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Hämoglobin-A1c (HbA1c) ist ein Hämoglobin (Hb)-Subtypus, der durch nicht-enzymatische Glykierung des N-terminalen Valinrestes der Hämoglobin-beta-Kette entsteht. Das gemessene Verhältnis von HbA1c zum Gesamt-Hämoglobin (5-20 % bei Diabetikern) repräsentiert den Mittelwert der Blutglucosekonzentration über einen zweimonatigen Zeitraum und stellt zur Beurteilung der diabetischen Stoffwechsellage eine Ergänzung zur Akutkontrolle der Glukosekonzentration dar. Ziel der vorliegenden Arbeit war es, einen amperometrischen Biosensor für die Bestimmung des medizinisch relevanten Parameters HbA1c zu entwickeln. Durch Selektion geeigneter Bioerkennungselemente und deren Immobilisierung unter Erhalt der Bindungsfunktion für die Zielmoleküle Hämoglobin bzw. HbA1c wurden spezifische, hochaffine und regenerationsstabile Sensoroberflächen geschaffen. Für die Entwicklung des HbA1c-Biosensors wurden zwei Konzepte - Enzymsensor und Immunosensor - miteinander verglichen. Die enzymatische Umsetzung von HbA1c erfolgte mit der Fructosylamin Oxidase (FAO) aus Pichia pastoris N 1-1 unter Freisetzung von H2O2, welches sowohl optisch über eine Indikatorreaktion als auch elektrochemisch nach Einschluss der FAO in PVA-SbQ und Fixierung des Immobilisats vor einer H2O2-Elektrode nachgewiesen wurde. Die Kalibration des Enzymsensors mit der HbA1c-Modellsubstanz Fructosyl-Valin ergab Nachweisgrenzen, die ausserhalb des physiologisch relevanten HbA1c-Konzentrationsbereich lagen. Aus der Umsetzung von glykierten Peptiden mit einer nicht HbA1c analogen Aminosäurensequenz, z.B. Fructosyl-Valin-Glycin wurde zudem eine geringe HbA1c-Spezifität abgeleitet. Für den Immunosensor wurden zwei heterogene Immunoassay-Formate unter Verwendung von hochaffinen und spezifischen Antikörpern in Kombination mit Glucose Oxidase (GOD) als Markerenzym zum Nachweis von HbA1c untersucht. Beim indirekt-kompetitiven Immunoassay wurde anstelle des kompletten HbA1c-Moleküls das glykierte Pentapeptid Fructosyl-Valin-Histidin-Leucin-Threonin-Prolin (glkPP) als Kompetitor und Affinitätsligand immobilisiert und so eine regenerierfähige Oberfläche geschaffen. Beim Sandwich-Immunoassay wurde im ersten Schritt Gesamt-Hämoglobin an die mit Haptoglobin (Hp) modifizierte Festphase angereichert und im zweiten Schritt der gebundene HbA1c-Anteil nachgewiesen. Für die Konstruktion des HbA1c-Immunosensors wurden Affinitätsmatrizen durch Modifizierung von Cellulose-Dialysemembranen mit glkPP bzw. Hp hergestellt. Grundlegend studiert wurde die Aktivierung der Cellulose-Membranen mit 1,1'-Carbonyldiimidazol (CDI) und 1-Cyano-4-dimethylaminopyridintetrafluoroborat (CDAP) als Aktivierungsagenzien. Eine gerichtete Immobilisierung der Liganden wurde realisiert, indem glkPP über dessen C-Terminus (einzige Carboxylatgruppe) und Hp über dessen periodat-oxidiertem Kohlenhydratrest an die amino- oder hydrazidfunktionalisierte Membranen kovalent gekoppelt wurden. Mit dem Einsatz der glkPP- und Hp-modifizierten Membranen in der elektrochemischen Messzelle war erstmalig der biosensorische Nachweis von HbA1c möglich. Als Transduktor diente eine Pt-Elektrode, an der das von der GOD generierte H2O2 umgesetzt und ein mit der HbA1c-Konzentration korrelierendes Stromsignal erzeugt wurde. Die Immunosensoren zeigten Ansprechzeiten von 3 s. Mit dem Immunosensor auf Basis des indirekt-kompetitiven Testprinzips wurde eine Kalibrationskurve für HbA1c im Bereich von 0,25-30 µg/ml (3,9-465 nM, CV 3-9 %) mit Assayzeiten von 60 min und mit dem Immunosensor im Sandwich-Format eine Kalibrationskurve im Bereich von 0,5-5 µg/ml (7,8-78 nM; 5-50 % HbA1c vom Gesamt-Hb, CV 6-10 %, 3 h) aufgenommen.
Bacteria are one of the most widespread kinds of microorganisms that play essential roles in many biological and ecological processes. Bacteria live either as independent individuals or in organized communities. At the level of single cells, interactions between bacteria, their neighbors, and the surrounding physical and chemical environment are the foundations of microbial processes. Modern microscopy imaging techniques provide attractive and promising means to study the impact of these interactions on the dynamics of bacteria. The aim of this dissertation is to deepen our understanding four fundamental bacterial processes – single-cell motility, chemotaxis, bacterial interactions with environmental constraints, and their communication with neighbors – through a live cell imaging technique. By exploring these processes, we expanded our knowledge on so far unexplained mechanisms of bacterial interactions.
Firstly, we studied the motility of the soil bacterium Pseudomonas putida (P. putida), which swims through flagella propulsion, and has a complex, multi-mode swimming tactic. It was recently reported that P. putida exhibits several distinct swimming modes – the flagella can push and pull the cell body or wrap around it. Using a new combined phase-contrast and fluorescence imaging set-up, the swimming mode (push, pull, or wrapped) of each run phase was automatically recorded, which provided the full swimming statistics of the multi-mode swimmer. Furthermore, the investigation of cell interactions with a solid boundary illustrated an asymmetry for the different swimming modes; in contrast to the push and pull modes, the curvature of runs in wrapped mode was not affected by the solid boundary. This finding suggested that having a multi-mode swimming strategy may provide further versatility to react to environmental constraints.
Then we determined how P. putida navigates toward chemoattractants, i.e. its chemotaxis strategies. We found that individual run modes show distinct chemotactic responses in nutrition gradients. In particular, P. putida cells exhibited an asymmetry in their chemotactic responsiveness; the wrapped mode (slow swimming mode) was affected by the chemoattractant, whereas the push mode (fast swimming mode) was not. These results can be seen as a starting point to understand more complex chemotaxis strategies of multi-mode swimmers going beyond the well-known paradigm of Escherichia coli, that exhibits only one swimming mode.
Finally we considered the cell dynamics in a dense population. Besides physical interactions with their neighbors, cells communicate their activities and orchestrate their population behaviors via quorum-sensing. Molecules that are secreted to the surrounding by the bacterial cells, act as signals and regulate the cell population behaviour. We studied P. putida’s motility in a dense population by exposing the cells to environments with different concentrations of chemical signals. We found that higher amounts of chemical signals in the surrounding influenced the single-cell behaviourr, suggesting that cell-cell communications may also affect the flagellar dynamics.
In summary, this dissertation studies the dynamics of a bacterium with a multi-mode swimming tactic and how it is affected by the surrounding environment using microscopy imaging. The detailed description of the bacterial motility in fundamental bacterial processes can provide new insights into the ecology of microorganisms.
Moss-microbe associations are often characterised by syntrophic interactions between the microorganisms and their hosts, but the structure of the microbial consortia and their role in peatland development remain unknown.
In order to study microbial communities of dominant peatland mosses, Sphagnum and brown mosses, and the respective environmental drivers, four study sites representing different successional stages of natural northern peatlands were chosen on a large geographical scale: two brown moss-dominated, circumneutral peatlands from the Arctic and two Sphagnum-dominated, acidic peat bogs from subarctic and temperate zones.
The family Acetobacteraceae represented the dominant bacterial taxon of Sphagnum mosses from various geographical origins and displayed an integral part of the moss core community. This core community was shared among all investigated bryophytes and consisted of few but highly abundant prokaryotes, of which many appear as endophytes of Sphagnum mosses. Moreover, brown mosses and Sphagnum mosses represent habitats for archaea which were not studied in association with peatland mosses so far. Euryarchaeota that are capable of methane production (methanogens) displayed the majority of the moss-associated archaeal communities. Moss-associated methanogenesis was detected for the first time, but it was mostly negligible under laboratory conditions. Contrarily, substantial moss-associated methane oxidation was measured on both, brown mosses and Sphagnum mosses, supporting that methanotrophic bacteria as part of the moss microbiome may contribute to the reduction of methane emissions from pristine and rewetted peatlands of the northern hemisphere.
Among the investigated abiotic and biotic environmental parameters, the peatland type and the host moss taxon were identified to have a major impact on the structure of moss-associated bacterial communities, contrarily to archaeal communities whose structures were similar among the investigated bryophytes. For the first time it was shown that different bog development stages harbour distinct bacterial communities, while at the same time a small core community is shared among all investigated bryophytes independent of geography and peatland type.
The present thesis displays the first large-scale, systematic assessment of bacterial and archaeal communities associated both with brown mosses and Sphagnum mosses. It suggests that some host-specific moss taxa have the potential to play a key role in host moss establishment and peatland development.
In der vorliegenden Arbeit wurden cDNAs, kodierend für bisher unbekannte stärkeabbauende Enzyme, aus Kartoffel isoliert und funktionell analysiert. Die Isolation der cDNAs erfolgte mit Hilfe eines Systems, welches sich der funktionellen Expression von cDNA-Bibliotheken in E. coli bediente. Die mit diesem System zur Expression gebrachten cDNA-Bibliotheken wurden im Rahmen dieser Arbeit hergestellt. Zum einen handelte es sich um eine blattspezifische Phagen-cDNA-Bibliothek (Proben wurden während des Tag/Nacht Übergangs genommen), zum anderen um eine knollenspezifische cDNA-Bibliothek aus kaltgelagerten Knollen. Nach der Überführung der Phagen-Bibliotheken in Plasmid-Bibliotheken wurden diese funktionell in dem E. coli Stamm KV832 exprimiert. Der Stamm KV832 wurde aufgrund seiner Fähigkeit, lineare Glucane zu akkumulieren, ausgewählt. Werden Glucan akkumulierende KV832 Kolonien mit Jod bedampft, so zeigen diese eine typische Blaufärbung. Nach der Expression der Plasmid-Bibliotheken in KV832 wurden solche Kolonien weiter untersucht, welche in ihrer Färbung von den blauen Kolonien abwichen. Mittels eines zweiten E. coli Stamms, PGM −, welcher ebenfalls in der Lage ist, lineare Glucane zu akkumulieren, wurden die Ergebnisse für KV832 bestätigt. Die funktionelle Expression der Bibliotheken führte zur Isolation einer Reihe von unbekannten cDNAs. Zwei dieser cDNAs wurden im Rahmen dieser Arbeit weiterführend untersucht. Zum einen handelte es sich um eine cDNA, die für eine bis dahin unbekannte β-Amylase aus Kartoffel kodierte und deren Homolog aus Arabidopsis (CT-BMY) im Laufe dieser Arbeit von Lao et al. (1999) veröffentlicht wurde, zum anderen um eine cDNA, die für ein unbekanntes Enzym kodierte (DSD10). Das Arabidopsis Homolog zu DSD10 wurde im Zuge der Arabidopsis Genominitiative Ende 2000 publiziert. Im Rahmen dieser Arbeit konnte gezeigt werden, dass die isolierte β-Amylase cDNA für eine funktionelle β-Amylase kodiert und dieses Enzym in der Lage ist, neben löslicher auch rohe Stärke anzugreifen. Lokalisationsexperimente zeigten, dass das Enzym in isolierte Erbsenchloroplasten importiert wurde und dass die 100 N-terminalen Aminosäuren für den Import in die Plastiden ausreichten. Die β-Amylase wurde als PCT-BMYI bezeichnet. Die »antisense«-Inhibierung von PCT-BMYI führte zu einem Hochstärke-Phänotyp der Blätter, sowie zu einem Anstieg der Trockenmasse. Der Hochstärke-Phänotyp ist auf eine Reduktion der Stärkemobilisierung und die daraus folgende Akkumulation der Stärke während der Vegetationsperiode zurückzuführen. Damit konnte erstmals die physiologische Bedeutung einer β-Amylase für den Abbau der transitorischen Stärke gezeigt werden. Kein Einfluss zeigte die »antisense« Inhibierung von PCT-BMYI auf den kälteinduzierten Abbau der Speicherstärke in Knollen. Es konnte auch kein Unterschied im Keimverhalten oder der Entwicklung der neuen Pflanze beobachtet werden. Ein Teil der Ergebnisse zu PCT-BMYI wurde bereits publiziert (Scheidig et al., 2002). Die isolierten cDNAs dsd10, sgeI (die Volllängen cDNA zu dsd10) und das Arabidopsis Homolog asgeI kodieren für Enzyme, welche α-Amylase-Aktivität besitzen, aber keine Homologie zu bekannten α-Amylasen aufweisen. Ein mögliches Glucoamylase Motiv erwies sich für die Aktivität des Proteins als essentiell. Lokalisationsexperimente deuteten auf den Import des SGEI Proteins in isolierte Erbsenchloroplasten hin. Die »antisense«-Inhibierung von sgeI führte in den entsprechenden Linien zu einem Hochstärke-Phänotyp in Blättern, einem Anstieg der Trockenmasse in Blättern, sowie zu größeren Stärkekörnern in einer der untersuchten Linien. Ein nicht erwarteter Effekt zeigte sich in Blättern der entsprechenden Linien, welche für längere Zeit dunkel gehalten wurden. Die Blätter der untransformierten Kontrolle waren abgestorben, wohingegen die Blätter der SGEI »antisense« Linien grün und vital erschienen. Die α- und β-Amylase-Aktivität war in Blättern der SGEI »antisense« Linien reduziert, weshalb eine genaue Zuordnung der Funktion von SGEI nicht möglich war. Die vorliegenden Ergebnisse zu den SGEI »antisense« Linien deuten aber darauf hin, dass der beobachtete Hochstärke-Phänotyp nicht alleine auf die Reduktion der β-Amylase-Aktivität zurückzuführen ist. Ein Einfluss von SGEI auf den kälteinduzierten Abbau der Speicherstärke konnte nicht beobachtet werden. Es konnte auch hier kein Unterschied im Keimverhalten oder der Entwicklung der neuen Pflanze beobachtet werden.
Die Honigbiene Apis mellifera gilt seit langem als Modell-Organismus zur Untersuchung von Lern- und Gedächtnisvorgängen sowie zum Studium des Sozialverhaltens und der Arbeitsteilung. Bei der Steuerung und Regulation dieser Verhaltensweisen spielt das Indolalkylamin Serotonin eine wesentliche Rolle. Serotonin entfaltet seine Wirkung durch die Bindung an G-Protein-gekoppelte Rezeptoren (GPCRs). In der vorliegenden Arbeit wird der erste Serotonin-Rezeptor aus der Honigbiene molekular charakterisiert. Durch die Anwendung zwei verschiedener Klonierungsstrategien konnten drei cDNA-Sequenzen isoliert werden, die für potentielle Serotonin-Rezeptoren kodieren. Die Sequenzen weisen die größte Ähnlichkeit zu dem 5-HT7- und 5-HT2-Rezeptor von Drosophila melanogaster bzw. dem 5-HT1-Rezeptor von Panulirus interruptus auf. Die isolierten Serotonin-Rezeptoren der Honigbiene wurden dementsprechend Am(Apis mellifera)5-HT1, Am5-HT2 und Am5-HT7 benannt. Das Hydropathieprofil des Am5-HT1-, Am5-HT2- und Am5-HT7-Rezeptors deutet auf das Vorhandensein des charakteristischen heptahelikalen Aufbaus G-Protein-gekoppelter Rezeptoren hin. Die abgeleiteten Aminosäuresequenzen zeigen typische Merkmale biogener Amin-Rezeptoren. Aminosäuren, die eine Bedeutung bei der Bildung der Liganden-Bindungstasche, der Rezeptor-Aktivierung und der Kopplung eines G-Proteins an den Rezeptor haben, sind in allen drei Rezeptoren konserviert. Interessanterweise ist jedoch das in den meisten biogenen Amin-Rezeptoren vorhandene DRY-Motiv in dem Am5-HT2- und Am5-HT7-Rezeptor nicht konserviert. Das Vorhandensein einer PDZ-Domäne in dem Am5-HT1- und Am5-HT7-Rezeptor lässt vermuten, dass diese Rezeptoren als Adapterproteine fungieren, die Signalmoleküle zu einem Signaltransduktionskomplex vereinigen. RT-PCR-Experimente zeigen die Expression der Rezeptoren in verschiedenen Geweben der Honigbiene. Auffallend ist die hohe Expression im Zentralgehirn. Des Weiteren konnte die Expression der Serotonin-Rezeptoren in den optischen Loben, Antennalloben sowie in der Peripherie, d.h. in der Flugmuskulatur und den Malpighischen Gefäßen nachgewiesen werden. Durch in situ Hybridisierungen wurde die Expression in Gefrierschnitten von Gehirnen adulter Sammlerinnen im Detail untersucht. Transkripte der Rezeptoren sind in den Somata von intrinsischen Pilzkörperzellen, Neuronen der optischen Loben und Neuronen der Antennalloben vorhanden. In einem heterologen Expressionssystem wurde der intrazelluläre Signalweg des Am5-HT7-Rezeptors untersucht. Die Aktivierung des stabil exprimierten Rezeptors durch Serotonin führt zur Bildung von cAMP. Der 5-HT7-Rezeptor spezifische Agonist 5-CT zeigt eine mit Serotonin vergleichbare Fähigkeit, die intrazelluläre cAMP-Konzentration zu erhöhen. Am5-HT7 gehört daher funktionell zu der Gruppe der 5-HT7-Rezeptoren. Der EC50-Wert von 1,06~nM (5-HT), ist im Vergleich zu anderen 5-HT7-Rezeptoren äußert niedrig. Des Weiteren wurde gezeigt, dass das basale cAMP-Niveau in den transfizierten Zellen im Vergleich zu nicht transfizierten Zellen deutlich erhöht ist. Das heißt, dass der Rezeptor auch in der Abwesenheit eines Liganden aktiv ist. Diese konstitutive Aktivität ist auch von anderen biogenen Amin-Rezeptoren bekannt. Methiothepin wurde als wirksamer inverser Agonist des Am5-HT7-Rezeptors identifiziert, da es in der Lage ist, der konstitutiven Aktivität entgegenzuwirken. Die Ergebnisse der vorliegenden Arbeit deuten darauf hin, dass die Serotonin-Rezeptoren in verschiedenen Regionen des ZNS der Honigbiene an der Informationsverarbeitung beteiligt sind. Es kann eine Beeinflussung von Lern- und Gedächtnisprozessen sowie des olfaktorischen und visuellen Systems durch diese Rezeptoren vermutet werden. Mit der Klonierung und funktionellen Charakterisierung des ersten Serotonin-Rezeptors der Honigbiene ist eine Grundlage für die Untersuchung der molekularen Mechanismen der serotonergen Signaltransduktion geschaffen worden.
Der Bittergeschmack dient Säugern vermutlich zur Wahrnehmung und Vermeidung toxischer Substanzen. Bitterstoffe können jedoch auch gesund sein oder werden oft bereitwillig mit der Nahrung aufgenommen. Ob sie geschmacklich unterschieden werden können, ist allerdings umstritten. Detektiert werden Bitterstoffe von oralen Bittergeschmacksrezeptoren, den TAS2R (human) bzw. Tas2r (murin). In der Literatur gibt es aber immer mehr Hinweise darauf, dass überdies Tas2r nicht nur in extragustatorischen Organen exprimiert werden, sondern dort auch wichtige Aufgaben erfüllen könnten, was wiederum die Aufklärung ihrer noch nicht vollständig entschlüsselten Funktionsweisen erfordert. So ist noch unbekannt, ob alle bisher als funktionell identifizierten Tas2r wirklich gustatorische Funktionen erfüllen.
Im Rahmen der Charakterisierung neu generierter, im Locus des Bittergeschmacksrezeptors Tas2r131 genetisch modifizierter Mauslinien, wurde in vorliegender Arbeit die gustatorische sowie extragustatorische Expression von Tas2r131 untersucht. Dass Tas2r131 nicht nur in Pilzpapillen, Wall- und Blätterpapillen (VP+FoP), Gaumen, Ductus nasopalatinus, Vomeronasalorgan und Kehldeckel, sondern auch in Thymus, Testes und Nebenhodenkopf, in Gehirnarealen sowie im Ganglion geniculatum nachgewiesen wurde, bildete die Grundlage für weiterführende Studien. Die vorliegende Arbeit zeigt außerdem, dass Tas2r108, Tas2r126, Tas2r135, Tas2r137 und Tas2r143 in Blut exprimiert werden, was auf eine heterogene Funktion der Tas2r hindeutet. Dass zusätzlich erstmals die Expression aller 35 als funktionell beschriebenen Tas2r im gustatorischen VP+FoP-Epithel von C57BL/6-Mäusen nachgewiesen wurde, verweist auf deren Relevanz als funktionelle Geschmacksrezeptoren.
Weiter zeigten Untersuchungen zur Aufklärung eines möglichen Bitter-Unterscheidungsvermögens in Geschmackspapillen von Mäusen mit fluoreszenzmarkierten oder ablatierten Tas2r131-Zellen, dass Tas2r131 exprimierende Zellen eine Tas2r-Zellsubpopulation bilden. Darüber hinaus existieren innerhalb der Bitterzellen geordnete Tas2r-Expressionsmuster, die sich nach der chromosomalen Lage ihrer Gene richten. Isolierte Bitterzellen reagieren heterogen auf bekannte Bitterstoffe. Und Mäuse mit ablatierter Tas2r131-Zellpopulation besitzen noch andere Tas2r-Zellen und schmecken damit einige Bitterstoffe kaum noch, andere aber noch sehr gut. Diese Befunde belegen die Existenz verschiedener gustatorischer Tas2r-Zellpopulationen, welche die Voraussetzung bilden, Bitterstoffe heterogen zu detektieren. Ob dies die Grundlage für ein divergierendes Verhalten gegenüber unverträglichen und harmlosen oder gar nützlichen Bitterstoffen darstellt, kann mit Hilfe der dargelegten Tas2r-Expressionsmuster künftig in Verhaltensexperimenten geprüft werden.
Die Bittergeschmackswahrnehmung in Säugetieren stellt sich als ein hochkomplexer Mechanismus dar, dessen Vielschichtigkeit durch die hier neu aufgezeigten heterogenen Tas2r-Expressions- und Funktionsmuster erneut verdeutlicht wird.
Lamine bilden zusammen mit laminassoziierten Proteinen die nukleäre Lamina. Diese ist notwendig für die mechanische Stabilität von Zellen, die Organisation des Chromatins, der Genexpression, dem Fortgang des Zellzyklus und der Zellmigration. Die vielfältigen Funktionen der Lamine werden durch die Pathogenese von Laminopathien belegt. Zu diesen Erkrankungen, welche ihre Ursache in Mutationen innerhalb der laminkodierenden Gene, oder der Gene laminassoziierter bzw. laminprozessierender Proteine haben, zählen unter anderem das „Hutchinson-Gilford Progerie Syndrom“, die „Emery-Dreifuss“ Muskeldystrophie und die dilatierte Kardiomyopathie. Trotz der fundamentalen Bedeutung der Lamine, wurden diese bisher nur in Metazoen und nicht in einzelligen Organismen detektiert. Der amöbide Organismus Dictyostelium discoideum ist ein haploider Eukaryot, der häufig als Modellorganismus in den verschiedensten Bereichen der Zellbiologie eingesetzt wird. Mit der Entdeckung von NE81, einem Protein das mit der inneren Kernhülle von Dictyostelium discoideum assoziiert ist, wurde erstmals ein Protein identifiziert, dass man aufgrund seiner Eigenschaften als laminähnliches Protein in einem niederen Eukaryoten bezeichnen kann. Diese Merkmale umfassen die Existenz lamintypischer Sequenzen, wie die CDK1-Phosphorylierungsstelle, direkt gefolgt von einer zentralen „Rod“-Domäne, sowie eine typische NLS und die hoch konservierte CaaX-Box. Für die Etablierung des NE81 als „primitives“ Lamin, wurden im Rahmen dieser Arbeit verschiedene Experimente durchgeführt, die strukturelle und funktionelle Gemeinsamkeiten zu den Laminen in anderen Organismen aufzeigen konnten. Die Herstellung eines polyklonalen Antikörpers ermöglichte die Verifizierung der subzellulären Lokalisation des NE81 durch Elektronenmikroskopie und gab Einblicke in das Verhalten des endogenen Proteins innerhalb des Zellzyklus. Mit der Generierung von NE81-Nullmutanten konnte demonstriert werden, dass NE81 eine wichtige Rolle bei der nukleären Integrität und der Chromatinorganisation von Zellen spielt. Des Weiteren führte die Expression von zwei CaaX-Box deletierten NE81 - Varianten dazu, den Einfluss des Proteins auf die mechanische Stabilität der Zellen nachweisen zu können. Auch die Bedeutung der hochkonservierten CaaX-Box für die Lokalisation des Proteins wurde durch die erhaltenen Ergebnisse deutlich. Mit der Durchführung von FRAP-Experimente konnte außerdem die strukturgebende Funktion von NE81 innerhalb des Zellkerns bekräftigt werden. Zusätzlich wurde im Rahmen dieser Arbeit damit begonnen, den Einfluss der Isoprenylcysteincarboxylmethyltransferase auf die Lokalisation des Proteins aufzuklären. Die Entdeckung eines laminähnlichen Proteins in einem einzelligen Organismus, der an der Schwelle zu den Metazoen steht, ist für die evolutionäre Betrachtung der Entwicklung der sozialen Amöbe und für die Erforschung der molekularen Basis von Laminopathien in einem einfachen Modellorganismus sehr interessant. Die Arbeit mit Dictyostelium discoideum könnte daher Wege aufzeigen, dass Studium der Laminopathien am Tiermodell drastisch zu reduzieren. In den letzten Jahren hat die Erforschung unbekannter Bestandteile des Centrosoms in Dictyostelium discoideum große Fortschritte gemacht. Eine zu diesem Zwecke von unserer Arbeitsgruppe durchgeführte Proteomstudie, führte zur Identifizierung weiterer, potentiell centrosomaler Kandidatenproteine. Der zweite Teil dieser Arbeit beschäftigt sich mit der Charakterisierung eines solchen Kandidatenproteins, dem CP75. Es konnte gezeigt werden, dass CP75 einen echten, centrosomalen Bestandteil darstellt, der mikrotubuli-unabhängig mit der Core Struktur des Zellorganells assoziiert ist. Weiterhin wurde deutlich, dass die Lokalisation am Centrosom in Abhängigkeit vom Zellzyklus erfolgt und CP75 vermutlich mit CP39, einem weiteren centrosomalen Core Protein, interagiert.
Molekulare Charakterisierung von CP75, einem neuen centrosomalen Protein in Dictyostelium discoideum
(2016)
Das Centrosom ist ein Zellkern-assoziiertes Organell, das nicht von einer Membran umschlossen ist. Es spielt eine wichtige Rolle in vielen Mikrotubuli- abhängigen Prozessen wie Organellenpositionierung, Zellpolarität oder die Organisation der mitotischen Spindel. Das Centrosom von Dictyostelium besteht aus einer dreischichtigen Core-Struktur umgeben von einer Corona, die Mikrotubuli-nukleierende Komplexe enthält. Die Verdoppelung des Centrosoms in Dictyostelium findet zu Beginn der Mitose statt. In der Prophase vergrößert sich die geschichtete Core-Struktur und die Corona löst sich auf. Anschließend trennen sich die beiden äußeren Lagen der Core-Struktur und bilden in der Metaphase die beiden Spindelpole, die in der Telophase zu zwei vollständigen Centrosomen heranreifen. Das durch eine Proteom-Analyse identifizierte Protein CP75 lokalisiert am Centrosom abhängig von den Mitosephasen. Es dissoziiert von der Core-Struktur in der Prometaphase und erscheint an den Spindelpolen in der Telophase wieder. Dieses Verhalten korreliert mit dem Verhalten der mittleren Lage der Core-Struktur in der Mitose, was darauf hinweist, dass CP75 eine Komponente dieser Schicht sein könnte. Die FRAP-Experimente am Interphase- Centrosom zeigen, dass GFP-CP75 dort nicht mobil ist. Das deutet darauf hin, dass das Protein wichtige Funktionen im Strukturerhalt der centrosomalen Core- Struktur übernehmen könnte. Sowohl die C- als auch die N-terminale Domäne von CP75 enthalten centrosomale Targeting-Domäne. Als GFP-Fusionsproteine (GFP-CP75-N und -C) lokalisieren die beiden Fragmente am Centrosom in der Interphase. Während GFP-CP75-C in der Mitose am Centrosom verbleibt, verschwindet GFP-CP75-N in der Metaphase und kehrt erst in der späten Telophase zurück. GFP-CP75-C und GFP-CP75O/E kolokalisieren mit F-Aktin am Zellcortex, zeigen aber keine Interaktion mit Aktin mit der BioID-Methode. Die N-terminale Domäne von CP75 enthält eine potentielle Plk1- Phosphorylierungssequenz. Die Überexpression der nichtphosphorylierbaren Punktmutante (GFP-CP75-Plk-S143A) ruft verschiedene Phänotypen wie verlängerte oder überzählige Centrosomen, vergrößerte Zellkerne und Anreicherung von detyrosinierten Mikrotubuli hervor. Die ähnlichen Phänotypen konnten auch bei GFP-CP75-N und CP75-RNAi beobachtet werden. Der
Phänotyp der detyrosinierten Mikrotubuli bringt erstmals den Beweis dafür, dass I
in Dictyostelium posttranslationale Modifikation an Tubulinen stattfindet. Außerdem zeigten CP75-RNAi-Zellen Defekte in der Organisation der mitotischen Spindel. Mittels BioID-Methode konnten drei potentielle Interaktionspartner von CP75 identifiziert werden. Diese drei Proteine CP39, CP91 und Cep192 sind ebenfalls Bestandteile des Centrosoms.
Molekulare Charakterisierung des Centrosom-assoziierten Proteins CP91 in Dictyostelium discoideum
(2016)
Das Dictyostelium-Centrosom ist ein Modell für acentrioläre Centrosomen. Es besteht aus einer dreischichtigen Kernstruktur und ist von einer Corona umgeben, welche Nukleationskomplexe für Mikrotubuli beinhaltet. Die Verdoppelung der Kernstruktur wird einmal pro Zellzyklus am Übergang der G2 zur M-Phase gestartet. Durch eine Proteomanalyse isolierter Centrosomen konnte CP91 identifiziert werden, ein 91 kDa großes Coiled-Coil Protein, das in der centrosomalen Kernstruktur lokalisiert. GFP-CP91 zeigte fast keine Mobilität in FRAP-Experimenten während der Interphase, was darauf hindeutet, dass es sich bei CP91 um eine Strukturkomponente des Centrosoms handelt. In der Mitose hingegen dissoziieren das GFP-CP91 als auch das endogene CP91 ab und fehlen an den Spindelpolen von der späten Prophase bis zur Anaphase. Dieses Verhalten korreliert mit dem Verschwinden der zentralen Schicht der Kernstruktur zu Beginn der Centrosomenverdopplung. Somit ist CP91 mit großer Wahrscheinlichkeit ein Bestandteil dieser Schicht. CP91-Fragmente der N-terminalen bzw. C-terminalen Domäne (GFP-CP91 N-Terminus, GFP-CP91 C-Terminus) lokalisieren als GFP-Fusionsproteine exprimiert auch am Centrosom, zeigen aber nicht die gleiche mitotische Verteilung des Volllängenproteins. Das CP91-Fragment der zentralen Coiled-Coil Domäne (GFP-CP91cc) lokalisiert als GFP-Fusionsprotein exprimiert, als ein diffuser cytosolische Cluster, in der Nähe des Centrosoms. Es zeigt eine partiell ähnliche mitotische Verteilung wie das Volllängenprotein. Dies lässt eine regulatorische Domäne innerhalb der Coiled-Coil Domäne vermuten. Die Expression der GFP-Fusionsproteine unterdrückt die Expression des endogenen CP91 und bringt überzählige Centrosomen hervor. Dies war auch eine markante Eigenschaft nach der Unterexpression von CP91 durch RNAi. Zusätzlich zeigte sich in CP91-RNAi Zellen eine stark erhöhte Ploidie verursacht durch schwere Defekte in der Chromosomensegregation verbunden mit einer erhöhten Zellgröße und Defekten im Abschnürungsprozess während der Cytokinese. Die Unterexpression von CP91 durch RNAi hatte auch einen direkten Einfluss auf die Menge an den centrosomalen Proteinen CP39, CP55 und CEP192 und dem Centromerprotein Cenp68 in der Interphase. Die Ergebnisse deuten darauf hin, dass CP91 eine zentrale centrosomale Kernkomponente ist und für den Zusammenhalt der beiden äußeren Schichten der Kernstruktur benötigt wird. Zudem spielt CP91 eine wichtige Rolle für eine ordnungsgemäße Centrosomenbiogenese und, unabhängig davon, bei dem Abschnürungsprozess der Tochterzellen während der Cytokinese.
Environmental stresses such as drought, high salt and low temperature affect plant growth and decrease crop productivity extremely. It is important to improve stress tolerance of the crop plant to increase crop yield under stress conditions. The Arabidopsis thaliana salt tolerance 1 gene (AtSTO1) was originally identified by Lippuner et al., (1996). In this study around 27 members of STO-like proteins were identified in Arabidopsis thaliana, rice and other plant species. The STO proteins have two consensus motifs (CCADEAAL and FCV(L)EDRA). The STO family members can be regarded as a distinct class of C2C2 proteins considering their low sequence similarity to other GATA like proteins and poor conservation in the C-terminus. AtSTO1 was found to be induced by salt, cold and drought in leaves and roots of 4-week-old Arabidopsis thaliana wild-type plants. The expression of AtSTO1 under salt and cold stress was more pronounced in roots than in leaves. The data provided here revealed that the AtSTO1 protein is localized in the nucleus. The observation that AtSTO1 localizes in the nucleus is consistent with its proposed function as a transcription factor. AtSTO1-dependent phenotypes were observed when plant were grown at 50 mM NaCl on agar plates. Leaves of AtSTO1 overexpression lines were bigger with dark green coloration, whereas stunted growth and yellowish leaves were observed in wild-type and RNAi plants. Also, the AtSTO1 overexpression plants when exposed to long-term cold stress had a red leaf coloration which was much stronger than in wild-type and RNAi lines. Growth of AtSTO1 overexpression lines in long term under salt and cold stress was always associated with long roots which was more pronounced than in wild-type and RNAi lines. Proline accumulation increased more strongly in leaves and roots of AtSTO1 overexpression lines than in tissues of wild-type and RNAi lines when treated with 200 mM NaCl, exposed to cold stress or when watering was prevented for one day or two weeks. Also, soluble sugar content increased to higher levels under salt, cold and drought stress in AtSTO1 overexpression lines when compared to wild-type and RNAi lines. The increase in soluble sugar content was detected in AtSTO1 overexpression lines after long-term (2 weeks) growth of plants under these stresses. Anthocyanins accumulated in leaves of AtSTO1 overexpression lines when exposed to long term salt stress (200 mM NaCl for 2 weeks) or to 4°C for 6 and 8 weeks. Also, anthocyanin content was increased in flowers of AtSTO1 overexpression plants kept at 4°C for 8 weeks. Taken together these data indicate that overexpression of AtSTO1 enhances abiotic stress toleranc via a more pronounced accumulation of compatible solutes under stress.
