8,799 research outputs found
Ruthenium metallotherapeutics: a targeted approach to combatting multidrug resistant pathogens
The discovery of antibiotics revolutionised healthcare practice. However due to overuse, inappropriate use, widespread prophylaxis therapy and the lack of new developments, the threat of antimicrobial resistance is now a major global threat to health. By 2050, it is estimated that mortality due to antimicrobial resistant infections will exceed 10 million people per annum, superseding cancer as the leading cause of global mortality. The use of drug repurposing to identify potential therapies which combat antimicrobial resistance is one potential solution. Metals have been used as antimicrobial agents throughout the history of medicine for a broad range of applications, including the use of Silver as an antimicrobial agent which dates back to antiquity. More recently, Ruthenium metallotherapeutic complexes have been shown to exhibit highly active antimicrobial properties by targeting a range of bacterial species, and in contrast to traditional antibiotics, these compounds are thought to elicit antibacterial activity at multiple sites within the bacterial cell, which may reduce the possibility of resistance evolution. This study aimed to evaluate the antimicrobial activity of a series of Ruthenium metallotherapeutic complexes against multidrug-resistant bacterial pathogens, with a focus on use within wound care applications.
Antimicrobial susceptibility assays identified two lead candidates, Hexaammineruthenium (III) chloride and [Chlorido(η6-p-cymene)(N-(4-chlorophenyl)pyridine-2-carbothioamide) ruthenium (II)] chloride which demonstrated activity against Pseudomonas aeruginosa and Staphylococcus aureus respectively with MIC values ranging between 4 Όg mL-1 and 16 Όg mL-1. Furthermore, Hexaammineruthenium (III) chloride demonstrated antibiofilm activity in both a time and concentration-dependent manner. Synergy studies combining lead complexes with antibiotics demonstrated the potential for use as resistance breakers. Subsequent in vitro infection modelling using scratch assays with skin cell lines, coupled with a 3D full thickness skin wound infection model was used to determine potential applied applications of Hexaammineruthenium (III) chloride for use as topical antimicrobial agent against P. aeruginosa infections.
Antimicrobial mechanistic studies demonstrated that Hexaammineruthenium (III) chloride targeted the bacterial cell ultrastructure of P. aeruginosa strain PAO1 as cell perturbations were observed when treated cells were analysed by scanning electron microscopy. Furthermore, exposure of P. aeruginosa PAO1 to Hexaammineruthenium (III) chloride also resulted in a concentration dependent membrane depolarisation, which further supported the antimicrobial mechanistic role.
Finally, global changes in gene expression following exposure of P. aeruginosa strain PAO1 to Hexaammineruthenium (III) chloride were explored by RNA sequencing. Genes involved in ribosome function, cofactor biosynthesis and membrane fusion were downregulated, which provided a further insight into the wider mechanisms of antibacterial activity.
