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Computational Modeling in Single-Cell Genomics: Linking Genetic Diversity to Cellular Identity and Function
No full textEn af de store udfordringer inden for biologi er at forstå, hvordan celler etablerer deres cellulære identitet. Meget af vores viden stammer fra studier, der ser på gennemsnittet af cellepopulationer, men sådanne studier kan overse vigtige detaljer, da celler kan eksistere i forskellige tilstande som reaktion på eksterne og interne stimuli. ’Single-cell genomics’ anses for at være den førende metode til at undersøge de processer, der bestemmer cellulær identitet og cellers udvikling til specifikke celletyper. Fordelen ved disse metoder ligger i evnen til at opfange forskelle mellem enkelte celler i en population gennem forskellige lag af information, såsom epigenetiske modifikationer, genudtryk og genetiske variationers indflydelse på cellulær identitet. Alle disse informationslag er vigtige for at kunne forstå de mekanismer, der ligger bag cellulær identitet.Formålet med dette Ph.d.-projekt er at forstå, hvordan celler udvikler deres identitet på enkeltcelleniveau. I den første del af projektet har vi udviklet et værktøj til automatisk at finde ’barcodes’ af høj kvalitet i single-cell- og single-nucleus RNA-sekventeringsdata ved hjælp af datatilpassede tærskelværdier. Metoden anvender forskellige statistiske modeller og sammenhænge mellem kvalitetsmetrikker til at fjerne ’barcodes’ af lav kvalitet. Derudover bruger metoden en ny grupperingsbaseret metode til at identificere celler med distinkte biologiske signaler, og forudsiger og markerer døde celler med høj nøjagtighed. I en sammenligning med seks andre almindeligt anvendte kvalitetskontrolmetoder rangerede vores værktøj favorabelt, da den havde bedre stabilitet og adskillelse af biologiske signaler fra celletyper efter filtrering. I den anden del af projektet har vi implementeret en arbejdsgang til at undersøge mekanismen bag almindelige genetiske varianter, der er associeret med forekomsten af celler i en bestemt tilstand under hepatogenese. Ved at bruge et ’village-in-a-dish’-system kortlagde vi transkriptomet af individuelle in vitro inducerede humane pluripotente stamceller på forskellige tidspunkter. Vi identificerede flere genetiske varianter, der er forbundet med cellekomposition, og påviste en central mekanisme, hvormed disse varianter påvirker cellulær commitment, involverer stamfader-specifikke transkriptionsfaktorer. Disse faktorer skubber differentieringen af humane inducerede pluripotente stamceller til specifikke cellelinjer.One of the grand challenges in biology is understanding how cells establish their cellular identity. Much of our current knowledge is based on the studies of the averaging cell populations. However, these studies can mask important biology of the cell subpopulations that may represent cells existing in different states as a response to external and internal stimuli. Single-cell genomics is currently considered at the frontier of studying cell commitment to specialized cell types and cellular identity processes. The advantage of those methods lies in the ability to capture cell-to-cell differences in a population of cells through the different layers, such as epigenetic modifications, the patterns of gene expression, and genetic variation effect on cellular identity. Those added layers of information are crucial in understanding the orchestrated regulation of cellular identity mechanisms.This PhD project aims to understand how cells develop their identity at single-cell resolution. First, we developed an automated toolbox to identify high-quality barcodes from single-cell RNA and single-cell nucleus RNA-sequencing data using data-adaptive thresholds. The method uses different statistical models and the relationships between quality metrics to remove low-quality barcodes. Additionally, the method uses a novel clustering-based approach to identify cells with distinct biological signals. Furthermore, our developed model predicts and flags dead cells with high accuracy. This model was benchmarked against six commonly used quality control methods and ranked favorably, showing higher consistency and separation of biological signals from cell types after the filtering. In the second part, we implemented a workflow to investigate the mechanism of common genetic variants associated with cell state abundance during hepatogenesis. Using a village-in-a-dish system, we profiled the single-cell transcriptome of human induced pluripotent stem cells in vitro at different time points. We identified several genetic variants associated with cellular composition and demonstrated that a key mechanism by which these variants influence cellular commitment involves progenitor-specific transcription factors. These factors bias the differentiation of human induced pluripotent stem cells into specific lineages
Computational Modeling in Single-Cell Genomics: Linking Genetic Diversity to Cellular Identity and Function