Despite general concern that the massive deposits of methane stored under permafrost underground and undersea could be released into the atmosphere due to rising temperatures attributed to global climate change, little is known about the methanogenic microorganisms in permafrost sediments, their role in methane emissions, and their phylogeny. The aim of this thesis was to increase knowledge of uncultivated methanogenic microorganisms in submarine and terrestrial permafrost deposits, their community composition, the role they play with regard to methane emissions, and their phylogeny. It is assumed that methanogenic communities in warmer submarine permafrost may serve as a model to anticipate the response of methanogenic communities in colder terrestrial permafrost to rising temperatures. The compositions of methanogenic communities were examined in terrestrial and submarine permafrost sediment samples. The submarine permafrost studied in this research was 10°C warmer than the terrestrial permafrost. By polymerase chain reaction (PCR), DNA was extracted from each of the samples and analyzed by molecular microbiological methods such as PCR-DGGE, RT-PCR, and cloning. Furthermore, these samples were used for in vitro experiment and FISH. The submarine permafrost analysis of the isotope composition of CH4 suggested a relationship between methane content and in situ active methanogenesis. Furthermore, active methanogenesis was proven using 13C-isotope measurements of methane in submarine permafrost sediment with a high TOC value and a high methane concentration. In the molecular-microbiological studies uncultivated lines of Methanosarcina, Methanomicrobiales, Methanobacteriacea and the Groups 1.3 and Marine Benthic from Crenarchaeota were found in all submarine and terrestrial permafrost samples. Methanosarcina was the dominant group of the Archaea in all submarine and terrestrial permafrost samples. The archaeal community composition, in particular, the methanogenic community composition showed diversity with changes in temperatures. Furthermore, cell count of methanogens in submarine permafrost was 10 times higher than in terrestrial permafrost. In vitro experiments showed that methanogens adapt quickly and well to higher temperatures. If temperatures rise due to climate change, an increase in methanogenic activity can be expected as long as organic material is sufficiently available and qualitatively adequate.
Research on novel and advanced biomaterials is an indispensable step towards their applications in desirable fields such as tissue engineering, regenerative medicine, cell culture, or biotechnology. The work presented here focuses on such a promising material: polyelectrolyte multilayer (PEM) composed of hyaluronic acid (HA) and poly(L-lysine) (PLL). This gel-like polymer surface coating is able to accumulate (bio-)molecules such as proteins or drugs and release them in a controlled manner. It serves as a mimic of the extracellular matrix (ECM) in composition and intrinsic properties. These qualities make the HA/PLL multilayers a promising candidate for multiple bio-applications such as those mentioned above. The work presented aims at the development of a straightforward approach for assessment of multi-fractional diffusion in multilayers (first part) and at control of local molecular transport into or from the multilayers by laser light trigger (second part).
The mechanism of the loading and release is governed by the interaction of bioactives with the multilayer constituents and by the diffusion phenomenon overall. The diffusion of a molecule in HA/PLL multilayers shows multiple fractions of different diffusion rate. Approaches, that are able to assess the mobility of molecules in such a complex system, are limited. This shortcoming motivated the design of a novel evaluation tool presented here.
The tool employs a simulation-based approach for evaluation of the data acquired by fluorescence recovery after photobleaching (FRAP) method. In this approach, possible fluorescence recovery scenarios are primarily simulated and afterwards compared with the data acquired while optimizing parameters of a model until a sufficient match is achieved. Fluorescent latex particles of different sizes and fluorescein in an aqueous medium are utilized as test samples validating the analysis results. The diffusion of protein cytochrome c in HA/PLL multilayers is evaluated as well.
This tool significantly broadens the possibilities of analysis of spatiotemporal FRAP data, which originate from multi-fractional diffusion, while striving to be widely applicable. This tool has the potential to elucidate the mechanisms of molecular transport and empower rational engineering of the drug release systems.
The second part of the work focuses on the fabrication of such a spatiotemporarily-controlled drug release system employing the HA/PLL multilayer. This release system comprises different layers of various functionalities that together form a sandwich structure. The bottom layer, which serves as a reservoir, is formed by HA/PLL PEM deposited on a planar glass substrate. On top of the PEM, a layer of so-called hybrids is deposited. The hybrids consist of thermoresponsive poly(N-isopropylacrylamide) (PNIPAM) -based hydrogel microparticles with surface-attached gold nanorods. The layer of hybrids is intended to serve as a gate that controls the local molecular transport through the PEM–solution-interface. The possibility of stimulating the molecular transport by near-infrared (NIR) laser irradiation is being explored.
From several tested approaches for the deposition of hybrids onto the PEM surface, the drying-based approach was identified as optimal. Experiments, that examine the functionality of the fabricated sandwich at elevated temperature, document the reversible volume phase transition of the PEM-attached hybrids while sustaining the sandwich stability. Further, the gold nanorods were shown to effectively absorb light radiation in the tissue- and cell-friendly NIR spectral region while transducing the energy of light into heat. The rapid and reversible shrinkage of the PEM-attached hybrids was thereby achieved. Finally, dextran was employed as a model transport molecule. It loads into the PEM reservoir in a few seconds with the partition constant of 2.4, while it spontaneously releases in a slower, sustained manner. The local laser irradiation of the sandwich, which contains the fluorescein isothiocyanate tagged dextran, leads to a gradual reduction of fluorescence intensity in the irradiated region.
The release system fabricated employs renowned photoresponsivity of the hybrids in an innovative setting. The results of the research are a step towards a spatially-controlled on-demand drug release system that paves the way to spatiotemporally controlled drug release.
The approaches developed in this work have the potential to elucidate the molecular dynamics in ECM and to foster engineering of multilayers with properties tuned to mimic the ECM. The work aims at spatiotemporal control over the diffusion of bioactives and their presentation to the cells.
Plants can be primed to survive the exposure to a severe heat stress (HS) by prior exposure to a mild HS. The information about the priming stimulus is maintained by the plant for several days. This maintenance of acquired thermotolerance, or HS memory, is genetically separable from the acquisition of thermotolerance itself and several specific regulatory factors have been identified in recent years.
On the molecular level, HS memory correlates with two types of transcriptional memory, type I and type II, that characterize a partially overlapping subset of HS-inducible genes. Type I transcriptional memory or sustained induction refers to the sustained transcriptional induction above non-stressed expression levels of a gene for a prolonged time period after the end of the stress exposure. Type II transcriptional memory refers to an altered transcriptional response of a gene after repeated exposure to a stress of similar duration and intensity. In particular, enhanced re-induction refers to a transcriptional pattern in which a gene is induced to a significantly higher degree after the second stress exposure than after the first.
This thesis describes the functional characterization of a novel positive transcriptional regulator of type I transcriptional memory, the heat shock transcription factor HSFA3, and compares it to HSFA2, a known positive regulator of type I and type II transcriptional memory. It investigates type I transcriptional memory and its dependence on HSFA2 and HSFA3 for the first time on a genome-wide level, and gives insight on the formation of heteromeric HSF complexes in response to HS. This thesis confirms the tight correlation between transcriptional memory and H3K4 hyper-methylation, reported here in a case study that aimed to reduce H3K4 hyper-methylation of the type II transcriptional memory gene APX2 by CRISPR/dCas9-mediated epigenome editing. Finally, this thesis gives insight into the requirements for a heat shock transcription factor to function as a positive regulator of transcriptional memory, both in terms of its expression profile and protein abundance after HS and the contribution of individual functional domains.
In summary, this thesis contributes to a more detailed understanding of the molecular processes underlying transcriptional memory and therefore HS memory, in Arabidopsis thaliana.
About 2,000 of the more than 27,000 genes of the genetic model plant Arabidopsis thaliana encode for transcription factors (TFs), proteins that bind DNA in the promoter region of their target genes and thus act as transcriptional activators and repressors. Since TFs play essential roles in nearly all biological processes, they are of great scientific and biotechnological interest. This thesis concentrated on the functional characterisation of four selected members of the Arabidopsis DOF-family, namely DOF1.2, DOF3.1, DOF3.5 and DOF5.2, which were selected because of their specific expression pattern in the root tip, a region that comprises the stem cell niche and cells for the perception of environmental stimuli. DOF1.2, DOF3.1 and DOF3.5 are previously uncharacterized members of the Arabidopsis DOF-family, while DOF5.2 has been shown to be involved in the phototrophic flowering response. However, its role in root development has not been described so far. To identify biological processes regulated by the four DOF proteins in detail, molecular and physiological characterization of transgenic plants with modified levels of DOF1.2, DOF3.1, DOF3.5 and DOF5.2 expression (constitutive and inducible over-expression, artificial microRNA) was performed. Additionally expression patterns of the TFs and their target genes were analyzed using promoter-GUS lines and publicly available microarray data. Finally putative protein-protein interaction partners and upstream regulating TFs were identified using the yeast two-hybrid and one-hybrid system. This combinatorial approach revealed distinct biological functions of DOF1.2, DOF3.1, DOF3.5 and DOF5.2 in the context of root development. DOF1.2 and DOF3.5 are specifically and exclusively expressed in the root cap, including the central root cap (columella) and the lateral root cap, organs which are essential to direct oriented root growth. It could be demonstrated that both genes work in the plant hormone auxin signaling pathway and have an impact on distal cell differentiation. Altered levels of gene expression lead to changes in auxin distribution, abnormal cell division patterns and altered root growth orientation. DOF3.1 and DOF5.2 share a specific expression pattern in the organizing centre of the root stem cell niche, called the quiescent centre. Both genes redundantly control cell differentiation in the root´s proximal meristem and unravel a novel transcriptional regulation pathway for genes enriched in the QC cells. Furthermore this work revealed a novel bipartite nuclear localisation signal being present in the protein sequence of the DOF TF family from all sequenced plant species. Summing up, this work provides an important input into our knowledge about the role of DOF TFs during root development. Future work will concentrate on revealing the exact regulatory networks of DOF1.2, DOF3.1, DOF3.5 and DOF5.2 and their possible biotechnological applications.
Die Ca2+/Calmodulin-aktivierte Serin/Threonin-Phosphatase Calcineurin ist ein Schlüsselmolekül des T-Zell-Rezeptorabhängigen Signalnetzwerkes. Calcineurin aktiviert die Transkriptionsfaktoren der NFATc-Familie durch Dephosphorylierung und reguliert darüber die Expression wichtiger Zytokine und Oberflächenproteine. Die Aktivität von Calcineurin wird durch zahlreiche endogene Proteine moduliert und ist Angriffspunkt der immunsuppressiven Substanzen Cyclosporin A und FK506. In dieser Arbeit wurde der alternative niedermolekulare Calcineurin-NFATc-Inhibitor NCI3 hinsichtlich seiner Effekte auf T-Zell-Rezeptor-abhängige Signalwege charakterisiert. Die Ergebnisse zeigen, daß das Pyrazolopyrimidinderivat NCI3 nichttoxisch und zellmembranpermeabel ist. In T-Zell-Rezeptor-stimulierten primären humanen TH-Zellen unterdrückt NCI3 die Proliferation und IL-2-Produktion (IC50-Wert ~4 µM), da die Dephosphorylierung von NFATc und die anschließende nukleäre Translokation gehemmt wird. NCI3 inhibiert die calcineurinabhängige NFAT- und NF-κB-, aber nicht die AP-1-kontrollierte Reprtergenexpression, in mikromolaren Konzentrationen (IC50-Werte 2 bzw. 7 µM). Im Gegensatz zu Cyclosporin A stört NCI3 nicht die Phosphataseaktivität von Calcineurin, sondern interferiert mit der Calcineurin-NFATc-Bindung. Ein wichtiges endogenes Modulatorprotein für die Calcineurinaktivität ist RCAN1, das vermutlich den Calcineurin-NFATc-Signalweg über einen negativen Rückkopplungsmechanismus reguliert. Hier wurde gezeigt, daß RCAN1 in humanen TH-Zellen exprimiert wird. Die Spleißvariante RCAN1-1 ist in ruhenden T-Zellen basal exprimiert und wird nicht durch T-Zell-Rezeptor-Stimulierung in seiner Expression verändert. RCAN1-4 dagegen ist in ruhenden Zellen kaum zu detektieren und wird stimulierungsabhängig induziert. Durch die Verwendung Calcineurin-NFATc-spezifischer Inhibitoren wie NCI3 wurde gezeigt, daß die RCAN1-4-Induktion durch diesen Signalweg limitiert ist. Die in dieser Arbeit gewonnenen Daten und Erkenntnisse tragen dazu bei, das Verständnis der Funktion und Regulation von Calcineurin in T-Zellen zu vertiefen.
Macrophages play an integral role for the innate immune system. It is critically important for basic research and therapeutic applications to find approaches to potentially modulate their function as the first line of defense. Transient genetic engineering via delivery of synthetic mRNA can serve for such purposes as a robust, reliable and safe technology to modulate macrophage functions. However, a major drawback particularly in the transfection of sensitive immune cells such as macrophages is the immunogenicity of exogenous IVT-mRNAs. Consequently, the direct modulation of human macrophage activity by mRNA-mediated genetic engineering was the aim of this work. The synthetic mRNA can instruct macrophages to synthesize specific target proteins, which can steer macrophage activity in a tailored fashion. Thus, the focus of this dissertation was to identify parameters triggering unwanted immune activation of macrophages, and to find approaches to minimize such effects. When comparing different carrier types as well as mRNA chemistries, the latter had unequivocally a more pronounced impact on activation of human macrophages and monocytes. Exploratory investigations revealed that the choice of nucleoside chemistry, particularly of modified uridine, plays a crucial role for IVT-mRNA-induced immune activation, in a dose-dependent fashion. Additionally, the contribution of the various 5’ cap structures tested was only minor. Moreover, to address the technical aspects of the delivery of multiple genes as often mandatory for advanced gene delivery studies, two different strategies of payload design were investigated, namely “bicistronic” delivery and “monocistronic” co-delivery. The side-by-side comparison of mRNA co-delivery via a bicistronic design (two genes, one mRNA) with a monocistronic design (two gene, two mRNAs) unexpectedly revealed that, despite the intrinsic equimolar nature of the bicistronic approach, it was outperformed by the monocistronic approach in terms of reliable co-expression when quantified on the single cell level. Overall, the incorporation of chemical modifications into IVT-mRNA by using respective building blocks, primarily with the aim to minimize immune activation as exemplified in this thesis, has the potential to facilitate the selection of the proper mRNA chemistry to address specific biological and clinical challenges. The technological aspects of gene delivery evaluated and validated by the quantitative methods allowed us to shed light on crucial process parameters and mRNA design criteria, required for reliable co-expression schemes of IVT-mRNA delivery.
Deoxyribonucleic acid (DNA) nanostructures enable the attachment of functional molecules to nearly any unique location on their underlying structure. Due to their single-base-pair structural resolution, several ligands can be spatially arranged and closely controlled according to the geometry of their desired target, resulting in optimized binding and/or signaling interactions.
This dissertation covers three main projects. All of them use variations of functionalized DNA nanostructures that act as platform for oligovalent presentation of ligands. The purpose of this work was to evaluate the ability of DNA nanostructures to precisely display different types of functional molecules and to consequently enhance their efficacy according to the concept of multivalency. Moreover, functionalized DNA structures were examined for their suitability in functional screening assays. The developed DNA-based compound ligands were used to target structures in different biological systems.
One part of this dissertation attempted to bind pathogens with small modified DNA nanostructures. Pathogens like viruses and bacteria are known for their multivalent attachment to host cells membranes. By blocking their receptors for recognition and/or fusion with their targeted host in an oligovalent manner, the objective was to impede their ability to adhere to and invade cells. For influenza A, only enhanced binding of oligovalent peptide-DNA constructs compared to the monovalent peptide could be observed, whereas in the case of respiratory syncytial virus (RSV), binding as well as blocking of the target receptors led to an increased inhibition of infection in vitro.
In the final part, the ability of chimeric DNA-peptide constructs to bind to and activate signaling receptors on the surface of cells was investigated. Specific binding of DNA trimers, conjugated with up to three peptides, to EphA2 receptor expressing cells was evaluated in flow cytometry experiments. Subsequently, their ability to activate these receptors via phosphorylation was assessed. EphA2 phosphorylation was significantly increased by DNA trimers carrying three peptides compared to monovalent peptide. As a result of activation, cells underwent characteristic morphological changes, where they "round up" and retract their periphery.
The results obtained in this work comprehensively prove the capability of DNA nanostructures to serve as stable, biocompatible, controllable platforms for the oligovalent presentation of functional ligands. Functionalized DNA nanostructures were used to enhance biological effects and as tool for functional screening of bio-activity. This work demonstrates that modified DNA structures have the potential to improve drug development and to unravel the activation of signaling pathways.
This thesis aims at a better mechanistic understanding of animal communities. Therefore, an allometry- and individual-based model has been developed which was used to simulate mammal and bird communities in heterogeneous landscapes, and to to better understand their response to landscape changes (habitat loss and fragmentation).
Mathematical modeling of biological systems is a powerful tool to systematically investigate the functions of biological processes and their relationship with the environment. To obtain accurate and biologically interpretable predictions, a modeling framework has to be devised whose assumptions best approximate the examined scenario and which copes with the trade-off of complexity of the underlying mathematical description: with attention to detail or high coverage. Correspondingly, the system can be examined in detail on a smaller scale or in a simplified manner on a larger scale. In this thesis, the role of photosynthesis and its related biochemical processes in the context of plant metabolism was dissected by employing modeling approaches ranging from kinetic to stoichiometric models. The Calvin-Benson cycle, as primary pathway of carbon fixation in C3 plants, is the initial step for producing starch and sucrose, necessary for plant growth. Based on an integrative analysis for model ranking applied on the largest compendium of (kinetic) models for the Calvin-Benson cycle, those suitable for development of metabolic engineering strategies were identified. Driven by the question why starch rather than sucrose is the predominant transitory carbon storage in higher plants, the metabolic costs for their synthesis were examined. The incorporation of the maintenance costs for the involved enzymes provided a model-based support for the preference of starch as transitory carbon storage, by only exploiting the stoichiometry of synthesis pathways. Many photosynthetic organisms have to cope with processes which compete with carbon fixation, such as photorespiration whose impact on plant metabolism is still controversial. A systematic model-oriented review provided a detailed assessment for the role of this pathway in inhibiting the rate of carbon fixation, bridging carbon and nitrogen metabolism, shaping the C1 metabolism, and influencing redox signal transduction. The demand of understanding photosynthesis in its metabolic context calls for the examination of the related processes of the primary carbon metabolism. To this end, the Arabidopsis core model was assembled via a bottom-up approach. This large-scale model can be used to simulate photoautotrophic biomass production, as an indicator for plant growth, under so-called optimal, carbon-limiting and nitrogen-limiting growth conditions. Finally, the introduced model was employed to investigate the effects of the environment, in particular, nitrogen, carbon and energy sources, on the metabolic behavior. This resulted in a purely stoichiometry-based explanation for the experimental evidence for preferred simultaneous acquisition of nitrogen in both forms, as nitrate and ammonium, for optimal growth in various plant species. The findings presented in this thesis provide new insights into plant system's behavior, further support existing opinions for which mounting experimental evidences arise, and posit novel hypotheses for further directed large-scale experiments.
East Africa is a natural laboratory: Studying its unique geological and biological history can help us better inform our theories and models. Studying its present and future can help us protect its globally important biodiversity and ecosystem services. East African vegetation plays a central role in all these aspects, and this dissertation aims to quantify its dynamics through computer simulations.
Computer models help us recreate past settings, forecast into the future or conduct simulation experiments that we cannot otherwise perform in the field. But before all that, one needs to test their performance. The outputs that the model produced using the present day-inputs, agreed well with present-day observations of East African vegetation. Next, I simulated past vegetation for which we have fossil pollen data to compare. With computer models, we can fill the gaps of knowledge between sites where we have fossil pollen data from, and create a more complete picture of the past. Good level of agreement between model and pollen data where they overlapped in space further validated our model performance.
Once the model was tested and validated for the region, it became possible to probe one of the long standing questions regarding East African vegetation: How did East Africa lose its tropical forests? The present-day vegetation in the tropics is mainly characterized by continuous forests worldwide except in tropical East Africa, where forests only occur as patches. In a series of simulation experiments, I was able to show under which conditions these forest patches could have been connected and fragmented in the past. This study showed the sensitivity of East African vegetation to climate change and variability such as those expected under future climate change.
El Niño Southern Oscillation (ENSO) events that result from the fluctuations in temperature between the ocean and atmosphere, bring further variability to East African climate and are predicted to increase in intensity in the future. But climate models are still not good at capturing the pattens of these events. In a study where I quantified the influence of ENSO events on East African vegetation, I showed how different the future vegetation could be from what we currently predict with these climate models that lack accurate ENSO contribution. Consideration of these discrepancies is important for our future global carbon budget calculations and management decisions.
The overall objective of the study is an elaboration of quantitative methods for national conservation planning, coincident with the international approach ('hotspots' approach). This objective requires a solution of following problems: 1) How to estimate large scale vegetation diversity from abiotic factors only? 2) How to adopt 'global hotspots' approach for bordering of national biodiversity hotspots? 3) How to set conservation targets, accounting for difference in environmental conditions and human threats between national biodiversity hotspots? 4) How to design large scale national conservation plan reflecting hierarchical nature of biodiversity? The case study for national conservation planning is Russia. Conclusions: · Large scale vegetation diversity can be predicted to a major extent by climatically determined latent heat for evaporation and geometrical structure of landscape, described as an altitudinal difference. The climate based model reproduces observed species number of vascular plant for different areas of the world with an average error 15% · National biodiversity hotspots can be mapped from biotic or abiotic data using corrected for a country the quantitative criteria for plant endemism and land use from the 'global hotspots' approach · Quantitative conservation targets, accounting for difference in environmental conditions and human threats between national biodiversity hotspots can be set using national data for Red Data book species · Large scale national conservation plan reflecting hierarchical nature of biodiversity can be designed by combination of abiotic method at national scale (identification of large scale hotspots) and biotic method at regional scale (analysis of species data from Red Data book)
Monoclonal antibodies (mAbs) are engineered immunoglobulins G (IgG) used for more than 20 years as targeted therapy in oncology, infectious diseases and (auto-)immune disorders. Their protein nature greatly influences their pharmacokinetics (PK), presenting typical linear and non-linear behaviors.
While it is common to use empirical modeling to analyze clinical PK data of mAbs, there is neither clear consensus nor guidance to, on one hand, select the structure of classical compartment models and on the other hand, interpret mechanistically PK parameters. The mechanistic knowledge present in physiologically-based PK (PBPK) models is likely to support rational classical model selection and thus, a methodology to link empirical and PBPK models is desirable. However, published PBPK models for mAbs are quite diverse in respect to the physiology of distribution spaces and the parameterization of the non-specific elimination involving the neonatal Fc receptor (FcRn) and endogenous IgG (IgGendo). The remarkable discrepancy between the simplicity of biodistribution data and the complexity of published PBPK models translates in parameter identifiability issues.
In this thesis, we address this problem with a simplified PBPK model—derived from a hierarchy of more detailed PBPK models and based on simplifications of tissue distribution model. With the novel tissue model, we are breaking new grounds in mechanistic modeling of mAbs disposition: We demonstrate that binding to FcRn is indeed linear and that it is not possible to infer which tissues are involved in the unspecific elimination of wild-type mAbs. We also provide a new approach to predict tissue partition coefficients based on mechanistic insights: We directly link tissue partition coefficients (Ktis) to data-driven and species-independent published antibody biodistribution coefficients (ABCtis) and thus, we ensure the extrapolation from pre-clinical species to human with the simplified PBPK model. We further extend the simplified PBPK model to account for a target, relevant to characterize the non-linear clearance due to mAb-target interaction.
With model reduction techniques, we reduce the dimensionality of the simplified PBPK model to design 2-compartment models, thus guiding classical model development with physiological and mechanistic interpretation of the PK parameters. We finally derive a new scaling approach for anatomical and physiological parameters in PBPK models that translates the inter-individual variability into the design of mechanistic covariate models with direct link to classical compartment models, specially useful for PK population analysis during clinical development.
Animal movement is a crucial aspect of life, influencing ecological and evolutionary processes. It plays an important role in shaping biodiversity patterns, connecting habitats and ecosystems. Anthropogenic landscape changes, such as in agricultural environments, can impede the movement of animals by affecting their ability to locate resources during recurring movements within home ranges and, on a larger scale, disrupt migration or dispersal. Inevitably, these changes in movement behavior have far-reaching consequences on the mobile link functions provided by species inhabiting such extensively altered matrix areas. In this thesis, I investigate the movement characteristics and activity patterns of the European hare (Lepus europaeus), aiming to understand their significance as a pivotal species in fragmented agricultural landscapes. I reveal intriguing results that shed light on the importance of hares for seed dispersal, the influence of personality traits on behavior and space use, the sensitivity of hares to extreme weather conditions, and the impacts of GPS collaring on mammals' activity patterns and movement behavior.
In Chapter I, I conducted a controlled feeding experiment to investigate the potential impact of hares on seed dispersal. By additionally utilizing GPS data of hares in two contrasting landscapes, I demonstrated that hares play a vital role, acting as effective mobile linkers for many plant species in small and isolated habitat patches. The analysis of seed intake and germination success revealed that distinct seed traits, such as density, surface area, and shape, profoundly affect hares' ability to disperse seeds through endozoochory. These findings highlight the interplay between hares and plant communities and thus provide valuable insights into seed dispersal mechanisms in fragmented landscapes.
By employing standardized behavioral tests in Chapter II, I revealed consistent behavioral responses among captive hares while simultaneously examining the intricate connection between personality traits and spatial patterns within wild hare populations. This analysis provides insights into the ecological interactions and dynamics within hare populations in agricultural habitats. Examining the concept of animal personality, I established a link between personality traits and hare behavior. I showed that boldness, measured through standardized tests, influences individual exploration styles, with shy and bold hares exhibiting distinct space use patterns. In addition to providing valuable insights into the role of animal personality in heterogeneous environments, my research introduced a novel approach demonstrating the feasibility of remotely assessing personality types using animal-borne sensors without additional disturbance of the focal individual.
While climate conditions severely impact the activity and, consequently, the fitness of wildlife species across the globe, in Chapter III, I uncovered the sensitivity of hares to temperature, humidity, and wind speed during their peak reproduction period. I found a strong response in activity to high temperatures above 25°C, with a particularly pronounced effect during temperature extremes of over 35°C. The non-linear relationship between temperature and activity was characterized by contrasting responses observed for day and night. These findings emphasize the vulnerability of hares to climate change and the potential consequences for their fitness and population dynamics with the ongoing rise of temperature.
Since such insights can only be obtained through capturing and tagging free-ranging animals, I assessed potential impacts and the recovery process post-collar attachment in Chapter IV. For this purpose, I examined the daily distances moved and the temporal-associated activity of 1451 terrestrial mammals out of 42 species during their initial tracking period. The disturbance intensity and the speed of recovery varied across species, with herbivores, females, and individuals captured and collared in relatively secluded study areas experiencing more pronounced disturbances due to limited anthropogenic influences.
Mobile linkers are essential for maintaining biodiversity as they influence the dynamics and resilience of ecosystems. Furthermore, their ability to move through fragmented landscapes makes them a key component for restoring disturbed sites. Individual movement decisions determine the scale of mobile links, and understanding variations in space use among individuals is crucial for interpreting their functions. Climate change poses further challenges, with wildlife species expected to adjust their behavior, especially in response to high-temperature extremes, and comprehending the anthropogenic influence on animal movements will remain paramount to effective land use planning and the development of successful conservation strategies.
This thesis provides a comprehensive ecological understanding of hares in agricultural landscapes. My research findings underscore the importance of hares as mobile linkers, the influence of personality traits on behavior and spatial patterns, the vulnerability of hares to extreme weather conditions, and the immediate consequences of collar attachment on mammalian movements. Thus, I contribute valuable insights to wildlife conservation and management efforts, aiding in developing strategies to mitigate the impact of environmental changes on hare populations. Moreover, these findings enable the development of methodologies aimed at minimizing the impacts of collaring while also identifying potential biases in the data, thereby benefiting both animal welfare and the scientific integrity of localization studies.
This dissertation aimed to determine differential expressed miRNAs in the context of chronic pain in polyneuropathy. For this purpose, patients with chronic painful polyneuropathy were compared with age matched healthy patients. Taken together, all miRNA pre library preparation quality controls were successful and none of the samples was identified as an outlier or excluded for library preparation. Pre sequencing quality control showed that library preparation worked for all samples as well as that all samples were free of adapter dimers after BluePippin size selection and reached the minimum molarity for further processing. Thus, all samples were subjected to sequencing. The sequencing control parameters were in their optimal range and resulted in valid sequencing results with strong sample to sample correlation for all samples. The resulting FASTQ file of each miRNA library was analyzed and used to perform a differential expression analysis. The differentially expressed and filtered miRNAs were subjected to miRDB to perform a target prediction. Three of those four miRNAs were downregulated: hsa-miR-3135b, hsa-miR-584-5p and hsa-miR-12136, while one was upregulated: hsa-miR-550a-3p. miRNA target prediction showed that chronic pain in polyneuropathy might be the result of a combination of miRNA mediated high blood flow/pressure and neural activity dysregulations/disbalances. Thus, leading to the promising conclusion that these four miRNAs could serve as potential biomarkers for the diagnosis of chronic pain in polyneuropathy.
Since TRPV1 seems to be one of the major contributors of nociception and is associated with neuropathic pain, the influence of PKA phosphorylated ARMS on the sensitivity of TRPV1 as well as the part of AKAP79 during PKA phosphorylation of ARMS was characterized. Therefore, possible PKA-sites in the sequence of ARMS were identified. This revealed five canonical PKA-sites: S882, T903, S1251/52, S1439/40 and S1526/27. The single PKA-site mutants of ARMS revealed that PKA-mediated ARMS phosphorylation seems not to influence the interaction rate of TRPV1/ARMS. While phosphorylation of ARMST903 does not increase the interaction rate with TRPV1, ARMSS1526/27 is probably not phosphorylated and leads to an increased interaction rate. The calcium flux measurements indicated that the higher the interaction rate of TRPV1/ARMS, the lower the EC50 for capsaicin of TRPV1, independent of the PKA phosphorylation status of ARMS. In addition, the western blot analysis confirmed the previously observed TRPV1/ARMS interaction. More importantly, AKAP79 seems to be involved in the TRPV1/ARMS/PKA signaling complex. To overcome the problem of ARMS-mediated TRPV1 sensitization by interaction, ARMS was silenced by shRNA. ARMS silencing resulted in a restored TRPV1 desensitization without affecting the TRPV1 expression and therefore could be used as new topical therapeutic analgesic alternative to stop ARMS mediated TRPV1 sensitization.
Mikroorganismen in geothermischen Aquiferen : Einfluss mikrobieller Prozesse auf den Anlagenbetrieb
(2012)
In Fluid-, Filter- und Sedimentproben von vier geothermischen Anlagen des Norddeutschen Beckens wurden mit molekulargenetischen Verfahren unterschiedliche mikrobielle Gemeinschaften nachgewiesen. Die mikrobielle Zusammensetzung in den Prozesswässern wurde dabei durch die Aquiferteufe, die Salinität, die Temperatur und den verfügbaren Elektronendonatoren und -akzeptoren beeinflusst. Die in den anoxischen Prozesswässern identifizierten Organismen zeichneten sich durch einen chemoheterotrophen oder chemoautotrophen Stoffwechsel aus, wobei Nitrat, Sulfat, Eisen (III) oder Bikarbonat als terminale Elektronenakzeptoren fungierten. Mikroorganismen beeinflussten den Betrieb von zwei Anlagen negativ. So reduzierten im Prozesswasser des Kältespeichers am Berliner Reichstag vorhandene Eisenoxidierer, nahe verwandt zu der Gattung Gallionella, die Injektivität der Bohrungen durch Eisenhydroxidausfällungen in den Filterschlitzen. Biofilme, die von schwefeloxidierenden Bakterien der Gattung Thiothrix in den Filtern der obertägigen Anlage gebildet wurden, führten ebenfalls zu Betriebsstörungen, indem sie die Injektion des Fluids in den Aquifer behinderten. Beim Wärmespeicher in Neubrandenburg waren Sulfatreduzierer vermutlich an der Bildung von Eisensulfidausfällungen in den obertägigen Filtern und im bohrlochnahen Bereich beteiligt und verstärkten Korrosionsprozesse an der Pumpe im Bohrloch der kalten Aquiferseite. Organische Säuren in den Fluiden sowie mineralische Ausfällungen in den Filtern der obertägigen Anlagen waren Belege für die Aktivität der in den verschiedenen Anlagen vorhandenen Mikroorganismen. Es wurde zudem deutlich, dass Mikroorganismen auf Grund der hohen Durchflussraten in den Anlagen chemische Veränderungen in den Prozesswässern deutlich sensitiver anzeigen als chemische Analyseverfahren. So deuteten Änderungen in der Zusammensetzung der mikrobiellen Biozönosen und speziell die Identifikation von Indikatororganismen wie Eisen- und Schwefeloxidierern, fermentativen Bakterien und Sulfatreduzierern auf eine erhöhte Verfügbarkeit von Elektronendonatoren oder akzeptoren in den Prozesswässern hin. Die Ursachen für die an den Geothermieanlagen auftretenden Betriebsstörungen konnten dadurch erkannt werden.
Bei konventionellen Mikroarray-Experimenten zur Genexpressionsanalyse wird fluoreszenz- oder radioaktiv-markierte cDNA oder RNA mit immobilisierten Proben hybridisiert. Für ein gut detektierbares und auswertbares Ergebnis werden jedoch pro Array mindestens 15 - 20 µg Hybridisierungstarget benötigt. Dazu müssen entweder 15 - 20 µg RNA direkt durch Reverse Transkription in markierte cDNA umgeschrieben werden oder bei Vorhandensein von weniger Startmaterial die RNA amplifiziert werden (Standard- Affymetrix-Protokolle, Klur et al. 2004). Oft sind damit zeit- und kostenintensive Probenpräparationen verbunden und das Ergebnis ist nicht immer reproduzierbar. Obwohl es inzwischen einige Protokolle gibt, die dieses Problem zu lösen versuchen (Zhang et al. 2001, Iscove et al. 2002, McClintick et al. 2003, Stirewalt et al. 2004), eine optimale, leicht handbare und reproduzierbare Methode gibt es weiterhin nicht, weshalb in dieser Arbeit ein weiterer Lösungsansatz gesucht wurde. In der vorgestellten Arbeit werden zwei einfache Methoden beschrieben, mit denen Gene aus geringen RNA-Mengen nachgewiesen werden können: erstens die On Chip- RT-PCR mit cDNA als Matrize und zweitens diese Methode als One-Step-Reaktion mit RNA als Matrize. Beide Methoden beruhen auf dem Prinzip der PCR an immobilisierten Primern auf einer Chipoberfläche. Diese Möglichkeit der exponentiellen Amplifikation ist reproduzierbar und sensitiv. In Experimenten zur Etablierung des On-Chip-PCR-Systems wurden für die Immobilisierung der Primer verschiedene Kopplungsmethoden verwendet. Die affine Kopplung über Biotin- Streptavidin erwies sich als geeignet. Die On-Chip-Reaktion an kovalent gebundenen Primern wurde für amino-modifizierte Primer auf Epoxy-Oberflächen sowie für EDC-Kopplung auf silanisierten Oberflächen gezeigt. Für die letztgenannte Methode wurde die On-Chip-PCR optimiert, dass Spottingkonzentrationen der Primer von 5 - 10µM schon ausreichend sind. Der Einsatz von fluoreszenz-markierten Primern während der PCR ermöglicht eine unmittelbare Auswertung nach der Synthese ohne zusätzliche Detektionsschritte. In dieser Arbeit konnte außerdem mit der vorgestellten Methode der simultane Nachweis zweier Gene gezeigt werden. Die Methode kann noch als Multiplex-Analyse ausgebaut werden, um so mehrere Gene in gleichzeitig einem Ansatz nachweisen zu können. Die Ergebnisse der Versuche mit Matrizen aus unterschiedlichen Zelltypen deuten darauf hin, dass die On-Chip-RT-PCR eine weitere optimale Methode für den Nachweis von gering exprimierten Genen bietet.
