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 181 / Background: 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 sleep in proper cuticle formation in C. elegans and potentially even a broader role in the regulation of precise gene expression timing.: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 181

Pre-clinical evaluation of novel antimicrobial coatings for blood-contacting devices commonly relies on the performance of animal studies since alternative in vitro models do not adequately represent the interactions between blood, bacteria, and material surfaces as they occur in vivo. To reduce the need of these cost-intensive and controversial animal tests, this project was dedicated to the development of a new model setup that overcomes this limitation and allows in vitro evaluation under in vivo-like conditions. This newly developed model was intended to be directly applied to evaluate recently in-house developed antimicrobial coatings, so-called anchor polymers. Therefore, the project was divided into two parts. The first part of the project focused on the evaluation of the anchor polymer coatings concerning their applicability in blood-contacting devices. For this purpose, the PEGylated styrene-maleic acid copolymers were intensively studied using established laboratory tests. These examinations showed very promising results regarding adsorption and stability on relevant polymer substrates, antimicrobial efficacy, and biological safety of the coatings, thus revealing their great potential for future applications in medical devices. Moreover, this basic characterization was meant to allow a subsequent comparison of the new in vitro model with state-of-the-art in vitro tests. The second part of the thesis focused on the development of the realistic in vitro model. Here, a single-pass flow system realized the implementation of adjustable flow conditions. Furthermore, incubation with freshly drawn human blood provided a physiological nutrient environment and included the influence of an immune response. Staphylococcus aureus were chosen as representative microorganisms, as they are responsible for a majority of device-related blood stream infections. The resulting blood flow model was validated with one anti-adhesive and one contact-killing anchor polymer coating, confirming the model’s ability to differentiate the investigated surfaces. Inflammatory and coagulant blood activation correlated slightly with bacterial coverage, which in turn was strongly dependent on the investigated material surface. Incubation with varying flow conditions demonstrated the model’s capability to reflect the well-documented dependence of bacterial colonization and occurring flow conditions. In contrast to the state-of-the-art in vitro tests, the simultaneous incubation of test surface, bacteria and whole blood allowed the analysis of mutual interactions of the three parameters. Thus, the model represents an excellent method for pre-clinical evaluation of novel antimicrobial coatings for blood-contacting devices.

Sensing of pathogens by pattern recognition receptors (PRR) is critical to initiate protective host defence reactions. However, activation of the immune system has to be carefully titrated to avoid tissue damage necessitating mechanisms to control and terminate PRR signalling. Dectin-1 is a PRR for fungal β-glucans on immune cells that is rapidly internalised after ligand-binding. Here, we demonstrate that pathogen recognition by the Dectin-1a isoform results in the formation of a stable receptor fragment devoid of the ligand binding domain. This fragment persists in phagosomal membranes and contributes to signal transduction which is terminated by the intramembrane proteases Signal Peptide Peptidase-like (SPPL) 2a and 2b. Consequently, immune cells lacking SPPL2b demonstrate increased anti-fungal ROS production, killing capacity and cytokine responses. The identified mechanism allows to uncouple the PRR signalling response from delivery of the pathogen to degradative compartments and identifies intramembrane proteases as part of a regulatory circuit to control anti-fungal immune responses.

No description available.

Salmonella spp. sind fakultative intrazelluläre Pathogene, die gastrointestinale und systemische Erkrankungen in einem umfassenden Wirtsbereich, einschließlich Tier und Mensch, hervorrufen. Salmonella benötigt verschiedene Virulenzgene für die Infektion welche auf sogenannten Salmonella Pathogenitäts-Inseln (SPI) kodiert sind. Hinzu kommt, dass auch zahlreiche im Salmonella Genom verstreuten Gene an verschiedenen Aspekten von Virulenz und Pathogenese beteiligt sind. In der vorliegenden Studie wurde die Funktion eines zuvor nicht beschriebenen putativen transkriptionellen Regulators (STM0029) charakterisiert und definiert. Dieser scheint für die Abwehr von zellulären bakterizid wirkenden Verbindungen und das Überleben des Bakteriums innerhalb einer intrazellulären Nische von entscheidender Bedeutung zu sein. Die STM0029-deletierte Mutante wies eine gesteigerte Sensitivität gegenüber antimikrobiellen Peptiden und bakteriziden Verbindungen auf. Dazu zählten α-Defensin-1, β- Defensin-1, β-Defensin-2, LL-37 und Polymyxin B sowie Komponenten des Komplementsystems. Unerwartet war die Beobachtung, dass die Expression von STM0029 durch das PmrA/B Zwei Komponenten System reprimiert vorlag, während das PhoP/Q Zwei Komponenten System keinen Einfluss auf die Expression von STM0029 zu scheinen hat. Beide Komponent Systeme spielen bekanntlich eine entscheidende Rolle bei der Expressionsregulation von Genen die für das intrazelluläre Überleben von Salmonella wichtig sind. Bemerkenswert ist, dass ein Set von Genen welche an der Biosynthese und/oder der Modifikation für das LPS O-Antigen sowie des Peptidoglykans in der bakteriellen Zellwand beteiligt ist, im STM0029 Deletionshintergrund herab reguliert vorlag. Dieses Ergebnis deutet darauf hin, dass das STM0029 Genprodukt die Persistenz des Pathogen in Wirtszellen beeinflusst. Möglicherweise geschieht dies durch das Umgehen von wirtseigenen Abwehrmechanismen. / Salmonella spp. are facultative intracellular pathogens, which cause gastrointestinal and systemic diseases in a broad range of hosts including animals and humans. In addition to virulence genes clustered within pathogenicity islands, numerous additional genes scattered throughout the genome are also involved in various aspects of Salmoenlla virulence and pathogenesis. In this study, I identified a Salmonella putative transcriptional regulator encoded by a previously uncharacterized open reading frame designated STM0029. Deletion of STM0029 altered the expression of genes involved in both the resistance to host bactericidal challenges, and bacterial cell wall biosynthesis in S. Tyhpimurium. The ΔSTM0029 strain showed a defect in the resistance to host antimicrobial peptides, including α-defensin-1, β-defensin-1, β-defensin-2, LL-37, and polymyxin B as well as serum challenges compared to the wildtype. Unexpectedly, expression of STM0029 was found to be repressed by the PmrA/B two component system, but appeared to be independent of the PhoP/Q two component system, both of which are well-known regulatory systems involved in the regulation of expression of genes involved in Salmonella intracellular survival. Notably, the expression of a set of genes involved in bacterial LPS O-antigen and peptidoglycan biosyntheses and modifications showed decreases in the absence of STM0029. These experimental results indicate that the STM0029 gene product in S. Typhimurium contributes to resistance against host cell defense mechanisms, likely through regulation of genes involved in LPS O-antigen and peptidoglycan biosynthesis and modifications.