No full textEn af de store udfordringer inden for biologi er at forstå, hvordan celler etablerer deres cellulære identitet. Meget af vores viden stammer fra studier, der ser på gennemsnittet af cellepopulationer, men sådanne studier kan overse vigtige detaljer, da celler kan eksistere i forskellige tilstande som reaktion på eksterne og interne stimuli. ’Single-cell genomics’ anses for at være den førende metode til at undersøge de processer, der bestemmer cellulær identitet og cellers udvikling til specifikke celletyper. Fordelen ved disse metoder ligger i evnen til at opfange forskelle mellem enkelte celler i en population gennem forskellige lag af information, såsom epigenetiske modifikationer, genudtryk og genetiske variationers indflydelse på cellulær identitet. Alle disse informationslag er vigtige for at kunne forstå de mekanismer, der ligger bag cellulær identitet.Formålet med dette Ph.d.-projekt er at forstå, hvordan celler udvikler deres identitet på enkeltcelleniveau. I den første del af projektet har vi udviklet et værktøj til automatisk at finde ’barcodes’ af høj kvalitet i single-cell- og single-nucleus RNA-sekventeringsdata ved hjælp af datatilpassede tærskelværdier. Metoden anvender forskellige statistiske modeller og sammenhænge mellem kvalitetsmetrikker til at fjerne ’barcodes’ af lav kvalitet. Derudover bruger metoden en ny grupperingsbaseret metode til at identificere celler med distinkte biologiske signaler, og forudsiger og markerer døde celler med høj nøjagtighed. I en sammenligning med seks andre almindeligt anvendte kvalitetskontrolmetoder rangerede vores værktøj favorabelt, da den havde bedre stabilitet og adskillelse af biologiske signaler fra celletyper efter filtrering. I den anden del af projektet har vi implementeret en arbejdsgang til at undersøge mekanismen bag almindelige genetiske varianter, der er associeret med forekomsten af celler i en bestemt tilstand under hepatogenese. Ved at bruge et ’village-in-a-dish’-system kortlagde vi transkriptomet af individuelle in vitro inducerede humane pluripotente stamceller på forskellige tidspunkter. Vi identificerede flere genetiske varianter, der er forbundet med cellekomposition, og påviste en central mekanisme, hvormed disse varianter påvirker cellulær commitment, involverer stamfader-specifikke transkriptionsfaktorer. Disse faktorer skubber differentieringen af humane inducerede pluripotente stamceller til specifikke cellelinjer.One of the grand challenges in biology is understanding how cells establish their cellular identity. Much of our current knowledge is based on the studies of the averaging cell populations. However, these studies can mask important biology of the cell subpopulations that may represent cells existing in different states as a response to external and internal stimuli. Single-cell genomics is currently considered at the frontier of studying cell commitment to specialized cell types and cellular identity processes. The advantage of those methods lies in the ability to capture cell-to-cell differences in a population of cells through the different layers, such as epigenetic modifications, the patterns of gene expression, and genetic variation effect on cellular identity. Those added layers of information are crucial in understanding the orchestrated regulation of cellular identity mechanisms.This PhD project aims to understand how cells develop their identity at single-cell resolution. First, we developed an automated toolbox to identify high-quality barcodes from single-cell RNA and single-cell nucleus RNA-sequencing data using data-adaptive thresholds. The method uses different statistical models and the relationships between quality metrics to remove low-quality barcodes. Additionally, the method uses a novel clustering-based approach to identify cells with distinct biological signals. Furthermore, our developed model predicts and flags dead cells with high accuracy. This model was benchmarked against six commonly used quality control methods and ranked favorably, showing higher consistency and separation of biological signals from cell types after the filtering. In the second part, we implemented a workflow to investigate the mechanism of common genetic variants associated with cell state abundance during hepatogenesis. Using a village-in-a-dish system, we profiled the single-cell transcriptome of human induced pluripotent stem cells in vitro at different time points. We identified several genetic variants associated with cellular composition and demonstrated that a key mechanism by which these variants influence cellular commitment involves progenitor-specific transcription factors. These factors bias the differentiation of human induced pluripotent stem cells into specific lineages
Microbial Adaptations to Pulses of Organic Matter in the Deep-Sea
No full textDen biologiske kulstofpumpe i havet henviser til eksporten af partikulært organisk materiale (POM)fra overfladen til dybere vandlag, en proces der bidrager til langvarig kulstoflagring i havet. Mikrobiel omdannelse af både partikulært organisk materiale (POM) og opløst organisk materiale(DOM) modulerer effektiviteten af denne pumpe ved at regulere skæbnen for kulstof på tværs af dybder og oceanografiske forhold. I den del af havet der henligger i mørke, særligt i den bathypelagiske zone (1000-4000 m), styres denne omdannelse af organisk stof af et komplekst samspil af miljømæssige faktorer, herunder temperatur, højt hydrostatisk tryk (HHP) og kvaliteten af organiske materiale. På trods af den store betydning af disse processer, er de samlede påvirkninger af disse faktorer på mikrobielt stofskifte og sammensætningen af de mikrobielle samfund dårligt belyst, hvilket begrænser anvendeligheden af globale modeller for kulstofkredsløbet i dybhavet. Denne afhandling undersøger, hvordan disse faktorer påvirker prokaryote mikroorganismers respiration (PR), produktion (PP), væksteffektivitet (PGE) og samfundssammensætning (PCC) over et bredt interval havdybder og oceanografiske forhold. Manuskript I indeholder en systematisk vurdering af hvordan temperatur (0 - 25℃), organisk material sammensætning (primært C:N-forholdet), og substratkompleksitet påvirker det mikrobielle stofskifte fra overflade til den bathypelagiske zone. Alanin (C:N = 3:1) understøttede den højeste PGE, mens glukose (Ingen N) stimulerede den mikrobielle