The research conducted in the present study indicated the potential use of Hexaammineruthenium (III) chloride (and derivatives) as a potential treatment option for chronic wounds infected with P. aeruginosa, which could be applied as either a direct treatment or used within antimicrobial wound care applications
Converging organoids and extracellular matrix::New insights into liver cancer biology
Primary liver cancer, consisting primarily of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), is a heterogeneous malignancy with a dismal prognosis, resulting in the third leading cause of cancer mortality worldwide [1, 2]. It is characterized by unique histological features, late-stage diagnosis, a highly variable mutational landscape, and high levels of heterogeneity in biology and etiology [3-5]. Treatment options are limited, with surgical intervention the main curative option, although not available for the majority of patients which are diagnosed in an advanced stage. Major contributing factors to the complexity and limited treatment options are the interactions between primary tumor cells, non-neoplastic stromal and immune cells, and the extracellular matrix (ECM). ECM dysregulation plays a prominent role in multiple facets of liver cancer, including initiation and progression [6, 7]. HCC often develops in already damaged environments containing large areas of inflammation and fibrosis, while CCA is commonly characterized by significant desmoplasia, extensive formation of connective tissue surrounding the tumor [8, 9]. Thus, to gain a better understanding of liver cancer biology, sophisticated in vitro tumor models need to incorporate comprehensively the various aspects that together dictate liver cancer progression. Therefore, the aim of this thesis is to create in vitro liver cancer models through organoid technology approaches, allowing for novel insights into liver cancer biology and, in turn, providing potential avenues for therapeutic testing. To model primary epithelial liver cancer cells, organoid technology is employed in part I. To study and characterize the role of ECM in liver cancer, decellularization of tumor tissue, adjacent liver tissue, and distant metastatic organs (i.e. lung and lymph node) is described, characterized, and combined with organoid technology to create improved tissue engineered models for liver cancer in part II of this thesis. Chapter 1 provides a brief introduction into the concepts of liver cancer, cellular heterogeneity, decellularization and organoid technology. It also explains the rationale behind the work presented in this thesis. In-depth analysis of organoid technology and contrasting it to different in vitro cell culture systems employed for liver cancer modeling is done in chapter 2. Reliable establishment of liver cancer organoids is crucial for advancing translational applications of organoids, such as personalized medicine. Therefore, as described in chapter 3, a multi-center analysis was performed on establishment of liver cancer organoids. This revealed a global establishment efficiency rate of 28.2% (19.3% for hepatocellular carcinoma organoids (HCCO) and 36% for cholangiocarcinoma organoids (CCAO)). Additionally, potential solutions and future perspectives for increasing establishment are provided. Liver cancer organoids consist of solely primary epithelial tumor cells. To engineer an in vitro tumor model with the possibility of immunotherapy testing, CCAO were combined with immune cells in chapter 4. Co-culture of CCAO with peripheral blood mononuclear cells and/or allogenic T cells revealed an effective anti-tumor immune response, with distinct interpatient heterogeneity. These cytotoxic effects were mediated by cell-cell contact and release of soluble factors, albeit indirect killing through soluble factors was only observed in one organoid line. Thus, this model provided a first step towards developing immunotherapy for CCA on an individual patient level. Personalized medicine success is dependent on an organoids ability to recapitulate patient tissue faithfully. Therefore, in chapter 5 a novel organoid system was created in which branching morphogenesis was induced in cholangiocyte and CCA organoids. Branching cholangiocyte organoids self-organized into tubular structures, with high similarity to primary cholangiocytes, based on single-cell sequencing and functionality. Similarly, branching CCAO obtain a different morphology in vitro more similar to primary tumors. Moreover, these branching CCAO have a higher correlation to the transcriptomic