765 research outputs found
Rational development of stabilized cyclic disulfide redox probes and bioreductive prodrugs to target dithiol oxidoreductases
Countless biological processes allow cells to develop, survive, and proliferate. Among these, tightly balanced regulatory enzymatic pathways that can respond rapidly to external impacts maintain dynamic physiological homeostasis. More specifically, redox homeostasis broadly affects cellular metabolism and proliferation, with major contributions by thiol/disulfide oxidoreductase systems, in particular, the Thioredoxin Reductase Thioredoxin (TrxR/Trx) and the Glutathione Reductase-Glutathione-Glutaredoxin (GR/GSH/Grx) systems.
These cascades drive vital cellular functions in many ways through signaling, regulating other proteins' activity by redox switches, and by stoichiometric reductant transfers in metabolism and antioxidant systems. Increasing evidence argues that there is a persistent alteration of the redox environment in certain pathological states, such as cancer, that heavily involve the Trx system: upregulation and/or overactivity of the Trx system may support or drive cancer progression, making both TrxR and Trx promising targets for anti-cancer drug development.
Understanding the biochemical mechanisms and connections between certain redox cascades requires research tools that interact with them. The state-of-the-art genetic tools are mostly ratiometric reporters that measure reduced:oxidized ratios of selected redox pairs or the general thiol pool. However, the precise cellular roles of the central oxidoreductase systems, including TrxR and Trx, remain inaccessible due to the lack of probes to selectively measure turnover by either of these proteins. However, such probes would allow measuring their effective reductive activity apart from expression levels in native systems, including in cells, animals, or patient samples. They are also of high interest to identify chemical inhibitors for TrxR/Trx in cells and to validate their potential use as anti-cancer agents (to date, there is no selective cellular Trx inhibitor, and most known TrxR inhibitors were not comprehensively evaluated considering selectivity and potential off-targets). However, small molecule redox imaging tools are underdeveloped: their protein specificity, spectral properties, and applicability remain poorly precedented.
This work aimed to address this opportunity gap and develop novel, small molecule diagnostic and therapeutic tools to selectively target the Trx system based on a modular trigger cargo design: artificial cyclic disulfide substrates (trigger) for oxidoreductases are tethered to molecular agents (cargo) such that the cargo’s activity is masked and is re-established only through reduction by a target protein.
The rational design of these novel reduction sensors to target the cell's strongest disulfide-reducing enzymes was driven by the following principles: (i) cyclic disulfide triggers with stabilized ring systems were used to gain low reduction potentials that should resist reduction except by the strongest cellular reductases, such as Trx; and (ii) the cyclic topology also offers the potential for kinetic reversibility that should select for dithiol-type redox proteins over the cellular monothiol background. Creating imaging agents based on such two-component designs to selectively measure redox protein activity in native cells required to combine the correct trigger reducibility, probe activation kinetics, and imaging modalities and to consider the overall molecular architecture.
The major prior art in this field has applied cyclic 5-membered disulfides (1,2 dithiolanes) as substrates for TrxR in a similar way to create such tools. However, this motif was described elsewhere as thermodynamically instable and was due to widely used for dynamic covalent cascade reactions. By comparing a novel 1,2 dithiolane-based probe to the state-of-the-art probes, including commercial TrxR sensors, by screening a conclusive assay panel of cellular TrxR modulations, I clarified that 1,2 dithiolanes are not selective substrates for TrxR in biological settings (Nat Commun 2022).
Instead, aiming for more stable ring systems and thus more robust redox probes, during this work, I developed bicyclic 6 membered disulfides (piperidine fused 1,2 dithianes) with remarkably low reduction potentials. I showed that molecular probes using them as reduction sensors can be mostly processed by thioredoxins while being stable against reduction by GSH. The thermodynamically stabilized decalin like topology of the cis-annelated 1,2 dithianes requires particularly strong reductants to be cleaved. They also select for dithiol type redox proteins, like Trx, based on kinetic reversibility and offer fast cyclization due to the preorganization by annelation (JACS 2021).
This work further expanded the system’s modularity with structural cores based on piperazine-fused 1,2 dithianes with the two amines allowing independent derivatization. Diagnostic tools using them as reduction sensors proved equally robust but with highly improved activation kinetics and were thus cellularly activated. Cellular studies evolved that they are substrates for both Trxs and their protein cousins Grxs, so measuring the cellular dithiol protein pool rather than solely Trx activity (preprint 2023).