respirationen. Q10 værdier varierede fra1,9-2,9, hvor de mikrobielle samfund i de dybere zoner viste større temperaturfølsomhed. Sekventering af 16S rRNA afslørede at forskydninger PCC (ændringer sammensætningen af i de mikrobielle samfund) afspejlede de metaboliske ændringer med dybden, og dybe samfund viste den mest udtalte taksonomiske omstrukturering med stigende temperatur. Dette indikerer en tæt kobling mellem metabolisme og samfundssammensætning i dybhavet. Disse resultater antyder, at selv bathypelagiske mikrober responderer på temperaturændringer, og er reguleret af OM kvalitet. Manuskript II behandlede, hvordan HHP og OM kompleksitet modulerer PR, PP, PGE, og PCC i dybhav (2000 - 4000 m) i forhold til i lavvandede (20 m) mikrobielle samfund. Overfladesamfund viste stærk trykfølsomhed, med næsten fuldstændig hæmning af PP og endda celledød ved tryk≥32,5 MPa. Forsøg med at genoprette den mikrobielle aktivitet efter trykhæmning, der involverede tryksænkning for at måle genopretning i metaboliske hastigheder ved atmosfærisk tryk efter HHP eksponering, bekræftede at denne trykinducerede undertrykkelse i overflademikrober ved tryk≥32,5 var irreversibel. I modsætning til dette, opretholdt dybhavssamfund deres metabolisk aktivitet under in-situ trykforhold, især når de blev forsynet med det komplekse polymere substrat chitin. Disse resultater viser hvordan det komplekse samspil af miljøfaktorer, der er karakteristiske for dybhavet, såsom HHP og tilgængelighed og kvalitet af OM, påvirker de mikrobielle processer. Manuskript III udvidede denne ramme ved at sammenligne effekten af OM kvalitet og HHP på metabolisme og sammensætning af mikrobielle samfund på to lokaliteter i den vestlige Nordatlanten med meget forskellige produktionsforhold (oligotrof gyre vs næringsrige lokalitet) på tværs af en vertikal dybdegradient med 6 dybder fra 10 m til 4000 m. Påvirkningen af de mikrobielle reaktioner varierede med OM-type (tilsætning af koncentrerede partikler vs POC og DOC), og tilsætningen af chitin understøttede en højere PR og PP under in-situ tryk, hvilket er i overensstemmelse med resultaterne fra Manuskript II. Kulstofremineralisering integreret overdybdeintervallet 0-4000 m var cirka 25 % lavere under in situ-tryk sammenlignet med atmosfærisktryk, hvor den næringsrige station havde omkring 1,4 gange højere rater end den oligotrofe gyre. Disse observationer fremhæver den stærke indflydelse af regionale biogeokemiske forhold på mikrobielt stofskifte og afslører, at biomasseproduktion respondere mere end respiration på de lokale miljømæssige variationer. Tilsammen demonstrerer disse opdagelser, at mikrobielkulstofomsætning i oceanets indre er stærkt afhængig af den lokale fysisk-kemiske forhold. Effekterne af tryk og temperatur moduleres af OM-kvalitet, med implikationer for modellering af kulstoffluxe. Denne afhandling demonstrerer behovet for undersøgelser betydningen af tryk, stedsspecifikke forhold og substratsammensætning for at opnå en mere detaljeret forståelse af hvordan den mikrobielle metabolisme reguleres og hvilken rolle de spiller in det globale kulstofkredsløb.The biological carbon pump of the ocean refers to the export of particulate organic matter (POM)from surface to depth, a process that contributes to long-term carbon sequestration in the ocean. Microbial transformation of both particulate organic matter (POM) and dissolved organic matter(DOM) modulates the efficiency of this pump by regulating the fate of carbon across depths and oceanographic settings. In the dark ocean, particularly the bathypelagic zone (1000-4000 m), these transformations are governed by a complex interplay of environmental factors including temperature, high hydrostatic pressure (HHP), and organic matter (OM) quality. Yet, the combined effects of these variables on microbial metabolism and community composition remain poorly resolved, limiting predictive models of deep-ocean carbon cycling. This thesis investigates how these factors shape prokaryotic respiration (PR), production (PP), growth efficiency (PGE), and community composition (PCC) across a range of depths and oceanographic settings. Manuscript Isystematically assessed how temperature (0 - 25℃), OM stoichiometry (C:N), and substrate complexity influence microbial metabolism from surface to bathypelagic waters. Alanine (C:N =3:1) supported the highest PGE, while glucose (N-poor) preferentially stimulated respiration. Q10values ranged from 1.9-2.9, with deeper communities exhibiting greater temperature sensitivity. Crucially, 16S rRNA sequencing revealed that PCC shifts mirrored metabolic trends, and deep communities showed the most pronounced taxonomic restructuring with increasing temperature,indicating tighter coupling between metabolism and community turnover at depth. These resultssuggest that even bathypelagic microbes retain temperature-responsive traits, modulated by OMquality. Manuscript II addressed how HHP and OM complexity modulate PR, PP, PGE and PCC indeep-sea (2000 - 4000 m) vs shallow (20 m) microbial communities. Surface communities exhibited strong pressure sensitivity, with near-complete suppression of PP and even cell-death at pressures≥32.5 MPa. Recovery experiments which involved pressurizing surface microbes followed by depressurization to measure recovery in metabolic rates at atmospheric pressure confirmed their reversibility of this pressure-induced suppression in surface microbes at pressures ≥32.5. In contrast, deep-sea communities maintained metabolic activity under in-situ pressure conditions, particularly when supplied with the complex polymeric substrate chitin. These results show the complex interplay of environmental factors characteristic of