profile of patient-paired tumor tissue and an increased drug resistance to gemcitabine and cisplatin, the standard chemotherapy regimen for CCA patients in the clinic. As discussed, CCAO represent the epithelial compartment of CCA. Proliferation, invasion, and metastasis of epithelial tumor cells is highly influenced by the interaction with their cellular and extracellular environment. The remodeling of various properties of the extracellular matrix (ECM), including stiffness, composition, alignment, and integrity, influences tumor progression. In chapter 6 the alterations of the ECM in solid tumors and the translational impact of our increased understanding of these alterations is discussed. The success of ECM-related cancer therapy development requires an intimate understanding of the malignancy-induced changes to the ECM. This principle was applied to liver cancer in chapter 7, whereby through a integrative molecular and mechanical approach the dysregulation of liver cancer ECM was characterized. An optimized agitation-based decellularization protocol was established for primary liver cancer (HCC and CCA) and paired adjacent tissue (HCC-ADJ and CCA-ADJ). Novel malignancy-related ECM protein signatures were found, which were previously overlooked in liver cancer transcriptomic data. Additionally, the mechanical characteristics were probed, which revealed divergent macro- and micro-scale mechanical properties and a higher alignment of collagen in CCA. This study provided a better understanding of ECM alterations during liver cancer as well as a potential scaffold for culture of organoids. This was applied to CCA in chapter 8 by combining decellularized CCA tumor ECM and tumor-free liver ECM with CCAO to study cell-matrix interactions. Culture of CCAO in tumor ECM resulted in a transcriptome closely resembling in vivo patient tumor tissue, and was accompanied by an increase in chemo resistance. In tumor-free liver ECM, devoid of desmoplasia, CCAO initiated a desmoplastic reaction through increased collagen production. If desmoplasia was already present, distinct ECM proteins were produced by the organoids. These were tumor-related proteins associated with poor patient survival. To extend this method of studying cell-matrix interactions to a metastatic setting, lung and lymph node tissue was decellularized and recellularized with CCAO in chapter 9, as these are common locations of metastasis in CCA. Decellularization resulted in removal of cells while preserving ECM structure and protein composition, linked to tissue-specific functioning hallmarks. Recellularization revealed that lung and lymph node ECM induced different gene expression profiles in the organoids, related to cancer stem cell phenotype, cell-ECM integrin binding, and epithelial-to-mesenchymal transition. Furthermore, the metabolic activity of CCAO in lung and lymph node was significantly influenced by the metastatic location, the original characteristics of the patient tumor, and the donor of the target organ. The previously described in vitro tumor models utilized decellularized scaffolds with native structure. Decellularized ECM can also be used for creation of tissue-specific hydrogels through digestion and gelation procedures. These hydrogels were created from both porcine and human livers in chapter 10. The liver ECM-based hydrogels were used to initiate and culture healthy cholangiocyte organoids, which maintained cholangiocyte marker expression, thus providing an alternative for initiation of organoids in BME. Building upon this, in chapter 11 human liver ECM-based extracts were used in combination with a one-step microfluidic encapsulation method to produce size standardized CCAO. The established system can facilitate the reduction of size variability conventionally seen in organoid culture by providing uniform scaffolding. Encapsulated CCAO retained their stem cell phenotype and were amendable to drug screening, showing the feasibility of scalable production of CCAO for throughput drug screening approaches. Lastly, Chapter 12 provides a global discussion and future outlook on tumor tissue engineering strategies for liver cancer, using organoid technology and decellularization. Combining multiple aspects of liver cancer, both cellular and extracellular, with tissue engineering strategies provides advanced tumor models that can delineate fundamental mechanistic insights as well as provide a platform for drug screening approaches.<br/
CANCER TREATMENT BY TARGETING HDAC4 TRANSLOCATION INDUCED BY MICROSECOND PULSED ELECTRIC FIELD EXPOSURE: MECHANISTIC INSIGHTS THROUGH KINASES AND PHOSPHATASES