Finally, a trigger based on a slightly adapted reduction sensor, a desymmetrized 1,2 thiaselenane, was designed for selective reduction by TrxR’s selenol/thiol active site, then combined with a precipitating large Stokes’ shift fluorophore and a solubilizing group, to evolve the first selective probe RX1 to measure cellular TrxR activity, which even allowed high throughput inhibitor screening (Chem 2022).
The central principle of this work was further advanced to therapeutic prodrugs based on the duocarmycin cargo (CBI) with tunable potency (JACS Au 2022) that can be used to create off-to-on therapeutic prodrugs. Such CBI prodrugs employing stabilized 1,2 dichalcogenide triggers proved to be cytotoxins that depend on Trx system activity in cells. They could further be exploited for cell-line dependent reductase activity profiling by screening their redox activation indices, the reduction-dependent part of total prodrug activation, in 177 cell lines. Beyond that, these prodrugs were well-tolerated in animals and showed anti-cancer efficacy in vivo in two distinct mouse tumor models (preprint 2022).
Taken together, I introduced unique monothiol-resistant reducible motifs to target the cellular Trx system with chemocompatible units for each for TrxR and Trx/Grx, where the cyclic nature of the dichalcogenides avoids activation by GSH. By using them with distinct molecular cargos, I developed novel selective fluorescent reporter probes; and introduced a new class of bioreductive therapeutic constructs based on a common modular design. These were either applied to selectively measure cellular reductase activity or to deliver cytotoxic anti cancer agents in vivo. Ongoing work aims to differentiate between the two major redox effector proteins Trx and Grx, requiring additional layers of selectivity that may be addressed by tuned molecular recognition. The flexible use of various molecular cargos allows harnessing the same cellular redox machinery by either probes or prodrugs. This allows predictive conclusions from diagnostics to be directly translated into therapy and offers great potential for future adaptation to other enzyme classes and therapeutic venues.Die zelluläre Redox-Homöostase hängt von Thiol/Disulfid-Oxidoreduktasen ab, die den Stoffwechsel, die Proliferation und die antioxidative Antwort von Zellen beeinflussen. Die wichtigsten Netzwerke sind die Thioredoxin Reduktase-Thioredoxin (TrxR/Trx) und Glutathion Reduktase-Glutathion-Glutaredoxin (GR/GSH/Grx) Systeme, die über Redox-Schalter in Substratproteinen lebenswichtige zelluläre Funktionen steuern und so an der Redox-Regulation und -Signalübertragung beteiligt sind. Persistente Veränderungen des Redoxmilieus in pathologischen Zuständen, wie z. B. bei Krebs, sind in hohem Maße mit dem Trx-System verbunden. Eine Hochregulierung und/oder Überaktivität des Trx-Systems, die bei vielen Krebsarten auftreten, unterstützt zudem das Fortschreiten des Krebswachstums, was TrxR/Trx zu vielversprechenden Zielproteinen für die Entwicklung neuer Krebsmedikamente macht.
Um die biochemischen Prozesse dahinter zu erforschen, sind spezielle Techniken zur Visualisierung und Messung enzymatischer Aktivität nötig. Die hierzu geeigneten, meist genetischen Sensoren messen ratiometrisch das Verhältnis reduzierter/oxidierter Spezies in zellulärem Umfeld oder spezifisch ausgewählte Redoxpaare. Die weitere Erforschung der exakten Funktion von TrxR/Trx und deren Substrate ist jedoch durch mangelnde Nachweismethoden limitiert. Diese sind außerdem zur Validierung chemischer Hemmstoffe für TrxR/Trx in Zellen und deren potenziellen Verwendung als Krebsmittel von großem Interesse. Bislang gibt es keinen selektiven zellulären Trx-Inhibitor und potenzielle Off-Target-Effekte der bekannten TrxR-Inhibitoren wurden nicht abschließend bewertet.
