216 research outputs found

    Rational development of stabilized cyclic disulfide redox probes and bioreductive prodrugs to target dithiol oxidoreductases

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    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 zellulĂ€re Redox-Homöostase hĂ€ngt 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 ĂŒber Redox-Schalter in Substratproteinen lebenswichtige zellulĂ€re Funktionen steuern und so an der Redox-Regulation und -SignalĂŒbertragung beteiligt sind. Persistente VerĂ€nderungen des Redoxmilieus in pathologischen ZustĂ€nden, wie z. B. bei Krebs, sind in hohem Maße mit dem Trx-System verbunden. Eine Hochregulierung und/oder ÜberaktivitĂ€t des Trx-Systems, die bei vielen Krebsarten auftreten, unterstĂŒtzt zudem das Fortschreiten des Krebswachstums, was TrxR/Trx zu vielversprechenden Zielproteinen fĂŒr die Entwicklung neuer Krebsmedikamente macht. Um die biochemischen Prozesse dahinter zu erforschen, sind spezielle Techniken zur Visualisierung und Messung enzymatischer AktivitĂ€t nötig. Die hierzu geeigneten, meist genetischen Sensoren messen ratiometrisch das VerhĂ€ltnis reduzierter/oxidierter Spezies in zellulĂ€rem Umfeld oder spezifisch ausgewĂ€hlte 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 fĂŒr TrxR/Trx in Zellen und deren potenziellen Verwendung als Krebsmittel von großem Interesse. Bislang gibt es keinen selektiven zellulĂ€ren 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) fĂŒr Oxidoreduktasen so mit molekularen Wirkstoffen (Cargo) verknĂŒpft, dass dabei die WirkstoffaktivitĂ€t 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 stĂ€rksten Reduktasen gespalten werden; und (ii) die zyklische Topologie ermöglicht die kinetische ReversibilitĂ€t der zwei Thiol-Disulfid-Austauschreaktionen, die eine erste Reaktion mit Monothiolen, wie z. B. GSH, sofort umkehrt und so eine vollstĂ€ndige Reduktion verhindert. Die meisten frĂŒheren Arbeiten auf diesem Gebiet verwendeten ein zyklisches, fĂŒnfgliedriges Disulfid (1,2 Dithiolan) als Substrat fĂŒr TrxR. Das gleiche Strukturmotiv wurde jedoch an anderer Stelle als thermodynamisch instabil beschrieben und aufgrund dieser Eigenschaft explizit fĂŒr dynamische Kaskadenreaktionen verwendet. Deshalb vergleicht diese Arbeit zu Beginn einen neuen 1,2 Dithiolan basierten fluorogenen Indikator mit bestehenden, z. T. kommerziellen, Redox Sonden fĂŒr TrxR in einer Reihe von Zellkultur-Experimenten unter Modulation der zellulĂ€ren TrxR AktivitĂ€t und stellt so einen Widerspruch in der Literatur klar: 1,2 Dithiolane eignen sich nicht als selektive Substrate fĂŒr 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 EnzymspezifitĂ€t, durch seine zyklische Natur und die Annelierung, auch unter Verwendung unterschiedlicher Farb-/Wirkstoffe. Auf dieser Grundlage konnte die molekulare Struktur durch einen weiteren Modifikationspunkt fĂŒr die flexible Verwendung weiterer funktioneller Einheiten ergĂ€nzt werden. Obwohl zellulĂ€re 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 MolekĂŒle wertvoll, um den katalytischen Umsatz zellulĂ€rer Dithiol-Reduktasen, der sogenannten Trx Superfamilie, selektiv anzuzeigen (Preprint 2023). BegĂŒnstigt durch das modulare MolekĂŒldesign stellt diese Arbeit zudem das erste Reportersystem RX1 zum selektiven Nachweis der TrxR-AktivitĂ€t 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 fĂŒr eine Hochdurchsatz-Validierung bestehender TrxR Inhibitoren und unterstreicht dadurch den kommerziellen Nutzen derartiger Diagnostika (Chem 2022). Das zentrale Trigger-Cargo Konzept dieser Arbeit wurde fĂŒr 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 fĂŒr Trx; 1,2 Thiaselenan fĂŒr TrxR) reagierten signifikant auf TrxR-Modulation in Zellen. Sie wurden darĂŒber hinaus durch das Referenzieren ihrer AktivitĂ€t gegenĂŒber nicht-reduzierbaren KontrollmolekĂŒle fĂŒr die Erstellung zelllinienabhĂ€ngiger Profile der ReduktaseaktivitĂ€t in 177 Zelllinien genutzt. Schließlich waren diese neuen Krebsmittel im Tiermodell gut vertrĂ€glich und zeigten in zwei verschiedenen Mausmodellen eine krebshemmende Wirkung (Preprint 2022b). Zusammenfassend prĂ€sentiert diese Dissertation monothiol-resistente reduzierbare Trigger-Einheiten fĂŒr das zellulĂ€re 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 zellulĂ€rer ProteinaktivitĂ€t oder zum Einsatz als Antikrebsmittel Verwendung. Es wurden chemokompatible Motive sowohl fĂŒr TrxR als auch fĂŒr 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 zusĂ€tzliche 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 zellulĂ€ren Redox-Maschinerie analog umgesetzt werden, und dadurch Schlussfolgerungen aus der Diagnostik direkt auf eine Therapie ĂŒbertragbar machen. Dies birgt großes Potenzial fĂŒr kĂŒnftige Entwicklungen bei einer potenziellen Übertragung des modularen Konzepts auf andere Enzymklassen und therapeutische Einsatzgebiete