Epigenetic modifications, arising from sub-cellular shifts in histone deacetylase (HDAC) activity and localization, present promising strategies for diverse cancer treatments. HDACs, enzymes responsible for post-translational histone modifications, induce these epigenetic changes by removing acetyl groups from Δ-N-acetyl-lysine residues on histones, thereby suppressing gene transcription. Within the HDAC group, class IIa HDACs are notable for their responsiveness to extracellular signals, bridging the gap between external stimuli, plasma membrane, and genome through nuclear-cytoplasmic translocation. This localization offers two significant mechanisms for cancer treatment: nuclear accumulation of HDACs represses oncogenic transcription factors, such as myocyte-specific enhancer factor 2C (MEF2C), triggering various cell death pathways. Conversely, cytoplasmic HDAC accumulation acts similarly to HDAC inhibitors by silencing genes. My dissertation introduces an innovative approach for glioblastoma and breast cancer treatment by investigating the application of microsecond pulsed electric fields. It particularly focuses on HDAC4, a class IIa HDAC overexpressed in these cancers. Beyond demonstrating HDAC4 translocation, my research delves into the intricate roles of kinases and phosphatases, shedding light on the underlying factors governing HDAC4 translocation
Enhancing clinical potential of liquid biopsy through a multi-omic approach: A systematic review
In the last years, liquid biopsy gained increasing clinical relevance for detecting and monitoring several cancer types, being minimally invasive, highly informative and replicable over time. This revolutionary approach can be complementary and may, in the future, replace tissue biopsy, which is still considered the gold standard for cancer diagnosis. âClassicalâ tissue biopsy is invasive, often cannot provide sufficient bioptic material for advanced screening, and can provide isolated information about disease evolution and heterogeneity. Recent literature highlighted how liquid biopsy is informative of proteomic, genomic, epigenetic, and metabolic alterations. These biomarkers can be detected and investigated using single-omic and, recently, in combination through multi-omic approaches. This review will provide an overview of the most suitable techniques to thoroughly characterize tumor biomarkers and their potential clinical applications, highlighting the importance of an integrated multi-omic, multi-analyte approach. Personalized medical investigations will soon allow patients to receive predictable prognostic evaluations, early disease diagnosis, and subsequent ad hoc treatments
Effects of municipal smoke-free ordinances on secondhand smoke exposure in the Republic of Korea
ObjectiveTo reduce premature deaths due to secondhand smoke (SHS) exposure among non-smokers, the Republic of Korea (ROK) adopted changes to the National Health Promotion Act, which allowed local governments to enact municipal ordinances to strengthen their authority to designate smoke-free areas and levy penalty fines. In this study, we examined national trends in SHS exposure after the introduction of these municipal ordinances at the city level in 2010.MethodsWe used interrupted time series analysis to assess whether the trends of SHS exposure in the workplace and at home, and the primary cigarette smoking rate changed following the policy adjustment in the national legislation in ROK. Population-standardized data for selected variables were retrieved from a nationally representative survey dataset and used to study the policy actionâs effectiveness.ResultsFollowing the change in the legislation, SHS exposure in the workplace reversed course from an increasing (18% per year) trend prior to the introduction of these smoke-free ordinances to a decreasing (â10% per year) trend after adoption and enforcement of these laws (ÎČ2â=â0.18, p-valueâ=â0.07; ÎČ3â=ââ0.10, p-valueâ=â0.02). SHS exposure at home (ÎČ2â=â0.10, p-valueâ=â0.09; ÎČ3â=ââ0.03, p-valueâ=â0.14) and the primary cigarette smoking rate (ÎČ2â=â0.03, p-valueâ=â0.10; ÎČ3â=â0.008, p-valueâ=â0.15) showed no significant changes in the sampled period. Although analyses stratified by sex showed that the allowance of municipal ordinances resulted in reduced SHS exposure in the workplace for both males and females, they did not affect the primary cigarette smoking rate as much, especially among females.ConclusionStrengthening the role of local governments by giving them the authority to enact and enforce penalties on SHS exposure violation helped ROK to reduce SHS exposure in the workplace. However, smoking behaviors and related activities seemed to shift to less restrictive areas such as on the streets and in apartment hallways, negating some of the effects due to these ordinances. Future studies should investigate how smoke-free policies beyond public places can further reduce the SHS exposure in ROK