Ziel dieser Arbeit ist die Entwicklung niedermolekularer, diagnostischer und therapeutischer Werkzeuge, die selektiv auf das Trx-System abzielen und auf einem modularen Trigger-Cargo Design basieren. Hierzu werden zyklische Disulfid-Substrate (Trigger) für Oxidoreduktasen so mit molekularen Wirkstoffen (Cargo) verknüpft, dass dabei die Wirkstoffaktivität maskiert, und erst nach Reduktion durch ein Zielprotein wiederhergestellt wird. Diese neuartigen, synthetischen Reduktionssensoren basieren auf den folgenden Grundprinzipien: (i) Zyklische Disulfide sind thermodynamisch stabilisiert und können nur durch die stärksten Reduktasen gespalten werden; und (ii) die zyklische Topologie ermöglicht die kinetische Reversibilität der zwei Thiol-Disulfid-Austauschreaktionen, die eine erste Reaktion mit Monothiolen, wie z. B. GSH, sofort umkehrt und so eine vollständige Reduktion verhindert.
Die meisten früheren Arbeiten auf diesem Gebiet verwendeten ein zyklisches, fünfgliedriges Disulfid (1,2 Dithiolan) als Substrat für TrxR. Das gleiche Strukturmotiv wurde jedoch an anderer Stelle als thermodynamisch instabil beschrieben und aufgrund dieser Eigenschaft explizit für dynamische Kaskadenreaktionen verwendet. Deshalb vergleicht diese Arbeit zu Beginn einen neuen 1,2 Dithiolan basierten fluorogenen Indikator mit bestehenden, z. T. kommerziellen, Redox Sonden für TrxR in einer Reihe von Zellkultur-Experimenten unter Modulation der zellulären TrxR Aktivität und stellt so einen Widerspruch in der Literatur klar: 1,2 Dithiolane eignen sich nicht als selektive Substrate für TrxR, da sie labil sowohl gegen die Reduktion durch andere Redoxproteine, als auch gegen den Monothiol Hintergrund in Zellen sind (Nat. Commun. 2022).
Als alternatives Strukturmotiv wird in dieser Arbeit ein bizyklisches sechsgliedriges Disulfid (anneliertes 1,2 Dithian) etabliert. Durch sein niedriges Reduktionspotenzial, also seine hohe Resistenz gegen Reduktion, werden molekulare Sonden basierend auf diesem 1,2 Dithian als Reduktionssensor fast ausschließlich von Trx aktiviert, nicht aber von TrxR oder GSH (JACS 2021). Dieses Kernmotiv bestimmt dabei die Reduzierbarkeit, und damit die Enzymspezifität, durch seine zyklische Natur und die Annelierung, auch unter Verwendung unterschiedlicher Farb-/Wirkstoffe. Auf dieser Grundlage konnte die molekulare Struktur durch einen weiteren Modifikationspunkt für die flexible Verwendung weiterer funktioneller Einheiten ergänzt werden. Obwohl zelluläre Studien ergaben, dass diese neuartigen 1,2 Dithian Einheiten in Zellen sowohl Trx als auch das strukturell verwandte Grx adressieren, sind die daraus resultierenden diagnostischen Moleküle wertvoll, um den katalytischen Umsatz zellulärer Dithiol-Reduktasen, der sogenannten Trx Superfamilie, selektiv anzuzeigen (Preprint 2023).
Begünstigt durch das modulare Moleküldesign stellt diese Arbeit zudem das erste Reportersystem RX1 zum selektiven Nachweis der TrxR-Aktivität in Zellen vor. Es basiert auf der Verwendung eines zyklischen, unsymmetrischen Selenenylsulfid-Sensors (1,2 Thiaselenan), der selektiv von dem einzigartigen Selenolat der TrxR angegriffen wird, und dadurch letztlich nur von TrxR reduziert werden kann. RX1 eignete sich zudem für eine Hochdurchsatz-Validierung bestehender TrxR Inhibitoren und unterstreicht dadurch den kommerziellen Nutzen derartiger Diagnostika (Chem 2022).