Microplastics in the environments are estimated to increase in the near future due to increasing consumption of plastic product and also due to further fragmentation in small pieces. The fate and effects of MP once released into the freshwater environment are still scarcely studied, compared to the marine environment. In order to understand possible effect and interaction of MPs in freshwater environment, planktonic zooplankton organisms are very useful for their crucial trophic role. In particular freshwater rotifers are one of the most abundant organisms and they are the interface between primary producers and secondary consumers. The aim of my thesis was to investigate the ingestion and the effect of MPs in rotifers from a more natural scenario and to individuate processes such as the aggregation of MPs, the food dilution effect and the increasing concentrations of MPs that could influence the final outcome of MPs in the environment. In fact, in a near natural scenario MPs interaction with bacteria and algae, aggregations together with the size and concentration are considered drivers of ingestion and effect. The aggregation of MPs makes smaller MPs more available for rotifers and larger MPs less ingested. The negative effect caused by the ingestion of MPs was modulated by their size but also by the quantity and the quality of food that cause variable responses. In fact, rotifers in the environment are subjected to food limitation and the presence of MPs could exacerbate this condition and decrease the population and the reproduction input. Finally, in a scenario incorporating an entire zooplanktonic community, MPs were ingested by most individuals taking into account their feeding mode but also the concentration of MPs, which was found to be essential for the availability of MPs. This study highlights the importance to investigate MPs from a more environmental perspective, this in fact could provide an alternative and realistic view of effect of MPs in the ecosystem.
Plastic pollution is ubiquitous on the planet since several millions of tons of plastic waste enter aquatic ecosystems each year. Furthermore, the amount of plastic produced is expected to increase exponentially shortly. The heterogeneity of materials, additives and physical characteristics of plastics are typical of these emerging contaminants and affect their environmental fate in marine and freshwaters. Consequently, plastics can be found in the water column, sediments or littoral habitats of all aquatic ecosystems. Most of this plastic debris will fragment as a product of physical, chemical and biological forces, producing particles of small size. These particles (< 5mm) are known as “microplastics” (MP). Given their high surface-to-volume ratio, MP stimulate biofouling and the formation of biofilms in aquatic systems.
As a result of their unique structure and composition, the microbial communities in MP biofilms are referred to as the “Plastisphere.” While there is increasing data regarding the distinctive composition and structure of the microbial communities that form part of the plastisphere, scarce information exists regarding the activity of microorganisms in MP biofilms. This surface-attached lifestyle is often associated with the increase in horizontal gene transfer (HGT) among bacteria. Therefore, this type of microbial activity represents a relevant function worth to be analyzed in MP biofilms. The horizontal exchange of mobile genetic elements (MGEs) is an essential feature of bacteria. It accounts for the rapid evolution of these prokaryotes and their adaptation to a wide variety of environments. The process of HGT is also crucial for spreading antibiotic resistance and for the evolution of pathogens, as many MGEs are known to contain antibiotic resistance genes (ARGs) and genetic determinants of pathogenicity.
In general, the research presented in this Ph.D. thesis focuses on the analysis of HGT and heterotrophic activity in MP biofilms in aquatic ecosystems. The primary objective was to analyze the potential of gene exchange between MP bacterial communities vs. that of the surrounding water, including bacteria from natural aggregates. Moreover, the thesis addressed the potential of MP biofilms for the proliferation of biohazardous bacteria and MGEs from wastewater treatment plants (WWTPs) and associated with antibiotic resistance. Finally, it seeks to prove if the physiological profile of MP biofilms under different limnological conditions is divergent from that of the water communities. Accordingly, the thesis is composed of three independent studies published in peer-reviewed journals. The two laboratory studies were performed using both model and environmental microbial communities. In the field experiment, natural communities from freshwater ecosystems were examined.
In Chapter I, the inflow of treated wastewater into a temperate lake was simulated with a concentration gradient of MP particles. The effects of MP on the microbial community structure and the occurrence of integrase 1 (int 1) were followed. The int 1 is a marker associated with mobile genetic elements and known as a proxy for anthropogenic effects on the spread of antimicrobial resistance genes. During the experiment, the abundance of int1 increased in the plastisphere with increasing MP particle concentration, but not in the surrounding water. In addition, the microbial community on MP was more similar to the original wastewater community with increasing microplastic concentrations. Our results show that microplastic particles indeed promote persistence of standard indicators of microbial anthropogenic pollution in natural waters.
In Chapter II, the experiments aimed to compare the permissiveness of aquatic bacteria towards model antibiotic resistance plasmid pKJK5, between communities that form biofilms on MP vs. those that are free-living. The frequency of plasmid transfer in bacteria associated with MP was higher when compared to bacteria that are free-living or in natural aggregates. Moreover, comparison increased gene exchange occurred in a broad range of phylogenetically-diverse bacteria. The results indicate a different activity of HGT in MP biofilms, which could affect the ecology of aquatic microbial communities on a global scale and the spread of antibiotic resistance.
Finally, in Chapter III, physiological measurements were performed to assess whether microorganisms on MP had a different functional diversity from those in water. General heterotrophic activity such as oxygen consumption was compared in microcosm assays with and without MP, while diversity and richness of heterotrophic activities were calculated by using Biolog® EcoPlates. Three lakes with different nutrient statuses presented differences in MP-associated biomass build up. Functional diversity profiles of MP biofilms in all lakes differed from those of the communities in the surrounding water, but only in the oligo-mesotrophic lake MP biofilms had a higher functional richness compared to the ambient water. The results support that MP surfaces act as new niches for aquatic microorganisms and can affect global carbon dynamics of pelagic environments.
Overall, the experimental works presented in Chapters I and II support a scenario where MP pollution affects HGT dynamics among aquatic bacteria. Among the consequences of this alteration is an increase in the mobilization and transfer efficiency of ARGs. Moreover, it supposes that changes in HGT can affect the evolution of bacteria and the processing of organic matter, leading to different catabolic profiles such as demonstrated in Chapter III. The results are discussed in the context of the fate and magnitude of plastic pollution and the importance of HGT for bacterial evolution and the microbial loop, i.e., at the base of aquatic food webs. The thesis supports a relevant role of MP biofilm communities for the changes observed in the aquatic microbiome as a product of intense human intervention.
The Arctic region is especially impacted by global warming as temperatures in high latitude regions have increased and are predicted to further rise at levels above the global average. This is crucial to Arctic soils and the shallow shelves of the Arctic Ocean as they are underlain by permafrost. Perennially frozen ground is a habitat for a large number and great diversity of viable microorganisms, which can remain active even under freezing conditions. Warming and thawing of permafrost makes trapped soil organic carbon more accessible to microorganisms. They can transform it to the greenhouse gases carbon dioxide, methane and nitrous oxide. On the other hand, it is assumed that thawing of the frozen ground stimulates microbial activity and carbon turnover. This can lead to a positive feedback loop of warming and greenhouse gas release.
Submarine permafrost covers most areas of the Siberian Arctic Shelf and contains a large though unquantified carbon pool. However, submarine permafrost is not only affected by changes in the thermal regime but by drastic changes in the geochemical composition as it formed under terrestrial conditions and was inundated by Holocene sea level rise and coastal erosion. Seawater infiltration into permafrost sediments resulted in an increase of the pore water salinity and, thus, in thawing of permafrost in the upper sediment layers even at subzero temperatures. The permafrost below, which was not affected by seawater, remained ice-bonded, but warmed through seawater heat fluxes.
The objective of this thesis was to study microbial communities in submarine permafrost with a focus on their response to seawater influence and long-term warming using a combined approach of molecular biological and physicochemical analyses. The microbial abundance, community composition and structure as well as the diversity were investigated in drill cores from two locations in the Laptev Sea, which were subjected to submarine conditions for centuries to millennia. The microbial abundance was measured through total cell counts and copy numbers of the 16S rRNA gene and of functional genes. The latter comprised genes which are indicative for methane production (mcrA) and sulfate reduction (dsrB). The microbial community was characterized by high-throughput-sequencing of the 16S rRNA gene. Physicochemical analyses included the determination of the pore water geochemical and stable isotopic composition, which were used to describe the degree of seawater influence. One major outcome of the thesis is that the submarine permafrost stratified into different so-called pore water units centuries as well as millennia after inundation: (i) sediments that were mixed with seafloor sediments, (ii) sediments that were infiltrated with seawater, and (iii) sediments that were unaffected by seawater. This stratification was reflected in the submarine permafrost microbial community composition only millennia after inundation but not on time-scales of centuries.
Changes in the community composition as well as abundance were used as a measure for microbial activity and the microbial response to changing thermal and geochemical conditions. The results were discussed in the context of permafrost temperature, pore water composition, paleo-climatic proxies and sediment age. The combination of permafrost warming and increasing salinity as well as permafrost warming alone resulted in a disturbance of the microbial communities at least on time-scales of centuries. This was expressed by a loss of microbial abundance and bacterial diversity. At the same time, the bacterial community of seawater unaffected but warmed permafrost was mainly determined by environmental and climatic conditions at the time of sediment deposition. A stimulating effect of warming was observed only in seawater unaffected permafrost after millennia-scale inundation, visible through increased microbial abundance and reduced amounts of substrate.
Despite submarine exposure for centuries to millennia, the community of bacteria in submarine permafrost still generally resembled the community of terrestrial permafrost. It was dominated by phyla like Actinobacteria, Chloroflexi, Firmicutes, Gemmatimonadetes and Proteobacteria, which can be active under freezing conditions.
Moreover, the archaeal communities of both study sites were found to harbor high abundances of marine and terrestrial anaerobic methane oxidizing archaea (ANME). Results also suggested ANME populations to be active under in situ conditions at subzero temperatures. Modeling showed that potential anaerobic oxidation of methane (AOM) could mitigate the release of almost all stored or microbially produced methane from thawing submarine permafrost.
Based on the findings presented in this thesis, permafrost warming and thawing under submarine conditions as well as permafrost warming without thaw are supposed to have marginal effects on the microbial abundance and community composition, and therefore likely also on carbon mobilization and the formation of methane. Thawing under submarine conditions even stimulates AOM and thus mitigates the release of methane.
The continuously increasing pollution of aquatic environments with microplastics (plastic particles < 5 mm) is a global problem with potential implications for organisms of all trophic levels. For microorganisms, trillions of these floating microplastics particles represent a huge surface area for colonization. Due to the very low biodegradability, microplastics remain years to centuries in the environment and can be transported over thousands of kilometers together with the attached organisms. Since also pathogenic, invasive, or otherwise harmful species could be spread this way, it is essential to study microplastics-associated communities.
For this doctoral thesis, eukaryotic communities were analyzed for the first time on microplastics in brackish environments and compared to communities in the surrounding water and on the natural substrate wood. With Illumina MiSeq high-throughput sequencing, more than 500 different eukaryotic taxa were detected on the microplastics samples. Among them were various green algae, dinoflagellates, ciliates, fungi, fungal-like protists and small metazoans such as nematodes and rotifers. The most abundant organisms was a dinoflagellate of the genus Pfiesteria, which could include fish pathogenic and bloom forming toxigenic species. Network analyses revealed that there were numerous interaction possibilities among prokaryotes and eukaryotes in microplastics biofilms. Eukaryotic community compositions on microplastics differed significantly from those on wood and in water, and compositions were additionally distinct among the sampling locations. Furthermore, the biodiversity was clearly lower on microplastics in comparison to the diversity on wood or in the surrounding water.
In another experiment, a situation was simulated in which treated wastewater containing microplastics was introduced into a freshwater lake. With increasing microplastics concentrations, the resulting bacterial communities became more similar to those from the treated wastewater. Moreover, the abundance of integrase I increased together with rising concentrations of microplastics. Integrase I is often used as a marker for anthropogenic environmental pollution and is further linked to genes conferring, e.g., antibiotic resistance.
This dissertation gives detailed insights into the complexity of prokaryotic and eukaryotic communities on microplastics in brackish and freshwater systems. Even though microplastics provide novel microhabitats for various microbes, they might also transport toxigenic, pathogenic, antibiotic-resistant or parasitic organisms; meaning their colonization can pose potential threats to humans and the environment. Finally, this thesis explains the urgent need for more research as well as for strategies to minimize the global microplastic pollution.
Übergewicht und Adipositas führen zu Insulinresistenz und erhöhen deutlich das Risiko für die Entwicklung von Typ-2-Diabetes und kardiovaskulären Erkrankungen. Sowohl Adipositas als auch die Suszeptibilität gegenüber Diabetes sind zu einem erheblichen Teil genetisch determiniert. Die relevanten Risikogene, deren Interaktion mit der Umwelt, insbesondere mit Bestandteilen der Nahrung, und die Pathomechanismen, die zur Insulinresistenz und Diabetes führen, sind nicht vollständig aufgeklärt. In der vorliegenden Arbeit sollte durch Genexpressionsanalysen des weißen Fettgewebes (WAT) und der Langerhansschen Inseln die Entstehung und Progression von Adipositas und Typ-2-Diabetes untersucht werden, um relevante Pathomechanismen und neue Kandidatengene zu identifizieren. Zu diesem Zweck wurden Diät-Interventionsstudien mit NZO- und verwandten NZL-Mäusen, zwei polygenen Mausmodellen für das humane metabolische Syndrom, durchgeführt. Eine kohlenhydrathaltige Hochfett-Diät (HF: 14,6 % Fettanteil) führte in beiden Mausmodellen zu früher Adipositas, Insulinresistenz und Typ 2 Diabetes. Eine fettreduzierte Standarddiät (SD: 3,3 % Fettanteil), welche die Entstehung von Adipositas und Diabetes stark verzögert, sowie eine diabetesprotektive kohlenhydratfreie Hochfett-Diät (CHF: 30,2 % Fettanteil) dienten als Kontrolldiäten. Mit Hilfe der Microarray-Technologie wurden genomweite Expressionsprofile des WAT erstellt. Pankreatische Inseln wurden durch laserbasierte Mikropräparation (Laser Capture Microdissection; LCM) isoliert und ebenfalls hinsichtlich ihres Expressionsprofils analysiert. Differenziell exprimierte Gene wurden durch Real-Time-PCR validiert. Im WAT der NZO-Maus bewirkte die HF-Diät eine reduzierte Expression nukleärer Gene der oxidativen Phosphorylierung und von lipogenen Enzymen. Dies deutet auf eine inadäquate Fettspeicherung und -verwertung in diesen Tieren hin. Die Reduktion in der Fettspeicherung und -oxidation ist spezifisch für das adipöse NZO-Modell und konnte bei der schlanken SJL Maus nicht beobachtet werden, was auf eine mögliche Beteiligung an der Entstehung der Insulinresistenz hinweist. Zusätzlich wurde bestätigt, dass die Expansion des Fettgewebes bei der adipösen NZO-Maus eine zeitlich verzögerte Infiltration von Makrophagen in das WAT und dort eine lokale Immunantwort auslöst. Darüber hinaus wurde die Methode der LCM etabliert und zur Gewinnung hochangereicherter RNA aus den Langerhansschen Inseln eingesetzt. In erstmalig durchgeführten genomweiten Expressionsanalysen wurde zu einem frühen Zeitpunkt in der Diabetesentwicklung der Einfluss einer diabetogenen HF-Diät und einer diabetesprotektiven CHF-Diät auf das Expressionsprofil von pankreatischen Inselzellen verglichen. Im Gegensatz zum WAT bewirkt die diabetogene HF-Diät in Inselzellen einerseits, eine erhöhte Expression von nukleären Genen für die oxidative Phosphorylierung und andererseits von Genen, die mit Zellproliferation assoziiert sind. Zudem wurden 37 bereits annotierte Gene identifiziert, deren differenzielle Expression mit der Diabetesentwicklung korreliert. Das Peptidhormon Cholecystokinin (Cck, 11,8-fach erhöht durch die HF) stellt eines der am stärksten herauf regulierten Gene dar. Die hohe Anreicherung der Cck-mRNA in Inselzellen deutet auf eine bisher unbekannte Funktion des Hormons in der Regulation der Inselzellproliferation hin. Der Transkriptionsfaktor Mlxipl (ChREBP; 3,8-fach erniedrigt durch die HF) stellt in Langerhansschen Inseln eines der am stärksten herunter regulierten Gene dar. Ferner wurde ChREBP, dessen Funktion als glucoseregulierter Transkriptionsfaktor für lipogene Enzyme bislang in der Leber, aber nicht in Inselzellen nachgewiesen werden konnte, erstmals immunhistochemisch in Inselzellen detektiert. Dies deutet auf eine neue, bisher unbekannte regulatorische Funktion von ChREBP im Glucosesensor-Mechanismus der Inselzellen hin. Eine durchgeführte Korrelation der mit der Diabetesentwicklung assoziierten, differenziell exprimierten Inselzellgene mit Genvarianten aus humanen genomweiten Assoziationsstudien für Typ-2-Diabetes (WTCCC, Broad-DGI-T2D-Studie) ermöglichte die Identifizierung von 24 neuartigen Diabetes-Kandidatengenen. Die Ergebnisse der erstmals am polygenen NZO-Mausmodell durchgeführten genomweiten Expressionsuntersuchungen bestätigen bisherige Befunde aus Mausmodellen für Adipositas und Diabetes (z.B. ob/ob- und db/db-Mäuse), zeigen in einigen Fällen aber auch Unterschiede auf. Insbesondere in der oxidativen Phosphorylierung könnten die Ergebnisse relevant sein für das Verständnis der Pathogenese des polygen-bedingten humanen metabolischen Syndroms.
Mars is one of the best candidates among planetary bodies for supporting life. The presence of water in the form of ice and atmospheric vapour together with the availability of biogenic elements and energy are indicators of the possibility of hosting life as we know it. The occurrence of permanently frozen ground – permafrost, is a common phenomenon on Mars and it shows multiple morphological analogies with terrestrial permafrost. Despite the extreme inhospitable conditions, highly diverse microbial communities inhabit terrestrial permafrost in large numbers. Among these are methanogenic archaea, which are anaerobic chemotrophic microorganisms that meet many of the metabolic and physiological requirements for survival on the martian subsurface. Moreover, methanogens from Siberian permafrost are extremely resistant against different types of physiological stresses as well as simulated martian thermo-physical and subsurface conditions, making them promising model organisms for potential life on Mars. The main aims of this investigation are to assess the survival of methanogenic archaea under Mars conditions, focusing on methanogens from Siberian permafrost, and to characterize their biosignatures by means of Raman spectroscopy, a powerful technology for microbial identification that will be used in the ExoMars mission. For this purpose, methanogens from Siberian permafrost and non-permafrost habitats were subjected to simulated martian desiccation by exposure to an ultra-low subfreezing temperature (-80ºC) and to Mars regolith (S-MRS and P-MRS) and atmospheric analogues. They were also exposed to different concentrations of perchlorate, a strong oxidant found in martian soils. Moreover, the biosignatures of methanogens were characterized at the single-cell level using confocal Raman microspectroscopy (CRM). The results showed survival and methane production in all methanogenic strains under simulated martian desiccation. After exposure to subfreezing temperatures, Siberian permafrost strains had a faster metabolic recovery, whereas the membranes of non-permafrost methanogens remained intact to a greater extent. The strain Methanosarcina soligelidi SMA-21 from Siberian permafrost showed significantly higher methane production rates than all other strains after the exposure to martian soil and atmospheric analogues, and all strains survived the presence of perchlorate at the concentration on Mars. Furthermore, CRM analyses revealed remarkable differences in the overall chemical composition of permafrost and non-permafrost strains of methanogens, regardless of their phylogenetic relationship. The convergence of the chemical composition in non-sister permafrost strains may be the consequence of adaptations to the environment, and could explain their greater resistance compared to the non-permafrost strains. As part of this study, Raman spectroscopy was evaluated as an analytical technique for remote detection of methanogens embedded in a mineral matrix. This thesis contributes to the understanding of the survival limits of methanogenic archaea under simulated martian conditions to further assess the hypothetical existence of life similar to methanogens on the martian subsurface. In addition, the overall chemical composition of methanogens was characterized for the first time by means of confocal Raman microspectroscopy, with potential implications for astrobiological research.
Permafrost-affected ecosystems including peat wetlands are among the most obvious regions in which current microbial controls on organic matter decomposition are likely to change as a result of global warming. Wet tundra ecosystems in particular are ideal sites for increased methane production because of the waterlogged, anoxic conditions that prevail in seasonally increasing thawed layers. The following doctoral research project focused on investigating the abundance and distribution of the methane-cycling microbial communities in four different polygons on Herschel Island and the Yukon Coast. Despite the relevance of the Canadian Western Arctic in the global methane budget, the permafrost microbial communities there have thus far remained insufficiently characterized. Through the study of methanogenic and methanotrophic microbial communities involved in the decomposition of permafrost organic matter and their potential reaction to rising environmental temperatures, the overarching goal of the ensuing thesis is to fill the current gap in understanding the fate of the organic carbon currently stored in Artic environments and its implications regarding the methane cycle in permafrost environments. To attain this goal, a multiproxy approach including community fingerprinting analysis, cloning, quantitative PCR and next generation sequencing was used to describe the bacterial and archaeal community present in the active layer of four polygons and to scrutinize the diversity and distribution of methane-cycling microorganisms at different depths. These methods were combined with soil properties analyses in order to identify the main physico-chemical variables shaping these communities. In addition a climate warming simulation experiment was carried-out on intact active layer cores retrieved from Herschel Island in order to investigate the changes in the methane-cycling communities associated with an increase in soil temperature and to help better predict future methane-fluxes from polygonal wet tundra environments in the context of climate change. Results showed that the microbial community found in the water-saturated and carbon-rich polygons on Herschel Island and the Yukon Coast was diverse and showed a similar distribution with depth in all four polygons sampled. Specifically, the methanogenic community identified resembled the communities found in other similar Arctic study sites and showed comparable potential methane production rates, whereas the methane oxidizing bacterial community differed from what has been found so far, being dominated by type-II rather than type-I methanotrophs. After being subjected to strong increases in soil temperature, the active-layer microbial community demonstrated the ability to quickly adapt and as a result shifts in community composition could be observed. These results contribute to the understanding of carbon dynamics in Arctic permafrost regions and allow an assessment of the potential impact of climate change on methane-cycling microbial communities. This thesis constitutes the first in-depth study of methane-cycling communities in the Canadian Western Arctic, striving to advance our understanding of these communities in degrading permafrost environments by establishing an important new observatory in the Circum-Arctic.
Domestication syndrome has resulted in the large loss of genetic variation of crop plants. Because of such genetic loss, productivity of various beneficial secondary (specialized) metabolites that protect against abiotic/biotic stresses, has been narrowed in many domesticated crops. Many key regulators or structural genes of secondary metabolic pathways in the domesticated as well as wild tomatoes are still largely unknown. In recent studies, metabolic quantitative trait loci (mQTL) analysis using the population of introgression lines (ILs), each containing a single introgression from Solanum pennellii (wild tomato) in the genetic background of domesticated tomato (M82, Solanum lycopersicum), has been used for investigation of metabolic regulation and key genes involved in both primary and secondary metabolism. In this thesis, three research projects, i) understanding of metabolic linkage between branched chain amino acids (BCAAs) and secondary metabolism using antisense lines of BCAAs metabolic genes, ii) investigation of novel key genes involved in tomato secondary metabolism and fruit ripening, iii) mapping of drought stress responsive mQTLs in tomato, are presented and discussed. In the first part, metabolic linkage between leucine and secondary metabolism is investigated by analyzing antisense lines of four key genes (ketol-acid reductoisomerase, KARI; dihydroxy-acid dehydratase, DHAD; isopropylmalate dehydratase, IPMD and branched chain aminotransferases1, BCAT1) found previously in mQTL of leucine contents. Obtained results indicate that KARI might be a rate limiting enzyme for iC5 acyl-sucrose synthesis in young leaf but not in red ripe fruits. By integrating obtained results with previous reports, inductive metabolic linkage between BCAAs and other secondary metabolic pathways at DHAD transcriptional levels in fruit is proposed. In the second part, candidate genes that are involved in secondary metabolism and fruit ripening in tomato were found by the approach of expression quantitative trait loci (eQTL) analysis. To predict functions of those candidate genes, functional validation by virus induced gene silencing and transient overexpression were performed. Results obtained by analyzing T0 overexpression and artificial miRNA lines for some of those candidates confirm their predicted functions, for example involved in fruit ripening (WD40, Solyc04g005020) and iC5 acyl-sucrose synthesis (P450, Solyc03g111940). In the third part, mapping of drought stress responsive mQTLs was performed using 57 S. pennellii ILs population. Evaluation of genetic architecture of mQTL analysis resulted in identifying drought responsive ILs (11-2, 8-3-1, 10-1-1 and 3-1). Location of well characterized regulators in these ILs helped to filter potential new key genes involved in drought stress tolerance. Obtained results suggests us our approaches could be viable for narrowing down potential candidates involved in creating interspecific variation in secondary metabolite content and at the level of fruit ripening.
Plant growth and survival depend on photosynthesis in the leaves. This involves the uptake of carbon dioxide from the atmosphere and the simultaneous capture of light energy to produce organic molecules, which enter metabolism and are converted to many other compounds which then serve as building blocks for biomass growth. Leaves are organs specialised for photosynthetic carbon dioxide fixation. The function of leaves involves many trade-offs which must be optimised in order to achieve effective use of resources and maximum photosynthesis. It is known that the morphology of leaves adjusts to the growth environment of plants and this is important for optimising their function for photosynthesis. However, it is unclear how this adjustment is regulated. The general aim of the work presented in this thesis is to understand how leaf growth and morphology are regulated in the model species Arabidopsis thaliana. Special attention was dedicated to the possibility that there might be internal metabolic signals within the plant which affect the growth and development of leaves. In order to investigate this question, leaf growth and development must be considered beyond the level of the single organ and in the context of the whole plant because leaves do not grow autonomously but depend on resources and regulatory influences delivered by the rest of the plant. Due to the complexity of this question, three complementary approaches were taken. In the first and most specific approach it was asked whether a proposed down-stream component of sucrose signalling, trehalose-6-phosphate (Tre-6-P), might influence leaf development and growth. To investigate this question, transgenic Arabidopsis lines with perturbed levels of Tre-6-P were generated using the constitutive 35S promoter to express bacterial enzymes involved in trehalose metabolism. These experiments also led to an unanticipated project concerning a possible role for Tre-6-P in stomatal function, which is another very important function in leaves. In a second and more general approach it was investigated whether changes in sugar levels in plants affect the morphogenesis of leaves in response to light. For this, a series of metabolic mutants impaired in central metabolism were grown in one light environment and their leaf morphology was analysed. In a third and even more general approach the natural variation in leaf and rosette morphological traits was investigated in a panel of wild Arabidopsis accessions with the aim of understanding how leaf morphology affects leaf function and whole plant growth and how different traits relate to each other. The analysis included measurements of leaf morphological traits as well as the number of leaves in the plant to put leaf morphology in a whole plant context. The variance in plant growth could not be explained by variation in photosynthetic rates and only to a small degree by variation in rates of dark respiration. There were four key axes of variation in rosette and leaf morphology – leaf area growth, leaf thickness, cell expansion and leaf number. These four processes were integrated in the context of whole plant growth by models that employed a multiple linear regression approach. This then led to a theoretical approach in which a simple allometric mathematical model was constructed, linking leaf number, leaf size and plant growth rate together in a whole plant context in Arabidopsis.
Biosensoren werden oft für die Messung einzelner Substanzen in komplexen Medien verwendet, wie z.B. bei der Blutzuckerbestimmung. Sie bestehen aus einem physikochemischen Sensor, dem Transduktionselement, und einer darauf immobilisierten biologischen Komponente, dem Erkennungselement. In dieser Arbeit wurde als Transduktionselement eine Elektrode und als Biokomponente das Enzym „Meerrettich Peroxidase“ (engl. horseradish peroxidase, HRP) verwendet. Solche HRP-Elektroden werden für die Messung von Wasserstoffperoxid (H2O2) eingesetzt. H2O2 wird im Körper von weißen Blutkörperchen produziert, um Bakterien abzutöten, wird teilweise ausgeatmet und kann in kondensierter Atemluft nachgewiesen werden. Da viele weiße Blutkörperchen bei einer Chemotherapie abgetötet und dadurch die Patienten anfälliger für Infektionen werden, muss ihre Anzahl regelmäßig überwacht werden. Dazu wird zurzeit Blut abgenommen. Im ersten Teil dieser Arbeit wurde untersucht, ob eine Überwachung der Anzahl an weißen Blutkörperchen ohne Blutabnahme durch eine H2O2-Messung erfolgen kann. Ein direkter Zusammenhang zwischen der ausgeatmeten H2O2-Menge und der Zahl der weißen Blutkörperchen konnte dabei nicht festgestellt werden. Für empfindliche H2O2-Messungen mit einer HRP-Elektrode ist ein schneller Austausch von Elektronen zwischen der Elektrode und dem Enzym notwendig. Eine Vorraussetzung dafür ist eine kurze Distanz zwischen dem aktiven Zentrum des Enzyms und der Elektrodenoberfläche. Um einen kurzen Abstand zu erreichen wurden im zweiten Teil dieser Arbeit verschiedene poröse graphitähnliche Materialien aus pyrolysierten Kobalt-Porphyrinen für die Elektrodenherstellung verwendet. Dabei stellte sich heraus, dass eines der untersuchten Materialien, welches Poren von etwa der Größe eines Enzyms hat, Elektronen etwa 200mal schneller mit dem Enzym austauscht als festes Graphit. Die HRP selbst enthält in seinem aktiven Zentrum ein Eisen-Protoporphyrin, also ein aus vier Ringen bestehendes flaches Molekül mit einem Eisenatom im Zentrum. Reagiert die HRP mit H2O2, so entzieht es dem Peroxid zwei Elektronen. Eines dieser Elektronen wird am Eisen, das andere im Ringsystem zwischengespeichert, bevor sie an ein anderes Molekül oder an die Elektrode weitergegeben werden. Im letzten Teil dieser Arbeit wurde das Eisen durch Osmium ausgetauscht. Das so veränderte Enzym entzieht Peroxiden nur noch ein Elektron. Dadurch reagiert es zwar langsamer mit Wasserstoffperoxid, dafür aber schneller mit tert-Butylhydroperoxid, einem organischen Vertreter der Peroxid-Familie.
Apoptose, der programmierte Zelltod, spielt eine wichtige Rolle für das Gleichgewicht zwischen Proliferation und Sterben von Zellen und ist außerdem an der Beseitigung von infizierten und geschädigten Zellen beteiligt. Apoptose kann durch Stimulation von Rezeptoren aus der Familie der TNF-Rezeptoren wie CD95, ausgelöst werden. Nach Liganden-induzierter Trimerisierung der Rezeptoren bindet FADD an den zytoplasmatischen Teil des Rezeptors und rekrutiert Caspase-8 und/oder -10. Die räumliche Nähe der Caspasen in diesem als DISC bezeichneten Komplex führt zu ihrer auto- und transkatalytischen Spaltung und damit Aktivierung. Dadurch wird das apoptotische Programm gestartet, welches zum Tod der Zelle führt. Kontrolliert wird dieser Vorgang von einer Vielzahl anti-apoptotischer Proteine. Störungen in diesem System sind an der Entstehung einer Reihe von Krankheiten beteiligt. Die Blockade der Apoptoseinduktion kann zur Entstehung von Tumoren beitragen. Das klassische Hodgkin Lymphom ist eine maligne Erkrankung des lymphatischen Systems. Die Tumorzellen sind große, einkernige Hodgkin- oder mehrkernige Reed/Sternbergzellen (HRS-Zellen). Sie leiten sich von Keimzentrum-B-Zellen ab. In HRS-Zellen fehlt die Expression einer Vielzahl von typischen B-Zellmarkern, darunter die des B-Zellrezeptors. Solche B-Zellen sterben normalerweise während der Keimzentrumsreaktion durch Apoptose. An diesem Prozess ist CD95 beteiligt. In einer Reihe von malignen Erkrankungen wurden eine Herunterregulation der CD95-Expression oder Mutationen im CD95-Gen beobachtet. Es wird daher vermutet, dass CD95-induzierte Apoptose zur Entfernung von Tumorzellen beiträgt. Im Gegensatz dazu exprimieren sowohl primäre HRS-Zellen als auch etablierte HRS-Zelllinien in der Regel Wildtyp-CD95, sind aber trotzdem CD95-resistent. In dieser Arbeit konnte gezeigt werden, dass Komponenten des CD95-Systems, im Gegensatz zu anderen malignen Erkrankungen, in den HRS-Zellen hochreguliert sind, darunter CD95 selbst. In immunpräzipitierten DISCs von CD95-stimulierten HRS-Zellen wurde neben FADD und Caspase-8/-10 auch c-FLIP nachgewiesen. c-FLIP ist ein Caspase-8/-10-Homolog, das ebenfalls an FADD bindet, aber aufgrund fehlender katalytischer Aktivität die Aktivierung der Caspasen im DISC und damit die Apoptoseinduktion verhindert. Eine starke c-FLIP-Expression konnte in allen HRS-Zelllinien und in den HRS-Zellen nahezu aller untersuchter primärer Hodgkinfälle (55/59) gezeigt werden. Durch siRNA-vermittelte Herunterregulation von c-FLIP war es möglich, HRS-Zelllinien gegenüber CD95-induzierter Apoptose zu sensitivieren. Dies zeigt, dass die CD95-Rezeptor-induzierte Apoptose in den HRS-Zellen nicht strukturell, sondern funktionell inhibiert ist und c-FLIP stark zu dieser Inhibition beiträgt. Darüber hinaus konnte gezeigt werden, dass die c-FLIP-Expression in den HRS-Zellen von der konstitutiven Aktivität des Transkriptionsfaktors NF-κB abhängt, die charakteristisch für diese Zellen ist. Normalerweise wird NF-κB von Inhibitorproteinen, den IκBs, im Zytoplasma zurückgehalten. Diverse Stimuli können den IKK-Komplex aktivieren, der die IκBs an bestimmten Serinresten phosphoryliert. Dies hat die Ubiquitinylierung und den Abbau der IκBs zur Folge, wodurch NF-κB frei wird, in den Kern wandert und dort seine Zielgene aktiviert. Es wird angenommen, dass in HRS-Zellen ein konstitutiv aktiver IKK-Komplex und teilweise Mutationen der IκB-Proteine zur konstitutiven NF-κB-Aktivität beitragen. Zu den NF-κB-abhängigen Genen in den HRS-Zellen gehören solche mit anti-apoptotischer und Zellzyklus-treibender Wirkung. Die Inhibition der NF-κB-Aktivität in den HRS-Zellen führt zu Apoptose und eingeschränkter Proliferation. Von dreiwertigem Arsen ist bekannt, dass es die Induzierbarkeit des IKK-Komplexes inhibieren kann und damit letztendlich die Aktivierung von NF-κB. In dieser Arbeit konnte gezeigt werden, dass Arsen in HRS-Zellen den konstitutiv aktiven IKK-Komplex inhibiert. In Zelllinien mit intakten IκB-Proteinen führte dies zur NF-κB-Inhibition und Apoptoseinduktion. Die Reduktion der NF-κB-Aktivität ging mit der Herunterregulation von anti-apoptotischen und Proliferations-fördernden Zielgenen einher. Die ektope Überexpression von NF-κB hob die Apoptose-induzierende Wirkung von Arsen teilweise auf. Durch Arsen-Behandlung von Mäusen konnte das Tumorwachstum xenotransplantierter HRS-Zellen stark verlangsamt werden. In explantierten Tumorzellen konnte ebenfalls eine NF-κB-Inhibition nachgewiesen werden. Die NF-κB-Inhibition durch Arsen trägt also stark zur Apoptoseinduktion in den HRS-Zellen bei. Zusammengefasst zeigen die Ergebnisse dieser Arbeit, dass die Modulation der Apoptoseresistenz neue therapeutische Ansätze für die Behandlung des Hodgkin Lymphoms bieten könnte. Der Einsatz von Arsen ist dabei besonders interessant, da Arsen schon für die Behandlung anderer maligner Erkrankungen eingesetzt wird.
The human immunodeficiency virus (HIV) has resisted nearly three decades of efforts targeting a cure. Sustained suppression of the virus has remained a challenge, mainly due
to the remarkable evolutionary adaptation that the virus exhibits by the accumulation of drug-resistant mutations in its genome. Current therapeutic strategies aim at achieving and maintaining a low viral burden and typically involve multiple drugs. The choice of optimal combinations of these drugs is crucial, particularly in the background of treatment failure having occurred previously with certain other drugs. An understanding of the dynamics of viral mutant genotypes aids in the assessment of treatment failure with a certain drug
combination, and exploring potential salvage treatment regimens.