the deep-sea such as HHP and OM availability and quality in shaping microbial responses. Manuscript III expanded this framework by comparing metabolic and community composition responses to OM quality and HHP at two contrasting sites in the Western North Atlantic (oligotrophic gyre vs nutrient-rich boundary current) across a vertical depth gradient with 6 depths from 10 m to 4000 m. Microbial responses varied with OM type (concentrated particles vs POC and DOC), and chitin addition supported higher PR and PP under in-situ pressures, consistent with the results from Manuscript II. Carbonremineralisation integrated over the 0-4000 m depth range was approximately 25% lower under insitu pressure compared to atmospheric conditions, with the nutrient-rich station showing rates about1.4 times higher than those in the oligotrophic gyre. These findings highlight the strong influence of regional biogeochemical conditions on microbial metabolism and reveal that biomass production responds far more dramatically than respiration to local environmental context. Together, these findings demonstrate that microbial carbon processing in the ocean interior is highly contingent upon local physicochemical context. The effects of pressure and temperature are modulated by OMquality, with implications for carbon flux modeling. This thesis demonstrates the need for pressureaware, site-specific and substrate-specific studies to refine our understanding of microbial dynamics and their role in the biological carbon pump
Investigation of cell surface biology and biophysics of a methanogenic Archaea capable of extracellular electron uptake
No full textMethanosarcina barkeri er en alsidig og modstandsdygtig metanogen, som benytter en variation a elektron donerer, så simpelt som elektroner selv, til at drive dens stofskifte. Ekstracellulær elektron overførsel (EET) er vidt udbredt og baner vej for mange interaktioner mellem miljøet og mikrobielle samfund. Selvom M. barkeri er ligesom mange andre mikrober, der kan udføre EET, er M. barkeri speciel i dens måde at udføre EET. M. barkeri benytter en ukendt, ikke-kanonisk EET-mekanisme, hvilket inspirerede dette projekt. EET-Interaktioner finder sted i en grænseflade mellem mikrober og miljøet. Fokusset var derfor at gennemsøge den cellulære overflade af M. barkeri for at finde elektroaktive ledninger, der gør at M. barkeri kan modtage ekstracellulære elektroner. Globale proteomiske studier af M. barkeri, med brug af en katodisk overflade som elektron doner til at drive elektrometanogenese, afslørede unikke udtrykte flavin-relaterede proteiner, som var ledsaget af metal- og ion transporterer samt overfladelags omarrangerings proteiner. Dette indikerer en flavin-basereret ekstracellulær elektron overførsels (FLEET) mekanisme, som aldrig før er blevet beskrevet for arkæer, samt en vigtighed i at kunne omarranger overfladelaget under elektrometanogenese. Overfladelaget er tykt og består af exopolysakkerider (methanochondroitin) og er antaget til at kunne bidrage til EET gennem sekvestrering af redox-aktive metaller og co-faktorer. I det følgende manuskript udforsker vi metallomet for M. barkeri under elektrometanogenese sammenlignet med metallomet under acetoklastisk metanogenese. Metallomet under elektrometanogenese-forhold viste signifikant bioakkumulering af metal-ioner. Vi undersøgte den sub-cellulære lokalisering af disse metaller via røntgenspektroskopi samt synkrotron nano-XRF, som viste at nogle af disse metaller er indlejret i methanochondroitin-laget hos cellerne. For at fremme elektrometanogenese i en bioteknologisk kontekst, er vi nødt til at stimulerer en aggregering af Methanosarcina på katoder og undersøge hvordan meget aggregerede Methanosarcina påvirker elektrometanogenese.I det sidste kapitel, sammenligner vi arter inden for slægten Methanosarcina, som før er blevet adskilt fra hindanen ud fra deres stofskifte. Vi viser her at flere forskelle i celleoverfladens komponenter også kan informere om deres EET-strategi under interaktion med ikke-opløste elektron donerer. Denne afhandling giver en indsigt i hvordan M. barkeri integrerer overfladestrukturer, flavoproteiner og metal co-faktorer til at opnå et cytochrome-frit elektron optagelse og således fremme feltet indenfor bioelektrokemi hos arkæer.Methanosarcina barkeri is a versatile and resilient methanogen that utilizes a variety of electron donors, as simple as electrons. Extracellular Electron Transfer (EET) is widespread, making way for various interactions between the environment and microbial communities. While M. barkeri is just another microbe that can perform EET, it is not just another microbe regarding how it performs EET. M. barkeri uses an unknown non-canonical EET mechanism, which inspired this project. Interactions between microbes and the environment take place at the interface. So, the focus point was to search the surface for electroactive conduits that allow M. barkeri to pick up extracellular electrons. Global proteomics of M. barkeri using a cathodic surface as an electron donor to drive electromethanogenesis revealed uniquely expressed flavin-related proteins, accompanied by metal and ion transporters and surface layer rearranging proteins. This points towards a flavin-based extracellular electron transfer (FLEET) mechanism, that has never been described before in archaea and hints at the importance of rearranging the surface layer during electromethanogenesis. The surface layer is a thick exopolysaccharide (methanochondroitin), hypothesized to contribute to EET by sequestering redox-active metals and cofactors. Therefore, in the following manuscript, we investigated the metallome of M. barkeri during electromethanogenesis