Das zentrale Trigger-Cargo Konzept dieser Arbeit wurde für therapeutische Zwecke weiterentwickelt und nutzt dabei den einzigartigen Wirkmechanismus der Duocarmycin-Naturstoffklasse (CBI) (JACS Au 2022) zur Entwicklung reduktiv aktivierbarer Therapeutika. CBI Prodrugs basierend auf stabilisierten Redox-Schaltern (1,2 Dithiane für Trx; 1,2 Thiaselenan für TrxR) reagierten signifikant auf TrxR-Modulation in Zellen. Sie wurden darüber hinaus durch das Referenzieren ihrer Aktivität gegenüber nicht-reduzierbaren Kontrollmoleküle für die Erstellung zelllinienabhängiger Profile der Reduktaseaktivität in 177 Zelllinien genutzt. Schließlich waren diese neuen Krebsmittel im Tiermodell gut verträglich und zeigten in zwei verschiedenen Mausmodellen eine krebshemmende Wirkung (Preprint 2022b).
Zusammenfassend präsentiert diese Dissertation monothiol-resistente reduzierbare Trigger-Einheiten für das zelluläre Trx-System zur Entwicklung neuartiger, selektiver Reporter-Sonden, sowie eine neue Klasse reduktiv aktivierbarer Krebsmittel auf Basis eines adaptierbaren Trigger-Cargo Designs. Diese fanden entweder zur selektiven Messung zellulärer Proteinaktivität oder zum Einsatz als Antikrebsmittel Verwendung. Es wurden chemokompatible Motive sowohl für TrxR als auch für Trx/Grx identifiziert, wobei deren zyklische Natur eine Aktivierung durch GSH verhindert. Eine weitere Differenzierung zwischen den beiden Redox-Proteinen Trx und Grx und anderen Proteinen der Trx-Superfamilie erfordert eine zusätzliche Ebene der Selektierung, z. B. durch molekulare Erkennung, und ist Gegenstand laufender Arbeiten.
Die flexible Verwendung verschiedener molekularer Wirkstoffe ermöglicht dabei die „Pipeline-Entwicklung“ von Diagnostika und Therapeutika, die von der zellulären Redox-Maschinerie analog umgesetzt werden, und dadurch Schlussfolgerungen aus der Diagnostik direkt auf eine Therapie übertragbar machen. Dies birgt großes Potenzial für künftige Entwicklungen bei einer potenziellen Übertragung des modularen Konzepts auf andere Enzymklassen und therapeutische Einsatzgebiete
Transcriptomics in Neurodegenerative Disease
Alzheimer’s (AD) and Parkinson’s (PD) are primary neurodegenerative diseases (NDs) worldwide. Their complex mechanisms, illuminated by neuropathological studies and modern biomedical tools, call for non-invasive biomarkers, preferably from blood, for early detection, differentiating from regular aging.
Omics advancements, especially genomics and transcriptomics, have deepened our molecular understanding of NDs, helping segregate patients based on distinct molecular attributes and refining biomarker clarity.
Our research used RNA sequencing (RNA-seq) to analyze transcriptomes from the dorsolateral prefrontal cortex (DLPFC/BA9), whole blood (WB), and peripheral blood mononuclear cells (PBMC) using data from the Sequence Read Archive (SRA), negating in-house sequencing. With single nucleotide polymorphisms (SNPs), we explored shared pathological occurrences between AD and PD, investigating if ND brain molecular routes manifest in blood.
Applying differential gene expression and a random forest machine-learning technique, our data revealed a scarce overlap between AD and PD. Noteworthy AD markers like Mef2C in AD-DLPFC/BA9 and TMED7 were identified. AD blood markers emphasized cilia-driven TLR4/NF-κB activation and Fyn. In PD, SNARE proteins, e.g., VAMP2 and STX1A, suggested connections between α-syn and SNARE protein reshuffling, implicating synaptic malfunction.
Genome-wide association studies (GWAS) affirmed certain discoveries. Both NDs showed overlaps in the brain and blood. Mainly, VEGF-A and inositol polyphosphates (InsPs) were detected. AD blood samples chiefly exhibited elements tied to Aβ aggregation and tau irregularities.
Further, eQTL and sQTL mapping illuminated the roles of GWAS-associated markers in AD and PD susceptibility, with shifts in gene expression and splicing events affecting both NDs.