Mathematical models of viral dynamics have proved invaluable in understanding the viral life cycle and the impact of antiretroviral drugs. However, such models typically use simplified and coarse-grained mutation schemes, that curbs the extent of their application to drug-specific clinical mutation data, in order to assess potential next-line therapies. Statistical
models of mutation accumulation have served well in dissecting mechanisms of resistance evolution by reconstructing mutation pathways under different drug-environments. While these models perform well in predicting treatment outcomes by statistical learning, they do not incorporate drug effect mechanistically. Additionally, due to an inherent lack of
temporal features in such models, they are less informative on aspects such as predicting mutational abundance at treatment failure. This limits their application in analyzing the
pharmacology of antiretroviral drugs, in particular, time-dependent characteristics of HIV therapy such as pharmacokinetics and pharmacodynamics, and also in understanding the impact of drug efficacy on mutation dynamics.
In this thesis, we develop an integrated model of in vivo viral dynamics incorporating drug-specific mutation schemes learned from clinical data. Our combined modelling
approach enables us to study the dynamics of different mutant genotypes and assess mutational abundance at virological failure. As an application of our model, we estimate in vivo
fitness characteristics of viral mutants under different drug environments. Our approach also extends naturally to multiple-drug therapies. Further, we demonstrate the versatility of our model by showing how it can be modified to incorporate recently elucidated mechanisms of drug action including molecules that target host factors.
Additionally, we address another important aspect in the clinical management of HIV disease, namely drug pharmacokinetics. It is clear that time-dependent changes in in vivo
drug concentration could have an impact on the antiviral effect, and also influence decisions on dosing intervals. We present a framework that provides an integrated understanding
of key characteristics of multiple-dosing regimens including drug accumulation ratios and half-lifes, and then explore the impact of drug pharmacokinetics on viral suppression.
Finally, parameter identifiability in such nonlinear models of viral dynamics is always a concern, and we investigate techniques that alleviate this issue in our setting.
Inflammatory bowel diseases (IBD), characterised by a chronic inflammation of the gut wall, develop as consequence of an overreacting immune response to commensal bacteria, caused by a combination of genetic and environmental conditions. Large inter-individual differences in the outcome of currently available therapies complicate the decision for the best option for an individual patient. Predicting the prospects of therapeutic success for an individual patient is currently only possible to a limited extent; for this, a better understanding of possible differences between responders and non-responders is needed.
In this thesis, we have developed a mathematical model describing the most important processes of the gut mucosal immune system on the cellular level. The model is based on literature data, which were on the one hand used (qualitatively) to choose which cell types and processes to incorporate and to derive the model structure, and on the other hand (quantitatively) to derive the parameter values. Using ordinary differential equations, it describes the concentration-time course of neutrophils, macrophages, dendritic cells, T cells and bacteria, each subdivided into different cell types and activation states, in the lamina propria and mesenteric lymph nodes. We evaluate the model by means of simulations of the healthy immune response to salmonella infection and mucosal injury.
A virtual population includes IBD patients, which we define through their initially asymptomatic, but after a trigger chronically inflamed gut wall. We demonstrate the model's usefulness in different analyses: (i) The comparison of virtual IBD patients with virtual healthy individuals shows that the disease is elicited by many small or fewer large changes, and allows to make hypotheses about dispositions relevant for development of the disease. (ii) We simulate the effects of different therapeutic targets and make predictions about the therapeutic outcome based on the pre-treatment state. (iii) From the analysis of differences between virtual responders and non-responders, we derive hypotheses about reasons for the inter-individual variability in treatment outcome. (iv) For the example of anti-TNF-alpha therapy, we analyse, which alternative therapies are most promising in case of therapeutic failure, and which therapies are most suited for combination therapies: For drugs also directly targeting the cytokine levels or inhibiting the recruitment of innate immune cells, we predict a low probability of success when used as alternative treatment, but a large gain when used in a combination treatment. For drugs with direct effects on T cells, via modulation of the sphingosine-1-phosphate receptor or inhibition of T cell proliferation, we predict a considerably larger probability of success when used as alternative treatment, but only a small additional gain when used in a combination therapy.
Complex networks have been successfully employed to represent different levels of biological systems, ranging from gene regulation to protein-protein interactions and metabolism. Network-based research has mainly focused on identifying unifying structural properties, including small average path length, large clustering coefficient, heavy-tail degree distribution, and hierarchical organization, viewed as requirements for efficient and robust system architectures. Existing studies estimate the significance of network properties using a generic randomization scheme - a Markov-chain switching algorithm - which generates unrealistic reactions in metabolic networks, as it does not account for the physical principles underlying metabolism. Therefore, it is unclear whether the properties identified with this generic approach are related to the functions of metabolic networks. Within this doctoral thesis, I have developed an algorithm for mass-balanced randomization of metabolic networks, which runs in polynomial time and samples networks almost uniformly at random. The properties of biological systems result from two fundamental origins: ubiquitous physical principles and a complex history of evolutionary pressure. The latter determines the cellular functions and abilities required for an organism’s survival. Consequently, the functionally important properties of biological systems result from evolutionary pressure. By employing randomization under physical constraints, the salient structural properties, i.e., the smallworld property, degree distributions, and biosynthetic capabilities of six metabolic networks from all kingdoms of life are shown to be independent of physical constraints, and thus likely to be related to evolution and functional organization of metabolism. This stands in stark contrast to the results obtained from the commonly applied switching algorithm. In addition, a novel network property is devised to quantify the importance of reactions by simulating the impact of their knockout. The relevance of the identified reactions is verified by the findings of existing experimental studies demonstrating the severity of the respective knockouts. The results suggest that the novel property may be used to determine the reactions important for viability of organisms. Next, the algorithm is employed to analyze the dependence between mass balance and thermodynamic properties of Escherichia coli metabolism. The thermodynamic landscape in the vicinity of the metabolic network reveals two regimes of randomized networks: those with thermodynamically favorable reactions, similar to the original network, and those with less favorable reactions. The results suggest that there is an intrinsic dependency between thermodynamic favorability and evolutionary optimization. The method is further extended to optimizing metabolic pathways by introducing novel chemically feasibly reactions. The results suggest that, in three organisms of biotechnological importance, introduction of the identified reactions may allow for optimizing their growth. The approach is general and allows identifying chemical reactions which modulate the performance with respect to any given objective function, such as the production of valuable compounds or the targeted suppression of pathway activity. These theoretical developments can find applications in metabolic engineering or disease treatment. The developed randomization method proposes a novel approach to measuring the significance of biological network properties, and establishes a connection between large-scale approaches and biological function. The results may provide important insights into the functional principles of metabolic networks, and open up new possibilities for their engineering.
During the last decades, the global change of the environment has caused a dramatic loss of habitats and species. In Central Europe, open habitats are particularly affected. The main objective of this thesis was to experimentally test the suitability of wild megaherbivore grazing as a conservation tool to manage open habitats. We studied the effect of wild ungulates in a 160 ha game preserve in NE Germany in three successional stages (i) Corynephorus canescens-dominated grassland, (ii) ruderal tall forb vegetation dominated by Tanacetum vulgare and (iii) Pinus sylvestris-pioneer forest over three years. Our results demonstrate that wild megaherbivores considerably affected species composition and delayed successional pathways in open habitats. Grazing effects differed considerably between successional stages: species richness was higher in grazed ruderal and pioneer forest plots, but not in the Corynephorus sites. Species composition changed significantly in the Corynephorus and ruderal sites. Grazed ruderal sites had turned into sites with very short vegetation dominated by Agrostis spp. and the moss Brachythecium albicans, most species did not flower. Woody plant cover was significantly affected only in the pioneer forest sites. Young pine trees were severely damaged and tree height was considerably reduced, leading to a “Pinus-macchie”-appearance. Ecological patterns and processes are known to vary with spatial scale. Since grazing by megaherbivores has a strong spatial component, the scale of monitoring success of grazing may largely differ among and within different systems. Thus, the second aim of this thesis was to test whether grazing effects are consistent over different spatial scales, and to give recommendations for appropriate monitoring scales. For this purpose, we studied grazing effects on plant community structure using multi-scale plots that included three nested spatial scales (0.25 m2, 4 m2, and 40 m2). Over all vegetation types, the scale of observation directly affected grazing effects on woody plant cover and on floristic similarity, but not on the proportion of open soil and species richness. Grazing effects manifested at small scales regarding floristic similarity in pioneer forest and ruderal sites and regarding species richness in ruderal sites. The direction of scale-effects on similarity differed between vegetation types: Grazing effects on floristic similarity in the Corynephorus sites were significantly higher at the medium and large scale, while in the pioneer forest sites they were significantly higher at the smallest scale. Disturbances initiate vegetation changes by creating gaps and affecting colonization and extinction rates. The third intention of the thesis was to investigate the effect of small-scale disturbances on the species-level. In a sowing experiment, we studied early establishment probabilities of Corynephorus canescens, a key species of open sandy habitats. Applying two different regimes of mechanical ground disturbance (disturbed and undisturbed) in the three successional stages mentioned above, we focused on the interactive effects of small-scale disturbances, successional stage and year-to-year variation. Disturbance led to higher emergence in a humid and to lower emergence in a very dry year. Apparently, when soil moisture was sufficient, the main factor limiting C. canescens establishment was competition, while in the dry year water became the limiting factor. Survival rates were not affected by disturbance. In humid years, C. canescens emerged in higher numbers in open successional stages while in the dry year, emergence rates were higher in late stages, suggesting an important role of late successional stages for the persistence of C. canescens. We conclude that wild ungulate grazing is a useful tool to slow down succession and to preserve a species-rich, open landscape, because it does not only create disturbances, thereby supporting early successional stages, but at the same time efficiently controls woody plant cover. However, wild ungulate grazing considerably changed the overall appearance of the landscape. Additional measures like shifting exclosures might be necessary to allow vulnerable species to flower and reproduce. We further conclude that studying grazing impacts on a range of scales is crucial, since different parameters are affected at different spatial scales. Larger scales are suitable for assessing grazing impact on structural parameters like the proportion of open soil or woody plant cover, whereas species richness and floristic similarity are affected at smaller scales. Our results further indicate that the optimal strategy for promoting C. canescens is to apply disturbances just before seed dispersal and not during dry years. Further, at the landscape scale, facilitation by late successional species may be an important mechanism for the persistence of protected pioneer species.
In the context of ecological risk assessment of chemicals, individual-based population models hold great potential to increase the ecological realism of current regulatory risk assessment procedures. However, developing and parameterizing such models is time-consuming and often ad hoc. Using standardized, tested submodels of individual organisms would make individual-based modelling more efficient and coherent. In this thesis, I explored whether Dynamic Energy Budget (DEB) theory is suitable for being used as a standard submodel in individual-based models, both for ecological risk assessment and theoretical population ecology. First, I developed a generic implementation of DEB theory in an individual-based modeling (IBM) context: DEB-IBM. Using the DEB-IBM framework I tested the ability of the DEB theory to predict population-level dynamics from the properties of individuals. We used Daphnia magna as a model species, where data at the individual level was available to parameterize the model, and population-level predictions were compared against independent data from controlled population experiments. We found that DEB theory successfully predicted population growth rates and peak densities of experimental Daphnia populations in multiple experimental settings, but failed to capture the decline phase, when the available food per Daphnia was low. Further assumptions on food-dependent mortality of juveniles were needed to capture the population dynamics after the initial population peak. The resulting model then predicted, without further calibration, characteristic switches between small- and large-amplitude cycles, which have been observed for Daphnia. We conclude that cross-level tests help detecting gaps in current individual-level theories and ultimately will lead to theory development and the establishment of a generic basis for individual-based models and ecology. In addition to theoretical explorations, we tested the potential of DEB theory combined with IBMs to extrapolate effects of chemical stress from the individual to population level. For this we used information at the individual level on the effect of 3,4-dichloroanailine on Daphnia. The individual data suggested direct effects on reproduction but no significant effects on growth. Assuming such direct effects on reproduction, the model was able to accurately predict the population response to increasing concentrations of 3,4-dichloroaniline. We conclude that DEB theory combined with IBMs holds great potential for standardized ecological risk assessment based on ecological models.
Light-triggered release of bioactive compounds from HA/PLL multilayer films for stimulation of cells
(2016)
The concept of targeting cells and tissues by controlled delivery of molecules is essential in the field of biomedicine. The layer-by-layer (LbL) technology for the fabrication of polymer multilayer films is widely implemented as a powerful tool to assemble tailor-made materials for controlled drug delivery. The LbL films can as well be engineered to act as mimics of the natural cellular microenvironment. Thus, due to the myriad possibilities such as controlled cellular adhesion and drug delivery offered by LbL films, it becomes easily achievable to direct the fate of cells by growing them on the films.
The aim of this work was to develop an approach for non-invasive and precise control of the presentation of bioactive molecules to cells. The strategy is based on employment of the LbL films, which function as support for cells and at the same time as reservoirs for bioactive molecules to be released in a controlled manner. UV light is used to trigger the release of the stored ATP with high spatio-temporal resolution. Both physico-chemical (competitive intermolecular interactions in the film) and biological aspects (cellular response and viability) are addressed in this study.
Biopolymers hyaluronic acid (HA) and poly-L-lysine (PLL) were chosen as the building blocks for the LbL film assembly. Poor cellular adhesion to native HA/PLL films as well as significant degradation by cells within a few days were shown. However, coating the films with gold nanoparticles not only improved cellular adhesion and protected the films from degradation, but also formed a size-exclusion barrier with adjustable cut-off in the size range of a few tens of kDa.
The films were shown to have high reservoir capacity for small charged molecules (reaching mM levels in the film). Furthermore, they were able to release the stored molecules in a sustained manner. The loading and release are explained by a mechanism based on interactions between charges of the stored molecules and uncompensated charges of the biopolymers in the film. Charge balance and polymer dynamics in the film play the pivotal role.
Finally, the concept of light-triggered release from the films has been proven using caged ATP loaded into the films from which ATP was released on demand. ATP induces a fast cellular response, i.e. increase in intracellular [Ca2+], which was monitored in real-time. Limitations of the cellular stimulation by the proposed approach are highlighted by studying the stimulation as a function of irradiation parameters (time, distance, light power). Moreover, caging molecules bind to the film stronger than ATP does, which opens new perspectives for the use of the most diverse chemical compounds as caging molecules.
Employment of HA/PLL films as a nouvelle support for cellular growth and hosting of bioactive molecules, along with the possibility to stimulate individual cells using focused light renders this approach highly efficient and unique in terms of precision and spatio-temporal resolution among those previously described. With its high potential, the concept presented herein provides the foundation for the design of new intelligent materials for single cell studies, with the focus on tissue engineering, diagnostics, and other cell-based applications.
Cellular membranes constantly experience remodeling, as exemplified by morphological changes during endo- and exocytosis. Regulation of membrane morphology is essential for these processes. In this work, we attempt to establish a regulation path based on the use of photoswitches exhibiting conformational changes in model membranes, namely, giant unilamellar vesicles (GUVs). The mechanism of the changes in the GUVs’ morphology caused by isomerization of the photosensitive molecules has been previously explored but still remains elusive. We examine the morphological reshaping of GUVs in the presence of the photoswitch o-tetrafluoroazobenzene (F-azo) and show that the mechanism behind the resulting morphological changes involves both an increase in the membrane area and generation of a positive spontaneous curvature. First, we characterize the partitioning of F-azo in a single-component membrane using both experimental and computational approaches. The partition coefficient calculated from molecular dynamic simulations agrees with experimental data obtained with size-exclusion chromatography. Then, we implement the approach of vesicle electrodeformation in order to assess the increase in the membrane area, which is observed as a result of the conformational change of F-azo. Finally, the local and the effective membrane spontaneous curvatures were estimated from the observed shapes of vesicles exhibiting outward budding. We then extend the application of the F-azo to multicomponent lipid membranes, which exhibit a coexistence of domains in different liquid phases due to a miscibility gap between the lipids. We perform initial experiments to investigate whether F-azo can be employed to modulate the lateral lipid packing and organization. We observe either complete mixing of the domains or the appearing of disordered domains within the domains of more ordered phase. The type of behavior observed in response to the photoisomerization of F-azo was dependent on the used lipid composition. We believe that the findings introduced here will have an impact in understanding and controlling both lipid phase modulation and regulation of the membrane morphology in membrane systems.
Light-induced pH cycle
(2019)
Background Many biochemical reactions depend on the pH of their environment and some are strongly accelerated in an acidic surrounding. A classical approach to control biochemical reactions non-invasivly is by changing the temperature. However, if the pH could be controlled by optical means using photo-active chemicals, this would mean to be able to accelerate suitable biochemical reactions. Optically switching the pH can be achieved by using photoacids. A photoacid is a molecule with a functional group that releases a proton upon irradiation with the suitable wavelength, acidifying the environmental aqueous surrounding. A major goal of this work was to establish a non-invasive method of optically controlling the pH in aqueous solutions, offering the opportunity to enhance the known chemical reactions portfolio. To demonstrate the photo-switchable pH cycling we chose an enzymatic assay using acid phosphatase, which is an enzyme with a strong pH dependent activity.
Results In this work we could demonstrate a light-induced, reversible control of the enzymatic activity of acid phosphatase non-invasivly. To successfully conduct those experiments a high power LED array was designed and built, suitable for a 96 well standard microtiter plate, not being commercially available. Heat management and a lateral ventilation system to avoid heat accumulation were established and a stable light intensity achieved. Different photoacids were characterised and their pH dependent absorption spectra recorded. By using the reversible photoacid G-acid as a proton donor, the pH can be changed reversibly using high power UV 365 nm LEDs. To demonstrate the pH cycling, acid phosphatase with hydrolytic activity under acidic conditions was chosen. An assay using the photoacid together with the enzyme was established, also providing that G-acid does not inhibit acid phosphatase. The feasibility of reversibly regulating the enzyme’s pH dependent activity by optical means was demonstrated, by controlling the enzymatic activity with light. It was demonstrated that the enzyme activity depends on the light exposure time only. When samples are not illuminated and left in the dark, no enzymatic activity was recorded. The process can be rapidly controlled by simply switching the light on and off and should be applicable to a wide range of enzymes and biochemical reactions.
Conclusions Reversible photoacids offer a light-dependent regulation of pH, making them extremely attractive for miniaturizable, non-invasive and time-resolved control of biochemical reactions. Many enzymes have a sharp pH dependent activity, thus the established setup in this thesis could be used for a versatile enzyme portfolio. Even though the demonstrated photo-switchable strategy could also be used for non-enzymatic assays, greatly facilitating the assay establishment. Photoacids have the potential for high throughput methods and automation. We demonstrated that it is possible to control photoacids using commonly available LEDs, making their use in highly integrated devices and instruments more attractive. The successfully designed 96 well high power UV LED array presents an opportunity for general combinatorial analysis in e.g. photochemistry, where a high light intensity is needed for the investigation of various reactions.
Die vorliegende Dissertation behandelt die Ökologie von Cnidium dubium (Schkuhr) Thell. (Sumpf-Brenndolde), Gratiola officinalis L. (Gottes-Gnadenkraut) und Juncus atratus Krocker (Schwarze Binse), drei gefährdeten Arten, die als sogenannte Stromtalpflanzen in Mitteleuropa in ihrem Vorkommen eng an die Flussauen gebunden sind. Die Arbeit basiert auf verschiedenen Simulationsexperimenten und Feldstudien in der Unteren Havelniederung, einem „Feuchtgebiet von internationaler Bedeutung“. Sie behandelt Themenkomplexe wie das Samenbankverhalten, die Samenkeimung, die Stickstofflimitierung, die Konkurrenzkraft, das Verhalten der Pflanzen nach einer Sommertrockenheit und nach einer Winter/Frühjahrsüberflutung. Ferner widmet sie sich der Populationsbiologie der Arten und dem Verhalten der Pflanzen nach besonderen Störungsereignissen wie Mahd, Herbivorie und der Sommerflut 2002. Der Leser erfährt, wie die Pflanzen in verschiedenen Lebensphasen auf die auentypische Umwelt reagieren und erhält umfassende Einblicke in physiologische Mechanismen, die der Anpassung an die typischen Bedingungen einer mitteleuropäischen Flussaue dienen. Eine Interpretation der Ergebnisse zeigt auf, welche der spezifischen Eigenschaften zur Gefährdung der drei Stromtalarten beitragen. Die Arbeit ist für den Arten-, Biotop- und Landschaftsschutz interessant. Darüber hinaus bietet sie zahlreiche Anknüpfungspunkte zur ökophysiologischen Grundlagenforschung. Die verstärkte Nutzung physiologischer Methoden bei der Klärung ökologischer Fragestellungen wird angeregt.
Die vorliegende Arbeit ist eine aktuelle Dokumentation von Körperbau, Körperzusammensetzung und Ernährungsgewohnheiten an 708 jüngeren und älteren Männern und Frauen aus dem Bundesland Brandenburg. Der Körperbau wurde über ein 42 Längen-, Breiten-, Tiefen- und Umfangsmaße umfassendes anthropometrisches Untersuchungsprogramm bestimmt. Die Einschätzung von Gesamtkörperfettanteil und Magermasse erfolgte mit zwei Feldmethoden, der Hautfaltendickenmessung und der bioelektrischen Impedanzanalyse. Mit Hilfe eines semiquantitativen Fragebogens zu den Ernährungsgewohnheiten wurde der Lebensmittelverzehr erfasst und daraus die Energie- und Nährstoffaufnahme berechnet. Die Ergebnisse zum Körperbau zeigen im Mittel eine Abnahme der Längenmaße, jedoch eine Zunahme der Breiten-, Tiefen- und Umfangsmaße mit steigendem Erwachsenenalter. Einfache Parameter zur Beurteilung des Ernährungszustandes, wie Körpermasse und Body-Mass-Index (BMI) nehmen im Alter geschlechtsspezifisch zu. Nach den Richtlinien der WHO für den BMI gelten 55,3% der untersuchten Männern als übergewichtig, davon 10% als adipös. Von allen untersuchten Frauen sind 41,6% übergewichtig, davon sind 14,3% adipös. Der Anteil der Übergewichtigen ist zwar beim weiblichen Geschlecht geringer, aber dafür haben mehr Frauen die Grenze zur Adipositas überschritten. Für eine wissenschaftlich exakte Beurteilung des Ernährungszustandes reichen Körpermasse und BMI nicht aus, da sie die Körperzusammensetzung nicht bzw. nicht genügend berücksichtigen. Die subkutane Fettschichtdicke nimmt insbesondere am Rumpf zu, was als zusätzliches Gesundheitsrisiko gilt. Der Gesamtkörperfettanteil steigt im Erwachsenenalter abhängig von der Berechnungsmethode an. Die untersuchten Frauen sind gegenüber den Männern in allen Altersgruppen durch einen etwa ein Drittel höheren Körperfettanteil gekennzeichnet. Die tägliche Nahrungsenergieaufnahme der untersuchten Personen lässt eine abnehmende Tendenz bis zum 65. Lebensjahr erkennen. Trotz einer sinkenden Nahrungsenergieaufnahme im Alter, nimmt der BMI zu. Mögliche Ursachen hierfür werden in der Arbeit diskutiert. Der Anteil der Grundnährstoffe an der Energiebereitstellung entspricht in keiner der untersuchten Gruppen den Empfehlungen der Deutschen Gesellschaft für Ernährung. Allgemein ist der Fettkonsum zu hoch und der Kohlenhydratanteil zu gering. Das zeigt sich besonders in den beiden mittleren untersuchten Altersgruppen und bei den Männern stärker als bei den Frauen.
Die ergonomische Anpassung von Produkten der körpernahen Umwelt an den menschlichen Körper in seiner gesamten Variabilität erfordert anthropometrische Grundlagen. Die vorliegende Arbeit beschreibt und analysiert die Körpermasse, 17 Längenmaße, 5 Skelettrobustizitätsmaße, 6 Korpulenzmaße, 3 Kopfmaße, 5 Handmaße, 3 Fußmaße, sowie 10 Beweglichkeitsmaße der Wirbelsäule, 8 Beweglichkeitsmaße der Hand, 2 Beweglichkeitsmaße des Beines und 7 Handkräfte von 295 Probanden der drei Altersgruppen 20 bis 29 Jahre, 50 bis 59 Jahre und 60 bis 69 Jahre. Die Untersuchungen wurden im Zeitraum von September 2006 bis April 2007 durchgeführt. Ziel der Arbeit ist es, für den überwiegenden Teil der untersuchten körperlichen Merkmale erstmals für die deutsche Bevölkerung geschlechts- und altersspezifische Mittelwerte und Variabilitätsbereiche bis zum vollendeten 70. Lebensjahr zur Verfügung zu stellen. Das gilt insbesondere für die untersuchten Beweglichkeitsmaße und Handkräfte. Erstmals werden Korrelationen zwischen der Körperform, wie sie sich im Maßzusammenhang der unterschiedlichen Körperbautypen darstellt, der Gelenkbeweglichkeit und den Handkräften vorgestellt. Darüber hinaus wird durch den Vergleich der Ergebnisse der jungen und der beiden älteren Erwachsenengruppen untersucht, welche Unterschiede zwischen den verschiedenen Altersgruppen bestehen. Im Hinblick auf die zeitliche Gültigkeit der aktuellen Untersuchungsergebnisse werden der Einfluss des säkularen Trends und der Einfluss der ontogenetischen Alternsprozesse auf Längenmaße und Korpulenzmaße diskutiert. Die Arbeit zeigt auf, dass innerhalb der untersuchten Probanden eine große Variationsbreite in den Körpermaßen auftritt. Es lassen sich typische Altersunterschiede erkennen. Die Älteren sind im Mittel kleiner, weisen jedoch größere Skelettrobustizitäts- und Korpulenzmaße auf. Die dynamischen Maße weisen auf eine geringere Beweglichkeit der Wirbelsäule, teilweise auch der Hand hin. Die Handkräfte der Frauen werden mit zunehmendem Alter geringer, bei den Männern sind die Älteren kräftiger als die jungen Erwachsenen. Die Ergebnisse deuten auf einen gegenüber früheren Generationen verzögerten Beginn von körperlichen Alterserscheinungen hin, der im Hinblick auf die steigende Lebenserwartung der Bevölkerung eingehender untersucht werden sollte.
Die Enzyme der Sulfotransferase-Gensuperfamilie (SULT) konjugieren nukleophile Gruppen von kleinen endogenen Verbindungen und Fremdstoffen mit der negativ geladenen Sulfo-Gruppe. Dadurch wird die Polarität dieser Verbindungen erhöht, ihre passive Permeation von Zellmembranen verhindert und somit ihre Ausscheidung erleichtert. Jedoch stellt die Sulfo-Gruppe in bestimmten chemischen Verbindungen eine gute Abgangsgruppen dar. Aus der Spaltung resultierende Carbenium- oder Nitreniumionen können mit DNA oder anderen zellulären Nukleophilen reagieren. In Testsystemen für Mutagenität wurden zahlreiche Verbindungen, darunter Nahrungsinhaltsstoffe und Umweltkontaminanten, durch SULT zu Mutagenen aktiviert. Dabei zeigten sich zum einen eine ausgeprägte Substratspezifität selbst orthologer SULT-Formen unterschiedlicher Spezies und zum anderen Interspezies-Unterschiede in der SULT-Gewebeverteilung. Daher könnten sich die Zielgewebe einer SULT-induzierten Krebsentstehung bei Mensch und Nager unterscheiden. Um die Beteiligung von humanen SULT an der Bioaktivierung von Fremdstoffen im Tiermodell untersuchen zu können, wurden transgene Mauslinien für den Cluster der humanen SULT1A1- und -1A2-Gene sowie für die humane SULT1B1 generiert. Zur Herstellung der transgenen Linien wurden große genomische Konstrukte verwendet, die die SULT-Gene sowie – zum Erreichen einer der Humansituation entsprechenden Gewebeverteilung der Proteinexpression – deren potentielle regulatorische Sequenzen enthielten. Es wurden je drei transgene Linien für hSULT1A1/hSULT1A2 und drei transgene Linien für hSULT1B1 etabliert. Die Expression der humanen Proteine konnte in allen Linien gezeigt werden und fünf der sechs Linien konnten zur Homozygotie bezüglich der Transgene gezüchtet werden. In der molekularbiologischen Charakterisierung der transgenen Linien wurde der chromosomale Integrationsort der Konstrukte bestimmt und die Kopienzahl pro Genom untersucht. Mit Ausnahme einer hSULT1A1/hSULT1A2-transgenen Linie, bei der Kopien des Konstrukts in zwei unterschiedliche Chromosomen integriert vorliegen, wiesen alle Linien nur einen Transgen-Integrationsort auf. Die Untersuchung der Transgen-Kopienzahl ergab, dass die Mauslinien zwischen einer und etwa 20 Kopien des Transgen-Konstrukts pro Genom trugen. In der proteinbiochemischen Charakterisierung wurde gezeigt, dass die transgenen Linien die humanen Proteine mit einer weitgehend der des Menschen entsprechenden Gewebeverteilung exprimieren. Die Intensität der im Immunblot nachgewiesenen Expression korrelierte mit der Kopienzahl der Transgene. Die zelluläre und subzelluläre Verteilung der Transgen-Expression wurden bei einer der hSULT1A1/hSULT1A2-transgenen Linien in Leber, Niere, Lunge, Pankreas, Dünndarm und Kolon und bei einer der hSULT1B1-transgenen Linien im Kolon untersucht. Sie stimmte ebenfalls mit der Verteilung der entsprechenden SULT-Formen im Menschen überein. Da sich die erzeugten transgenen Linien aufgrund ihrer mit dem Menschen vergleichbaren Gewebeverteilung der SULT-Expression als Modellsystem zur Untersuchung der menschlichen SULT-vermittelten metabolischen Aktivierung eigneten, wurde eine der hSULT1A1/hSULT1A2-transgenen Linien für zwei erste toxikologische Untersuchungen eingesetzt. Den Mäusen wurden chemische Verbindungen verabreicht, für die in in-vitro-Versuchen eine hSULT1A1/hSULT1A2-vermittelte Bioaktivierung zu Mutagenen gezeigt worden war. In beiden Untersuchungen wurde die Gewebeverteilung der entstandenen DNA-Addukte als Endpunkt einer gewebespezifischen genotoxischen Wirkung ermittelt. In der ersten Untersuchung wurden 90 mg/kg Körpergewicht 2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridin – ein in gebratenem Fleisch gebildetes heterozyklisches aromatisches Amin – transgenen sowie Wildtyp-Mäusen oral verabreicht. Acht Stunden nach Applikation wiesen die transgenen Mäuse signifikant höhere Adduktniveaus als die Wildtyp-Mäuse in Leber, Lunge, Niere, Milz und Kolon auf. In der Leber der transgen Mäuse war das Adduktniveau 17fach höher als in der Leber der Wildtyp-Mäuse. Die Leber war bei den transgenen Tieren das Organ mit dem höchsten, bei den Wildtyp-Tieren hingegen mit dem niedrigsten DNA-Adduktniveau. In der zweiten Untersuchung (Pilotstudie mit geringer Tierzahl) wurde transgenen und Wildtyp-Mäusen 19 mg/kg Körpergewicht des polyzyklischen aromatischen Kohlenwasserstoffs 1-Hydroxymethylpyren – ein Metabolit der Nahrungs- und Umweltkontaminante 1-Methylpyren – intraperitoneal verabreicht. Nach 30 Minuten wurden, verglichen mit den Wildtyp-Mäusen, bis zu 25fach erhöhte Adduktniveaus bei den transgenen Mäusen in Leber, Niere, Lunge und Jejunum nachgewiesen. Somit konnte anhand einer in dieser Arbeit generierten transgenen Mauslinie erstmals gezeigt werden, dass die Expression der humanen SULT1A1/hSULT1A2 tatsächlich sowohl auf die Stärke als auch die Zielgewebe der DNA-Adduktbildung in vivo eine Auswirkung hat.
Die Tailspike Proteine (TSP) der Bakteriophagen P22, Sf6 und HK620 dienen der Erkennung von Kohlenhydratstrukturen auf ihren gram-negativen Wirtsbakterien und zeigen, von den ersten 110 Aminosäuren des N-Terminus abgesehen, keine Sequenzübereinstimmung. Mit Röntgenkristallstrukturanalyse konnte gezeigt werden, dass HK620TSP und Sf6TSP ebenfalls zu einer parallelen, rechtsgängigen beta-Helix falten, wie dies schon für P22TSP bekannt war. Die Kohlenhydratbindestelle ist bei Sf6TSP im Vergleich zu P22TSP zwischen die Untereinheiten verschoben.
Biogene Amine sind kleine organische Verbindungen, die sowohl bei Vertebraten als auch bei Invertebraten als Neurotransmitter, Neuromodulatoren und/oder Neurohormone wirken. Sie bilden eine bedeutende Gruppe von Botenstoffen und entfalten ihre Wirkungen vornehmlich über die Bindung an G-Protein-gekoppelte Rezeptoren. Bei Insekten wurde eine Vielzahl von Wirkungen biogener Amine beschrieben. Das führte schon frühzeitig zur Vermutung, dass Insekten (u. a. Invertebraten) wie die Wirbeltiere ein diverses Repertoire an aminergen Rezeptoren besitzen. Für ein umfassendes Verständnis der komplexen physiologischen Wirkungen biogener Amine fehlten jedoch wichtige Informationen über die molekulare Identität der entsprechenden Rezeptorproteine und ihrer pharmakologischen Eigenschaften, ihre Lokalisation und ihre intrazellulären Reaktionspartner. Viele bei Schaben gut untersuchte (neuro)physiologische Prozesse sowie Verhaltensweisen werden durch Serotonin und Dopamin gesteuert bzw. moduliert. Über die beteiligten Rezeptoren ist jedoch bisher vergleichsweise wenig bekannt. Die Klonierung und Charakterisierung von Serotonin- und Dopaminrezeptoren der Amerikanischen Schabe P. americana ist damit ein längst überfälliger Schritt auf dem Weg zu einem umfassenden Verständnis der vielfältigen Wirkungen biogener Amine bei Insekten. Durch die Anwendung verschiedener Klonierungsstrategien konnten cDNAs isoliert werden, die für potentielle Serotoninrezeptoren und einen Dopaminrezeptor kodieren. Die Sequenzen weisen die größte Ähnlichkeit zu Mitgliedern der 5-HT1- und 5-HT7-Rezeptorklassen bzw. den Invertebratentyp-Dopaminrezeptoren auf. Die isolierten Rezeptoren der Amerikanischen Schabe wurden dementsprechend Pea(Periplaneta americana)5-HT1, Pea5-HT7 und PeaDop2 benannt. Das Hydropathieprofil dieser Rezeptoren postuliert das Vorhandensein der charakteristischen heptahelikalen Architektur G-Protein-gekoppelter Rezeptoren. Die abgeleiteten Aminosäuresequenzen zeigen typische Merkmale aminerger Rezeptoren. So sind Aminosäuren, die bedeutend für die Ligandenbindung, die Rezeptoraktivierung und die Kopplung an GProteine sind, in den Rezeptoren konserviert. Expressionsstudien zeigten eine auffallend hohe Expression aller drei Rezeptor-mRNAs im Gehirn sowie in den Speicheldrüsen. Im Rahmen dieser Arbeit wurden polyklonale Antikörper gegen den Pea5-HT1-Rezeptor sowie den PeaDop2-Rezeptor hergestellt. Der anti-Pea5-HT1-Antikörper detektiert im Homogenat von Schabengehirnen, Speicheldrüsen und Pea5-HT1-exprimierenden HEK 293-Zellen die glykosylierte Form des Rezeptors. In Gehirnschnitten markiert der anti-Pea5-HT1-Antikörper spezifisch einige Zellkörper in der Pars intercerebralis und deren Axone, welche in den Corpora cardiaca Nerv I projizieren. Der PeaDop2-Rezeptor wurde durch den spezifischen anti-PeaDop2-Antikörper in Neuronen mit Somata im anterioren Randbereich der Medulla nachgewiesen. Diese Neurone innervieren die optischen Loben und projizieren in das ventrolaterale Protocerebrum. Die intrazellulären Signalwege der heterolog exprimierten Pea5-HT1- und PeaDop2-Rezeptoren wurden in HEK 293-Zellen untersucht. Die Aktivierung des Pea5-HT1-Rezeptors durch Serotonin führt zur Hemmung der cAMP-Synthese. Des Weiteren wurde gezeigt, dass der Rezeptor konstitutive Aktivität besitzt. WAY 100635, ein hoch selektiver 5-HT1A-Rezeptorantagonist, wurde als wirksamer inverser Agonist am Pea5-HT1-Rezeptor identifiziert. Der stabil exprimierte PeaDop2-Rezeptor antwortet auf eine Aktivierung durch Dopamin mit einer Erhöhung der cAMP-Konzentration. Eine C-terminal trunkierte Variante dieses Rezeptors ist eigenständig nicht funktional. Die Ergebnisse der vorliegenden Arbeit indizieren, dass die untersuchten aminergen Rezeptoren im zentralen Nervensystems der Schabe an der Informationsverarbeitung beteiligt sind und verschiedene physiologische Prozesse in peripheren Organen regulieren. Mit der Klonierung und funktionellen Charakterisierung der ersten Serotoninrezeptoren und eines Dopaminrezeptors ist damit eine wichtige Grundlage für die Untersuchung ihrer Funktionen geschaffen worden.