as compared to acetoclastic methanogenesis. The metallome under electromethanogenesis-conditions showed significant bioaccumulation of metal ions. We examined the subcellular localization of these metals via energy-dispersive X-ray and synchrotron nano-XRF and showed that some of these metals, are embedded within the methanochondroitin layer. To promote electromethanogenesis for biotechnological applications, we stimulated the aggregation of Methanosarcina on cathodes and examined the effect of highly aggregated Methanosarcina on electromethanogenesis. In a final chapter, we compared species within the genus Methanosarcina, which have been previously distinguished by their energy metabolism. We demonstrate that additional differences in surface features may also inform their EET strategy when interacting with insoluble electron donors. Together, this thesis provides insights into how M. barkeri integrates surface architecture, flavoproteins and metal cofactors to achieve cytochrome-free electron uptake, thereby advancing the field of archaeal bioelectrochemistry
Microbial Adaptations to Pulses of Organic Matter in the Deep-Sea
No full textDen biologiske kulstofpumpe i havet henviser til eksporten af partikulært organisk materiale (POM)fra overfladen til dybere vandlag, en proces der bidrager til langvarig kulstoflagring i havet. Mikrobiel omdannelse af både partikulært organisk materiale (POM) og opløst organisk materiale(DOM) modulerer effektiviteten af denne pumpe ved at regulere skæbnen for kulstof på tværs af dybder og oceanografiske forhold. I den del af havet der henligger i mørke, særligt i den bathypelagiske zone (1000-4000 m), styres denne omdannelse af organisk stof af et komplekst samspil af miljømæssige faktorer, herunder temperatur, højt hydrostatisk tryk (HHP) og kvaliteten af organiske materiale. På trods af den store betydning af disse processer, er de samlede påvirkninger af disse faktorer på mikrobielt stofskifte og sammensætningen af de mikrobielle samfund dårligt belyst, hvilket begrænser anvendeligheden af globale modeller for kulstofkredsløbet i dybhavet. Denne afhandling undersøger, hvordan disse faktorer påvirker prokaryote mikroorganismers respiration (PR), produktion (PP), væksteffektivitet (PGE) og samfundssammensætning (PCC) over et bredt interval havdybder og oceanografiske forhold. Manuskript I indeholder en systematisk vurdering af hvordan temperatur (0 - 25℃), organisk material sammensætning (primært C:N-forholdet), og substratkompleksitet påvirker det mikrobielle stofskifte fra overflade til den bathypelagiske zone. Alanin (C:N = 3:1) understøttede den højeste PGE, mens glukose (Ingen N) stimulerede den mikrobielle respirationen. Q10 værdier varierede fra1,9-2,9, hvor de mikrobielle samfund i de dybere zoner viste større temperaturfølsomhed. Sekventering af 16S rRNA afslørede at forskydninger PCC (ændringer sammensætningen af i de mikrobielle samfund) afspejlede de metaboliske ændringer med dybden, og dybe samfund viste den mest udtalte taksonomiske omstrukturering med stigende temperatur. Dette indikerer en tæt kobling mellem metabolisme og samfundssammensætning i dybhavet. Disse resultater antyder, at selv bathypelagiske mikrober responderer på temperaturændringer, og er reguleret af OM kvalitet. Manuskript II behandlede, hvordan HHP og OM kompleksitet modulerer PR, PP, PGE, og PCC i dybhav (2000 - 4000 m) i forhold til i lavvandede (20 m) mikrobielle samfund. Overfladesamfund viste stærk trykfølsomhed, med næsten fuldstændig hæmning af PP og endda celledød ved tryk≥32,5 MPa. Forsøg med at genoprette den mikrobielle aktivitet efter trykhæmning, der involverede tryksænkning for at måle genopretning i metaboliske hastigheder ved atmosfærisk tryk efter HHP eksponering, bekræftede at denne trykinducerede undertrykkelse i overflademikrober ved tryk≥32,5 var irreversibel. I modsætning til dette, opretholdt dybhavssamfund deres metabolisk aktivitet under in-situ trykforhold, især når de blev forsynet med det komplekse polymere substrat chitin. Disse resultater viser hvordan det komplekse samspil af miljøfaktorer, der er karakteristiske for dybhavet, såsom HHP og tilgængelighed og kvalitet af OM, påvirker de mikrobielle processer. Manuskript III udvidede denne ramme ved at sammenligne effekten af OM kvalitet og HHP på metabolisme og sammensætning af mikrobielle samfund på to lokaliteter i den vestlige Nordatlanten med meget forskellige produktionsforhold (oligotrof gyre vs næringsrige lokalitet) på tværs af en vertikal dybdegradient med 6 dybder fra 10 m til 4000 m. Påvirkningen af de mikrobielle reaktioner varierede med OM-type (tilsætning af koncentrerede partikler vs POC og DOC), og tilsætningen af chitin understøttede en højere PR og PP under in-situ tryk, hvilket er i overensstemmelse med resultaterne fra Manuskript II. Kulstofremineralisering integreret overdybdeintervallet 0-4000 m var cirka 25 % lavere under in situ-tryk sammenlignet med atmosfærisktryk, hvor den næringsrige station havde omkring 1,4 gange højere rater end den oligotrofe gyre. Disse observationer fremhæver den stærke indflydelse af regionale biogeokemiske forhold på mikrobielt stofskifte og afslører, at biomasseproduktion respondere mere end respiration på de lokale miljømæssige variationer. Tilsammen demonstrerer disse opdagelser, at mikrobielkulstofomsætning i oceanets indre er stærkt afhængig af den lokale fysisk-kemiske forhold. Effekterne af tryk og temperatur moduleres af OM-kvalitet, med implikationer for modellering af kulstoffluxe. Denne afhandling demonstrerer behovet for undersøgelser betydningen af tryk, stedsspecifikke forhold og substratsammensætning for at opnå en mere detaljeret forståelse