The present work emphasizes harnessing GWAS and QTL data to decipher ND complexities, urging continued research for therapeutic ND solutions
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TRANSCRIPTIONAL REGULATION OF BONE FORMATION, MECHANOSENSING, AND EVOLUTION
Given that bone remodeling is a dynamic process, the output of which is dependent upon time, levels of mechanical input, and the ability of bones to respond to that mechanical stimulus, I assessed regulation of gene expression in the craniofacial region to determine how a response happens during mechanosensing and bone remodeling. To do this, I used African cichlids as a model, as they known for their rapid speciation rates, high phenotypic variation within and between species, and ability to remodel their bones in response to mechanical loading. In chapter 2, I combined RNA-seq and ATAC-seq datasets to determine with high confidence genes are responsible for plasticity and shape differences in cichlid species with different feeding morphologies. In particular, I found genes that were both differentially expressed and differentially accessible to transcriptional machinery that were implicated in cell cycle progression. In chapter 3, using qPCR, I was able to determine that time is a critical factor in assessing plasticity and the response of certain species to mechanical input. This was paired with 2D morphometrics for shape analysis over time to show that species that do not fall on the extreme end of phenotypic variation are more genetically plastic, and gives insights into the underpinnings of evolution in cichlid jaw morphology. Results from both chapters 2 and 3 suggested that certain environments facilitate larger changes in gene expression than others. In chapter 4, using molecular techniques such as qPCR coupled with enzymatic staining, I found that when mechanosensitive structures in the cell are ablated, gene expression regulation collapses over time, and specific sites of bone remodeling activity are less predictive. Taken together, this body of work supports previous research in the field and gives insight into the regulation of gene expression during bone remodeling, plasticity, and evolution
ACARORUM CATALOGUS IX. Acariformes, Acaridida, Schizoglyphoidea (Schizoglyphidae), Histiostomatoidea (Histiostomatidae, Guanolichidae), Canestrinioidea (Canestriniidae, Chetochelacaridae, Lophonotacaridae, Heterocoptidae), Hemisarcoptoidea (Chaetodactylidae, Hyadesiidae, Algophagidae, Hemisarcoptidae, Carpoglyphidae, Winterschmidtiidae)
The 9th volume of the series Acarorum Catalogus contains lists of mites of 13 families, 225 genera and 1268 species of the superfamilies Schizoglyphoidea, Histiostomatoidea, Canestrinioidea and Hemisarcoptoidea. Most of these mites live on insects or other animals (as parasites, phoretic or commensals), some inhabit rotten plant material, dung or fungi. Mites of the families Chetochelacaridae and Lophonotacaridae are specialised to live with Myriapods (Diplopoda). The peculiar aquatic or intertidal mites of the families Hyadesidae and Algophagidae are also included.Publishe
Modelling complexity and redundancy in endocytic actin polymerisation
Actin is one of the most ubiquitous proteins of life and can form filaments which play crucial roles in a wide range of processes from cell division to intracellular trafficking. Formation of these filament networks is tightly controlled using a wide array of protein types, chief among them being nucleators. Nucleators facilitate the unfavourable first steps of filament formation and thus their regulation dictates when and where filamentous networks are produced. The central proline-rich region of Las17 (yeast homologue of human WASp) is thought to nucleate actin “mother filaments” at the endocytic sites. Arp2/3 – a potent nucleator activated by Las17 – can branch these mother filaments. Proline also constitutes the core binding region of SH3 domains which leaves the nucleating region of Las17 open to competitive regulation. Eleven Las17-binding SH3 domains are recruited to yeast endocytic sites. Five of these bind via a tandem of domains (three SH3s in Sla1 and two SH3s in Bzz1). We hypothesise that this “cloud” of SH3 domains can regulate the access of actin to the proline-rich region of Las17. However, the high number of proteins and interactions involved renders a purely experimental approach challenging.