Biomakromoleküle sind in der Natur für viele Abläufe in lebenden Organismen verantwortlich. Dies reicht vom Aufbau der extrazellulären Matrix und dem Cytoskelett über die Erkennung von Botenstoffen durch Rezeptoren bis hin zur Katalyse der verschiedensten Reaktionen in den Zellen selbst. Diese Aufgaben werden zum größten Teil von Proteinen übernommen, und besonders das spezifische Erkennen der Interaktionspartner ist für alle diese Moleküle äußerst wichtig, um eine fehlerfreie Funktion zu gewährleisten. Als Alternative zur evolutiven Erzeugung von optimalen Bindern und Katalysatoren auf der Basis von Aminosäuren und Nukleotiden wurden von Wulff, Shea und Mosbach synthetische molekular geprägte Polymere (molecularly imprinted polymers, MIPs) konzipiert. Das Prinzip dieser künstlichen Erkennungselemente beruht auf der Tatsache, dass sich funktionelle Monomere spezifisch um eine Schablone (Templat) anordnen. Werden diese Monomere dann vernetzend polymerisiert, entsteht ein Polymer mit molekularen Kavitäten, in denen die Funktionalitäten komplementär zum Templat fixiert sind. Dadurch ist die selektive Bindung des Templats in diese Kavitäten möglich. Aufgrund ihrer hohen chemischen und thermischen Stabilität und ihrer geringen Kosten haben “bio-inspirierte” molekular geprägte Polymere das Potential, biologische Erkennungselemente in der Affinitätschromatographie sowie in Biosensoren und Biochips zu ersetzen. Trotz einiger publizierter Sensorkonfigurationen steht der große Durchbruch noch aus. Ein Hindernis für Routineanwendungen ist die Signalgenerierung bei Bindung des Analyten an das Polymer. Eine Möglichkeit für die markerfreie Detektion ist die Benutzung von Kalorimetern, die Bindungs- oder Reaktionswärmen direkt messen können. In der Enzymtechnologie wird der Enzym-Thermistor für diesen Zweck eingesetzt, da enzymatische Reaktionen eine Enthalpie in einer Größenordnung von 5 – 100 kJ/mol besitzen. In dieser Arbeit wird die Herstellung von katalytisch geprägten Polymeren nach dem Verfahren des Oberflächenprägens erstmalig beschrieben. Die Methode zur Immobilisierung des Templats auf der Oberfläche von porösem Kieselgel sowie die Polymerzusammensetzung wurden optimiert. Weiter wird die Evaluation der katalytischen Eigenschaften über einen optischen Test, sowie das erste Mal die Kombination eines kalorimetrischen Transduktors – des Thermistors – mit der Analyterkennung durch ein katalytisch aktives MIP gezeigt. Bei diesen Messungen konnte zum ersten Mal gleichzeitig die Bindung/Desorption, sowie die katalytische Umwandlung des Substrats durch konzentrationsabhängige Wärmesignale nachgewiesen werden.
Hintergrund: Etablierte Protein- und Nukleinsäure-basierte Methoden für den spezifischen Pathogennachweis sind nur unter standardisierten Laborbedingungen von geschultem Personal durchführbar und daher mit einem hohen Zeit- und Kostenaufwand verbunden. In der Nukleinsäure-basierten Diagnostik kann durch die Einführung der isothermalen Amplifikation eine schnelle und kostengünstige Alternative zur Polymerase-Kettenreaktion (PCR) verwendet werden. Die Loop-mediated isothermal amplification (LAMP) bietet aufgrund der hohen Amplifikationseffizienz vielfältige Detektionsmöglichkeiten, die sowohl für Schnelltest- als auch für Monitoring-Anwendungen geeignet sind.
Ein wesentliches Ziel dieser Arbeit war die Verbesserung der Anwendbarkeit der LAMP und die Entwicklung einer neuen Methode für den einfachen, schnellen und günstigen Nachweis von Pathogenen mittels alternativer DNA- oder Pyrophosphat-abhängiger Detektionsverfahren. Hier wurden zunächst direkte und indirekte Detektionsmethoden untersucht und darauf aufbauend ein Verfahren entwickelt, mit dem neue Metallionen-abhängige Fluoreszenzfarbstoffe für die selektive Detektion von Pyrophosphat in der LAMP und anderen enzymatischen Reaktionen identifiziert werden können. Als Alternative für die DNA-basierte Detektion in der digitalen LAMP sollten die zuvor etablierten Farbstoffe für den Pyrophosphatnachweis in einer Emulsion getestet werden. Abschließend wurde ein neuer Reaktionsmechanismus für die effiziente Generierung hochmolekularer DNA unter isothermalen Bedingungen als Alternative zur LAMP entwickelt.
Ergebnisse: Für den Nachweis RNA- und DNA-basierter Phythopathogene konnte die Echtzeit- und Endpunktdetektion mit verschiedenen Farbstoffen in einem geschlossenen System etabliert werden. Hier wurde Berberin als DNA-interkalierender Fluoreszenzfarbstoff mit vergleichbarer Sensitivität zu SYBR Green und EvaGreen erfolgreich in der LAMP mit Echtzeitdetektion eingesetzt. Ein Vorteil von Berberin gegenüber den anderen Farbstoffen ist die Toleranz der DNA-Polymerase auch bei hohen Farbstoffkonzentrationen. Berberin kann daher auch in der geschlossenen LAMP-Reaktion ohne zusätzliche Anpassung der Reaktionsbedingungen für die Endpunktdetektion verwendet werden. Darüber hinaus konnte Hydroxynaphtholblau (HNB), das für den kolorimetrischen Endpunktnachweis bekannt ist, erstmals auch für die fluorimetrische Detektion der LAMP in Echtzeit eingesetzt werden. Zusätzlich konnten in der Arbeit weitere Metallionen-abhängige Farbstoffe zur indirekten Detektion der LAMP über das Pyrophosphat identifiziert werden. Dafür wurde eine iterative Methode entwickelt, mit der potenzielle Farbstoffe hinsichtlich ihrer Enzymkompatibilität und ihrer spektralen Eigenschaften bei An- oder Abwesenheit von Manganionen selektiert werden können. Mithilfe eines kombinatorischen Screenings im Mikrotiterplattenformat konnte die komplexe Konzentrationsabhängigkeit zwischen den einzelnen Komponenten für einen fluorimetrischen Verdrängungsnachweis untersucht werden. Durch die Visualisierung des Signal-Rausch-Verhältnis’ als Intensitätsmatrix (heatmap) konnten zunächst Alizarinrot S und Tetrazyklin unter simulierten Reaktionsbedingungen selektiert werden. In der anschließenden enzymatischen LAMP-Reaktion konnte insbesondere Alizarinrot S als günstiger, nicht-toxischer und robuster Fluoreszenzfarbstoff identifiziert werden und zeigte eine Pyrophosphat-abhängige Zunahme der Fluoreszenzintensität. Die zuvor etablierten Farbstoffe (HNB, Calcein und Alizarinrot S) konnten anschließend erfolgreich für die indirekte, fluorimetrische Detektion von Pyrophosphat in einer LAMP-optimierten Emulsion eingesetzt werden. Die Stabilität und Homogenität der generierten Emulsion wurde durch den Zusatz des Emulgators Poloxamer 188 verbessert. Durch die fluoreszenzmikroskopische Analyse der Emulsion war eine eindeutige Diskriminierung der positiven und negativen Tröpfchen vor allem bei Einsatz von Calcein und Alizarinrot S möglich. Aufgrund des komplexen Primer-Designs und der hohen Wahrscheinlichkeit unspezifischer Amplifikation in der LAMP wurde eine neue Bst DNA-Polymerase-abhängige isothermale Amplifikationsreaktion entwickelt. Durch die Integration einer spezifischen Linkerstruktur (abasische Stelle oder Hexaethylenglykol) zwischen zwei Primersequenzen konnte ein bifunktioneller Primer die effiziente Regenerierung der Primerbindungsstellen gewährleisten. Der neue Primer induziert nach der spezifischen Hybridisierung auf dem Templat die Rückfaltung zu einer Haarnadelstruktur und blockiert gleichzeitig die Polymeraseaktivität am Gegenstrang, wodurch eine autozyklische Amplifikation trotz konstanter Reaktionstemperatur möglich ist. Die Effizienz der „Hinge-initiated Primer dependent Amplification“ (HIP) konnte abschließend durch die Verkürzung der Distanz zwischen einem modifizierten Hinge-Primer und einem PCR-ähnlichen Primer verbessert werden.
Schlussfolgerung: Die LAMP hat sich aufgrund der hohen Robustheit und Effizienz zu einer leistungsfähigen Alternative für die klassische PCR in der molekularbiologischen Diagnostik entwickelt. Unterschiedliche Detektionsverfahren verbessern die Leistungsfähigkeit der qualitativen und quantitativen LAMP für die Feldanwendungen und für die Diagnostik, da die neuen DNA- und Pyrophosphat-abhängigen Nachweismethoden in einer geschlossenen Reaktion eingesetzt werden können und so eine einfache Pathogendiagnostik ermöglichen. Die gezeigten Methoden können darüber hinaus zu einer Kostensenkung und Zeitersparnis gegenüber den herkömmlichen Methoden beitragen. Ein attraktives Ziel stellt die Weiterentwicklung der HIP für den Pathogennachweis als Alternative zur LAMP dar. Hierbei können die neuen LAMP-Detektionsverfahren ebenfalls Anwendung finden. Die Verwendung von Bst DNA-Polymerase-abhängigen Reaktionen ermöglicht darüber hinaus die Integration einer robusten isothermalen Amplifikation in mikrofluidische Systeme. Durch die Kombination der Probenvorbereitung, Amplifikation und Detektion sind zukünftige Anwendungen mit kurzer Analysezeit und geringem apparativen Aufwand insbesondere in der Pathogendiagnostik möglich.
The NAC transcription factor (TF) JUNGBRUNNEN1 (JUB1) is an important negative regulator of plant senescence, as well as of gibberellic acid (GA) and brassinosteroid (BR) biosynthesis in Arabidopsis thaliana. Overexpression of JUB1 promotes longevity and enhances tolerance to drought and other abiotic stresses. A similar role of JUB1 has been observed in other plant species, including tomato and banana. Our data show that JUB1 overexpressors (JUB1-OXs) accumulate higher levels of proline than WT plants under control conditions, during the onset of drought stress, and thereafter. We identified that overexpression of JUB1 induces key proline biosynthesis and suppresses key proline degradation genes. Furthermore, bZIP63, the transcription factor involved in proline metabolism, was identified as a novel downstream target of JUB1 by Yeast One-Hybrid (Y1H) analysis and Chromatin immunoprecipitation (ChIP). However, based on Electrophoretic Mobility Shift Assay (EMSA), direct binding of JUB1 to bZIP63 could not be confirmed. Our data indicate that JUB1-OX plants exhibit reduced stomatal conductance under control conditions. However, selective overexpression of JUB1 in guard cells did not improve drought stress tolerance in Arabidopsis. Moreover, the drought-tolerant phenotype of JUB1 overexpressors does not solely depend on the transcriptional control of the DREB2A gene. Thus, our data suggest that JUB1 confers tolerance to drought stress by regulating multiple components. Until today, none of the previous studies on JUB1´s regulatory network focused on identifying protein-protein interactions. We, therefore, performed a yeast two-hybrid screen (Y2H) which identified several protein interactors of JUB1, two of which are the calcium-binding proteins CaM1 and CaM4. Both proteins interact with JUB1 in the nucleus of Arabidopsis protoplasts. Moreover, JUB1 is expressed with CaM1 and CaM4 under the same conditions. Since CaM1.1 and CaM4.1 encode proteins with identical amino acid sequences, all further experiments were performed with constructs involving the CaM4 coding sequence. Our data show that JUB1 harbors multiple CaM-binding sites, which are localized in both the N-terminal and C-terminal regions of the protein. One of the CaM-binding sites, localized in the DNA-binding domain of JUB1, was identified as a functional CaM-binding site since its mutation strongly reduced the binding of CaM4 to JUB1. Furthermore, JUB1 transactivates expression of the stress-related gene DREB2A in mesophyll cells; this effect is significantly reduced when the calcium-binding protein CaM4 is expressed as well. Overexpression of both genes in Arabidopsis results in early senescence observed through lower chlorophyll content and an enhanced expression of senescence-associated genes (SAGs) when compared with single JUB1 overexpressors. Our data also show that JUB1 and CaM4 proteins interact in senescent leaves, which have increased Ca2+ levels when compared to young leaves. Collectively, our data indicate that JUB1 activity towards its downstream targets is fine-tuned by calcium-binding proteins during leaf senescence.
‘Heterosis’ is a term used in genetics and breeding referring to hybrid vigour or the superiority of hybrids over their parents in terms of traits such as size, growth rate, biomass, fertility, yield, nutrient content, disease resistance or tolerance to abiotic and abiotic stress. Parental plants which are two different inbred (pure) lines that have desired traits are crossed to obtain hybrids. Maximum heterosis is observed in the first generation (F1) of crosses. Heterosis has been utilised in plant and animal breeding programs for at least 90 years: by the end of the 21st century, 65% of worldwide maize production was hybrid-based. Generally, it is believed that an understanding of the molecular basis of heterosis will allow the creation of new superior genotypes which could either be used directly as F1 hybrids or form the basis for the future breeding selection programmes. Two selected accessions of a research model plant Arabidopsis thaliana (thale cress) were crossed to obtain hybrids. These typically exhibited a 60-80% increase of biomass when compared to the average weight of both parents. This PhD project focused on investigating the role of selected regulatory genes given their potentially key involvement in heterosis. In the first part of the project, the most appropriate developmental stage for this heterosis study was determined by metabolite level measurements and growth observations in parents and hybrids. At the selected stage, around 60 candidate regulatory genes (i.e. differentially expressed in hybrids when compared to parents) were identified. Of these, the majority were transcription factors, genes that coordinate the expression of other genes. Subsequent expression analyses of the candidate genes in biomass-heterotic hybrids of other Arabidopsis accessions revealed a differential expression in a gene subset, highlighting their relevance for heterosis. Moreover, a fraction of the candidate regulatory genes were found within DNA regions closely linked to the genes that underlie the biomass or growth heterosis. Additional analyses to validate the role of selected candidate regulatory genes in heterosis appeared insufficient to establish their role in heterosis. This uncovered a need for using novel approaches as discussed in the thesis. Taken together, the work provided an insight into studies on the molecular mechanisms underlying heterosis. Although studies on heterosis date back to more than one hundred years, this project as many others revealed that more investigations will be needed to uncover this phenomenon.
More than a century ago the phenomenon of non-Mendelian inheritance (NMI), defined as any type of inheritance pattern in which traits do not segregate in accordance with Mendel’s laws, was first reported. In the plant kingdom three genomic compartments, the nucleus, chloroplast, and mitochondrion, can participate in such a phenomenon. High-throughput sequencing (HTS) proved to be a key technology to investigate NMI phenomena by assembling and/or resequencing entire genomes. However, generation, analysis and interpretation of such datasets remain challenging by the multi-layered biological complexity. To advance our knowledge in the field of NMI, I conducted three studies involving different HTS technologies and implemented two new algorithms to analyze them.
In the first study I implemented a novel post-assembly pipeline, called Semi-Automated Graph-Based Assembly Curator (SAGBAC), which visualizes non-graph-based assemblies as graphs, identifies recombinogenic repeat pairs (RRPs), and reconstructs plant mitochondrial genomes (PMG) in a semiautomated workflow. We applied this pipeline to assemblies of three Oenothera species resulting in a spatially folded and circularized model. This model was confirmed by PCR and Southern blot analyses and was used to predict a defined set of 70 PMG isoforms. With Illumina Mate Pair and PacBio RSII data, the stoichiometry of the RRPs was determined quantitatively differing up to three-fold.
In the second study I developed a post-multiple sequence alignment algorithm, called correlation mapping (CM), which correlates segment-wise numbers of nucleotide changes to a numeric ascertainable phenotype. We applied this algorithm to 14 wild type and 18 mutagenized plastome assemblies within the Oenothera genus and identified two genes, accD and ycf2 that may cause the competitive behavior of plastid genotypes as plastids can be biparental inherited in Oenothera. Moreover, lipid composition of the plastid envelope membrane is affected by polymorphisms within these two genes.
For the third study, I programmed a pipeline to investigate a NMI phenomenon, known as paramutation, in tomato by analyzing DNA and bisulfite sequencing data as well as microarray data. We identified the responsible gene (Solyc02g0005200) and were able to fully repress its caused phenotype by heterologous complementation with a paramutation insensitive transgene of the Arabidopsis thaliana orthologue. Additionally, a suppressor mutant shows a globally altered DNA methylation pattern and carries a large deletion leading to a gene fusion involving a histone deacetylase.
In conclusion, my developed and implemented algorithms and data analysis pipelines are suitable to investigate NMI and led to novel insights about such phenomena by reconstructing PMGs (SAGBAC) as a requirement to study mitochondria-associated phenotypes, by identifying genes (CM) causing interplastidial competition as well by applying a DNA/Bisulfite-seq analysis pipeline to shed light in a transgenerational epigenetic inheritance phenomenon.
Fire prone Mediterranean-type vegetation systems like those in the Mediterranean Basin and South-Western Australia are global hot spots for plant species diversity. To ensure management programs act to maintain these highly diverse plant communities, it is necessary to get a profound understanding of the crucial mechanisms of coexistence. In the current literature several mechanisms are discussed. The objective of my thesis is to systematically explore the importance of potential mechanisms for maintaining multi-species, fire prone vegetation by modelling. The model I developed is spatially-explicit, stochastic, rule- and individual-based. It is parameterised on data of population dynamics collected over 18 years in the Mediterranean-type shrublands of Eneabba, Western Australia. From 156 woody species of the area seven plant traits have been identified to be relevant for this study: regeneration mode, annual maximum seed production, seed size, maximum crown diameter, drought tolerance, dispersal mode and seed bank type. Trait sets are used for the definition of plant functional types (PFTs). The PFT dynamics are simulated annual by iterating life history processes. In the first part of my thesis I investigate the importance of trade-offs for the maintenance of high diversity in multi-species systems with 288 virtual PFTs. Simulation results show that the trade-off concept can be helpful to identify non-viable combinations of plant traits. However, the Shannon Diversity Index of modelled communities can be high despite of the presence of ‘supertypes’. I conclude, that trade-offs between two traits are less important to explain multi-species coexistence and high diversity than it is predicted by more conceptual models. Several studies show, that seed immigration from the regional seed pool is essential for maintaining local species diversity. However, systematical studies on the seed rain composition to multi-species communities are missing. The results of the simulation experiments, as presented in part two of this thesis, show clearly, that without seed immigration the local species community found in Eneabba drifts towards a state with few coexisting PFTs. With increasing immigration rates the number of simulated coexisting PFTs and Shannon diversity quickly approaches values as also observed in the field. Including the regional seed input in the model is suited to explain more aggregated measures of the local plant community structure such as species richness and diversity. Hence, the seed rain composition should be implemented in future studies. In the third part of my thesis I test the sensitivity of Eneabba PFTs to four different climate change scenarios, considering their impact on both local and regional processes. The results show that climate change clearly has the potential to alter the number of dispersed seeds for most of the Eneabba PFTs and therefore the source of the ‘immigrants’ at the community level. A classification tree analysis shows that, in general, the response to climate change was PFT-specific. In the Eneabba sand plains sensitivity of a PFT to climate change depends on its specific trait combination and on the scenario of environmental change i.e. development of the amount of rainfall and the fire frequency. This result emphasizes that PFT-specific responses and regional process seed immigration should not be ignored in studies dealing with the impact of climate change on future species distribution. The results of the three chapters are finally analysed in a general discussion. The model is discussed and improvements and suggestions are made for future research. My work leads to the following conclusions: i) It is necessary to support modelling with empirical work to explain coexistence in species-rich plant communities. ii) The chosen modelling approach allows considering the complexity of coexistence and improves the understanding of coexistence mechanisms. iii) Field research based assumptions in terms of environmental conditions and plant life histories can relativise the importance of more hypothetic coexistence theories in species-rich systems. In consequence, trade-offs can play a lower role than predicted by conceptual models. iv) Seed immigration is a key process for local coexistence. Its alteration because of climate change should be considered for prognosis of coexistence. Field studies should be carried out to get data on seed rain composition.
It is well documented that transcriptionally coordinated genes tend to be functionally related, and that such relationships may be conserved across different species, and even kingdoms. (Ihmels et al., 2004). Such relationships was initially utilized to reveal functional gene modules in yeast and mammals (Ihmels et al., 2004), and to explore orthologous gene functions between different species and kingdoms (Stuart et al., 2003; Bergmann et al., 2004). Model organisms, such as Arabidopsis, are readily used in basic research due to resource availability and relative speed of data acquisition. A major goal is to transfer the acquired knowledge from these model organisms to species that are of greater importance to our society. However, due to large gene families in plants, the identification of functional equivalents of well characterized Arabidopsis genes in other plants is a non-trivial task, which often returns erroneous or inconclusive results. In this thesis, concepts of utilizing co-expression networks to help infer (i) gene function, (ii) organization of biological processes and (iii) knowledge transfer between species are introduced. An often overlooked fact by bioinformaticians is that a bioinformatic method is as useful as its accessibility. Therefore, majority of the work presented in this thesis was directed on developing freely available, user-friendly web-tools accessible for any biologist.
Plant cell walls are complex structures that underpin plant growth and are widely exploited in diverse human activities thus placing them with a central importance in biology. Cell walls have been a prominent area of research for a long time, but the chemical complexity and diversity of cell walls not just between species, but also within plants, between cell-types, and between cell wall micro-domains pose several challenges. Progress accelerated several-fold in cell wall biology owing to advances in sequencing technology, aided soon thereafter by advances in omics and imaging technologies. This development provides additional perspectives of cell walls across a rapidly growing number of species, highlighting a myriad of architectures, compositions, and functions.
Furthermore, rather than the component centric view, integrative analysis of the different cell wall components across system-levels help to gain a more in-depth understanding of the structure and biosynthesis of the cell envelope and its interactions with the environment.
To this end, in this work three case studies are detailed, all pertaining to the integrative analysis of heterogeneous cell wall related data arising from different system-levels and analytical techniques. A detailed account of multiblock methods is provided and in particular canonical correlation and regression methods of data integration are discussed. In the first integrative analysis, by employing canonical correlation analysis - a multivariate statistical technique to study the association between two datasets - novel insight to the relationship between glycans and phenotypic traits is gained. In addition, sparse partial least squares regression approach that adapts Lasso penalization and allows for the selection of a subset of variables was employed. The second case study focuses on an integrative analysis of images obtained from different spectroscopic techniques. By employing yet another multiblock approach - multiple co-inertia analysis, insitu biochemical composition of cell walls from different cell-types is studied thereby highlighting the common and complementary parts of the two hyperspectral imaging techniques. Finally, the third integrative analysis facilitates gene expression analysis of the Arabidopsis root transcriptome and translatome for the identification of cell wall related genes and compare expression patterns of cell wall synthesis genes. The computational analysis considered correlation and variation of expression across cell-types at both system-levels, and also provides insight into the degree of co-regulatory relationships that are preserved between the two processes.
The integrative analysis of glycan data and phenotypic traits in cotton fibers using canonical methods led to the identification of specific polysaccharides which may play a major role during fiber development for the final fiber characteristics. Furthermore, this analysis provides a base for future studies on glycan arrays in case of developing cotton fibers. The integrative analysis of images from infrared and Raman spectroscopic approaches allowed the coupling of different analytical techniques to characterize complex biological material, thereby, representing various facets of their chemical properties. Moreover, the results from the co-inertia analysis demonstrated that the study was well adapted as it is relevant for coupling data tables in a symmetric way. Several indicators are proposed to investigate how the global and block scores are related. In addition, studying the root cells of \textit{Arabidopsis thaliana} allowed positing a novel pipeline to systematically investigate and integrate the different levels of information available at the global and single-cell level. The conducted analysis also confirms that previously identified key transcriptional activators of secondary cell wall development display highly conserved patterns of transcription and translation across the investigated cell-types. Moreover, the biological processes that display conserved and divergent patterns based on the cell-type-specific expression and translation levels are identified.
Influenza A virus (IAV) is a pathogen responsible for severe seasonal epidemics threatening human and animal populations every year. During the viral assembly process in the infected cells, the plasma membrane (PM) has to bend in localized regions into a vesicle towards the extracellular side. Studies in cellular models have proposed that different viral proteins might be responsible for inducing membrane curvature in this context (including M1), but a clear consensus has not been reached. M1 is the most abundant protein in IAV particles. It plays an important role in virus assembly and budding at the PM. M1 is recruited to the host cell membrane where it associates with lipids and other viral proteins. However, the details of M1 interactions with the cellular PM, as well as M1-mediated membrane bending at the budozone, have not been clarified.
In this work, we used several experimental approaches to analyze M1-lipids and M1-M1 interactions. By performing SPR analysis, we quantified membrane association for full-length M1 and different genetically engineered M1 constructs (i.e., N- and C-terminally truncated constructs and a mutant of the polybasic region). This allowed us to obtain novel information on the protein regions mediating M1 binding to membranes. By using fluorescence microscopy, cryogenic transmission electron microscopy (cryo-TEM), and three-dimensional (3D) tomography (cryo-ET), we showed that M1 is indeed able to cause membrane deformation on vesicles containing negatively-charged lipids, in the absence of other viral components. Further, sFCS analysis proved that simple protein binding is not sufficient to induce membrane restructuring. Rather, it appears that stable M1-M1 interactions and multimer formation are required to alter the bilayer three-dimensional structure through the formation of a protein scaffold.
Finally, to mimic the budding mechanism in cells that arise by the lateral organization of the virus membrane components on lipid raft domains, we created vesicles with lipid domains. Our results showed that local binding of M1 to spatial confined acidic lipids within membrane domains of vesicles led to local M1 inward curvature.
This thesis contains quantum chemical models and force field calculations for the RuBisCO isotope effect, the spectral characteristics of the blue-light sensor BLUF and the light harvesting complex II. The work focuses on the influence of the environment on the corresponding systems. For RuBisCO, it was found that the isotopic effect is almost unaffected by the environment. In case of the BLUF domain, an amino acid was found to be important for the UV/vis spectrum, but unaccounted for in experiments so far (Ser41). The residue was shown to be highly mobile and with a systematic influence on the spectral shift of the BLUF domain chromophore (flavin). Finally, for LHCII it was found that small changes in the geometry of a Chlorophyll b/Violaxanthin chromophore pair can have strong influences regarding the light harvesting mechanism. Especially here it was seen that the proper description of the environment can be critical. In conclusion, the environment was observed to be of often unexpected importance for the molecular properties, and it seems not possible to give a reliable estimate on the changes created by the presence of the environment.
The Cheb Basin (CZ) is a shallow Neogene intracontinental basin located in the western Eger Rift. The Cheb Basin is characterized by active seismicity and diffuse degassing of mantle-derived CO2 in mofette fields. Within the Cheb Basin, the Hartoušov mofette field shows a daily CO2 flux of 23–97 tons. More than 99% of CO2 released over an area of 0.35 km2. Seismic active periods have been observed in 2000 and 2014 in the Hartoušov mofette field. Due to the active geodynamic processes, the Cheb Basin is considered to be an ideal region for the continental deep biosphere research focussing on the interaction of biological processes with geological processes.
To study the influence of CO2 degassing on microbial community in the surface and subsurface environments, two 3-m shallow drillings and a 108.5-m deep scientific drilling were conducted in 2015 and 2016 respectively. Additionally, the fluid retrieved from the deep drilling borehole was also recovered. The different ecosystems were compared regarding their geochemical properties, microbial abundances, and microbial community structures. The geochemistry of the mofette is characterized by low pH, high TOC, and sulfate contents while the subsurface environment shows a neutral pH, and various TOC and sulfate contents in different lithological settings. Striking differences in the microbial community highlight the substantial impact of elevated CO2 concentrations and high saline groundwater on microbial processes. In general, the microorganisms had low abundance in the deep subsurface sediment compared with the shallow mofette. However, within the mofette and the deep subsurface sediment, the abundance of microbes does not show a typical decrease with depth, indicating that the uprising CO2-rich groundwater has a strong influence on the microbial communities via providing sufficient substrate for anaerobic chemolithoautotrophic microorganisms. Illumina MiSeq sequencing of the 16S rRNA genes and multivariate statistics reveals that the pH strongly influences the microbial community composition in the mofette, while the subsurface microbial community is significantly influenced by the groundwater which motivated by the degassing CO2. Acidophilic microorganisms show a much higher relative abundance in the mofette. Meanwhile, the OTUs assigned to family Comamonadaceae are the dominant taxa which characterize the subsurface communities. Additionally, taxa involved in sulfur cycling characterizing the microbial communities in both mofette and CO2 dominated subsurface environments.
Another investigated important geo–bio interaction is the influence of the seismic activity. During seismic events, released H2 may serve as the electron donor for microbial hydrogenotrophic processes, such as methanogenesis. To determine whether the seismic events can potentially trigger methanogenesis by the elevated geogenic H2 concentration, we performed laboratory simulation experiments with sediments retrieved from the drillings. The simulation results indicate that after the addition of hydrogen, substantial amounts of methane were produced in incubated mofette sediments and deep subsurface sediments. The methanogenic hydrogenotrophic genera Methanobacterium was highly enriched during the incubation. The modeling of the in-situ observation of the earthquake swarm period in 2000 at the Novy Kostel focal area/Czech Republic and our laboratory simulation experiments reveals a close relation between seismic activities and microbial methane production via earthquake-induced H2 release. We thus conclude that H2 – which is released during seismic activity – can potentially trigger methanogenic activity in the deep subsurface. Based on this conclusion, we further hypothesize that the hydrogenotrophic early life on Earth was boosted by the Late Heavy Bombardment induced seismic activity in approximately 4.2 to 3.8 Ga.
The prevalence of diseases associated with misfolded proteins increases with age. When cellular defense mechanisms become limited, misfolded proteins form aggregates and may also develop more stable cross-β structures ultimately forming amyloid aggregates. Amyloid aggregates are associated with neurodegenerative diseases such as Alzheimer’s disease and Huntington’s disease. The formation of amyloid deposits, their toxicity and cellular defense mechanisms have been intensively studied. However, surprisingly little is known about the effects of protein aggregates on cellular signal transduction. It is also not understood whether the presence of aggregation-prone, but still soluble proteins affect signal transduction.
In this study, the still soluble aggregation-prone HttExon1Q74 and its amyloid aggregates were used to analyze the effect of amyloid aggregates on internalization and receptor activation of G protein-coupled receptors (GPCRs), the largest protein family of mammalian cell surface receptors involved in signal transduction. The aggregated HttExon1Q74, but not its soluble form, could inhibit ligand-induced clathrin-mediated endocytosis (CME) of various GPCRs. Most likely this inhibitory effect is based on a terminal sequestration of the HSC70 chaperone to the aggregates which is necessary for CME. Using the vasopressinV1a receptor (V1aR) and the corticotropin-releasing factor receptor 1 (CRF1R) as a model, it could be shown that the presence of HttExon1Q74 aggregates and the inhibition of ligand-induced CME leads to an accumulation of desensitized receptors at the plasma membrane. In turn, this disrupts Gq-mediated Ca2+ signaling and Gs-mediated cAMP signaling of the V1aR and the CRF1R respectively. In contrast to HttExon1Q74 amyloid aggregates, soluble HttExon1Q74 as well as amorphous aggregates did not inhibit GPCR internalization and signaling demonstrating that cellular signal transduction mechanisms are specifically impaired in response to the formation of amyloid aggregates.
In addition, preliminary experiments could show that HttExon1Q74 aggregates provoke an increase in membrane expression of a protein from a structurally and functionally unrelated membrane protein family, namely the serotonin transporter SERT. As SERT is the main pharmacological target to treat depression this could shed light on this commonly occurring comorbidity in neurodegenerative diseases, in particular in early disease states.
The past decades are characterized by various efforts to provide complete sequence information of genomes regarding various organisms. The availability of full genome data triggered the development of multiplex high-throughput assays allowing simultaneous measurement of transcripts, proteins and metabolites. With genome information and profiling technologies now in hand a highly parallel experimental biology is offering opportunities to explore and discover novel principles governing biological systems. Understanding biological complexity through modelling cellular systems represents the driving force which today allows shifting from a component-centric focus to integrative and systems level investigations. The emerging field of systems biology integrates discovery and hypothesis-driven science to provide comprehensive knowledge via computational models of biological systems. Within the context of evolving systems biology, investigations were made in large-scale computational analyses on transcript co-response data through selected prokaryotic and plant model organisms. CSB.DB - a comprehensive systems-biology database - (http://csbdb.mpimp-golm.mpg.de/) was initiated to provide public and open access to the results of biostatistical analyses in conjunction with additional biological knowledge. The database tool CSB.DB enables potential users to infer hypothesis about functional interrelation of genes of interest and may serve as future basis for more sophisticated means of elucidating gene function. The co-response concept and the CSB.DB database tool were successfully applied to predict operons in Escherichia coli by using the chromosomal distance and transcriptional co-responses. Moreover, examples were shown which indicate that transcriptional co-response analysis allows identification of differential promoter activities under different experimental conditions. The co-response concept was successfully transferred to complex organisms with the focus on the eukaryotic plant model organism Arabidopsis thaliana. The investigations made enabled the discovery of novel genes regarding particular physiological processes and beyond, allowed annotation of gene functions which cannot be accessed by sequence homology. GMD - the Golm Metabolome Database - was initiated and implemented in CSB.DB to integrated metabolite information and metabolite profiles. This novel module will allow addressing complex biological questions towards transcriptional interrelation and extent the recent systems level quest towards phenotyping.
The aim of this study was to provide deeper insights in passerine phylogenetic relationships using new molecular markers. The monophyly of the largest avian order Passeriformes (~59% of all living birds) and the division into its suborders suboscines and oscines are well established. Phylogenetic relationships within the group have been extremely puzzling, as most of the evolutionary lineages originated through rapid radiation. Numerous studies have hypothesised conflicting passerine phylogenies and have repeatedly stimulated further research with new markers. In the present study, I used three different approaches to contribute to the ongoing phylogenetic debate in Passeriformes. I investigated the recently introduced gene ZENK for its phylogenetic utility for passerine systematics in combination and comparison to three already established nuclear markers. My phylogenetic analyses of a comprehensive data set yielded highly resolved, consistent and strongly supported trees. I was able to show the high utility of ZENK for elucidating phylogenetic relationships within Passeriformes. For the second and third approach, I used chicken repeat 1 (CR1) retrotransposons as phylogenetic markers. I presented two specific CR1 insertions as apomorphic characters, whose presence/absence pattern significantly contributed to the resolution of a particular phylogenetic uncertainty, namely the position of the rockfowl species Picathartes spp. in the passerine tree. Based on my results, I suggest a closer relationship of these birds to crows, ravens, jays, and allies. For the third approach, I showed that CR1 sequences contain phylogenetic signal and investigated their applicability in more detail. In this context, I screened for CR1 elements in different passerine birds, used sequences of several loci to construct phylogenetic trees, and evaluated their reliability. I was able to corroborate existing hypotheses and provide strong evidence for some new hypotheses, e.g. I suggest a revision of the taxa Corvidae and Corvinae as vireos are closer related to crows, ravens, and allies. The subdivision of the Passerida into three superfamilies, Sylvioidea, Passeroidea, and Muscicapoidea was strongly supported. I found evidence for a split within Sylvioidea into two clades, one consisting of tits and the other comprising warblers, bulbuls, laughingthrushes, whitethroats, and allies. Whereas Passeridae appear to be paraphyletic, monophyly of weavers and estrild finches as a separate clade was strongly supported. The sister taxon relationships of dippers and the thrushes/flycatcher/chat assemblage was corroborated and I suggest a closer relationship of waxwings and kinglets to wrens, tree-creepers, and nuthatches.