Signaling Mechanisms Behind the Benefits of Sleep
Hintergrund: Schlaf ist ein streng regulierter Zustand körperlicher Ruhe und reduzierten Bewusstseins, der evolutionĂ€r im ganzen Tierreich konserviert ist. Schlafmangel ist in der modernen Gesellschaft weit verbreitet und betrifft 10 â 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 SĂ€ugetieren 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 fĂŒr die Schlafforschung. Er schlĂ€ft sowohl im Rhythmus seiner Larvenentwicklung immer jeweils wĂ€hrend des Lethargus kurz vor der HĂ€utung, 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 genĂŒgt, um Schlaf zu induzieren. Eine Mutation des AP2 Transkriptionsfaktors APTF-1 verhindert die Expression von FLP-11, dem schlafinduzierenden Neuropeptid von RIS. Dies fĂŒhrt praktisch zu völliger Schlaflosigkeit, die in C. elegans in der Regel nicht tödlich ist, und deshalb ein nĂŒtzliches Modell fĂŒr genetisch-chronischen Schlafmangel darstellt. Unser Labor fand heraus, dass eine Gain-of-function-Mutation in der Kollagenase NAS-38 ĂŒber Signalwege der angeborenen ImmunitĂ€t und RIS-Aktivierung zu vermehrtem Schlaf wĂ€hrend des Lethargus fĂŒhrt. Gleichzeitig wird dabei die Expression einer ganzen Familie antimikrobieller Peptide (AMP) hochreguliert. Derselbe Signalweg, einschlieĂlich der AMP, sowie das Schlafverhalten werden auch durch Verletzungen induziert. Interessanterweise sterben nicht-schlafende WĂŒrmer nach einer Verletzung hĂ€ufiger. Insgesamt deutet dies darauf hin, dass AMP als SignalmolekĂŒle fungieren könnten, die Schlaf als Teil einer globalen Schutzreaktion vom peripheren Gewebe zum Nervensystem signalisieren. FĂŒr diese Hypothese fehlten bisher jedoch die Beweise. Fragestellungen und Hypothesen: Mein Ziel war es, den molekularen Mechanismus zu entschlĂŒsseln, durch den verschiedene Reize der angeborenen ImmunitĂ€t, das heiĂt NAS-38 sowie epidermale Verletzungen, Schlaf induzieren. Zwei Fragen habe ich hierbei im Speziellen adressiert: Welche DomĂ€nen des NAS 38-Proteins sind an der Schlafregulation beteiligt? Da die Astacin-DomĂ€ne als aktive ProteasedomĂ€ne von NAS-38 angesehen wird, erwartete ich eine SchlĂŒsselrolle dieser DomĂ€ne auch in der Schlafinduktion. Zweitens, welche Rolle spielen AMP bei der Signalisierung von immunitĂ€tsinduziertem Schlaf? Da gezeigt wurde, dass AMP wĂ€hrend 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 fĂŒr das Ăberleben von Verletzungen zugrunde liegen. Auch hier habe ich speziell zwei Fragestellungen untersucht: VerĂ€ndert genetischer Schlafentzug die transkriptionelle Reaktion auf epidermale Verletzungen? Da Schlaf fĂŒr viele fundamentale Prozesse wichtig ist und Schlaflosigkeit die Sterblichkeit nach Verletzungen erhöht, vermutete ich, dass genetischer Schlafentzug die transkriptionelle Reaktion auf Verletzungen beeintrĂ€chtigt. Zweitens, ist Schlaf wichtig fĂŒr die Entwicklung von Robustheit, um im Falle einer Verletzung weniger Schaden zu nehmen? WĂ€hrend der Larvenentwicklung fĂ€llt die Cuticula-Synthese mit Schlaf zeitlich zusammen. Daher stellte ich die Hypothese auf, dass Schlafentzug die korrekte Bildung einer Cuticula beeintrĂ€chtigt. 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 fĂŒhrte ich eine Struktur-Funktions-Analyse mit verschiedenen nas-38 Mutanten durch, in denen jeweils eine andere NAS-38 DomĂ€ne deletiert war. DarĂŒber hinaus testete ich verschiedene Suppressoren fĂŒr 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 SuppressionsphĂ€notyp 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 Ăberexpression des AMP NLP 29 unter der Kontrolle eines Hitzeschock-Promotors und analysierte die Sschlafsuppression durch verschiedene Knockout-Mutanten. Im zweiten Projekt beschĂ€ftigte ich mich mit der Frage, wie genau Schlaf das Ăberleben nach Verletzungen unterstĂŒtzt. Ich verglich die Expression von literaturbekannten Reportern fĂŒr verschiedene Aspekte der Verwundungsreaktion mittels Langzeit-Fluoreszenzmikroskopie im Wildtyp sowie dem Modell fĂŒr chronisch-genetischen Schlafmangel. DarĂŒber 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 auĂerdem rasterelektronen-mikroskopische Aufnahmen. Ergebnisse: Im ersten Projekt konnte ich zeigen, dass NAS-38 Schlaf durch seine Astacin-DomĂ€ne verlĂ€ngert. Dieser Prozess wird moderiert durch die TSP-1-DomĂ€ne. 