nlp-29p::GFP in L4 larvae 52 4.5.4.3. nlp-29p::GFP after heat shock-induced lin-3 overexpression 52 4.5.4.4. Imaging fluorescent markers in (wounded) young adults 52 4.5.4.5. Functional Ca2+ imaging in young adults 52 4.5.4.6. Fluorescence imaging across the whole developmental time 54 4.5.4.7. Nuclear decompaction assays 55 4.5.4.8. Transcription factor localization with spinning disc confocal microscopy 55 4.5.4.9. Imaging DPY-13::mKate2 in young adults 56 4.6. Image analysis 56 4.6.1. Assessment of developmental time and lethargus detection 56 4.6.2. Sleep detection in DIC mode 56 4.6.3. Analyzing functional Ca2+ images 57 4.6.4. Fluorescent reporter analysis during long-term imaging 57 4.7. RNAi-by-feeding 58 4.8. Transcriptome analysis 59 4.8.1. Analysis of the nas-38(ok3407) transcriptome 59 4.8.2. Analysis of the wounding transcriptome 59 4.9. Epidermal wounding 62 4.9.1. Laser wounding 62 4.9.2. Needle wounding 62 4.9.3. Survival assay 63 4.10. Scanning Electron Microscopy (SEM) 63 4.11. Histamine-inducible hyperpolarization of RIS 64 4.12. Cuticle integrity test with Sodium hypochlorite 64 4.13. NPR-12 receptor modeling 64 4.14. Quantification and statistical analysis 65 5. Results 66 5.1. Aim 1 – Characterizing the pathway through which NAS 38, wounding and innate immunity induce sleep 66 5.1.1. The loss of function mutation nas-38(tm2655) shows the opposite phenotype to the gain of function mutation nas-38(ok3407) 66 5.1.2. nas-38 gain-of-function mutants act through their astacin protease domain and are semi-dominant 66 5.1.3. Transcriptome analysis of nas-38(ok3407) reveals upregulation of genes associated with secretion, innate immunity and cuticle formation 69 5.1.4. nas-38(knu568) increased movement quiescence can be suppressed by mutations of innate immunity pathways 72 5.1.5. Multiple NLPs and CNCs act in parallel to mediate nas-38(ok3407) induced sleep 75 5.1.6. Wounding-induced sleep requires RIS, ALA, EGFR and immune signaling 77 5.1.7. NLP-29 signals via the NPR-12 receptor in neurons upstream of RIS 80 5.1.8. NLP-29 requires neuronal EGFR signaling to induce sleep 81 5.1.9. Simple in silico models suggest that many different NLPs can bind to NPR-12 83 5.1.10. AMPs contribute to the survival after wounding 85 5.2. Aim 2 – Identifying the advantages sleep provides that help to survive harmful conditions 87 5.2.1. Wounding decreases the lifespan in the wild type and the aptf 1(gk794) mutant 87 5.2.2. Histamine-inducible RIS hyperpolarization suppresses wounding sleep 87 5.2.3. Genetic sleep deprivation decreases translocation of DAF-16 into the nucleus immediately after wounding 89 5.2.4. Genetic sleep deprivation hardly changes the transcriptional wounding response 95 5.2.5. Genetic sleep deprivation and wounding increase nuclear PHA 4 101 5.2.6. Oscillating genes and genes associated with the cuticle and the unfolded protein response are upregulated in young adult aptf 1(gk794) mutants 106 5.2.7. Genetic sleep deprivation leads to a malformation of cuticular furrows 109 5.2.8. Genetic sleep deprivation leads to an increased transcription of lethargus specific oscillating genes in young adults 114 5.2.9. Genetic sleep deprivation does not significantly affect development time or body size 120 5.2.10. Expression of fluorescent reporters of oscillating genes is not phase-shifted in the aptf-1(gk794) mutant 122 6. Discussion and Outlook 128 6.1. NAS-38 acts through its astacin domain to increase sleep via innate immunity pathways 128 6.2. NAS-38 during larval lethargus and epidermal wounding in the adult signal sleep via many AMPs as part of a peripheral immune response 130 6.3. Epidermal AMPs activate a neuronal circuit to induce sleep 131 6.4. Genetically sleep deprived worms can mount a proper wounding response in many ways, except for DAF-16/FOXO regulation 132 6.5. Genetic sleep deprivation alters cuticle formation 135 6.6. The role of PHA-4/FOXA in genetically sleep-deprived animals 137 6.7. Conclusion 139 7. References 140 8. Acknowledgements 163 9. Appendix 166 9.1. Standard reagents 166 9.2. Sequence summary of PHX3754 167 9.3. MATLAB script to analyze the intensity of fluorescent reporters over time 171 9.4. Permissions to reprint figures 174 9.5. Experimental author contributions 175 9.6. Predicted interactions between the NPR-12 receptor and peptides of the nlp and cnc families 176 9.7. Overlap of the adult wounding transcriptome with other data sets 179 9.8. Curriculum Vitae – Marina Patricia Sinner 181Background: Sleep is a tightly regulated state of behavioral quiescence and reduced consciousness, which is conserved throughout the animal kingdom. In modern societies 10 – 30 % of the adult population suffer from insufficient sleep, which poses a serious health problem as sleep deprivation is associated with a variety of diseases including depression, cancer, and cardiovascular diseases. Conversely, sickness and the immune system also influence sleep patterns. Despite the important role of this interrelationship between sleep and immunity, basic molecular mechanisms that link both vital functions are only poorly understood yet. As sleep regulation is complex in mammals and is thus difficult to address experimentally, it is reasonable to investigate its basic conserved mechanisms in simpler models first. The nematode C. elegans is such a well-established, simple, and powerful model organism for sleep research. It displays stress-induced sleep, for example upon starvation or heat shock, but also developmentally-timed sleep during lethargus prior to each larval molt. C. elegans possesses an invariant nervous system in which rapid depolarization of the single RIS interneuron is sufficient to induce sleep. Mutation