af hvordan den mikrobielle metabolisme reguleres og hvilken rolle de spiller in det globale kulstofkredsløb.The biological carbon pump of the ocean refers to the export of particulate organic matter (POM)from surface to depth, a process that contributes to long-term carbon sequestration in the ocean. Microbial transformation of both particulate organic matter (POM) and dissolved organic matter(DOM) modulates the efficiency of this pump by regulating the fate of carbon across depths and oceanographic settings. In the dark ocean, particularly the bathypelagic zone (1000-4000 m), these transformations are governed by a complex interplay of environmental factors including temperature, high hydrostatic pressure (HHP), and organic matter (OM) quality. Yet, the combined effects of these variables on microbial metabolism and community composition remain poorly resolved, limiting predictive models of deep-ocean carbon cycling. This thesis investigates how these factors shape prokaryotic respiration (PR), production (PP), growth efficiency (PGE), and community composition (PCC) across a range of depths and oceanographic settings. Manuscript Isystematically assessed how temperature (0 - 25℃), OM stoichiometry (C:N), and substrate complexity influence microbial metabolism from surface to bathypelagic waters. Alanine (C:N =3:1) supported the highest PGE, while glucose (N-poor) preferentially stimulated respiration. Q10values ranged from 1.9-2.9, with deeper communities exhibiting greater temperature sensitivity. Crucially, 16S rRNA sequencing revealed that PCC shifts mirrored metabolic trends, and deep communities showed the most pronounced taxonomic restructuring with increasing temperature,indicating tighter coupling between metabolism and community turnover at depth. These resultssuggest that even bathypelagic microbes retain temperature-responsive traits, modulated by OMquality. Manuscript II addressed how HHP and OM complexity modulate PR, PP, PGE and PCC indeep-sea (2000 - 4000 m) vs shallow (20 m) microbial communities. Surface communities exhibited strong pressure sensitivity, with near-complete suppression of PP and even cell-death at pressures≥32.5 MPa. Recovery experiments which involved pressurizing surface microbes followed by depressurization to measure recovery in metabolic rates at atmospheric pressure confirmed their reversibility of this pressure-induced suppression in surface microbes at pressures ≥32.5. In contrast, deep-sea communities maintained metabolic activity under in-situ pressure conditions, particularly when supplied with the complex polymeric substrate chitin. These results show the complex interplay of environmental factors characteristic of the deep-sea such as HHP and OM availability and quality in shaping microbial responses. Manuscript III expanded this framework by comparing metabolic and community composition responses to OM quality and HHP at two contrasting sites in the Western North Atlantic (oligotrophic gyre vs nutrient-rich boundary current) across a vertical depth gradient with 6 depths from 10 m to 4000 m. Microbial responses varied with OM type (concentrated particles vs POC and DOC), and chitin addition supported higher PR and PP under in-situ pressures, consistent with the results from Manuscript II. Carbonremineralisation integrated over the 0-4000 m depth range was approximately 25% lower under insitu pressure compared to atmospheric conditions, with the nutrient-rich station showing rates about1.4 times higher than those in the oligotrophic gyre. These findings highlight the strong influence of regional biogeochemical conditions on microbial metabolism and reveal that biomass production responds far more dramatically than respiration to local environmental context. Together, these findings demonstrate that microbial carbon processing in the ocean interior is highly contingent upon local physicochemical context. The effects of pressure and temperature are modulated by OMquality, with implications for carbon flux modeling. This thesis demonstrates the need for pressureaware, site-specific and substrate-specific studies to refine our understanding of microbial dynamics and their role in the biological carbon pump
Investigation of cell surface biology and biophysics of a methanogenic Archaea capable of extracellular electron uptake
No full textMethanosarcina barkeri er en alsidig og modstandsdygtig metanogen, som benytter en variation a elektron donerer, så simpelt som elektroner selv, til at drive dens stofskifte. Ekstracellulær elektron overførsel (EET) er vidt udbredt og baner vej for mange interaktioner mellem miljøet og mikrobielle samfund. Selvom M. barkeri er ligesom mange andre mikrober, der kan udføre EET, er M. barkeri speciel i dens måde at udføre EET. M. barkeri benytter en ukendt, ikke-kanonisk EET-mekanisme, hvilket inspirerede dette projekt. EET-Interaktioner finder sted i en grænseflade mellem mikrober og miljøet. Fokusset var derfor at gennemsøge den cellulære overflade af M. barkeri for at finde elektroaktive ledninger, der gør at M. barkeri kan modtage ekstracellulære elektroner. Globale proteomiske studier af M. barkeri, med brug af en katodisk overflade som elektron doner til at drive elektrometanogenese, afslørede unikke udtrykte flavin-relaterede proteiner, som var ledsaget af metal- og ion transporterer samt overfladelags omarrangerings proteiner. Dette indikerer en flavin-basereret ekstracellulær elektron overførsels (FLEET) mekanisme, som aldrig før er blevet beskrevet for arkæer, samt en vigtighed i at kunne omarranger overfladelaget under elektrometanogenese. Overfladelaget er tykt og består af exopolysakkerider (methanochondroitin) og er antaget