In der humanen Haut spielen antimikrobielle Peptide (AP) bei Entzündungsgeschehen bakteriellen und nicht-bakteriellen Ursprungs eine bedeutende Rolle. Neben einer konstitutiven Expression AP können Zytokine deren vermehrte oder abgeschwächte Expression bewirken. In dieser Arbeit wurden die AP humanes β-Defensin (HBD) -2, HBD-3 und Psoriasin (PSO) in Bezug auf deren Expression in gesunder Haut und deren Modulation durch Zytokine in vivo anhand des xenogenen NOD-SCID-Maus-Transplantationsmodells untersucht. Nach erfolgreicher Transplantation von humaner Haut auf NOD-SCID Mäuse wurden die Zytokine TNF-α, IFN-γ und IL-13 in unterschiedlicher Dosierung einzeln und in Kombination intradermal appliziert. Für TNF-α konnte eine erhöhte Expression von HBD-2, HBD-3 und PSO auf RNA-Ebene mittels in-situ-Hybridisierung und Protein-Ebene mittels immunhistochemischer Nachweismethoden gezeigt werden. Eine erhöhte Expression nach Injektion von IFN-γ ließ sich für HBD-3 auf RNA-Ebene und Protein-Ebene und für HBD-2 auf RNA-Ebene erfolgreich belegen. PSO zeigte auf Protein-Ebene nach Modulation mit IFN-γ eine bei höherer Dosierung leicht abnehmende Expression. Eine Änderung der Expression durch IL-13 ließ sich nicht eindeutig belegen. In dieser Arbeit konnte die in der Literatur in vitro beschriebene Modulationsfähigkeit der untersuchten AP durch die verwendeten Zytokine in vivo belegt werden.

Solar-driven photocatalysis is of great interest in terms of a sustainable use of energy and its application in wastewater treatment. The UV light-driven photogeneration of H2O2 by solar irradiation is an advanced strategy for the treatment of bacteria and recalcitrant pollutants in wastewater, but suffers from low efficiencies. In this work, a solar-driven multifunctional nanocomposite consisting of Tm3+ upconverting nanoparticles, poly(vinyl alcohol), poly(acrylic acid) and hydroxylated sulfonated poly(ether ether ketone) was prepared. The components were crosslinked via a heating treatment at 170 °C, resulting in a non-leaching porous material. This nanocomposite exhibited excellent adsorption ability (89 % in 150 min) toward a 100 mg/L ciprofloxacin aqueous solution and proved to photodegrade it (50 %) upon 4 h artificial solar irradiation, exploiting photon upconversion processes. Moreover, an 80 % bactericidal effect against E. coli was registered upon sunlight irradiation. Altogether, these results suggest the feasibility of a solar-driven wastewater treatment based on upconverting nanoparticles.

Over the course of the last decades, the continuous exposure of bacteria to antibiotics—at least in parts due to misprescription, misuse, and misdosing—has led to the widespread development of antimicrobial resistances. This development poses a threat to the available medication in losing their effectiveness in treating bacterial infections. On the drug development side, only minor advances have been made to bring forward novel therapeutics. In addition to increasing the efforts and approaches of tapping the natural sources of new antibiotics, synthetic approaches to developing novel antimicrobials are being pursued. In this study, BDTL049 was rationally designed using knowledge based on the properties of natural antibiotics. BDTL049 is a carbosilane dendritic system with bow-tie type topology, which has antimicrobial activity at concentrations comparable to clinically established natural antibiotics. In this report, we describe its mechanism of action on the Gram-positive model organism Bacillus subtilis. Exposure to BDTL049 resulted in a complex transcriptional response, which pointed toward disturbance of the cell envelope homeostasis accompanied by disruption of other central cellular processes of bacterial metabolism as the primary targets of BDTL049 treatment. By applying a combination of whole-cell biosensors, molecular staining, and voltage sensitive dyes, we demonstrate that the mode of action of BDTL049 comprises membrane depolarization concomitant with pore formation. As a result, this new molecule kills Gram-positive bacteria within minutes. Since BDTL049 attacks bacterial cells at different targets simultaneously, this might decrease the chances for the development of bacterial resistances, thereby making it a promising candidate for a future antimicrobial agent.