    Measuring the impact of COVID-19 on hospital care pathways

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    Care pathways in hospitals around the world reported significant disruption during the recent COVID-19 pandemic but measuring the actual impact is more problematic. Process mining can be useful for hospital management to measure the conformance of real-life care to what might be considered normal operations. In this study, we aim to demonstrate that process mining can be used to investigate process changes associated with complex disruptive events. We studied perturbations to accident and emergency (A &E) and maternity pathways in a UK public hospital during the COVID-19 pandemic. Co-incidentally the hospital had implemented a Command Centre approach for patient-flow management affording an opportunity to study both the planned improvement and the disruption due to the pandemic. Our study proposes and demonstrates a method for measuring and investigating the impact of such planned and unplanned disruptions affecting hospital care pathways. We found that during the pandemic, both A &E and maternity pathways had measurable reductions in the mean length of stay and a measurable drop in the percentage of pathways conforming to normative models. There were no distinctive patterns of monthly mean values of length of stay nor conformance throughout the phases of the installation of the hospital’s new Command Centre approach. Due to a deficit in the available A &E data, the findings for A &E pathways could not be interpreted

    Reconfigurable Computing Systems for Robotics using a Component-Oriented Approach

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    Robotic platforms are becoming more complex due to the wide range of modern applications, including multiple heterogeneous sensors and actuators. In order to comply with real-time and power-consumption constraints, these systems need to process a large amount of heterogeneous data from multiple sensors and take action (via actuators), which represents a problem as the resources of these systems have limitations in memory storage, bandwidth, and computational power. Field Programmable Gate Arrays (FPGAs) are programmable logic devices that offer high-speed parallel processing. FPGAs are particularly well-suited for applications that require real-time processing, high bandwidth, and low latency. One of the fundamental advantages of FPGAs is their flexibility in designing hardware tailored to specific needs, making them adaptable to a wide range of applications. They can be programmed to pre-process data close to sensors, which reduces the amount of data that needs to be transferred to other computing resources, improving overall system efficiency. Additionally, the reprogrammability of FPGAs enables them to be repurposed for different applications, providing a cost-effective solution that needs to adapt quickly to changing demands. FPGAs' performance per watt is close to that of Application-Specific Integrated Circuits (ASICs), with the added advantage of being reprogrammable. Despite all the advantages of FPGAs (e.g., energy efficiency, computing capabilities), the robotics community has not fully included them so far as part of their systems for several reasons. First, designing FPGA-based solutions requires hardware knowledge and longer development times as their programmability is more challenging than Central Processing Units (CPUs) or Graphics Processing Units (GPUs). Second, porting a robotics application (or parts of it) from software to an accelerator requires adequate interfaces between software and FPGAs. Third, the robotics workflow is already complex on its own, combining several fields such as mechanics, electronics, and software. There have been partial contributions in the state-of-the-art for FPGAs as part of robotics systems. However, a study of FPGAs as a whole for robotics systems is missing in the literature, which is the primary goal of this dissertation. Three main objectives have been established to accomplish this. (1) Define all components required for an FPGAs-based system for robotics applications as a whole. (2) Establish how all the defined components are related. (3) With the help of Model-Driven Engineering (MDE) techniques, generate these components, deploy them, and integrate them into existing solutions. The component-oriented approach proposed in this dissertation provides a proper solution for designing and implementing FPGA-based designs for robotics applications. The modular architecture, the tool 'FPGA Interfaces for Robotics Middlewares' (FIRM), and the toolchain 'FPGA Architectures for Robotics' (FAR) provide a set of tools and a comprehensive design process that enables the development of complex FPGA-based designs more straightforwardly and efficiently. The component-oriented approach contributed to the state-of-the-art in FPGA-based designs significantly for robotics applications and helps to promote their wider adoption and use by specialists with little FPGA knowledge