til at kunne bidrage til EET gennem sekvestrering af redox-aktive metaller og co-faktorer. I det følgende manuskript udforsker vi metallomet for M. barkeri under elektrometanogenese sammenlignet med metallomet under acetoklastisk metanogenese. Metallomet under elektrometanogenese-forhold viste signifikant bioakkumulering af metal-ioner. Vi undersøgte den sub-cellulære lokalisering af disse metaller via røntgenspektroskopi samt synkrotron nano-XRF, som viste at nogle af disse metaller er indlejret i methanochondroitin-laget hos cellerne. For at fremme elektrometanogenese i en bioteknologisk kontekst, er vi nødt til at stimulerer en aggregering af Methanosarcina på katoder og undersøge hvordan meget aggregerede Methanosarcina påvirker elektrometanogenese.I det sidste kapitel, sammenligner vi arter inden for slægten Methanosarcina, som før er blevet adskilt fra hindanen ud fra deres stofskifte. Vi viser her at flere forskelle i celleoverfladens komponenter også kan informere om deres EET-strategi under interaktion med ikke-opløste elektron donerer. Denne afhandling giver en indsigt i hvordan M. barkeri integrerer overfladestrukturer, flavoproteiner og metal co-faktorer til at opnå et cytochrome-frit elektron optagelse og således fremme feltet indenfor bioelektrokemi hos arkæer.Methanosarcina barkeri is a versatile and resilient methanogen that utilizes a variety of electron donors, as simple as electrons. Extracellular Electron Transfer (EET) is widespread, making way for various interactions between the environment and microbial communities. While M. barkeri is just another microbe that can perform EET, it is not just another microbe regarding how it performs EET. M. barkeri uses an unknown non-canonical EET mechanism, which inspired this project. Interactions between microbes and the environment take place at the interface. So, the focus point was to search the surface for electroactive conduits that allow M. barkeri to pick up extracellular electrons. Global proteomics of M. barkeri using a cathodic surface as an electron donor to drive electromethanogenesis revealed uniquely expressed flavin-related proteins, accompanied by metal and ion transporters and surface layer rearranging proteins. This points towards a flavin-based extracellular electron transfer (FLEET) mechanism, that has never been described before in archaea and hints at the importance of rearranging the surface layer during electromethanogenesis. The surface layer is a thick exopolysaccharide (methanochondroitin), hypothesized to contribute to EET by sequestering redox-active metals and cofactors. Therefore, in the following manuscript, we investigated the metallome of M. barkeri during electromethanogenesis as compared to acetoclastic methanogenesis. The metallome under electromethanogenesis-conditions showed significant bioaccumulation of metal ions. We examined the subcellular localization of these metals via energy-dispersive X-ray and synchrotron nano-XRF and showed that some of these metals, are embedded within the methanochondroitin layer. To promote electromethanogenesis for biotechnological applications, we stimulated the aggregation of Methanosarcina on cathodes and examined the effect of highly aggregated Methanosarcina on electromethanogenesis. In a final chapter, we compared species within the genus Methanosarcina, which have been previously distinguished by their energy metabolism. We demonstrate that additional differences in surface features may also inform their EET strategy when interacting with insoluble electron donors. Together, this thesis provides insights into how M. barkeri integrates surface architecture, flavoproteins and metal cofactors to achieve cytochrome-free electron uptake, thereby advancing the field of archaeal bioelectrochemistry
Professionalisation and performance of Airbnb hosts in rural regions
No full textThis paper explores the professionalisation and performance aspects of Airbnb hosts in rural regions in Denmark, Iceland, and Norway. More specifically, based upon the professionalisation of hosts, which represents a proxy for the scale of their entrepreneurial engagement, the host landscape in the rural regions is investigated, resulting in different host profiles, including individual single- and multiple-listing hosts, and small and large tourism companies. The paper subsequently estimates the service quality performance of Airbnb hosts in relation to their professionalisation in rural regions through a u-shaped relationship, with the professionalisation influencing the performance evaluation of the hosts by the users. This twofold empirical analysis amends the extant literature, as it provides both a more nuanced and more comprehensive description of the nature and scale of Airbnb host engagement in rural regions, and points to the vast entrepreneurial opportunities for private households and companies on the platform
Biological invasions as burdens to primary economic sectors