Predator-forager interactions are a major factor in evolutionary adaptation of many species, as predators need to gain energy by consuming prey species, and foragers needs to avoid the worst fate of mortality while still consuming resources for energetic gains. In this evolutionary arms race, the foragers have constantly evolved anti-predator behaviours (e.g. foraging activity changes). To describe all these complex changes, researchers developed the framework of the landscape of fear, that is, the spatio-temporal variation of perceived predation risk. This concept simplifies all the involved ecological processes into one framework, by integrating animal biology and distribution with habitat characteristics. Researchers can then evaluate the perception of predation risk in prey species, what are the behavioural responses of the prey and, therefore, understand the cascading effects of landscapes of fear at the resource levels (tri-trophic effects). Although tri-trophic effects are well studied at the predator-prey interaction level, little is known on how the forager-resource interactions are part of the overall cascading effects of landscapes of fear, despite the changes of forager feeding behaviour - that occur with perceived predation risk - affecting directly the level of the resources.
This thesis aimed to evaluate the cascading effects of the landscape of fear on biodiversity of resources, and how the feeding behaviour and movement of foragers shaped the final resource species composition (potential coexistence mechanisms). We studied the changes caused by landscapes of fear on wild and captive rodent communities and evaluated: the cascading effects of different landscapes of fear on a tri-trophic system (I), the effects of fear on a forager’s movement patterns and dietary preferences (II) and cascading effects of different types of predation risk (terrestrial versus avian, III).
In Chapter I, we applied a novel measure to evaluate the cascading effects of fear at the level of resources, by quantifying the diversity of resources left after the foragers gave-up on foraging (diversity at the giving-up density). We tested the measure at different spatial levels (local and regional) and observed that with decreased perceived predation risk, the density and biodiversity of resources also decreased. Foragers left a very dissimilar community of resources based on perceived risk and resources functional traits, and therefore acted as an equalising mechanism.
In Chapter II, we wanted to understand further the decision-making processes of rodents in different landscapes of fear, namely, in which resource species rodents decided to forage on (based on three functional traits: size, nutrients and shape) and how they moved depending on perceived predation risk. In safe landscapes, individuals increased their feeding activity and movements and despite the increased costs, they visited more often patches that were further away from their central-place. Despite a preference for the bigger resources regardless of risk, when perceived predation risk was low, individuals changed their preference to fat-rich resources.
In Chapter III, we evaluated the cascading effects of two different types of predation risk in rodents: terrestrial (raccoon) versus avian predation risk. Raccoon presence or absence did not alter the rodents feeding behaviour in different landscapes of fear. Rodent’s showed risk avoidance behaviours towards avian predators (spatial risk avoidance), but not towards raccoons (lack of temporal risk avoidance).
By analysing the effects of fear in tri-trophic systems, we were able to deepen the knowledge of how non-consumptive effects of predators affect the behaviour of foragers, and quantitatively measure the cascading effects at the level of resources with a novel measure. Foragers are at the core of the ecological processes and responses to the landscape of fear, acting as variable coexistence agents for resource species depending on perceived predation risk. This newly found measures and knowledge can be applied to more trophic chains, and inform researchers on biodiversity patterns originating from landscapes of fear.
Measuring the metabolite profile of plants can be a strong phenotyping tool, but the changes of metabolite pool sizes are often difficult to interpret, not least because metabolite pool sizes may stay constant while carbon flows are altered and vice versa. Hence, measuring the carbon allocation of metabolites enables a better understanding of the metabolic phenotype. The main challenge of such measurements is the in vivo integration of a stable or radioactive label into a plant without perturbation of the system. To follow the carbon flow of a precursor metabolite, a method is developed in this work that is based on metabolite profiling of primary metabolites measured with a mass spectrometer preceded by a gas chromatograph (Wagner et al. 2003; Erban et al. 2007; Dethloff et al. submitted). This method generates stable isotope profiling data, besides conventional metabolite profiling data. In order to allow the feeding of a 13C sucrose solution into the plant, a petiole and a hypocotyl feeding assay are developed. To enable the processing of large numbers of single leaf samples, their preparation and extraction are simplified and optimised. The metabolite profiles of primary metabolites are measured, and a simple relative calculation is done to gain information on carbon allocation from 13C sucrose. This method is tested examining single leaves of one rosette in different developmental stages, both metabolically and regarding carbon allocation from 13C sucrose. It is revealed that some metabolite pool sizes and 13C pools are tightly associated to relative leaf growth, i.e. to the developmental stage of the leaf. Fumaric acid turns out to be the most interesting candidate for further studies because pool size and 13C pool diverge considerably. In addition, the analyses are also performed on plants grown in the cold, and the initial results show a different metabolite pool size pattern across single leaves of one Arabidopsis rosette, compared to the plants grown under normal temperatures. Lastly, in situ expression of REIL genes in the cold is examined using promotor-GUS plants. Initial results suggest that single leaf metabolite profiles of reil2 differ from those of the WT.
In silico identification of genes regulated by abscisic acid in Arabidopsis thaliana (L.) Heynh.
(2005)
Abscisic acid (ABA) is a major plant hormone that plays an important role during plant growth and development. During vegetative growth ABA mediates (in part) responses to various environmental stresses such as cold, drought and high salinity. The response triggered by ABA includes changes in the transcript level of genes involved in stress tolerance. The aim of this project was the In silico identification of genes putatively regulated by ABA in A. thaliana. In silico predictions were combined with experimental data in order to evaluate the reliability of computational predictions. Taking advantage of the genome sequence of A. thaliana publicly available since 2000, 1 kb upstream sequences were screened for combinations of cis-elements known to be involved in the regulation of ABA-responsive genes. It was found that around 10 to 20 percent of the genes of A. thaliana might be regulated by ABA. Further analyses of the predictions revealed that certain combinations of cis-elements that confer ABA-responsiveness were significantly over-represented compared with results in random sequences and with random expectations. In addition, it was observed that other combinations that confer ABA-responsiveness in monocotyledonous species might not be functional in A. thaliana. It is proposed that ABA-responsive genes in A. thaliana show pairs of ABRE (abscisic acid responsive element) with MYB binding sites, DRE (dehydration responsive element) or with itself. The analysis of the distances between pairs of cis-elements suggested that pairs of ABREs are bound by homodimers of ABRE binding proteins. In contrast, pairs between MYB binding sites and ABRE, or DRE and ABRE showed a distance between cis-elements that suggested that the binding proteins interact through protein complexes and not directly. The comparison of computational predictions with experimental data confirmed that the regulatory mechanisms leading to the induction or repression of genes by ABA is very incompletely understood. It became evident that besides the cis-elements proposed in this study to be present in ABA-responsive genes, other known and unknown cis-elements might play an important role in the transcriptional regulation of ABA-responsive genes. For example, auxin-related cis elements, or the cis-elements recognized by the NAM-family of transcription factors (Non-Apical meristem). This work documents the use of computational and experimental approaches to analyse possible interactions between cis-elements involved in the regulation of ABA-responsive genes. The computational predictions allowed the distinction between putatively relevant combinations of cis-elements from irrelevant combinations of cis-elements in ABA-responsive genes. The comparison with experimental data allowed to identify certain cis-elements that have not been previously associated to the ABA-mediated transcriptional regulation, but that might be present in ABA-responsive genes (e.g. auxin responsive elements). Moreover, the efforts to unravel the gene regulatory network associated with the ABA-signalling pathway revealed that NAM-transcription factors and their corresponding binding sequences are important components of this network.
Import and decomposition of dissolved organic carbon in pre-dams of drinking water reservoirs
(2017)
Dissolved organic carbon (DOC) depicts a key component in the aquatic carbon cycle as well as for drinking water production from surface waters. DOC concentrations increased in water bodies of the northern hemisphere in the last decades, posing ecological consequences and water quality problems. Within the pelagic zone of lakes and reservoirs, the DOC pool is greatly affected by biological activity as DOC is simultaneously produced and decomposed. This thesis aimed for a conceptual understanding of organic carbon cycling and DOC quality changes under differing hydrological and trophic conditions. Further, the occurrence of aquatic priming was investigated, which has been proposed as a potential process facilitating the microbial decomposition of stable allochthonous DOC within the pelagic zone.
To study organic carbon cycling under different hydrological conditions, quantitative and qualitative investigations were carried out in three pre-dams of drinking water reservoirs exhibiting a gradient in DOC concentrations and trophic states. All pre-dams were mainly autotrophic in their epilimnia. Discharge and temperature were identified as the key factors regulating net production and respiration in the upper water layers of the pre-dams. Considerable high autochthonous production was observed during the summer season under higher trophic status and base flow conditions. Up to 30% of the total gained organic carbon was produced within the epilimnia. Consequently, this affected the DOC quality within the pre-dams over the year and enhanced characteristics of algae-derived DOC were observed during base flow in summer. Allochthonous derived DOC dominated at high discharges and oligotrophic conditions when production and respiration were low. These results underline that also small impoundments with typically low water residence times are hotspots of carbon cycling, significantly altering water quality in dependence of discharge conditions, temperature and trophic status. Further, it highlights that these factors need to be considered in future water management as increasing temperatures and altered precipitation patterns are predicted in the context of climate change.
Under base flow conditions, heterotrophic bacteria preferentially utilized older DOC components with a conventional radiocarbon age of 195-395 years before present (i.e. before 1950). In contrast, younger carbon components (modern, i.e. produced after 1950) were mineralized following a storm flow event. This highlights that age and recalcitrance of DOC are independent from each other. To assess the ages of the microbially consumed DOC, a simplified method was developed to recover the respired CO2 from heterotrophic bacterioplankton for carbon isotope analyses (13C, 14C). The advantages of the method comprise the operation of replicate incubations at in-situ temperatures using standard laboratory equipment and thus enabling an application in a broad range of conditions.
Aquatic priming was investigated in laboratory experiments during the microbial decomposition of two terrestrial DOC substrates (peat water and soil leachate). Thereby, natural phytoplankton served as a source of labile organic matter and the total DOC pool increased throughout the experiments due to exudation and cell lysis of the growing phytoplankton. A priming effect for both terrestrial DOC substrates was revealed via carbon isotope analysis and mixing models. Thereby, priming was more pronounced for the peat water than for the soil leachate. This indicates that the DOC source and the amount of the added labile organic matter might influence the magnitude of a priming effect. Additional analysis via high-resolution mass spectrometry revealed that oxidized, unsaturated compounds were more strongly decomposed under priming (i.e. in phytoplankton presence). Given the observed increase in DOC concentrations during the experiments, it can be concluded that aquatic priming is not easily detectable via net concentration changes alone and could be considered as a qualitative effect.
The knowledge gained from this thesis contributes to the understanding of aquatic carbon cycling and demonstrated how DOC dynamics in freshwaters vary with hydrological, seasonal and trophic conditions. It further demonstrated that aquatic priming contributes to the microbial transformation of organic carbon and the observed decay of allochthonous DOC during transport in inland waters.
Cyanobacteria produce about 40 percent of the world’s primary biomass, but also a variety of often toxic peptides such as microcystin. Mass developments, so called blooms, can pose a real threat to the drinking water supply in many parts of the world. This study aimed at characterizing the biological function of microcystin production in one of the most common bloom-forming cyanobacterium Microcystis aeruginosa.
In a first attempt, the effect of elevated light intensity on microcystin production and its binding to cellular proteins was studied. Therefore, conventional microcystin quantification techniques were combined with protein-biochemical methods. RubisCO, the key enzyme for primary carbon fixation was a major microcystin interaction partner. High light exposition strongly stimulated microcystin-protein interactions. Up to 60 percent of the total cellular microcystin was detected bound to proteins, i.e. inaccessible for standard quantification procedures. Underestimation of total microcystin contents when neglecting the protein fraction was also demonstrated in field samples. Finally, an immuno-fluorescence based method was developed to identify microcystin producing cyanobacteria in mixed populations.
The high light induced microcystin interaction with proteins suggested an impact of the secondary metabolite on the primary metabolism of Microcystis by e.g. modulating the activity of enzymes. For addressing that question, a comprehensive GC/MS-based approach was conducted to compare the accumulation of metabolites in the wild-type of Microcystis aeruginosa PCC 7806 and the microcystin deficient ΔmcyB mutant. From all 501 detected non-redundant metabolites 85 (17 percent) accumulated significantly different in either of both genotypes upon high light exposition. Accumulation of compatible solutes in the ΔmcyB mutant suggests a role of microcystin in fine-tuning the metabolic flow to prevent stress related to excess light, high oxygen concentration and carbon limitation.
Co-analysis of the widely used model cyanobacterium Synechocystis PCC 6803 revealed profound metabolic differences between species of cyanobacteria. Whereas Microcystis channeled more resources towards carbohydrate synthesis, Synechocystis invested more in amino acids. These findings were supported by electron microscopy of high light treated cells and the quantification of storage compounds. While Microcystis accumulated mainly glycogen to about 8.5 percent of its fresh weight within three hours, Synechocystis produced higher amounts of cyanophycin. The results showed that the characterization of species-specific metabolic features should gain more attention with regard to the biotechnological use of cyanobacteria.
Anthropogenic activities such as continuous landscape changes threaten biodiversity at both local and regional scales. Metacommunity models attempt to combine these two scales and continuously contribute to a better mechanistic understanding of how spatial processes and constraints, such as fragmentation, affect biodiversity. There is a strong consensus that such structural changes of the landscape tend to negatively effect the stability of metacommunities. However, in particular the interplay of complex trophic communities and landscape structure is not yet fully understood.
In this present dissertation, a metacommunity approach is used based on a dynamic and spatially explicit model that integrates population dynamics at the local scale and dispersal dynamics at the regional scale. This approach allows the assessment of complex spatial landscape components such as habitat clustering on complex species communities, as well as the analysis of population dynamics of a single species. In addition to the impact of a fixed landscape structure, periodic environmental disturbances are also considered, where a periodical change of habitat availability, temporally alters landscape structure, such as the seasonal drying of a water body.
On the local scale, the model results suggest that large-bodied animal species, such as predator species at high trophic positions, are more prone to extinction in a state of large patch isolation than smaller species at lower trophic levels.
Increased metabolic losses for species with a lower body mass lead to increased energy limitation for species on higher trophic levels and serves as an explanation for a predominant loss of these species. This effect is particularly pronounced for food webs, where species are more sensitive to increased metabolic losses through dispersal and a change in landscape structure.
In addition to the impact of species composition in a food web for diversity, the strength of local foraging interactions likewise affect the synchronization of population dynamics. A reduced predation pressure leads to more asynchronous population dynamics, beneficial for the stability of population dynamics as it reduces the risk of correlated extinction events among habitats. On the regional scale, two landscape aspects, which are the mean patch isolation and the formation of local clusters of two patches, promote an increase in $\beta$-diversity. Yet, the individual composition and robustness of the local species community equally explain a large proportion of the observed diversity patterns.
A combination of periodic environmental disturbance and patch isolation has a particular impact on population dynamics of a species. While the periodic disturbance has a synchronizing effect, it can even superimpose emerging asynchronous dynamics in a state of large patch isolation and unifies trends in synchronization between different species communities.
In summary, the findings underline a large local impact of species composition and interactions on local diversity patterns of a metacommunity. In comparison, landscape structures such as fragmentation have a negligible effect on local diversity patterns, but increase their impact for regional diversity patterns. In contrast, at the level of population dynamics, regional characteristics such as periodic environmental disturbance and patch isolation have a particularly strong impact and contribute substantially to the understanding of the stability of population dynamics in a metacommunity. These studies demonstrate once again the complexity of our ecosystems and the need for further analysis for a better understanding of our surrounding environment and more targeted conservation of biodiversity.
Cyanobacteria are an abundant bacterial group and are found in a variety of ecological niches all around the globe. They can serve as a real threat for fish or mammals and can restrict the use of lakes or rivers for recreational purposes or as a source of drinking water, when they form blooms. One of the most abundant bloom-forming cyanobacteria is Microcystis aeruginosa.
In the first part of the study, the role and possible dynamics of RubisCO in M. aeruginosa during high-light irradiation were examined. Its response was analyzed on the protein and peptide level via immunoblotting, immunofluorescence microscopy and with high performance liquid chromatography (HPLC). It was revealed that large amounts of RubisCO were located outside of carboxysomes under the applied high light stress. RubisCO aggregated mainly underneath the cytoplasmic membrane. There it forms a putative Calvin-Benson-Bassham (CBB) super complex together with other enzymes of photosynthesis. This complex could be part of an alternative carbon-concentrating mechanism (CCM) in M. aeruginosa, which enables a faster, and energy saving adaptation to high light stress of the whole bloom.
Furthermore, the re-localization of RubisCO was delayed in the microcystin-deficient mutant ΔmcyB and RubisCO was more evenly distributed over the cell in comparison to the wild type. Since ΔmcyB is not harmed in its growth, possibly other produced cyanopeptides as aeruginosin or cyanopeptolin also play a role in the stabilization of RubisCO and the putative CBB complex, especially in the microcystin-free mutant.
In the second part of this work, the possible role of microcystin as an extracellular signaling peptide during the diurnal cycle was studied. HPLC analysis showed a strong increase of extracellular microcystin in the wild type when the population entered nighttime and it resumed into the next day as well. Together with the increase of extracellular microcystin, a strong decrease of protein-bound intracellular microcystin was observed via immunoblot analysis. Interestingly, the signal of the large subunit of RubisCO (RbcL) also diminished when high amounts of microcystin were present in the surrounding medium. Microcystin addition experiments to M. aeruginosa WT and ΔmcyB cultures support this observation, since the immunoblot signal of both subunits of RubisCO and CcmK, a shell protein of carboxysomes, diminished after the addition of microcystin. In addition, the fluctuation of cyanopeptolin during the diurnal cycle indicates a more prominent role of other cyanopeptides besides microcystin as a signaling peptide, intracellularly as well as extracellularly.
The life cycle of higher plants is based on recurring phases of growth and development based on repetitive sequences of cell division, cell expansion and cell differentiation. This dissertation deals with two projects, each of them investigating two different topics that are related to cell expansion. The first project is examining an Arabidopsis thaliana mutant exhibiting overall cell enlargement and the second project is analysing two naturally occurring floral morphs of Amsinckia spectabilis (Boraginaceae) differing (amongst others) in style length and anther heights due to differences in longitudinal cell elongation. The EMS-mutant eop1 was shown to exhibit a petal size increase of 26% caused by cell enlargement. Further phenotypes were detected, such as cotyledon size increase (based on larger cells) as well as increased carpel, sepal, leaf and pollen sizes. Plant height was shown to be increased and more highly branched trichomes explained the hairy eop1 phenotype. Fine mapping revealed the causal SNP to be a C to T transition at the last nucleotide of intron 7 of the INCURVATA11 (ICU11) gene, a 2-oxoglutarate /Fe(II)-dependant dioxygenase, and thus causing missplicing of the mRNA. Two T-DNA insertion lines (icu11-2 & icu11-4) confirmed ICU11 as causal gene by exhibiting increased petal size. A comparison of three icu11 alleles, which possessed different mutation-related changes, either overexpressing ICU11 or modified mRNAs, was the base for investigating the molecular mechanism that underlies the observed phenotype. Different approaches revealed contradictory results regarding ICU11 protein functionality in the icu11 mutants. A complementation assay proved the three mutants to be exchangeable and ICU11 overexpression in the wild-type led to an icu11-like phenotype, arguing for all three icu11 mutants to be GOF mutants. Contradicting this conclusion, the icu11-4 line could be rescued by a genomic ICU11 transgene. A model, based on the assumption that an overexpression of ICU11 is inhibiting the function of the protein, and thus causing the same effect as a LOF protein was proposed. Further, icu11-3 (eop1) mutants were shown to have an increased resistance towards paclobutrazol, a gibberellin (GA) inhibitor and an upregulation of AtGA20ox2, a main GA biosynthesis gene. Additionally, ICU11 subcellular localization was discovered to be cytoplasmic, supporting the assumption, that ICU11 affects GA biosynthesis and overall GA level, possibly explaining the observed (GA-overdose) phenotype.
The second project aimed to identify the genetic base of the S-locus in Amsinckia spectabilis, as the Amsinckia genus represents untypical characteristics for a heterostylous species, such as no obvious self-incompatibility (SI) and the repeated transition towards homostylous and fully selfing variants. The work was based on three Amsinckia spectabilis forms: a heterostylous form, consisting of two floral morphs with reciprocal positioning of sexual organs (S-morph: high anthers and a short style and L-morph: low anthers and a long style), and two homostylous forms, one large-flowered and partially selfing and the other small-flowered and fully selfing. The maintenance of the two floral morphs is genetically based on the S-locus region, containing genes that encode for the morph-specific traits, which are marked by a tight linkage due to suppressed recombination. Natural populations are found to possess a 1:1 S:L morph ratio, that can be explained by predominant disassortative mating of the two morphs, causing the occurrence of the dominant S-allele only in the heterozygous state (heterozygous (Ss) for the S-morph and homozygous recessive (ss) for the L-morph). Investigation of morph-specific phenotypes detected 56% elongated L-morph styles and 58% higher positioned S-morph anthers. Approximately 50% of the observed size differences were explained by an increase in cell elongation. Moreover, additional phenotypes were found, such as 21% enlarged S-morph pollen and no obvious SI, confirmed by hand pollinated seed counts, in vivo pollen tube growth and the development of homozygous dominant SS individuals via selfing. The Amsinckia spec. S-locus was assumed to at least consist of the G- (style length), the A- (anther height) and the P- (pollen size) locus. Comparative Transcriptomics of the two morphs revealed 22 differentially expressed markers that were found to be located within two contigs of a SS individual PacBio genome assembly, allowing the localization of the S-locus to be delimited to a region of approximately 23 Mb. Contradictory to revealed S-loci within the plant kingdom, no strong argument for a present hemizygous region was found to be causal for the suppressed recombination of the S-locus, so that an inversion was assumed to be the causal mechanism.
The unprecedented increase in atmospheric concentrations of carbon dioxide (CO2) and other greenhouse gases (GHG) by anthropogenic activities since the Industrial Revolution impacts on various earth system processes, commonly referred to as `climate change´ (CC). CC faces aquatic ecosystems with extreme abiotic perturbations that potentially alter the interrelations between functional autotrophic and heterotrophic plankton groups. These relations, however, modulate biogeochemical cycling and mediate the functioning of aquatic ecosystems as C sources or sinks to the atmosphere. The aim of this thesis was therefore to investigate how different aspects of CC influence community composition and functioning of pelagic heterotrophic bacteria. These organisms constitute a major component of biogeochemical cycling and largely determine the balance between autotrophic and heterotrophic processes.
Due to the vast amount of potential CC impacts, this thesis focuses on the following two aspects: (1) Increased exchange of CO2 across the atmosphere-water interface and reaction of CO2 with seawater leads to profound shifts in seawater carbonate chemistry, commonly termed as `ocean acidification´ (OA), with consequences for organism physiology and the availability of dissolved inorganic carbon (DIC) in seawater. (2) The increase in atmospheric GHG concentration impacts on the efficiency with which the Earth cools to space, affecting global surface temperature and climate. With ongoing CC, shifts in frequency and severity of episodic weather events, such as storms, are expected that in particular might affect lake ecosystems by disrupting thermal summer stratification. Both aspects of CC were studied at the ecosystem-level in large-volume mesocosm experiments by using the Kiel Off-shore Mesocosms for Future Ocean Simulations (KOSMOS) deployed at different coastal marine locations, and the LakeLab facility in Lake Stechlin.
We evaluated the impact of OA on heterotrophic bacterial metabolism in a brackish coastal ecosystem during low-nutrient summer months in the Baltic Sea. There are several in situ experiments that already assessed potential OA-induced changes in natural plankton communities at diverse spatial and seasonal conditions. However, most studies were performed at high phytoplankton biomass conditions, partly provoked by nutrient amendments. Our study highlights potential OA effects at low-nutrient conditions that are representative for most parts of the ocean and of particular interest in current OA research. The results suggest that during extended periods at low-nutrient concentrations, increasing pCO2 levels indirectly impact the growth balance of heterotrophic bacteria via trophic bacteria-phytoplankton interactions and shift the ecosystem to a more autotrophic system.
Further work investigated how OA affects heterotrophic bacterial dissolved organic matter (DOM) transformation in two mesocsom studies, performed at different nutrient conditions. We observed similar succession patterns for individual compound pools during a phytoplankton bloom and subsequent accumulation of these compounds irrespective of the pCO2 treatment. Our results indicate that OA-induced changes in the dynamics of bacterial DOM transformation and potential impacts on DOM quality are unlikely. In addition, there have been no indications that in dependence of nutrient conditions, different amounts of photosynthetic organic matter are channelled into the more recalcitrant DOM pool. This provides novel insights into the general dynamics of the marine DOM pool.
A fourth enclosure experiment in oligo-mesotrophic Lake Stechlin assessed the impact of a severe summer storm on lake bacterial communities during thermal stratification by artificially mixing. Mixing disrupted and lowered the thermocline, increasing the upper mixed layer and substantially changed water physical-chemical variables. Deep water entrainment and associated changes in water physical-chemical variables significantly affected relative bacterial abundances for about one week. Afterwards a pronounced cyanobacterial bloom developed in response to mixing which affected community assembly of heterotrophic bacteria. Colonization and mineralization of senescent phytoplankton cells by heterotrophic bacteria largely determined C-sequestration to the sediment. About six weeks after mixing, bacterial communities and measured activity parameters converged to control conditions. As such, summer storms have the potential to affect bacterial communities for a prolonged period during summer stratification. The results highlight effects on community assembly and heterotrophic bacterial metabolism that are associated to entrainment of deep water into the mixed water layer and assess consequences of an episodic disturbance event for the coupling between bacterial metabolism and autochthonous DOM production in large volume clear-water lakes.
Altogether, this doctoral thesis reveales substantial sensitivities of heterotrophic bacterial metabolism and community structure in response to OA and a simulated summer storm event, which should be considered when assessing the impact of climate change on marine and lake ecosystems.
Homogene Immunoassays sind immunologische Testverfahren, bei deren Durchführung vollständig auf Separations- und Waschschritte verzichtet werden kann. Der Substrate Channeling Immunoassay beruht auf der Weitergabe eines Substrates in einem immunologischen Komplex aus zwei Enzymen. Das Produkt des ersten Enzyms dient dem zweiten Enzym als Substrat zur Generierung eines photometrisch nachweisbaren Produktes. Voraussetzung für diese Weitergabe ist die enge räumliche Nähe beider Enzyme. Diese Nähe wird durch eine Bindung zwischen Analyt und anti-Analyt Antikörper vermittelt. Ein solcher Substrate Channeling Immunoassay wurde unter Verwendung der Enzyme Glucoseoxidase und Peroxidase aufgebaut. Das so etablierte System war funktionstüchtig, jedoch blieb seine Sensitivität hinter der normaler, heterogener Immunoassays zurück. Die Grundlage eines Fluorescence Quenching Immunoassays ist der gegenseitige Ausschluß zweier Antikörper bei der Bindung eines Dihapten-Konjugates. Das Konjugat besteht dabei aus dem Analyten und einem Fluorophor. Die beiden um die Konjugatbindung konkurrierenden Antikörper sind ein anti-Analyt Antikörper und ein anti-Fluorophor Antikörper, der zudem über die Eigenschaft verfügt, bei Bindung des Fluorophors dessen Fluoreszenz zu löschen. Externe Gaben des freien Analyten verschieben das eingestellte Gleichgewicht in Richtung Fluorophor-Bindung und damit Fluoreszenz-Löschung. Die Änderung der Fluoreszenz ist direkt an die Konzentration des freien Analyten gekoppelt und dient zu deren Bestimmung. Ein solcher Fluorescence Quenching Immunoassays wurde für die Konzentrationsbestimmung des Herbizides Diuron etabliert. Die erreichten Sensitivitäten erlauben die praktische, immundiagnostische Anwendung des Systems. Ein Dihapten-Konjugat wurde ebenfalls zum Aufbau eines Verfahrens zur Selektion Antikörper produzierender Zellen eingesetzt. Die Selektion der Antikörper produzierenden Zellen erfolgt unter Verwendung eines Toxinkonjugates. Dieses Konjugat besteht aus einem Liganden und einem Toxin. Die Antikörperbindung des Liganden behindert sterisch die Wechselwirkung der Toxinkomponente im Konjugat mit deren Zielstruktur in oder auf der Zelle. Nur Zellen die einen geeigneten Antikörper sezernieren, überleben die Selektion und reichern sich in der Kultur an. Das Selektionsverfahren wurde erfolgreich für die Selektion von E.coli Zellen eingesetzt, die einen rekombinanten, Fluorescein bindenden Antikörper produzierten. Das hierfür synthetisierte Toxinkonjugat bestand aus Fluorescein (Ligand) und Ampicillin (Toxinkomponente). Eine Ablösung der bisher für diese Aufgabe gebräuchlichen, außerordentlich kostenintensiven, Screening Methoden wird damit möglich.
Die Folgen einer lebensmittelbedingten Erkrankung sind zum Teil gravierend, insbesondere für Kinder und immunsupprimierte Menschen. Hierbei gehören Salmonella und Campylobacter zu den häufigsten Erregern, die verantwortlich für gastrointestinale Erkrankungen in Deutschland sind. Trotz umfassender Maßnahmen der EU zur Prävention und Bekämpfung von Salmonellen in Geflügelbeständen und der Lebensmittel-Industrie, wird von einem stagnierenden Trend von Infektionszahlen berichtet. Zoonose-Erreger wie Salmonellen können über Nutztiere in die Nahrungskette des Menschen gelangen, wodurch sich Infektionsherde schnell ausbreiten können. Dabei sind bestehende Präventionsstrategien für Geflügel vorhanden, die aber nicht auf den Menschen übertragbar sind. Folglich sind Diagnostik und Prävention in der Lebensmittelindustrie essentiell. Deshalb besteht ein hoher Bedarf für spezifische, sensitive und zuverlässige Nachweismethoden, die eine Point-of-care Diagnostik gewährleisten. Durch ein wachsendes Verständnis der wirtsspezifischen Faktoren von S. enterica Serovaren kann die Entwicklung sowohl neuartiger diagnostischer Methoden, als auch neuartiger Therapien und Impfstoffe maßgeblich vorangetrieben werden.
Infolgedessen wurde in dieser Arbeit ein infektionsähnliches in vitro Modell für S. Enteritidis etabliert und darauf basierend eine umfassende Untersuchung zur Identifizierung neuer Zielstrukturen für den Erreger durchgeführt. Während einer Salmonellen-Infektion ist die erste zelluläre Barriere im Wirt die Epithelschicht. Dementsprechend wurde eine humane Zelllinie (CaCo 2, Darmepithel) für die Pathogen-Wirt-Studie ausgewählt. Das Salmonellen-Transkriptom und morphologische Eigenschaften der Epithelzellen wurden in verschiedenen Phasen der Salmonellen-Infektion untersucht und mit bereits gut beschriebenen Virulenzfaktoren und Beobachtungen in Bezug gesetzt. Durch dieses Infektionsmodell konnte ein spezifischer Phänotyp für die intrazellulären Salmonellen in den Epithelzellen nachgewiesen werden. Zudem wurde aufgezeigt, dass bereits die Kultivierung in Flüssigmedium einen invasionsaktiven Zustand der Salmonellen erzeugt. Allerdings wurde durch die Kokultivierung mit Epithelzellen eine zusätzliche Expression relevanter Gene induziert, um eine effiziente Adhäsion und Transmembran-Transport zu gewährleisten. Letzterer ist charakteristisch für die intrazelluläre Limitierung von Nährstoffen und prägt den infektionsrelevanten Status. Unter Berücksichtigung dieser Faktoren ergab sich ein Phänotyp, der eindeutig Mechanismen zur Wirtsadaptation und möglicherweise auch Pathogenese aufzeigt. Die intrazellulären Bakterien müssen vom Wirt separiert werden, was ein wesentlicher Schritt für Pathogen-bestimmende Analysen ist. Hierbei wurde mithilfe einer Detergenz-basierten Lyse der eukaryotischen Zellmembran und differentieller Zentrifugation, der eukaryotische Eintrag minimal gehalten. Unter Verwendung der Virulenz-adaptierten Salmonellen wurden Untersuchungen in Hinblick auf die Identifizierung neuer Zielstrukturen für S. Enteritidis durchgeführt. Mithilfe eines immunologischen Screenings wurden neue potentielle Antigene entdeckt. Zu diesem Zweck wurden bakterielle cDNA-basierte Expressionsbibliotheken hergestellt, die durch eine vereinfachte Microarray-Anwendung ein Hochdurchsatzscreening von Proteinen als potentielle Binder ermöglichen. Folglich konnten neue unbeschriebene Proteine identifiziert werden, die sich durch eine Salmonella-Spezifität oder Membranständigkeit auszeichnen. Ebenso wurde ein Vergleich der im Screening identifizierten Proteine mit der Regulation der kodierenden Gene im infektionsähnlichen Modell durchgeführt. Dabei wurde deutlich, dass die Häufigkeit von Transkripten einen Einfluss auf die Verfügbarkeit in der cDNA-Bibliothek und folglich auch auf die Expressionsbibliothek nimmt. Angesichts eines Ungleichgewichts zwischen der Gesamtzahl protein-kodierender Gene in S. Enteritidis zu möglichen Klonen, die während des Microarray-Screenings untersucht werden können, besteht der Bedarf einer Anreicherung von Proteinen in der Expressionsbibliothek. Das infektionsähnliche Modell zeigte, dass nicht nur Virulenz-assoziierte, sondern auch Stress- und Metabolismus-relevante Gene hochreguliert werden. Durch die Konstruktion dieser spezifischen cDNA-Bibliotheken ist die Erkennung von charakteristischen molekularen Markern gegeben.
Weiterhin wurden anhand der Transkriptomanalyse spezifisch hochregulierte Gene identifiziert, die relevant für das intrazelluläre Überleben von S. Enteritidis in humanen Epithelzellen sind. Hiervon wurden drei Gene näher untersucht, indem ihr Einfluss im infektionsähnlichen Modell mittels entsprechender Gen-Knockout-Stämme analysiert wurde. Dabei wurde für eine dieser Mutanten ein reduziertes Wachstum in der späten intrazellulären Phase nachgewiesen. Weiterführende in vitro Analysen sind für die Charakterisierung des Knockout-Stamms notwendig, um den Einsatz als potenzielles Therapeutikum zu verifizieren.
Zusammenfassend wurde ein in vitro Infektionsmodell für S. Enteritidis etabliert, wodurch neue Zielstrukturen des Erregers identifiziert wurden. Diese sind für diagnostische oder therapeutische Anwendungen interessant. Das Modell lässt sich ebenso für andere intrazelluläre Pathogene übertragen und gewährleistet eine zuverlässige Identifizierung von potentiellen Antigenen.