    Proof-theoretic Semantics for Intuitionistic Multiplicative Linear Logic

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    This work is the first exploration of proof-theoretic semantics for a substructural logic. It focuses on the base-extension semantics (B-eS) for intuitionistic multiplicative linear logic (IMLL). The starting point is a review of Sandqvist’s B-eS for intuitionistic propositional logic (IPL), for which we propose an alternative treatment of conjunction that takes the form of the generalized elimination rule for the connective. The resulting semantics is shown to be sound and complete. This motivates our main contribution, a B-eS for IMLL , in which the definitions of the logical constants all take the form of their elimination rule and for which soundness and completeness are established

    Revista da Associação Portuguesa de Linguística

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    N.Âș 10 (2023) da Revista da Associação Portuguesa de LinguĂ­sticainfo:eu-repo/semantics/publishedVersio

    Research Paper: Process Mining and Synthetic Health Data: Reflections and Lessons Learnt

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    Analysing the treatment pathways in real-world health data can provide valuable insight for clinicians and decision-makers. However, the procedures for acquiring real-world data for research can be restrictive, time-consuming and risks disclosing identifiable information. Synthetic data might enable representative analysis without direct access to sensitive data. In the first part of our paper, we propose an approach for grading synthetic data for process analysis based on its fidelity to relationships found in real-world data. In the second part, we apply our grading approach by assessing cancer patient pathways in a synthetic healthcare dataset (The Simulacrum provided by the English National Cancer Registration and Analysis Service) using process mining. Visualisations of the patient pathways within the synthetic data appear plausible, showing relationships between events confirmed in the underlying non-synthetic data. Data quality issues are also present within the synthetic data which reflect real-world problems and artefacts from the synthetic dataset’s creation. Process mining of synthetic data in healthcare is an emerging field with novel challenges. We conclude that researchers should be aware of the risks when extrapolating results produced from research on synthetic data to real-world scenarios and assess findings with analysts who are able to view the underlying data

    PROCEEDINGS 5th PLATE Conference

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    The 5th international PLATE conference (Product Lifetimes and the Environment) addressed product lifetimes in the context of sustainability. The PLATE conference, which has been running since 2015, has successfully been able to establish a solid network of researchers around its core theme. The topic has come to the forefront of current (political, scientific & societal) debates due to its interconnectedness with a number of recent prominent movements, such as the circular economy, eco-design and collaborative consumption. For the 2023 edition of the conference, we encouraged researchers to propose how to extend, widen or critically re-construct thematic sessions for the PLATE conference, and the paper call was constructed based on these proposals. In this 5th PLATE conference, we had 171 paper presentations and 238 participants from 14 different countries. Beside of paper sessions we organized workshops and REPAIR exhibitions

    Uncovering the Electronic Structure of Systems Vital to Nuclear Energy, Life, and Space Exploration via Anion Photoelectron Spectroscopy

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    Photoelectron spectroscopy provides a window into the complicated world of electronic structure and interactions on a molecular level. More specifically, anion photoelectron spectroscopy (aPES) allows one to obtain information regarding an anionic atom, molecule, or cluster as well as its neutral counterpart, with the additional advantages of improved resolution and species selection through the direct manipulation of the ion beam. A combination of mass spectrometry, anion photoelectron spectroscopy (aPES) and theory were used harmoniously in this work to study the electronic structure of several atomic and clusters anions, from atoms to large heterogeneous metal clusters. Studies of biological molecules are presented in Chapter 2, including the correlation-bound anion p-chloroaniline and several antioxidants. Chapter 3 (as well as Appendix A) focus on uranium and thorium species, including the experimentally measured electron affinity of the uranium atom: a fundamental property of the element. Chapter 4 focuses on systems related to propulsion applications, including systematic studies of Al3Hn− (n=1–9) clusters and of Ir n− with hydroxylamine, offering a wealth of information regarding reactions leading to combustion and ignition in jet engines

    1996 July, University of Memphis bulletin

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    Vol. 85, No. 4 of the University of Memphis bulletin containing the graduate catalog for 1996-97, 1996 July.https://digitalcommons.memphis.edu/speccoll-ua-pub-bulletins/1183/thumbnail.jp
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