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 ĂŒber 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 auĂerdem heraus, dass fĂŒr NLP-29-vermittelten Schlaf der EGFR Signalweg notwendig ist. Im zweiten Projekt entdeckte ich, dass Schlaflosigkeit die transkriptionelle Reaktion auf Verletzungen nicht dramatisch verĂ€ndert. Allerdings ist das Transkriptionsprofil bereits in der unverletzten schlaflosen Mutante verĂ€ndert. 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 fĂŒr eine fehlerfreie Cuticula-Bildung ist, analysierte ich die Cuticula der schlaflosen Mutante. Ich konnte zeigen, dass die Cuticula des adulten Tieres tatsĂ€chlich einen strukturellen Defekt aufweist. Dieser betrifft speziell Furchen in der Region nahe den Alae und könnte möglicherweise die StrapazierfĂ€higkeit der Cuticula gegenĂŒber 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 gewebeĂŒbergreifend 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, einschlieĂlich des Menschen, Schlaf zur Genesung fördern. DarĂŒber hinaus habe ich die Grundlagen fĂŒr die Analyse molekularer Mechanismen geschaffen, die den essentiellen Funktionen des Schlafes fĂŒr Heilung und Ăberleben zugrunde liegen. Obwohl Schlaflosigkeit die transkriptionelle Reaktion auf Verletzungen nicht drastisch zu verĂ€ndern scheint, deuten meine Ergebnisse auf eine Rolle des Schlafes bei der richtigen Cuticula-Bildung und möglicherweise sogar auf eine vielfĂ€ltigere 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 â Characterizing the molecular mechanism through which NAS-38, innate immunity, and wounding induce sleep 34
3.2. Aim 2 â 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 â 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 â 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 â 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 â 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
Design of new algorithms for gene network reconstruction applied to in silico modeling of biomedical data
Programa de Doctorado en BiotecnologĂa, IngenierĂa y TecnologĂa QuĂmicaLĂnea de InvestigaciĂłn: IngenierĂa, Ciencia de Datos y BioinformĂĄticaClave Programa: DBICĂłdigo LĂnea: 111The root causes of disease are still poorly understood. The success of current therapies is limited because persistent diseases are frequently treated based on their symptoms rather than the underlying cause of the disease. Therefore, biomedical research is experiencing a technology-driven shift to data-driven holistic approaches to better characterize the molecular mechanisms causing disease. Using omics data as an input, emerging disciplines like network biology attempt to model the relationships between biomolecules. To this effect, gene co- expression networks arise as a promising tool for deciphering the relationships between genes in large transcriptomic datasets. However, because of their low specificity and high false positive rate, they demonstrate a limited capacity to retrieve the disrupted mechanisms that lead to disease onset, progression, and maintenance. Within the context of statistical modeling, we dove deeper into the reconstruction of gene co-expression networks with the specific goal of discovering disease-specific features directly from expression data. Using ensemble techniques, which combine the results of various metrics, we were able to more precisely capture biologically significant relationships between genes. We were able to find de novo potential disease-specific features with the help of prior biological knowledge and the development of new network inference techniques.
Through our different approaches, we analyzed large gene sets across multiple samples and used gene expression as a surrogate marker for the inherent biological processes, reconstructing robust gene co-expression networks that are simple to explore. By mining disease-specific gene co-expression networks we come up with a useful framework for identifying new omics-phenotype associations from conditional expression datasets.In this sense, understanding diseases from the perspective of biological network perturbations will improve personalized medicine, impacting rational biomarker discovery, patient stratification and drug design, and ultimately leading to more targeted therapies.Universidad Pablo de Olavide de Sevilla. Departamento de Deporte e InformĂĄtic
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