No full textMany human-introduced alien species economically impact industries worldwide. Management prioritisation and coordination efforts towards biological invasions are hampered by a lack of comprehensive quantification of costs to key economic sectors. Here, we quantify and estimate global invasion costs to seven major sectors and unravel the introduction pathways of species causing these costs — focusing mainly on primary economic sectors: agriculture, fisheries and forestry. From 1970 to 2020, costs reported in the InvaCost database as pertaining to Agriculture, Fisheries, and Forestry totaled 1.3 bn, and 365 bn), followed by China (36 bn). We further identified 19 countries highly economically reliant on Agriculture, Fisheries, and Forestry that are experiencing massive economic impacts from biological invasions, especially in the Global South. Based on an extrapolation to fill cost data gaps, we estimated total global costs ranging from at least 5.7–6.5 bn for Fisheries, and 600 bn in impacts has been incurred across Agriculture, Fisheries and Forestry, with the largest share reported in Agriculture. We further identify 19 countries, which rely heavily on primary sectors, facing comparatively high impacts from invasions, requiring urgent action. However, gaps in cost reporting across invasive taxa and countries suggest that these impacts are grossly underestimated. Proactive prioritisation by policymakers is needed to mitigate future impacts to primary sectors.</p
The effects of physical activity interventions on prereading, early word recognition and spelling development in children:A systematic review and meta-analysis
No full textBackground: Reading and spelling skills are important abilities to acquire for later success in school and therefore it is highly relevant to examine strategies of facilitating these academic skills. Physical activity integrated with the learning sessions or physical activity added but not integrated with the learning sessions has been identified as possible strategies. Objective: To identify, categorize and examine the characteristics and effects of interventions investigating the effect of physical activity on children's early reading skills. Design: This systematic review follows the PRISMA guidelines. Studies were separated into two categories based on whether physical activity integrates academic content or not, i.e., 1) low integration interventions or 2) high integration interventions. Additionally, high integration interventions were further subgrouped based on the congruency of the activity (i.e., whether the movement supported the learning content). Low integration interventions were subgrouped based on the complexity of the activity (i.e., if the physical activity was cognitively/coordinatively demanding or not). Study quality was assessed, and effects were summarized narratively for each category. Furthermore, a meta-analysis was conducted to quantify both types of interventions. Only studies involving children with an intervention investigating the long-term effects of physical activity on prereading, word recognition, and spelling skills were included. Data sources: PubMed, Web of Science, PsychINFO, Linguistics and Language Behavior Abstracts, ERIC and SportDiscuss. Results: Twenty-three studies were identified and categorized into two groups (thirteen high integration interventions and ten low integration interventions). Across outcome measures, eight of the thirteen high-integration intervention studies reported statistically significant effects on at least one measure of prereading, word recognition, or spelling skills favoring the intervention. In contrast, only two out of ten studies categorized as low integration interventions reported a statistically significant effect. Multilevel meta-analysis showed an insignificant average random-effects model size of Hedges’ g = 0.03 (95% CI: 0.02, 0.09) for low integration and a significant effect size of g = 0.69 (95% CI: 0.12, 1.26) for high integration interventions. The distinction between low and high complexity within the low integration group, and between levels of congruency in high integration interventions, revealed no significant differences between these subgroups. Conclusions: Results from the present review and meta-analysis suggest that the integration of physical activity into the classroom, using activities with a close temporal connection to the academic task (i.e., high integration interventions), appears to be an effective strategy to improve children's word recognition and spelling skills. Due to the limited number and heterogeneity across studies included these findings call for additional studies to clarify specific characteristics of the physical activity needed to support the process of learning to read and spell for children.</p
Comparison of all types of loop-diuretics for chronic heart failure:A protocol for a systematic review and network meta-analysis
No full textBackground: Patients with chronic heart failure (CHF) experience congestion-related symptoms such as dyspnoea and oedema, both of which are associated with poor outcomes. Loop diuretics are the cornerstone to treat congestion and to maintain euvolemia. While furosemide is the most used loop diuretic, other loop diuretics may exhibit theoretical advantages. We aim to compare all types of loop diuretics for patients with CHF. Methods: This protocol for a systematic review is conducted with guidance from the Cochrane Handbook and the Preferred Reporting Items for Systematic Reviews and Meta-Analysis Protocols. We will include randomised clinical trials (RCTs) of loop diuretics with other loop diuretics, placebo, or standard of care in CHF patients. The search will be conducted across the major medical databases (including Medline, Embase, and Cochrane Central Register of Controlled Trials). The searches will begin in February 2025. The primary outcome will be all-cause mortality. Secondary outcomes will be all-cause hospitalization, serious adverse events (SAEs), and changes in body weight (kg). Data will be analysed by traditional meta-analyses, Trial Sequential Analyses (TSA), and network meta analysis. The risk of bias will be assessed using the Cochrane Risk of Bias tool—version 2. The certainty of the evidence will be assessed using the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) and Confidence in Network Meta-Analysis (CINEMA) approach.</p