Genetic variation is crucial for the long-term survival of the species as it provides the potential for adaptive responses to environmental changes such as emerging diseases. The Major Histocompatibility Complex (MHC) is a gene family that plays a central role in the vertebrate’s immune system by triggering the adaptive immune response after exposure to pathogens. MHC genes have become highly suitable molecular markers of adaptive significance. They synthesize two primary cell surface molecules namely MHC class I and class II that recognize short fragments of proteins derived respectively from intracellular (e.g. viruses) and extracellular (e.g. bacteria, protozoa, arthropods) origins and present them to immune cells. High levels of MHC polymorphism frequently observed in natural populations are interpreted as an adaptation to detect and present a wide array of rapidly evolving pathogens. This variation appears to be largely maintained by positive selection driven mainly by pathogenic selective pressures. For my doctoral research I focused on MHC I and II variation in free-ranging cheetahs (Acinonyx jubatus) and leopards (Panthera pardus) on Namibian farmlands. Both felid species are sympatric thus subject to similar pathogenic pressures but differ in their evolutionary and demographic histories. The main aims were to investigate 1) the extent and patterns of MHC variation at the population level in both felids, 2) the association between levels of MHC variation and disease resistance in free-ranging cheetahs, and 3) the role of selection at different time scales in shaping MHC variation in both felids. Cheetahs and leopards represent the largest free-ranging carnivores in Namibia. They concentrate in unprotected areas on privately owned farmlands where domestic and other wild animals also occur and the risk of pathogen transmission is increased. Thus, knowledge on adaptive genetic variation involved in disease resistance may be pertinent to both felid species’ conservation. The cheetah has been used as a classic example in conservation genetics textbooks due to overall low levels of genetic variation. Reduced variation at MHC genes has been associated with high susceptibility to infectious diseases in cheetahs. However, increased disease susceptibility has only been observed in captive cheetahs whereas recent studies in free-ranging Namibian cheetahs revealed a good health status. This raised the question whether the diversity at MHC I and II genes in free-ranging cheetahs is higher than previously reported. In this study, a total of 10 MHC I alleles and four MHC II alleles were observed in 149 individuals throughout Namibia. All alleles but one likely belong to functional MHC genes as their expression was confirmed. The observed alleles belong to four MHC I and three MHC II genes in the species as revealed by phylogenetic analyses. Signatures of historical positive selection acting on specific sites that interact directly with pathogen-derived proteins were detected in both MHC classes. Furthermore, a high genetic differentiation at MHC I was observed between Namibian cheetahs from east-central and north-central regions known to differ substantially in exposure to feline-specific viral pathogens. This suggests that the patterns of MHC I variation in the current population mirrors different pathogenic selective pressure imposed by viruses. Cheetahs showed low levels of MHC diversity compared with other mammalian species including felids, but this does not seem to influence the current immunocompetence of free-ranging cheetahs in Namibia and contradicts the previous conclusion that the cheetah is a paradigm species of disease susceptibility. However, it cannot be ruled out that the low MHC variation might limit a prosperous immunocompetence in the case of an emerging disease scenario because none of the remaining alleles might be able to recognize a novel pathogen. In contrast to cheetahs, leopards occur in most parts of Africa being perhaps the most abundant big cat in the continent. Leopards seem to have escaped from large-scale declines due to epizootics in the past in contrast to some free-ranging large carnivore populations in Africa that have been afflicted by epizootics. Currently, no information about the MHC sequence variation and constitution in African leopards exists. In this study, I characterized genetic variation at MHC I and MHC II genes in free-ranging leopards from Namibia. A total of six MHC I and six MHC II sequences were detected in 25 individuals from the east-central region. The maximum number of sequences observed per individual suggests that they likely correspond to at least three MHC I and three MHC II genes. Hallmarks of MHC evolution were confirmed such as historical positive selection, recombination and trans-species polymorphism. The low MHC variation detected in Namibian leopards is not conclusive and further research is required to assess the extent of MHC variation in different areas of its geographic range. Results from this thesis will contribute to better understanding the evolutionary significance of MHC and conservation implications in free-ranging felids. Translocation of wildlife is an increasingly used management tool for conservation purposes that should be conducted carefully as it may affect the ability of the translocated animals to cope with different pathogenic selective pressures.
MHC genes encode proteins that are responsible for the recognition of foreign antigens and the triggering of a subsequent, adequate immune response of the organism. Thus they hold a key position in the immune system of vertebrates. It is believed that the extraordinary genetic diversity of MHC genes is shaped by adaptive selectional processes in response to the reoccurring adaptations of parasites and pathogens. A large number of MHC studies were performed in a wide range of wildlife species aiming to understand the role of immune gene diversity in parasite resistance under natural selection conditions. Methodically, most of this work with very few exceptions has focussed only upon the structural, i.e. sequence diversity of regions responsible for antigen binding and presentation. Most of these studies found evidence that MHC gene variation did indeed underlie adaptive processes and that an individual’s allelic diversity explains parasite and pathogen resistance to a large extent. Nevertheless, our understanding of the effective mechanisms is incomplete. A neglected, but potentially highly relevant component concerns the transcriptional differences of MHC alleles. Indeed, differences in the expression levels MHC alleles and their potential functional importance have remained unstudied. The idea that also transcriptional differences might play an important role relies on the fact that lower MHC gene expression is tantamount with reduced induction of CD4+ T helper cells and thus with a reduced immune response. Hence, I studied the expression of MHC genes and of immune regulative cytokines as additional factors to reveal the functional importance of MHC diversity in two free-ranging rodent species (Delomys sublineatus, Apodemus flavicollis) in association with their gastrointestinal helminths under natural selection conditions. I established the method of relative quantification of mRNA on liver and spleen samples of both species in our laboratory. As there was no available information on nucleic sequences of potential reference genes in both species, PCR primer systems that were established in laboratory mice have to be tested and adapted for both non-model organisms. In the due course, sets of stable reference genes for both species were found and thus the preconditions for reliable measurements of mRNA levels established. For D. sublineatus it could be demonstrated that helminth infection elicits aspects of a typical Th2 immune response. Whereas mRNA levels of the cytokine interleukin Il4 increased with infection intensity by strongyle nematodes neither MHC nor cytokine expression played a significant role in D. sublineatus. For A. flavicollis I found a negative association between the parasitic nematode Heligmosomoides polygyrus and hepatic MHC mRNA levels. As a lower MHC expression entails a lower immune response, this could be evidence for an immune evasive strategy of the nematode, as it has been suggested for many micro-parasites. This implies that H. polygyrus is capable to interfere actively with the MHC transcription. Indeed, this parasite species has long been suspected to be immunosuppressive, e.g. by induction of regulatory T-helper cells that respond with a higher interleukin Il10 and tumor necrosis factor Tgfb production. Both cytokines in turn cause an abated MHC expression. By disabling recognition by the MHC molecule H. polygyrus might be able to prevent an activation of the immune system. Indeed, I found a strong tendency in animals carrying the allele Apfl-DRB*23 to have an increased infection intensity with H. polygyrus. Furthermore, I found positive and negative associations between specific MHC alleles and other helminth species, as well as typical signs of positive selection acting on the nucleic sequences of the MHC. The latter was evident by an elevated rate of non-synonymous to synonymous substitutions in the MHC sequences of exon 2 encoding the functionally important antigen binding sites whereas the first and third exons of the MHC DRB gene were highly conserved. In conclusion, the studies in this thesis demonstrate that valid procedures to quantify expression of immune relevant genes are also feasible in non-model wildlife organisms. In addition to structural MHC diversity, also MHC gene expression should be considered to obtain a more complete picture on host-pathogen coevolutionary selection processes. This is especially true if parasites are able to interfere with systemic MHC expression. In this case advantageous or disadvantageous effects of allelic binding motifs are abated. The studies could not define the role of MHC gene expression in antagonistic coevolution as such but the results suggest that it depends strongly on the specific parasite species that is involved.
Heat stress (HS) is a major abiotic stress that negatively affects plant growth and productivity. However, plants have developed various adaptive mechanisms to cope with HS, including the acquisition and maintenance of thermotolerance, which allows them to respond more effectively to subsequent stress episodes. HS memory includes type II transcriptional memory which is characterized by enhanced re-induction of a subset of HS memory genes upon recurrent HS. In this study, new regulators of HS memory in A. thaliana were identified through the characterization of rein mutants.
The rein1 mutant carries a premature stop in CYCLIN-DEPENDENT-KINASE 8 (CDK8) which is part of the cyclin kinase module of the Mediator complex. Rein1 seedlings show impaired type II transcriptional memory in multiple heat-responsive genes upon re-exposure to HS. Additionally, the mutants exhibit a significant deficiency in HS memory at the physiological level. Interaction studies conducted in this work indicate that CDK8 associates with the memory HEAT SHOCK FACTORs HSAF2 and HSFA3. The results suggest that CDK8 plays a crucial role in HS memory in plants together with other memory HSFs, which may be potential targets of the CDK8 kinase function. Understanding the role and interaction network of the Mediator complex during HS-induced transcriptional memory will be an exciting aspect of future HS memory research.
The second characterized mutant, rein2, was selected based on its strongly impaired pAPX2::LUC re-induction phenotype. In gene expression analysis, the mutant revealed additional defects in the initial induction of HS memory genes. Along with this observation, basal thermotolerance was impaired similarly as HS memory at the physiological level in rein2. Sequencing of backcrossed bulk segregants with subsequent fine mapping narrowed the location of REIN2 to a 1 Mb region on chromosome 1. This interval contains the At1g65440 gene, which encodes the histone chaperone SPT6L. SPT6L interacts with chromatin remodelers and bridges them to the transcription machinery to regulate nucleosome and Pol II occupancy around the transcriptional start site. The EMS-induced missense mutation in SPT6L may cause altered HS-induced gene expression in rein2, possibly triggered by changes in the chromatin environment resulting from altered histone chaperone function.
Expanding research on screen-derived factors that modify type II transcriptional memory has the potential to enhance our understanding of HS memory in plants. Discovering connections between previously identified memory factors will help to elucidate the underlying network of HS memory. This knowledge can initiate new approaches to improve heat resilience in crops.
Die Mykorrhiza (griechisch: mýkēs für „Pilz”; rhiza für „Wurzel”) stellt eine Symbiose zwischen Pilzen und einem Großteil der Landpflanzen dar. Der Pilz verbessert durch die Symbiose die Versorgung der Pflanze mit Nährstoffen, während die Pflanze den Pilz mit Kohlenhydraten versorgt. Die arbuskuläre Mykorrhiza (AM) stellt dabei einen beson-dere Form der Mykorrhiza dar. Der AM-Pilz bildet dabei während der Symbiose die namensgebenden Arbuskeln innerhalb der Wurzelzellen als Ort des primären Nährstoff- austausches aus. Die AM-Symbiose (AMS) ist der Forschungsschwerpunkt dieser Arbeit. Als Modellorganismen wurden Medicago truncatula und Glomus intraradices verwendet. Es wurden Transkriptionsanalysen durchgeführt um u.a. AMS regulierte Transkriptions- faktoren (TFs) zu identifizieren. Die Aktivität der Promotoren von drei der so identifizier-ten AMS-regulierten TFs (MtOFTN, MtNTS, MtDES) wurde mit Hilfe eine Reportergens visualisiert. Der Bereich der größten Promotoraktivität waren in einem Fall nur die ar- buskelhaltigen Zellen (MtOFTN). Im zweiten Fall war der Promotor auch aktiv in nicht arbuskelhaltigen Zellen, jedoch am stärksten aktiv in den arbuskelhaltigen Zellen (MtNTS). Ein weiterer Promotor war in arbuskelhaltigen Zellen und den diesen benach-barten Zellen gleich aktiv (MtDES). Zusätzlich wurden weitere Gene als AMS-reguliert identifiziert und es wurde für drei dieser Gene (MtPPK, MtAmT, MtMDRL) ebenfalls eine Promotor::Reporter-Aktivitäts- studie durchgeführt. Die Promotoren der Kinase (MtPPK) und des Ammoniumtrans-porters (MtAmt) waren dabei ausschließlich in arbuskelhaltigen Zellen aktiv, während die Aktivität des ABC-Transporters (MtMDRL) keinem bestimmten Zelltyp zuzuordnen war. Für zwei weitere identifizierte Gene, ein Kupfertransporter (MtCoT) und ein Zucker- bzw. Inositoltransporter (MtSuT), wurden RNA-Interferenz (RNAi)-Untersuchungen durchgeführt. Dabei stellte sich in beiden Fällen heraus, dass, sobald ein RNAi-Effekt in den transformierten Wurzeln vorlag, diese in einem deutlich geringerem Ausmaß wie in der Wurzelkontrolle von G. intraradices kolonisiert worden sind. Im Falle von MtCoT könnte das aus dem selben Grund geschehen, wie im Falle von MtPt4. Welche Rolle MtSuT genau in der Ausbildung der AMS spielt und welche Rolle Inositol in der Aus- bildung der AMS spielt müsste durch weitere Untersuchungen am Protein untersucht werden. Weitere Untersuchen an den in dieser Arbeit als spezifisch für arbuskelhaltige Zellen gezeigten Genen MtAmT, MtPPK und MtOFTN könnten ebenfalls aufschlussreich für das weitere Verständnis der AMS sein. Dies trifft auch auf die TFs MtNTS und MtDES zu, die zwar nicht ausschließlich arbuskelspezifisch transkribiert werden, aber auch eine Rolle in der Regulation der AMS innerhalb von M. truncatula Wurzeln zu spielen scheinen.
Für alle Organismen ist die Aufrechterhaltung ihres energetischen Gleichgewichts unter fluktuierenden Umweltbedingungen lebensnotwendig. In Eukaryoten steuern evolutionär konservierte Proteinkinasen, die in Pflanzen als SNF1-RELATED PROTEIN KINASE1 (SnRK1) bezeichnet werden, die Adaption an Stresssignale aus der Umwelt und an die Limitierung von Nährstoffen und zellulärer Energie. Die Aktivierung von SnRK1 bedingt eine umfangreiche transkriptionelle Umprogrammierung, die allgemein zu einer Repression energiekonsumierender Prozesse wie beispielsweise Zellteilung und Proteinbiosynthese und zu einer Induktion energieerzeugender, katabolischer Stoffwechselwege führt. Wie unterschiedliche Signale zu einer generellen sowie teilweise gewebe- und stressspezifischen SnRK1-vermittelten Antwort führen ist bisher noch nicht ausreichend geklärt, auch weil bislang nur wenige Komponenten der SnRK1-Signaltransduktion identifiziert wurden. In dieser Arbeit konnte ein Protein-Protein-Interaktionsnetzwerk um die SnRK1αUntereinheiten aus Arabidopsis AKIN10/AKIN11 etabliert werden. Dadurch wurden zunächst Mitglieder der pflanzenspezifischen DUF581-Proteinfamilie als Interaktionspartner der SnRK1α-Untereinheiten identifiziert. Diese Proteine sind über ihre konservierte DUF581Domäne, in der ein Zinkfinger-Motiv lokalisiert ist, fähig mit AKIN10/AKIN11 zu interagieren. In planta Ko-Expressionsanalysen zeigten, dass die DUF581-Proteine eine Verschiebung der nucleo-cytoplasmatischen Lokalisierung von AKIN10 hin zu einer nahezu ausschließlichen zellkernspezifischen Lokalisierung begünstigen sowie die Ko-Lokalisierung von AKIN10 und DUF581-Proteinen im Nucleus. In Bimolekularen Fluoreszenzkomplementations-Analysen konnte die zellkernspezifische Interaktion von DUF581-Proteinen mit SnRK1α-Untereinheiten in planta bestätigt werden. Außerhalb der DUF581-Domäne weisen die Proteine einander keine große Sequenzähnlichkeit auf. Aufgrund ihrer Fähigkeit mit SnRK1 zu interagieren, dem Fehlen von SnRK1Phosphorylierungsmotiven sowie ihrer untereinander sehr variabler gewebs-, entwicklungs- und stimulusspezifischer Expression wurde für DUF581-Proteine eine Funktion als Adaptoren postuliert, die unter bestimmten physiologischen Bedingungen spezifische Substratproteine in den SnRK1-Komplex rekrutieren. Auf diese Weise könnten DUF581Proteine die Interaktion von SnRK1 mit deren Zielproteinen modifizieren und eine Feinjustierung der SnRK1-Signalweiterleitung ermöglichen. Durch weiterführende Interaktionsstudien konnten DUF581-interagierende Proteine darunter Transkriptionsfaktoren, Proteinkinasen sowie regulatorische Proteine gefunden werden, die teilweise ebenfalls Wechselwirkungen mit SnRK1α-Untereinheiten aufzeigten. Im Rahmen dieser Arbeit wurde eines dieser Proteine für das eine Beteiligung an der SnRK1Signalweiterleitung als Transkriptionsregulator vermutet wurde näher charakterisiert. STKR1 (STOREKEEPER RELATED 1), ein spezifischer Interaktionspartner von DUF581-18, gehört zu einer pflanzenspezifischen Leucin-Zipper-Transkriptionsfaktorfamilie und interagiert in Hefe sowie in planta mit SnRK1. Die zellkernspezifische Interaktion von STKR1 und AKIN10 in Pflanzen unterstützt die Vermutung der kooperativen Regulation von Zielgenen. Weiterhin stabilisierte die Anwesenheit von AKIN10 die Proteingehalte von STKR1, das wahrscheinlich über das 26S Proteasom abgebaut wird. Da es sich bei STKR1 um ein Phosphoprotein mit SnRK1-Phosphorylierungsmotiv handelt, stellt es sehr wahrscheinlich ein SnRK1-Substrat dar. Allerdings konnte eine SnRK1-vermittelte Phosphorylierung von STKR1 in dieser Arbeit nicht gezeigt werden. Der Verlust von einer Phosphorylierungsstelle beeinflusste die Homo- und Heterodimerisierungsfähigkeit von STKR1 in Hefeinteraktionsstudien, wodurch eine erhöhte Spezifität der Zielgenregulation ermöglicht werden könnte. Außerdem wurden Arabidopsis-Pflanzen mit einer veränderten STKR1-Expression phänotypisch, physiologisch und molekularbiologisch charakterisiert. Während der Verlust der STKR1-Expression zu Pflanzen führte, die sich kaum von Wildtyp-Pflanzen unterschieden, bedingte die konstitutive Überexpression von STKR1 ein stark vermindertes Pflanzenwachstum sowie Entwicklungsverzögerungen hinsichtlich der Blühinduktion und Seneszenz ähnlich wie sie auch bei SnRK1α-Überexpression beschrieben wurden. Pflanzen dieser Linien waren nicht in der Lage Anthocyane zu akkumulieren und enthielten geringere Gehalte an Chlorophyll und Carotinoiden. Neben einem erhöhten nächtlichen Stärkeumsatz waren die Pflanzen durch geringere Saccharosegehalte im Vergleich zum Wildtyp gekennzeichnet. Eine Transkriptomanalyse ergab, dass in den STKR1-überexprimierenden Pflanzen unter Energiemangelbedingungen, hervorgerufen durch eine verlängerte Dunkelphase, eine größere Anzahl an Genen im Vergleich zum Wildtyp differentiell reguliert war als während der Lichtphase. Dies spricht für eine Beteiligung von STKR1 an Prozessen, die während der verlängerten Dunkelphase aktiv sind. Ein solcher ist beispielsweise die SnRK1-Signaltransduktion, die unter energetischem Stress aktiviert wird. Die STKR1Überexpression führte zudem zu einer verstärkten transkriptionellen Induktion von Abwehrassoziierten Genen sowie NAC- und WRKY-Transkriptionsfaktoren nach verlängerter Dunkelphase. Die Transkriptomdaten deuteten auf eine stimulusunabhängige Induktion von Abwehrprozessen hin und konnten eine Erklärung für die phänotypischen und physiologischen Auffälligkeiten der STKR1-Überexprimierer liefern.
Zur Detektion neuer IgE- reaktiver Proteine wurde in dieser Arbeit ein zweidimensionales Proteintrennverfahren verwendet. Resultierende Proteinfraktionen wurden mithilfe von 18 tomatensensibiliesierten Patientenseren im Immunoblot getestet. Detektierte Proteine in der SDS-PAGE wurden mittels LC-MS/MS identifiziert. Dadurch konnten 2 Tomatensamenproteine, die im Immunoblot ein IgE- reaktives Signal zeigten eindeutig mittels Massenspektrometrie identifiziert werden. Diese Proteine sind Legumin und Vicilin. Durch Sequenzabgleich und Proteinstrukturmodellierung im Vergleich zu bereits bekannten Allergenen (Erdnuss und Cashewnuss), konnte eine hohe Homologie gezeigt werden.
Seit der Einführung von Antibiotika in die medizinische Behandlung von bakteriellen Infektionskrankheiten existiert ein Wettlauf zwischen der Evolution von Bakterienresistenzen und der Entwicklung wirksamer Antibiotika. Während bis in die 80er Jahre verstärkt an neuen Antibiotika geforscht wurde, gewinnen multiresistente Keime heute zunehmend die Oberhand. Um einzelne Pathogene erfolgreich nachzuweisen und zu bekämpfen, ist ein grundlegendes Wissen über den Erreger unumgänglich. Bakterielle Proteine, die bei einer Infektion vorrangig vom Immunsystem prozessiert und präsentiert werden, könnten für die Entwicklung von Impfstoffen oder gezielten Therapeutika nützlich sein. Auch für die Diagnostik wären diese immundominanten Proteine interessant. Allerdings herrscht ein Mangel an Wissen über spezifische Antigene vieler pathogener Bakterien, die eine eindeutige Diagnostik eines einzelnen Erregers erlauben würden.
Daher wurden in dieser Arbeit vier verschiedene Humanpathogene mittels Phage Display untersucht: Neisseria gonorrhoeae, Neisseria meningitidis, Borrelia burgdorferi und Clostridium difficile. Hierfür wurden aus der genomischen DNA der vier Erreger Bibliotheken konstruiert und durch wiederholte Selektion und Amplifikation, dem sogenannten Panning, immunogene Proteine isoliert. Für alle Erreger bis auf C. difficile wurden immunogene Proteine aus den jeweiligen Bibliotheken isoliert. Die identifizierten Proteine von N. meningitidis und B. burgdorferi waren größtenteils bekannt, konnten aber in dieser Arbeit durch Phage Display verifiziert werden. Für N. gonorrhoeae wurden 21 potentiell immunogene Oligopeptide isoliert, von denen sechs Proteine als neue zuvor unbeschriebene Proteine mit immunogenem Charakter identifiziert wurden. Von den Phagen-präsentierten Oligopeptide der 21 immunogenen Proteine wurden Epitopmappings mit verschiedenen polyklonalen Antikörpern durchgeführt, um immunogene Bereiche näher zu identifizieren und zu charakterisieren. Bei zehn Proteinen wurden lineare Epitope eindeutig mit drei polyklonalen Antikörpern identifiziert, von fünf weiteren Proteinen waren Epitope mit mindestens einem Antikörper detektierbar. Für eine weitere Charakterisierung der ermittelten Epitope wurden Alaninscans durchgeführt, die eine detaillierte Auskunft über kritische Aminosäuren für die Bindung des Antikörpers an das Epitop geben.
Ausgehend von dem neu identifizierten Protein mit immunogenem Charakter NGO1634 wurden 26 weitere Proteine aufgrund ihrer funktionellen Ähnlichkeit ausgewählt und mithilfe bioinformatischer Analysen auf ihre Eignung zur Entwicklung einer diagnostischen Anwendung analysiert. Durch Ausschluss der meisten Proteine aufgrund ihrer Lokalisation, Membrantopologie oder unspezifischen Proteinsequenz wurden scFv-Antikörper gegen acht Proteine mittels Phage Display generiert und anschließend als scFv-Fc-Fusionsantikörper produziert und charakterisiert.
Die hier identifizierten Proteine und linearen Epitope könnten einen Ansatzpunkt für die Entwicklung einer diagnostischen oder therapeutischen Anwendung bieten. Lineare Epitopsequenzen werden häufig für die Impfstoffentwicklung eingesetzt, sodass vor allem die in dieser Arbeit bestimmten Epitope von Membranproteinen interessante Kandidaten für weitere Untersuchungen in diese Richtung sind. Durch weitere Untersuchungen könnten möglicherweise unbekannte Virulenzfaktoren entdeckt werden, deren Inhibierung einen entscheidenden Einfluss auf Infektionen haben könnten.
Die immunologische Kontrazeption mittels Zona pellucida (ZP) Proteinen gilt als vielversprechender Ansatz für die Reproduktionskontrolle verwilderter Haus- und Wildtierbestände. Da die Applikation von nativer ZP mit Nebenwirkungen verbunden ist, wird die Verwendung einzelner ZP Peptide als Bestandteil kontrazeptiver Vakzine als besonders aussichtsreich erachtet. Das Prinzip dieser nebenwirkungsfreien ZP Immunisierung ist die gezielte Trennung der Entzündungsreaktionen auslösenden T-Zell-Epitope der ZP von den kontrazeptiv wirkenden B-Zell-Epitopen. Niedermolekulare synthetische oder rekombinante Peptide allein sind gering immunogen und können somit keine ausreichende Immunantwort induzieren. Die Verwendung von Peptiden für die immunologische Kontrazeption erfordert daher ein „Vakzin-Design“, d. h. die gezielte Kombination der Peptide mit immunstimulierenden Substanzen (Liposomen, Carrierproteinen, Adjuvantien). Zielstellung der vorliegenden Arbeit war die Untersuchung des Potentials synthetischer Peptide für die Immunokontrazeption von verwilderten Hauskatzen (Felis catus). Dazu wurden zunächst relevante B-Zell-Epitope des felinen Zona pellucida Proteins, ZPB2, identifiziert und synthetisiert. Zwei der synthetischen Peptide (P3, P6) wurden zur Herstellung von Antikörpern an BSA konjugiert und zusammen mit Freundschem Adjuvans in Ratten verimpft. Die kontrazeptive Relevanz beider Peptide sowie der Ratten Anti-Peptid Antiseren wurde im in vitro Befruchtungssystem der Hauskatze geprüft. Zur Untersuchung der Immunogenität der Peptide in der Zielspezies Hauskatze erfolgte die Entwicklung von Vakzin-Prototypen für die einmalige Applikation. Neben der Eruierung der Stärke und Dauer der Immunantwort wurde durch Verpaarung der Tiere auch das kontrazeptive Potential in vivo abgeschätzt.
In order to function properly, organisms have a complex control mechanism, in which a given gene is expressed at a particular time and place. One way to achieve this control is to regulate the initiation of transcription. This step requires the assembly of several components, i.e., a basal/general machinery common to all expressed genes, and a specific/regulatory machinery, which differs among genes and is the responsible for proper gene expression in response to environmental or developmental signals. This specific machinery is composed of transcription factors (TFs), which can be grouped into evolutionarily related gene families that possess characteristic protein domains. In this work we have exploited the presence of protein domains to create rules that serve for the identification and classification of TFs. We have modelled such rules as a bipartite graph, where families and protein domains are represented as nodes. Connections between nodes represent that a protein domain should (required rule) or should not (forbidden rule) be present in a protein to be assigned into a TF family. Following this approach we have identified putative complete sets of TFs in plant species, whose genome is completely sequenced: Cyanidioschyzon merolae (red algae), Chlamydomonas reinhardtii (green alga), Ostreococcus tauri (green alga), Physcomitrella patens (moss), Arabidopsis thaliana (thale cress), Populus trichocarpa (black cottonwood) and Oryza sativa (rice). The identification of the complete sets of TFs in the above-mentioned species, as well as additional information and reference literature are available at http://plntfdb.bio.uni-potsdam.de/. The availability of such sets allowed us performing detailed evolutionary studies at different levels, from a single family to all TF families in different organisms in a comparative genomics context. Notably, we uncovered preferential expansions in different lineages, paving the way to discover the specific biological roles of these proteins under different conditions. For the basic leucine zipper (bZIP) family of TFs we were able to infer that in the most recent common ancestor (MRCA) of all green plants there were at least four bZIP genes functionally involved in oxidative stress and unfolded protein responses that are bZIP-mediated processes in all eukaryotes, but also in light-dependent regulations. The four founder genes amplified and diverged significantly, generating traits that benefited the colonization of new environments. Currently, following the approach described above, up to 57 TF and 11 TR families can be identified, which are among the most numerous transcription regulatory families in plants. Three families of putative TFs predate the split between rhodophyta (red algae) and chlorophyta (green algae), i.e., G2-like, PLATZ, and RWPRK, and may have been of particular importance for the evolution of eukaryotic photosynthetic organisms. Nine additional families, i.e., ABI3/VP1, AP2-EREBP, ARR-B, C2C2-CO-like, C2C2-Dof, PBF-2-like/Whirly, Pseudo ARR-B, SBP, and WRKY, predate the split between green algae and streptophytes. The identification of putative complete list of TFs has also allowed the delineation of lineage-specific regulatory families. The families SBP, bHLH, SNF2, MADS, WRKY, HMG, AP2-EREBP and FHA significantly differ in size between algae and land plants. The SBP family of TFs is significantly larger in C. reinhardtii, compared to land plants, and appears to have been lost in the prasinophyte O. tauri. The families bHLH, SNF2, MADS, WRKY, HMG, AP2-EREBP and FHA preferentially expanded with the colonisation of land, and might have played an important role in this great moment in evolution. Later, after the split of bryophytes and tracheophytes, the families MADS, AP2-EREBP, NAC, AUX/IAA, PHD and HRT have significantly larger numbers in the lineage leading to seed plants. We identified 23 families that are restricted to land plants and that might have played an important role in the colonization of this new habitat. Based on the list of TFs in different species we have started to develop high-throughput experimental platforms (in rice and C. reinhardtii) to monitor gene expression changes of TF genes under different genetic, developmental or environmental conditions. In this work we present the monitoring of Arabidopsis thaliana TFs during the onset of senescence, a process that leads to cell and tissue disintegration in order to redistribute nutrients (e.g. nitrogen) from leaves to reproductive organs. We show that the expression of 185 TF genes changes when leaves develop from half to fully expanded leaves and finally enter partial senescence. 76% of these TFs are down-regulated during senescence, the remaining are up-regulated. The identification of TFs in plants in a comparative genomics setup has proven fruitful for the understanding of evolutionary processes and contributes to the elucidation of complex developmental programs.
Cellulose is the most abundant biopolymer on earth and the main load-bearing structure in plant cell walls. Cellulose microfibrils are laid down in a tight parallel array, surrounding plant cells like a corset. Orientation of microfibrils determines the direction of growth by directing turgor pressure to points of expansion (Somerville et al., 2004). Hence, cellulose deficient mutants usually show cell and organ swelling due to disturbed anisotropic cell expansion (reviewed in Endler and Persson, 2011). How do cellulose microfibrils gain their parallel orientation? First experiments in the 1960s suggested, that cortical microtubules aid the cellulose synthases on their way around the cell (Green, 1962; Ledbetter and Porter, 1963). This was proofed in 2006 through life cell imaging (Paredez et al., 2006). However, how this guidance was facilitated, remained unknown. Through a combinatory approach, including forward and reverse genetics together with advanced co-expression analysis, we identified pom2 as a cellulose deficient mutant. Map- based cloning revealed that the gene locus of POM2 corresponded to CELLULOSE SYNTHASE INTERACTING 1 (CSI1). Intriguingly, we previously found the CSI1 protein to interact with the putative cytosolic part of the primary cellulose synthases in a yeast-two-hybrid screen (Gu et al., 2010). Exhaustive cell biological analysis of the POM2/CSI1 protein allowed to determine its cellular function. Using spinning disc confocal microscopy, we could show that in the absence of POM2/CSI1, cellulose synthase complexes lose their microtubule-dependent trajectories in the plasma membrane. The loss of POM2/CSI1, however does not influence microtubule- dependent delivery of cellulose synthases (Bringmann et al., 2012). Consequently, POM2/CSI1 acts as a bridging protein between active cellulose synthases and cortical microtubules. This thesis summarizes three publications of the author, regarding the identification of proteins that connect cellulose synthases to the cytoskeleton. This involves the development of bioinformatics tools allowing candidate gene prediction through co-expression studies (Mutwil et al., 2009), identification of candidate genes through interaction studies (Gu et al., 2010), and determination of the cellular function of the candidate gene (Bringmann et al., 2012).
In a very simplified view, the plant leaf growth can be reduced to two processes, cell division and cell expansion, accompanied by expansion of their surrounding cell walls. The vacuole, as being the largest compartment of the plant cell, plays a major role in controlling the water balance of the plant. This is achieved by regulating the osmotic pressure, through import and export of solutes over the vacuolar membrane (the tonoplast) and by controlling the water channels, the aquaporins. Together with the control of cell wall relaxation, vacuolar osmotic pressure regulation is thought to play an important role in cell expansion, directly by providing cell volume and indirectly by providing ion and pH homestasis for the cytosoplasm. In this thesis the role of tonoplast protein coding genes in cell expansion in the model plant Arabidopsis thaliana is studied and genes which play a putative role in growth are identified. Since there is, to date, no clearly identified protein localization signal for the tonoplast, there is no possibility to perform genome-wide prediction of proteins localized to this compartment. Thus, a series of recent proteomic studies of the tonoplast were used to compile a list of cross-membrane tonoplast protein coding genes (117 genes), and other growth-related genes from notably the growth regulating factor (GRF) and expansin families were included (26 genes). For these genes a platform for high-throughput reverse transcription quantitative real time polymerase chain reaction (RT-qPCR) was developed by selecting specific primer pairs. To this end, a software tool (called QuantPrime, see http://www.quantprime.de) was developed that automatically designs such primers and tests their specificity in silico against whole transcriptomes and genomes, to avoid cross-hybridizations causing unspecific amplification. The RT-qPCR platform was used in an expression study in order to identify candidate growth related genes. Here, a growth-associative spatio-temporal leaf sampling strategy was used, targeting growing regions at high expansion developmental stages and comparing them to samples taken from non-expanding regions or stages of low expansion. Candidate growth related genes were identified after applying a template-based scoring analysis on the expression data, ranking the genes according to their association with leaf expansion. To analyze the functional involvement of these genes in leaf growth on a macroscopic scale, knockout mutants of the candidate growth related genes were screened for growth phenotypes. To this end, a system for non-invasive automated leaf growth phenotyping was established, based on a commercially available image capture and analysis system. A software package was developed for detailed developmental stage annotation of the images captured with the system, and an analysis pipeline was constructed for automated data pre-processing and statistical testing, including modeling and graph generation, for various growth-related phenotypes. Using this system, 24 knockout mutant lines were analyzed, and significant growth phenotypes were found for five different genes.
Red cell development in adult humans results in the mean daily production of 2x1011 erythrocytes. Mature hemoglobinized and enucleated erythrocytes develop from multipotent hematopoietic stem/progenitor cells through more committed progenitor cell types such as BFU-E and CFU-E. The studies on the molecular mechanisms of erythropoiesis in the human system require a sufficient number of purified erythroid progenitors of the different stages of erythropoiesis. Primary human erythroid progenitors are difficult to obtain as a homogenous population in sufficiently high cell numbers. Various culture conditions for the in vitro cell culture of primary human erythroid progenitors have been previously described. Mainly, the culture resulted in the generation of rather mature stages of Epo-dependent erythroid progenitors. In this study our efforts were directed towards the isolation and characterization of more early red cell progenitors that are Epo-independent. To identify such progenitors, CD34+ cells were purified from cord blood and cultured under serum free conditions in the presence of the growth factors SCF, IL-3 and hyper-IL-6, referred to as SI2 culture conditions. By immunomagnetic bead selection of E-cadherin (E-cad) positive cells, E-cad+ progenitors were isolated. These Epo-independent E-cad+ progenitors have been amplified under SI2 conditions to large cell numbers. The E-cad+ progenitors were characterized for surface antigen expression by flow cytometry, response to growth factors in proliferation assay and for their differentiation potential into mature red cells. Additionally, the properties of E-cad+ progenitors were compared to those of two other erythroid progenitors: Epo-dependent progenitors described by Panzenböck et al. (referred to as SCF/Epo progenitor), and CD36+ progenitors described by Freyssinier et al. (Panzenböck et al., 1998; Freyssinier et al., 1999). Finally, the gene expression profile of E-cad+ progenitors was compared to the profiles of SCF/Epo progenitors and CD36+ progenitors using the DNA microarray technique. Based on our studies we propose that Epo-independent E-cad+ progenitors are early stage, BFU-E like progenitors. They respond to Epo, despite the fact that they were generated in the absence of Epo, and can completely undergo erythroid differentiation. Furthermore, we demonstrate that the growth properties, the growth factor response and the surface marker expression of E-cad+ progenitors are similar to those of the SCF/Epo progenitors and the CD36+ progenitors. By the comparison of gene profiles, we were also able to demonstrate that the Epo-dependent and Epo-independent red cell progenitors are very similar. Analyzing the molecular differences between E-cad+ and SCF/Epo progenitors revealed several candidate genes such as galectin-3, cyclin D1, AMHR, PDF and IGFBP4, which are potential regulators involved in red cell development. We also demonstrate that the CD36+ progenitors, isolated by immunomagentic bead selection, are a heterogeneous progenitor population containing an E-cad+ and an E-cad- subpopulation. Based on their gene expression profile, CD36+ progenitors seem to exhibit both erythroid and megakaryocytic features. These studies led to a more updated model of erythroid cell development that should pave the way for further studies on molecular mechanisms of erythropoiesis.