Throughout this thesis, two agent-based models are built (one being a progression of the other) to test the veracity of our regulatory cloud hypothesis. Binding affinities were experimentally obtained to build the model, demonstrate the power of avidity conferred through tandem SH3 binding, and refine our Las17 nucleating mechanism. We identify that the weak interactions of the SH3 cloud can combine in effect – particularly complemented by the tandem binding of Sla1 and Bzz1 – to define a window of Las17 nucleating activity. This work suggests how endocytic SH3 domains can regulate endocytic progression whilst also furthering our understanding of the relatively unexplored nucleating mechanisms employed by Las1
LIPIcs, Volume 261, ICALP 2023, Complete Volume
LIPIcs, Volume 261, ICALP 2023, Complete Volum
The effect of the genetic background on phage infectivity in an encapsulated host
Los bacteriĂłfagos desempeñan un papel clave en la ecologĂa y evoluciĂłn bacterianas y son potenciales antimicrobianos. Sin embargo, los factores determinantes de la especificidad fago-hospedador siguen siendo poco conocidos. Debido a la prevalencia de bacterias encapsuladas, la cápsula es la primera barrera que encuentran muchos fagos. Esta capa, normalmente de polisacáridos, puede bloquear el acceso de los fagos a los receptores de su pared celular. Para superarla, algunos fagos codifican dominios depolimerasa (Dpos) especĂficos en sus proteĂnas de uniĂłn a receptores. En este trabajo, hemos cuantificado la predictibilidad de las interacciones fago-bacteria basándonos en el tipo de locus capsular del hospedador (CLT) y Dpos. Para ello, utilizamos Klebsiella pneumoniae como modelo debido a su alta diversidad capsular y a su importancia en salud pĂşblica. En primer lugar, establecimos una colecciĂłn de 138 cepas clĂnicas de K. pneumoniae representativas de la diversidad genĂłmica de la especie. Estas cepas se testaron con 46 fagos ambientales diferentes que comprendĂan 13 grupos filogenĂ©ticos. Las pruebas de spot revelaron que la mayorĂa de los fagos (42 de los 46) mostraban especificidad capsular, con un patrĂłn de infecciĂłn predicho con precisiĂłn (92%) por el CLT del hospedador. En consecuencia, las Dpos codificadas por los fagos, que a menudo han sufrido transferencia horizontal a largas escalas taxonĂłmicas, eran determinantes clave del tropismo del hospedador. La tolerancia a la identidad de los Dpos de los fagos era alta, como mostrĂł el tropismo capsular predicho con profagos de RefSeq para 13 CLT importantes epidemiolĂłgicamente. Aunque las cápsulas y el Dpos predijeron los primeros pasos del reconocimiento del viriĂłn y la adsorciĂłn, su precisiĂłn disminuyĂł para las infecciones productivas (53%). Aparte de los fagos especializados en cápsulas, encontramos que los fagos de los grupos S8/S9 tenĂan un rango de hospedador más amplio como resultado de un tropismo dependiente e independiente de la cápsula. El tropismo capsular de los fagos S8/S9 no estaba mediado por depolimerasas, sino que estos fagos codificaban proteĂnas de cola larga con mĂşltiples dominios de uniĂłn a carbohidratos. Es importante destacar que >90% de las cepas bacterianas presentaban heterogeneidad fenotĂpica capsular, con la coexistencia de clones encapsulados y no encapsulados. Esta heterogeneidad afectĂł al tropismo de los fagos, ya que los fagos de amplio espectro infectaron preferentemente bacterias con un mayor nĂşmero de bacterias acapsulares, al contrario que los fagos especialistas en cápsulas. Se observĂł que los mutantes acapsulares adquirĂan resistencia en 286 de las 406 combinaciones analizadas (70%). Ni la especificidad capsular de los fagos ni la infecciĂłn de bacterias acapsulares se correlacionaron con la clasificaciĂłn filogenĂ©tica de los fagos. Sorprendentemente, el 43% de los fagos mostraron un comportamiento de uniĂłn capsular obligado o facultativo en funciĂłn de la cepa bacteriana y su mecanismo de inactivaciĂłn capsular, lo que a su vez dio lugar a diferentes tasas de emergencia de resistencia. Estos resultados amplĂan nuestro conocimiento de las complejas interacciones entre bacterias y sus virus y señalan la viabilidad de predecir los primeros pasos de la infecciĂłn fágica utilizando secuencias genĂłmicas de bacterias y fagos.Bacteriophages play key roles in bacterial ecology and evolution and are potential antimicrobials. However, the determinants of phage-host specificity remain elusive. Due to the prevalence of encapsulated bacteria, the capsule is the first barrier encountered by many phages. This layer, usually made of