of the AP2 transcription factor APTF 1 deprives RIS of its sleep-inducing neuropeptide FLP-11 and thus virtually abolishes sleep. This is not per se lethal in C. elegans, thereby presenting a powerful model for genetic sleep deprivation. Our lab found that a gain-of-function mutation in the collagenase NAS-38 strongly increases RIS-dependent sleep during lethargus with a concomitant upregulation of a large family of antimicrobial peptides (AMPs) via immunity pathways. Epidermal wounding also triggers AMP expression via immune signaling and induces sleep in the adult worm. Moreover, genetic sleep deprivation increases mortality upon epidermal injury. Together, this suggests AMPs to act as somnogens from peripheral tissues to the nervous system as part of a protective response. This hypothesis, however, was hitherto lacking final evidence and pathway components. Research questions and hypotheses: I aimed to characterize the molecular mechanism by which separate triggers of innate immunity, i. e. NAS-38 and wounding, induce sleep. I specifically addressed two questions: Firstly, which domains of the NAS-38 protein are involved in sleep regulation? As the astacin domain is predicted to be the active protease domain of NAS-38, I expected a role for it also in sleep induction by NAS-38. Secondly, what is the role of AMPs in signaling immunity-induced sleep? As they have been shown to be upregulated during times of increased sleep in the nas-38 mutant and after wounding, I expected AMPs to be involved in signaling sleep from the epidermis to the nervous system. In a second step, I investigated the molecular mechanisms underlying the benefits of sleep for surviving injury. Again, I addressed two questions: Firstly, does genetic sleep deprivation alter the transcriptional wounding response? As sleep has a role in many fundamental processes and sleeplessness increases mortality upon wounding, I hypothesized that genetic sleep deprivation impairs wounding-induced changes of transcriptional activity. Secondly, does sleep help building robustness before encountering injury? During larval development the synthesis of a new cuticle coincides with sleep. Thus, I hypothesized that genetic sleep deprivation impairs proper cuticle formation. Methods: To dissect the signaling mechanisms by which NAS-38 and wounding induced sleep, I followed sleep behavior of C. elegans by long-term imaging in agarose microchambers. I performed a structure-function analysis with different nas-38 mutants, each carrying a deletion of a different domain. Moreover, I screened for suppressors of sleep induced by NAS 38 or wounding. To test for redundancy of the AMP family, I investigated the suppression-phenotype of a CRISPR/Cas9 edited multi-knockout mutant lacking 19 AMPs. To identify downstream effectors of the AMP NLP 29, I induced sleep by overexpressing NLP 29 from a heat-shock promoter and analyzed the suppression-phenotype of different knockout mutants. For the second project, I addressed the question how sleep aids recovery from injury. I followed fluorescent reporters of previously described wounding response pathways by fluorescent long-term imaging in wild-type and genetically sleep-deprived animals. Moreover, I compared the transcriptomes of adult wild-type and genetically sleep-deprived worms both wounded and unwounded. To investigate the structure of the cuticle, I analyzed scanning electron microscopy images. Results: In the first project, I could show that NAS-38 indeed increases sleep via its astacin domain in a process that is modulated by the TSP-1 domain. Moreover, I could show that many AMPs act redundantly in mediating immunity-induced sleep downstream of NAS-38 and after wounding. I demonstrated that the AMP NLP-29 signals sleep via the neuropeptide receptor NPR 12. This receptor can mediate sleep when it is specifically expressed in command interneurons of a circuit that has been shown to activate RIS. Interestingly, I also found that EGFR signaling is required to mediate NLP-29-induced sleep. In the second project, I found that sleeplessness does not dramatically alter the transcriptional wounding response. However, I could show that transcription is altered already in the unwounded non-sleeping mutant. This affects, among others, a specific subset of oscillating collagen-coding genes, whose expression usually peaks around the end of lethargus. As the timing of expression of collagens is thought to be highly important for proper cuticle formation, I characterized the cuticle of the aptf-1(gk794) mutant. I could show that young adult aptf 1(gk794) worms indeed have a structural defect affecting cuticular furrows in the region adjacent to the alae, which could potentially decrease specific aspects of resilience of the cuticle. Thus, sleep might be required to build robustness in the form of a properly structured cuticle. Conclusion: In this PhD project, I completed the characterization of a novel mechanism by which wounding signals sleep from the periphery to the nervous system as part of the immune response in C. elegans. I could show that AMPs act as cross-tissue signals from the epidermis to a neuronal RIS-controlling circuit that ultimately leads to sleep induction. As components of this molecular pathway are highly conserved, AMPs might also induce sleep to promote recovery from injury in other organisms, including humans. Moreover, I laid the foundations for dissecting the molecular mechanisms behind the functions of sleep for healing and survival. Even though the disability to sleep did not seem to drastically change the transcriptional response to wounding, my results indicate a role for
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