Cells are built from a variety of macromolecules and metabolites. Both, the proteome and the metabolome are highly dynamic and responsive to environmental cues and developmental processes. But it is not their bare numbers, but their interactions that enable life. The protein-protein (PPI) and protein-metabolite interactions (PMI) facilitate and regulate all aspects of cell biology, from metabolism to mitosis. Therefore, the study of PPIs and PMIs and their dynamics in a cell-wide context is of great scientific interest. In this dissertation, I aim to chart a map of the dynamic PPIs and PMIs across metabolic and cellular transitions. As a model system, I study the shift from the fermentative to the respiratory growth, known as the diauxic shift, in the budding yeast Saccharomyces cerevisiae. To do so, I am applying a co-fractionation mass spectrometry (CF-MS) based method, dubbed protein metabolite interactions using size separation (PROMIS). PROMIS, as well as comparable methods, will be discussed in detail in chapter 1.
Since PROMIS was developed originally for Arabidopsis thaliana, in chapter 2, I will describe the adaptation of PROMIS to S. cerevisiae. Here, the obtained results demonstrated a wealth of protein-metabolite interactions, and experimentally validated 225 previously predicted PMIs. Applying orthogonal, targeted approaches to validate the interactions of a proteogenic dipeptide, Ser-Leu, five novel protein-interactors were found. One of those proteins, phosphoglycerate kinase, is inhibited by Ser-Leu, placing the dipeptide at the regulation of glycolysis.
In chapter 3, I am presenting PROMISed, a novel web-tool designed for the analysis of PROMIS- and other CF-MS-datasets. Starting with raw fractionation profiles, PROMISed enables data pre-processing, profile deconvolution, scores differences in fractionation profiles between experimental conditions, and ultimately charts interaction networks. PROMISed comes with a user-friendly graphic interface, and thus enables the routine analysis of CF-MS data by non-computational biologists.
Finally, in chapter 4, I applied PROMIS in combination with the isothermal shift assay to the diauxic shift in S. cerevisiae to study changes in the PPI and PMI landscape across this metabolic transition. I found a major rewiring of protein-protein-metabolite complexes, exemplified by the disassembly of the proteasome in the respiratory phase, the loss of interaction of an enzyme involved in amino acid biosynthesis and its cofactor, as well as phase and structure specific interactions between dipeptides and enzymes of central carbon metabolism.
In chapter 5, I am summarizing the presented results, and discuss a strategy to unravel the potential patterns of dipeptide accumulation and binding specificities. Lastly, I recapitulate recently postulated guidelines for CF-MS experiments, and give an outlook of protein interaction studies in the near future.
After endosymbiosis, chloroplasts lost most of their genome. Many former endosymbiotic genes are now nucleus-encoded and the products are re-imported post-translationally. Consequently, photosynthetic complexes are built of nucleus- and plastid-encoded subunits in a well-defined stoichiometry. In Chlamydomonas, the translation of chloroplast-encoded photosynthetic core subunits is feedback-regulated by the assembly state of the complexes they reside in. This process is called Control by Epistasy of Synthesis (CES) and enables the efficient production of photosynthetic core subunits in stoichiometric amounts. In chloroplasts of embryophytes, only Rubisco subunits have been shown to be feedback-regulated. That opens the question if there is additional CES regulation in embryophytes. I analyzed chloroplast gene expression in tobacco and Arabidopsis mutants with assembly defects for each photosynthetic complex to broadly answer this question. My results (i) confirmed CES within Rubisco and hint to potential translational feedback regulation in the synthesis of (ii) cytochrome b6f (Cyt b6f) and (iii) photosystem II (PSII) subunits. This work suggests a CES network in PSII that links psbD, psbA, psbB, psbE, and potentially psbH expression by a feedback mechanism that at least partially differs from that described in Chlamydomonas. Intriguingly, in the Cyt b6f complex, a positive feedback regulation that coordinates the synthesis of PetA and PetB was observed, which was not previously reported in Chlamydomonas. No evidence for CES interactions was found in the expression of NDH and ATP synthase subunits of embryophytes. Altogether, this work provides solid evidence for novel assembly-dependent feedback regulation mechanisms controlling the expression of photosynthetic genes in chloroplasts of embryophytes.
In order to obtain a comprehensive inventory of the rbcL and psbA RNA-binding proteomes (including factors that regulate their expression, especially factors involved in CES), an aptamer based affinity purification method was adapted and refined for the specific purification these transcripts from tobacco chloroplasts. To this end, three different aptamers (MS2, Sephadex ,and streptavidin binding) were stably introduced into the 3’ UTRs of psbA and rbcL by chloroplast transformation. RNA aptamer based purification and subsequent chip analysis (RAP Chip) demonstrated a strong enrichment of psbA and rbcL transcripts and currently, ongoing mass spectrometry analyses shall reveal potential regulatory factors. Furthermore, the suborganellar localization of MS2 tagged psbA and rbcL transcripts was analyzed by a combined affinity, immunology, and electron microscopy approach and demonstrated the potential of aptamer tags for the examination of the spatial distribution of chloroplast transcripts.
Identification of chemical mediators that regulate the specialized metabolism in Nostoc punctiforme
(2021)
Specialized metabolites, so-called natural products, are produced by a variety of different organisms, including bacteria and fungi. Due to their wide range of different biological activities, including pharmaceutical relevant properties, microbial natural products are an important source for drug development. They are encoded by biosynthetic gene clusters (BGCs), which are a group of locally clustered genes. By screening genomic data for genes encoding typical core biosynthetic enzymes, modern bioinformatical approaches are able to predict a wide range of BGCs. To date, only a small fraction of the predicted BGCs have their associated products identified.
The phylum of the cyanobacteria has been shown to be a prolific, but largely untapped source for natural products. Especially multicellular cyanobacterial genera, like Nostoc, harbor a high amount of BGCs in their genomes.
A main goal of this study was to develop new concepts for the discovery of natural products in cyanobacteria. Due to its diverse setup of orphan BGCs and its amenability to genetic manipulation, Nostoc punctiforme PCC 73102 (N. punctiforme) appeared to be a promising candidate to be established as a model organism for natural product discovery in cyanobacteria. By utilizing a combination of genome-mining, bioactivity-screening, variations of culture conditions, as well as metabolic engineering, not only two new polyketides were discovered, but also first-time insights into the regulation of the specialized metabolism in N. punctiforme were gained during this study.
The cultivation of N. punctiforme to very high densities by utilizing increasing light intensities and CO2 levels, led to an enhanced metabolite production, causing rather complex metabolite extracts. By utilizing a library of CFP reporter mutant strains, each strain reporting for one of the predicted BGCs, it was shown that eight out of 15 BGCs were upregulated under high density (HD) cultivation conditions. Furthermore, it could be demonstrated that the supernatant of an HD culture can increase the expression of four of the influenced BGCs, even under conventional cultivation conditions. This led to the hypothesis that a chemical mediator encoded by one of the affected BGCs is accumulating in the HD supernatant and is able to increase the expression of other BGCs as part of a cell-density dependent regulatory circuit. To identify which of the BGCs could be a main trigger of the presumed regulatory circuit, it was tried to activate four BGCs (pks1, pks2, ripp3, ripp4) selectively by overexpression of putative pathway-specific regulatory genes that were found inside the gene clusters. Transcriptional analysis of the mutants revealed that only the mutant strain targeting the pks1 BGC, called AraC_PKS1, was able to upregulate the expression of its associated BGC. From an RNA sequencing study of the AraC_PKS1 mutant strain, it was discovered that beside pks1, the orphan BGCs ripp3 and ripp4 were also upregulated in the mutant strain. Furthermore, it was observed that secondary metabolite production in the AraC_PKS1 mutant strain is further enhanced under high-light and high-CO2 cultivation conditions. The increased production of the pks1 regulator NvlA also had an impact on other regulatory factors, including sigma factors and the RNA chaperone Hfq. Analysis of the AraC_PKS1 cell and supernatant extracts led to the discovery of two novel polyketides, nostoclide and nostovalerolactone, both encoded by the pks1 BGC. Addition of the polyketides to N. punctiforme WT demonstrated that the pks1-derived compounds are able to partly reproduce the effects on secondary metabolite production found in the AraC_PKS1 mutant strain. This indicates that both compounds are acting as extracellular signaling factors as part of a regulatory network. Since not all transcriptional effects that were found in the AraC_PKS1 mutant strain could be reproduced by the pks1 products, it can be assumed that the regulator NvlA has a global effect and is not exclusively specific to the pks1 pathway.
This study was the first to use a putative pathway specific regulator for the specific activation of BGC expression in cyanobacteria. This strategy did not only lead to the detection of two novel polyketides, it also gave first-time insights into the regulatory mechanism of the specialized metabolism in N. punctiforme. This study illustrates that understanding regulatory pathways can aid in the discovery of novel natural products. The findings of this study can guide the design of new screening strategies for bioactive compounds in cyanobacteria and help to develop high-titer production platforms for cyanobacterial natural products.
Aptamers are single-stranded DNA (ssDNA) or RNA molecules that can bind specifically and with high affinity to target molecules due to their unique three-dimensional structure. For this reason, they are often compared to antibodies and sometimes even referred to as “chemical antibodies”. They are simple and inexpensive to synthesize, easy to modify, and smaller than conventional antibodies. Enzymes, especially hydrolases, are interesting targets in this context. This class of enzymes is capable of hydrolytically cleaving various macromolecules such as proteins, as well as smaller molecules such as antibiotics. Hence, they play an important role in many biological processes including diseases and their treatment. Hydrolase detection as well as the understanding of their function is therefore of great importance for diagnostics and therapy. Due to their various desirable features compared to antibodies, aptamers are being discussed as alternative agents for analytical and diagnostic use in various applications. The use of aptamers in therapy is also frequently investigated, as the binding of aptamers can have effects on the catalytic activity, protein-protein interactions, or proteolytic cascades. Aptamers are generated by an in vitro selection process. Potential aptamer candidates are selected from a pool of enriched nucleic acid sequences with affinity to the target, and their binding affinity and specificity is investigated. This is one of the most important steps in aptamer generation to obtain specific aptamers with high affinity for use in analytical and diagnostic applications. The binding properties or binding domains and their effects on enzyme functions form the basis for therapeutic applications.
In this work, the binding properties of DNA aptamers against two different hydrolases were investigated. In view of their potential utility for analytical methods, aptamers against human urokinase (uPA) and New Delhi metallo-β-lactamase-1 (NDM-1) were evaluated for their binding affinity and specificity using different methods. Using the uPA aptamers, a protocol for measuring the binding kinetics of an aptamer-protein-interaction by surface plasmon resonance spectroscopy (SPR) was developed. Based on the increased expression of uPA in different types of cancer, uPA is discussed as a prognostic and diagnostic tumor marker. As uPA aptamers showed different binding sites on the protein, microtiter plate-based aptamer sandwich assay systems for the detection of uPA were developed. Because of the function of urokinase in cancer cell proliferation and metastasis, uPA is also discussed as a therapeutic target. In this regard, the different binding sites of aptamers showed different effects on uPA function. In vitro experiments demonstrated both inhibition of uPA binding to its receptor as well as the inhibition of uPA catalytic activity for different aptamers. Thus, in addition to their specificity and affinity for their targets, the utility of the aptamers for potential diagnostic and therapeutic applications was demonstrated. First, as an alternative inhibitor of human urokinase for therapeutic purposes, and second, as valuable recognition molecules for the detection of urokinase, as a prognostic and diagnostic marker for cancer, and for NDM-1 to detect resistance to carbapenem antibiotics.
Plants are the primary producers of biomass and thereby the basis of all life. Many varieties are cultivated, mainly to produce food, but to an increasing amount as a source of renewable energy. Because of the limited acreage available, further improvements of cultivated species both with respect to yield and composition are inevitable. One approach to further progress in developing improved plant cultivars is a systems biology oriented approach. This work aimed to investigate the primary metabolism of the model plant A.thaliana and its relation to plant growth using quantitative genetics methods. A special focus was set on the characterization of heterosis, the deviation of hybrids from their parental means for certain traits, on a metabolic level. More than 2000 samples of recombinant inbred lines (RILs) and introgression lines (ILs) developed from the two accessions Col-0 and C24 were analyzed for 181 metabolic traces using gas-chromatography/ mass-spectrometry (GC-MS). The observed variance allowed the detection of 157 metabolic quantitative trait loci (mQTL), genetic regions carrying genes, which are relevant for metabolite abundance. By analyzing several hundred test crosses of RILs and ILs it was further possible to identify 385 heterotic metabolic QTL (hmQTL). Within the scope of this work a robust method for large scale GC-MS analyses was developed. A highly significant canonical correlation between biomass and metabolic profiles (r = 0.73) was found. A comparable analysis of the results of the two independent experiments using RILs and ILs showed a large agreement. The confirmation rate for RIL QTL in ILs was 56 % and 23 % for mQTL and hmQTL respectively. Candidate genes from available databases could be identified for 67 % of the mQTL. To validate some of these candidates, eight genes were re-sequenced and in total 23 polymorphisms could be found. In the hybrids, heterosis is small for most metabolites (< 20%). Heterotic QTL gave rise to less candidate genes and a lower overlap between both populations than was determined for mQTL. This hints that regulatory loci and epistatic effects contribute to metabolite heterosis. The data described in this thesis present a rich source for further investigation and annotation of relevant genes and may pave the way towards a better understanding of plant biology on a system level.
The major aim of this work was the identification of new phloem sap proteins and a metabolic characterisation of this transport fluid. The experiments were performed on the three plant species C. sativus, C. maxima and B. napus. To characterise the phloem samples from B. napus, a new model plant for phloem analysis, western blot tests together with metabolite profiling were performed. GC-MS metabolite profiling and enzyme assays were used for measuring metabolites in the phloem of B. napus. Results from the phloem sap measurements showed, as expected, a typical sugar distribution for apoplasmic phloem loaders with sucrose being the predominant sugar. In stem extracts, the most abundant sugar was glucose with much lower fructose and sucrose levels. With the GC-MS approach it was possible to identify a number of metabolites which showed a differential distribution when phloem and stem tissue extracts were compared. For protein identification, two different approaches were employed (i) screening expression libraries with total phloem protein specific antisera and (ii) protein separation on 2 DE gels followed by ESI-MS/MS sequence analyses. For the first approach, three different phloem protein-specific antisera were produced and expression libraries were constructed. Phloem protein antisera were tested for specificity and some attempts to estimate specific epitopes were undertaken. Screening of the libraries resulted in the identification of 14 different proteins from all investigated species. Analyses of B. napus phloem sap proteins from 2 DE with ESI-MS/MS resulted in the identification of 5 different proteins. The phloem localisation of the identified proteins was additionally confirmed by western blot tests using specific antibodies. In order to functionally characterise some selected phloem proteins from B. napus, the group of potential calcium-binding polypeptides was analysed for functional Ca<sup>+2 binding properties and several Ca<sup>+2–binding proteins could be isolated. However, their sequences could as yet not be determined. Another approach used for functional protein characterisation was the analysis of Arabidopsis T-DNA insertion mutants. Four available mutants with insertions in phloem protein-specific genes were chosen from the SALK and GABI-Kat collections and selected homozygous lines were tested for the presence of the investigated proteins. In order to verify if the product of one of the mutated gene (GRP 7) is transported through the phloem, grafting experiments were performed followed by western blot analyses. Although the employed antiserum against GRP 7 protein did not allow distinguishing between the mutant and the wild type plants, successful Arabidopsis grafting could be established as a promising method for further studies on protein translocation through the phloem.
Tierische und menschliche Fäkalien aus Landwirtschaft und Haushalten enthalten zahlreiche obligat und opportunistisch pathogene Mikroorganismen, deren Konzentration u. a. je nach Gesundheitszustand der betrachteten Gruppe schwankt. Neben den Krankheitserregern enthalten Fäkalien aber auch essentielle Pflanzennährstoffe (276) und dienen seit Jahrtausenden (63) als Dünger für Feldfrüchte. Mit der unbedarften Verwendung von pathogenbelastetem Fäkaldünger steigt jedoch auch das Risiko einer Infektion von Mensch und Tier. Diese Gefahr erhöht sich mit der globalen Vernetzung der Landwirtschaft, z. B. durch den Import von kontaminierten Futter- bzw. Lebensmitteln (29).
Die vorliegende Arbeit stellt die milchsaure Fermentation von Rindergülle und Klärschlamm als alternative Hygienisierungsmethode gegenüber der Pasteurisation in Biogasanlagen bzw. gebräuchlichen Kompostierung vor.
Dabei wird ein Abfall der Gram-negativen Bakterienflora sowie der Enterokokken, Schimmel- und Hefepilze unter die Nachweisgrenze von 3 log10KbE/g beobachtet, gleichzeitig steigt die Konzentration der Lactobacillaceae um das Tausendfache. Darüber hinaus wird gezeigt, dass pathogene Bakterien wie Staphylococcus aureus, Salmonella spp., Listeria monocytogenes, EHEC O:157 und vegetative Clostridum perfringens-Zellen innerhalb von 3 Tagen inaktiviert werden. Die Inaktivierung von ECBO-Viren und Spulwurmeiern erfolgt innerhalb von 7 bzw. 56 Tagen. Zur Aufklärung der Ursache der beobachteten Hygienisierung wurde das fermentierte Material auf flüchtige Fettsäuren sowie pH-Wertänderungen untersucht. Es konnte festgestellt werden, dass die gemessenen Werte nicht die alleinige Ursache für das Absterben der Erreger sind, vielmehr wird eine zusätzliche bakterizide Wirkung durch eine mutmaßliche Bildung von Bakteriozinen in Betracht gezogen. Die parasitizide Wirkung wird auf die physikalischen Bedingungen der Fermentation zurückgeführt.
Die methodischen Grundlagen basieren auf Analysen mittels zahlreicher klassisch-kultureller Verfahren, wie z. B. der Lebendkeimzahlbestimmung. Darüber hinaus findet die MALDI-TOF-Massenspektrometrie und die klassische PCR in Kombination mit der Gradienten-Gelelektrophorese Anwendung, um kultivierbare Bakterienfloren zu beschreiben bzw. nicht kultivierbare Bakterienfloren stichprobenartig zu erfassen.
Neben den Aspekten der Hygienisierung wird zudem die Eignung der Methode für die landwirtschaftliche Nutzung berücksichtigt. Dies findet sich insbesondere in der Komposition des zu fermentierenden Materials wieder, welches für die verstärkte Humusakkumulation im Ackerboden optimiert wurde. Darüber hinaus wird die Masseverlustbilanz während der milchsauren Fermentation mit denen der Kompostierung sowie der Verarbeitung in der Biogasanlage verglichen und als positiv bewertet, da sie mit insgesamt 2,45 % sehr deutlich unter den bisherigen Alternativen liegt (73, 138, 458). Weniger Verluste an organischem Material während der Hygienisierung führen zu einer größeren verwendbaren Düngermenge, die auf Grund ihres organischen Ursprungs zu einer Verstärkung des Humusanteiles im Ackerboden beitragen kann (56, 132).
Today, analytical chemistry does not longer consist of only the big measuring devices and methods which are time consuming and expensive, which can furthermore only be handled by the qualified staff and in addition the results can also only be evaluated by this qualified staff. Usually, this technique, which shall be described in the following as 'classic analytic measuring technique', requires also rooms equipped especially and often a relative big quantity of the test compounds which should be prepared especially. Beside this classic analytic measuring technique, limited on definite substance groups and requests, a new measuring technique has gained acceptance particularly within the last years, which one can often be used by a layman, too. Often the new measuring technique has very little pieces of equipment. The needed sample volumes are also small and a special sample preparation isn't required. In addition, the new measuring instruments are simple to handle. They are cheap both in their production and in the use and they permit even a continuous measurement recording usually. Numerous of this new measuring instruments base on the research in the field of Biosensorik during the last 40 years. Since Clark and Lyon in the year 1962 were able to measure glucose with a simple oxygen electrode, completed by an enzyme the development of the new measuring technique did not have to be held back any longer. Biosensors, special pickups which consists of a combination from a biological component (permits a specific recognition of the analyte also without purification of the sample previously) and a physical pickup (convert the primary physicochemical effect into an electronically measurable signal), conquered the market. In the context of this thesis different tyrosinasesensors were developed which fulfilling the various requests, depending on origin and features of the used tyrosinase. One of the tyrosinasesensors for example was used for quantification of phenolic compounds in river and sea water and the results could correlated very well with the corresponding DIN-test for the determination of phenolic compounds. An other developed tyrosinasesensor showed a very high sensitiveness for catecholamines, substances which are of special importance in the medical diagnostics. In addition, the investigations of two different tyrosinases, which were carried out also in the context of this thesis, have shown, that a special tyrosinase (tyrosinase from Streptomyces antibioticus) will be the better choice as tyrosinase from Agaricus bisporus, which is used in the area of biosensor research till now, if one wants to develop in future even more sensitive tyrosinasesensors. Furthermore, first successes became reached on a molecular biological field, the production of tyrosinasemutants with special, before well-considered features. These successes can be used to develop a new generation of tyrosinasesensors, tyrosinasesensors in which tyrosinase can be bound directionally both to the corresponding physical pickup or also to another enzyme. From this one expects to achieve ways minimized which the substance to be determined (or whose product) otherwise must cover. Finally, this should result in an clearly visible increase of sensitivity of the Biosensor.
For recombinant production of proteins for structural and functional analyses, the E. coli expression system is the most widely used due to high yields and straightforward processing. However, particularly the expression of eukaryotic proteins in E. coli is often problematic, e.g. when the protein is not folded correctly and is deposited in insoluble inclusion bodies. In some cases it is favourable to analyse deletion constructs of a protein or an individual protein domain instead of the full-length protein. This implies the generation of a set of expression constructs that need to be characterised. In this work methods to optimise and evaluate in vitro folding of inclusion body proteins as well as high-throughput characterisation of expression constructs were developed. Transferring inclusion body proteins to their native state involves two steps: (a) solubilisation with a chaotropic reagent or a strong ionic detergent and (b) folding of the protein by removal of the chaotrop accompanied by the transfer into an appropriate buffer. The yield of natively folded protein is often substantially reduced due to aggregation or misfolding; it may, however, be improved by certain additives to the folding buffer. These additives need to be identified empirically. In this thesis a screening procedure for folding conditions was developed. To reduce the number of possible combinations of screening additives, empirical observations documented in the literature as well as well known properties of certain screening additives were considered. To decrease the amount of protein and work invested, the screen was miniaturised and automated using a pipetting robot. Twenty rapid dilution conditions for the denatured protein are tested and two conditions for folding of proteins using the detergent/cyclodextrin protein folding system of Rozema et al. (1996). 100 µg protein is used per condition. In addition, eight conditions can be tested for folding of His-tagged proteins (approx. 200 µg) immobilised on metal chelate resins. The screen was successfully applied to fold a human protein, the p22 subunit of dynactin that is expressed in inclusion bodies in E. coli. For p22 dynactin – as is the case for many proteins – there was no biological assay available to assess the success of the folding screen. Protein solubility can not be used as a stringent criterion because beside natively folded protein, soluble misfolded species and microaggregates may occur. This work evaluates methods to detect small amounts of natively folded protein after automated folding screening. Before folding screening with p22 dynactin, two model enzymes, bovine carbonic anhydrase II (CAB) and pig heart mitochondrial malate dehydrogenase, were used for evaluation. Recovered activity after refolding was correlated to different biophysical methods. 8-anilino-1-naphtalenesulfonic acid binding-experiments gave no useful information when refolding CAB, due to low sensitivity and because misfolded protein could not be readily distinguished from native protein. Tryptophan fluorescence spectra of refolded CAB were used to assess the success of refolding. The shift of the intensity maximum to a shorter wavelength, compared to the denaturant unfolded protein, as well as the fluorescence intensity correlated to recovered enzymatic activity. For both model enzymes, analytical hydrophobic interaction chromatography (HIC) was useful to identify refolded samples that contain active enzyme. Compactly folded, active enzyme eluted in a distinct peak in a decreasing ammonium sulfate gradient. The detection limit of analytical HIC was approx. 5 µg. In case of CAB, tryptophan fluorescence spectroscopy and analytical HIC showed that both methods in combination can be useful to rule out false positives or false negatives obtained with one method. These two methods were also useful to identify conditions for folding of p22 dynactin. However, tryptophan fluorescence spectroscopy can lead to false positives because in some cases spectra of soluble microaggregates are not well distinguishable from spectra of natively folded protein. In summary, a fast and reliable screening procedure was developed to make inclusion body proteins accessible to structural or functional analyses. In a separate project, 88 different E. coli expression constructs for 17 human protein domains that had been identified by sequence analysis were analysed using high-throughput purification and folding analysis in order to obtain candidates suitable for structural analysis. After 96 deep-well microplate expression and automated protein purification, solubly expressed protein domains were directly analysed using 1D ¹H-NMR spectroscopy. It was found that isolated methyl group signals below 0.5 ppm are particularly sensitive and reliable probes for folded protein. In addition – similar to the evaluation of a folding screen – analytical HIC proved to be an efficient tool for identifying constructs that yield compactly folded protein. Both methods, 1D ¹H-NMR spectroscopy and analytical HIC, provided complementary results. Six constructs, representing two domains, could be quickly identified as targets that are well suitable for structural analysis. The structure of one of these domains was solved recently by co-workers, the other structure was published by another group during this project.
Chloroplasts are the photosynthetic organelles in plant and algae cells that enable photoautotrophic growth. Due to their prokaryotic origin, modern-day chloroplast genomes harbor 100 to 200 genes. These genes encode for core components of the photosynthetic complexes and the chloroplast gene expression machinery, making most of them essential for the viability of the organism. The regulation of those genes is predominated by translational adjustments. The powerful technique of ribosome profiling was successfully used to generate highly resolved pictures of the translational landscape of Arabidopsis thaliana cytosol, identifying translation of upstream open reading frames and long non-coding transcripts. In addition, differences in plastidial translation and ribosomal pausing sites were addressed with this method. However, a highly resolved picture of the chloroplast translatome is missing. Here, with the use of chloroplast isolation and targeted ribosome affinity purification, I generated highly enriched ribosome profiling datasets of the chloroplasts translatome for Nicotiana tabacum in the dark and light. Chloroplast isolation was found unsuitable for the unbiased analysis of translation in the chloroplast but adequate to identify potential co-translational import. Affinity purification was performed for the small and large ribosomal subunit independently. The enriched datasets mirrored the results obtained from whole-cell ribosome profiling. Enhanced translational activity was detected for psbA in the light. An alternative translation initiation mechanism was not identified by selective enrichment of small ribosomal subunit footprints. In sum, this is the first study that used enrichment strategies to obtain high-depth ribosome profiling datasets of chloroplasts to study ribosome subunit distribution and chloroplast associated translation.
Ever-changing light intensities are challenging the photosynthetic capacity of photosynthetic organism. Increased light intensities may lead to over-excitation of photosynthetic reaction centers resulting in damage of the photosystem core subunits. Additional to an expensive repair mechanism for the photosystem II core protein D1, photosynthetic organisms developed various features to reduce or prevent photodamage. In the long-term, photosynthetic complex contents are adjusted for the efficient use of experienced irradiation. However, the contribution of chloroplastic gene expression in the acclimation process remained largely unknown. Here, comparative transcriptome and ribosome profiling was performed for the early time points of high-light acclimation in Nicotiana tabacum chloroplasts in a genome-wide scale. The time- course data revealed stable transcript level and only minor changes in translational activity of specific chloroplast genes during high-light acclimation. Yet, psbA translation was increased by two-fold in the high light from shortly after the shift until the end of the experiment. A stress-inducing shift from low- to high light exhibited increased translation only of psbA. This study indicate that acclimation fails to start in the observed time frame and only short-term responses to reduce photoinhibition were observed.
Biofilms are complex living materials that form as bacteria get embedded in a matrix of self-produced protein and polysaccharide fibres. The formation of a network of extracellular biopolymer fibres contributes to the cohesion of the biofilm by promoting cell-cell attachment and by mediating biofilm-substrate interactions. This sessile mode of bacteria growth has been well studied by microbiologists to prevent the detrimental effects of biofilms in medical and industrial settings. Indeed, biofilms are associated with increased antibiotic resistance in bacterial infections, and they can also cause clogging of pipelines or promote bio-corrosion. However, biofilms also gained interest from biophysics due to their ability to form complex morphological patterns during growth. Recently, the emerging field of engineered living materials investigates biofilm mechanical properties at multiple length scales and leverages the tools of synthetic biology to tune the functions of their constitutive biopolymers.
This doctoral thesis aims at clarifying how the morphogenesis of Escherichia coli (E. coli) biofilms is influenced by their growth dynamics and mechanical properties. To address this question, I used methods from cell mechanics and materials science. I first studied how biological activity in biofilms gives rise to non-uniform growth patterns. In a second study, I investigated how E. coli biofilm morphogenesis and its mechanical properties adapt to an environmental stimulus, namely the water content of their substrate. Finally, I estimated how the mechanical properties of E. coli biofilms are altered when the bacteria express different extracellular biopolymers.
On nutritive hydrogels, micron-sized E. coli cells can build centimetre-large biofilms. During this process, bacterial proliferation and matrix production introduce mechanical stresses in the biofilm, which release through the formation of macroscopic wrinkles and delaminated buckles. To relate these biological and mechanical phenomena, I used time-lapse fluorescence imaging to track cell and matrix surface densities through the early and late stages of E. coli biofilm growth. Colocalization of high cell and matrix densities at the periphery precede the onset of mechanical instabilities at this annular region. Early growth is detected at this outer annulus, which was analysed by adding fluorescent microspheres to the bacterial inoculum. But only when high rates of matrix production are present in the biofilm centre, does overall biofilm spreading initiate along the solid-air interface. By tracking larger fluorescent particles for a long time, I could distinguish several kinematic stages of E. coli biofilm expansion and observed a transition from non-linear to linear velocity profiles, which precedes the emergence of wrinkles at the biofilm periphery. Decomposing particle velocities to their radial and circumferential components revealed a last kinematic stage, where biofilm movement is mostly directed towards the radial delaminated buckles, which verticalize. The resulting compressive strains computed in these regions were observed to substantially deform the underlying agar substrates. The co-localization of higher cell and matrix densities towards an annular region and the succession of several kinematic stages are thus expected to promote the emergence of mechanical instabilities at the biofilm periphery. These experimental findings are predicted to advance future modelling approaches of biofilm morphogenesis.
E. coli biofilm morphogenesis is further anticipated to depend on external stimuli from the environment. To clarify how the water could be used to tune biofilm material properties, we quantified E. coli biofilm growth, wrinkling dynamics and rigidity as a function of the water content of the nutritive substrates. Time-lapse microscopy and computational image analysis revealed that substrates with high water content promote biofilm spreading kinetics, while substrates with low water content promote biofilm wrinkling. The wrinkles observed on biofilm cross-sections appeared more bent on substrates with high water content, while they tended to be more vertical on substrates with low water content. Both wet and dry biomass, accumulated over 4 days of culture, were larger in biofilms cultured on substrates with high water content, despite extra porosity within the matrix layer. Finally, the micro-indentation analysis revealed that substrates with low water content supported the formation of stiffer biofilms. This study shows that E. coli biofilms respond to the water content of their substrate, which might be used for tuning their material properties in view of further applications.
Biofilm material properties further depend on the composition and structure of the matrix of extracellular proteins and polysaccharides. In particular, E. coli biofilms were suggested to present tissue-like elasticity due to a dense fibre network consisting of amyloid curli and phosphoethanolamine-modified cellulose. To understand the contribution of these components to the emergent mechanical properties of E. coli biofilms, we performed micro-indentation on biofilms grown from bacteria of several strains. Besides showing higher dry masses, larger spreading diameters and slightly reduced water contents, biofilms expressing both main matrix components also presented high rigidities in the range of several hundred kPa, similar to biofilms containing only curli fibres. In contrast, a lack of amyloid curli fibres provides much higher adhesive energies and more viscoelastic fluid-like material behaviour. Therefore, the combination of amyloid curli and phosphoethanolamine-modified cellulose fibres implies the formation of a composite material whereby the amyloid curli fibres provide rigidity to E. coli biofilms, whereas the phosphoethanolamine-modified cellulose rather acts as a glue. These findings motivate further studies involving purified versions of these protein and polysaccharide components to better understand how their interactions benefit biofilm functions.
All three studies depict different aspects of biofilm morphogenesis, which are interrelated. The first work reveals the correlation between non-uniform biological activities and the emergence of mechanical instabilities in the biofilm. The second work acknowledges the adaptive nature of E. coli biofilm morphogenesis and its mechanical properties to an environmental stimulus, namely water. Finally, the last study reveals the complementary role of the individual matrix components in the formation of a stable biofilm material, which not only forms complex morphologies but also functions as a protective shield for the bacteria it contains. Our experimental findings on E. coli biofilm morphogenesis and their mechanical properties can have further implications for fundamental and applied biofilm research fields.
Glycosylphosphatidylinositols (GPIs) are highly complex glycolipids that serve as membrane anchors to a large variety of eukaryotic proteins. These are covalently attached to a group of peripheral proteins called GPI-anchored proteins (GPI-APs) through a post-translational modification in the endoplasmic reticulum. The GPI anchor is a unique structure composed of a glycan, with phospholipid tail at one end and a phosphoethanolamine linker at the other where the protein attaches. The glycan part of the GPI comprises a conserved pseudopentasaccharide core that could branch out to carry additional glycosyl or phosphoethanolamine units. GPI-APs are involved in a diverse range of cellular processes, few of which are signal transduction, protein trafficking, pathogenesis by protozoan parasites like the malaria- causing parasite Plasmodium falciparum. GPIs can also exist freely on the membrane surface without an attached protein such as those found in parasites like Toxoplasma gondii, the causative agent of Toxoplasmosis. These molecules are both structurally and functionally diverse, however, their structure-function relationship is still poorly understood. This is mainly because no clear picture exists regarding how the protein and the glycan arrange with respect to the lipid layer. Direct experimental evidence is rather scarce, due to which inconclusive pictures have emerged, especially regarding the orientation of GPIs and GPI-APs on membrane surfaces and the role of GPIs in membrane organization. It appears that computational modelling through molecular dynamics simulations would be a useful method to make progress. In this thesis, we attempt to explore characteristics of GPI anchors and GPI-APs embedded in lipid bilayers by constructing molecular models at two different resolutions – all-atom and coarse-grained.
First, we show how to construct a modular molecular model of GPIs and GPI-anchored proteins that can be readily extended to a broad variety of systems, addressing the micro-heterogeneity of GPIs. We do so by creating a hybrid link to which GPIs of diverse branching and lipid tails of varying saturation with their optimized force fields, GLYCAM06 and Lipid14 respectively, can be attached. Using microsecond simulations, we demonstrate that GPI prefers to “flop-down” on the membrane, thereby, strongly interacting with the lipid heads, over standing upright like a “lollipop”. Secondly, we extend the model of the GPI core to carry out a systematic study of the structural aspects of GPIs carrying different side chains (parasitic and human GPI variants) inserted in lipid bilayers. Our results demonstrate the importance of the side branch residues as these are the most accessible, and thereby, recognizable epitopes. This finding qualitatively agrees with experimental observations that highlight the role of the side branches in immunogenicity of GPIs and the specificity thereof. The overall flop-down orientation of the GPIs with respect to the bilayer surface presents the side chain residues to face the solvent. Upon attaching the green fluorescent protein (GFP) to the GPI, it is seen to lie in close proximity to the bilayer, interacting both with the lipid heads and glycan part of the GPI. However the orientation of GFP is sensitive to the type of GPI it is attached to. Finally, we construct a coarse-grained model of the GPI and GPI-anchored GFP using a modified version of the MARTINI force-field, using which the timescale is enhanced by at least an order of magnitude compared to the atomistic system.
This study provides a theoretical perspective on the conformational behavior of the GPI core and some of its branched variations in presence of lipid bilayers, as well as draws comparisons with experimental observations. Our modular atomistic model of GPI can be further employed to study GPIs of variable branching, and thereby, aid in designing future experiments especially in the area of vaccines and drug therapies. Our coarse-grained model can be used to study dynamic aspects of GPIs and GPI-APs w.r.t plasma membrane organization. Furthermore, the backmapping technique of converting coarse-grained trajectory back to the atomistic model would enable in-depth structural analysis with ample conformational sampling.