polysaccharides, can block phage access to their cell wall receptors. To overcome this, some phages encode specific depolymerase (Dpos) domains in their receptor-binding proteins. Here, we have quantified the predictability of phage-bacteria interactions based on the capsular locus type of host (CLT) and Dpos. For this, we used Klebsiella pneumoniae as a model due to its high capsular diversity and importance in global health. First, we established a collection of 138 clinical strains of K.pneumoniae representative of the genomic diversity of the species. These were challenged with 46 different environmental phages comprising 13 phylogenetic groups. Spot tests revealed that most of the phages (42 out of the 46) showed capsular specificity, with their infection pattern accurately predicted (92%) by the host CLT. Consequently, phage-encoded Dpos, which have often undergone horizontal gene transfer across large taxonomic scales, were key determinants of host tropism. The identity tolerance of phage Dpos was high, as shown by the predicted capsular tropism of RefSeq prophages for 13 important CLTs. Even though capsules and Dpos predicted the first steps of virion recognition and adsorption, their accuracy dropped for productive infections (53%). Apart from capsule-specialist phages, we found that the phages of groups S8/S9 had a broader host-range resulting from both capsule-dependent and independent tropism. Capsular tropism of S8/S9 phages was not mediated by depolymerases, instead these phages encoded long tail proteins with multiple carbohydrate-binding domains. Importantly, >90% of bacterial strains presented capsular phenotypic heterogeneity, with the co-existence of both encapsulated and non-encapsulated clones. This heterogeneity affected phage tropism, as broad-range phages preferentially infected bacteria with a higher number of acapsular bacteria, contrary to capsular-specialist phages. We found that acapsular mutants gained resistance in 286 of the 406 combinations tested (70%). Neither phage capsular specificity nor infection of acapsular bacteria was correlated with the phylogenetic classification of phages. Remarkably, 43% of the phages showed an obligate or facultative capsule attachment behavior depending on the bacterial strain and its mechanism of capsule inactivation, which in turn led to different trade-offs in resistance. These findings expand our knowledge of the complex interactions between bacteria and their viruses and point out the feasibility of predicting the first steps of phage infection using bacterial and phage genome sequences
In Silico Design funktionaler Moleküle: Vorhersage und Erklärung molekularen Verhaltens durch Dichtefunktionaltheorie
In dieser Dissertation wurden unterschiedliche Methoden der Dichtefunktionaltheorie verwendet, um eine Reihe von funktionalen Moleküle quantenmechanisch zu beschreiben. Dabei reichen die Themen vom Design neuer Verbindungen über die Vorhersage thermodynamischer, elektronischer und photophysikalischer Eigenschaften bis zur Erklärung von experimentellen Phänomenen. Der Hauptfokus der Arbeit liegt auf der Untersuchung und Entwicklung von Azo-basierten Photoschaltern. So konnte die durch Goldoberflächen beschleunigte Relaxation von Azobenzol (AB) aufgeklärt werden. Diese Spin-Switch-Katalyse konnte durch intelligentes Moleküldesign und die somit einstellbare Kopplung zur Oberfläche nachgewiesen werden. AB erfährt bei ausreichender Kopplung zum Leitungsband des Substrates ein intersystem crossing und die Relaxation wird nicht mehr adiabatisch, sondern durch die quantenmechanische Durchtrittswahrscheinlichkeit bestimmt. Um zu untersuchen, ob dieser Mechanismus auch für ähnliche Systeme gilt, wurden weitere Konzepte mit Diazocine (DACs) entwickelt. Da das Stammsystem jedoch einige Schwachstellen bezüglich Synthese und Substitution aufweist, wurden mit Hilfe von quantenchemischen Rechnungen verschiedene neue DACs entwickelt. Neben der Untersuchung von Photoschaltern wurde die Anisotropie der induzierten Stromdichte (ACID) Methode verwendet, um verschiedene Konzepte von elektronischer Delokalisierung aufzudecken und zu beweisen. Durch die quantitative und qualitative Auswertung mittels ACID konnte so die Stereo- und Regiochemie von Diels-Alder-Reaktionen an Porphyrinen und Derivaten vorausgesagt und erklärt werden. Zusätzlich half die Methode, Aromatizität in dreifach verdrillte Möbiusstrukturen nachzuweisen sowie Konjugation durch den freien Raum ohne kovalente Bindung visuell aufzuzeigen und somit zu belegen
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