38 research outputs found

    Towards a circular economy: fabrication and characterization of biodegradable plates from sugarcane waste

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    Bagasse pulp is a promising material to produce biodegradable plates. Bagasse is the fibrous residue that remains after sugarcane stalks are crushed to extract their juice. It is a renewable resource and is widely available in many countries, making it an attractive alternative to traditional plastic plates. Recent research has shown that biodegradable plates made from Bagasse pulp have several advantages over traditional plastic plates. For example, they are more environmentally friendly because they are made from renewable resources and can be composted after use. Additionally, they are safer for human health because they do not contain harmful chemicals that can leach into food. The production process for Bagasse pulp plates is also relatively simple and cost-effective. Bagasse is first collected and then processed to remove impurities and extract the pulp. The pulp is then molded into the desired shape and dried to form a sturdy plate. Overall, biodegradable plates made from Bagasse pulp are a promising alternative to traditional plastic plates. They are environmentally friendly, safe for human health, and cost-effective to produce. As such, they have the potential to play an important role in reducing plastic waste and promoting sustainable practices. Over the years, the world was not paying strict attention to the impact of rapid growth in plastic use. As a result, uncontrollable volumes of plastic garbage have been released into the environment. Half of all plastic garbage generated worldwide is made up of packaging materials. The purpose of this article is to offer an alternative by creating bioplastic goods that can be produced in various shapes and sizes across various sectors, including food packaging, single-use tableware, and crafts. Products made from bagasse help address the issue of plastic pollution. To find the optimum option for creating bagasse-based biodegradable dinnerware in Egypt and throughout the world, researchers tested various scenarios. The findings show that bagasse pulp may replace plastics in biodegradable packaging. As a result of this value-added utilization of natural fibers, less waste and less of it ends up in landfills. The practical significance of this study is to help advance low-carbon economic solutions and to produce secure bioplastic materials that can replace Styrofoam in tableware and food packaging production

    Biomimetic Based Applications

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    The interaction between cells, tissues and biomaterial surfaces are the highlights of the book "Biomimetic Based Applications". In this regard the effect of nanostructures and nanotopographies and their effect on the development of a new generation of biomaterials including advanced multifunctional scaffolds for tissue engineering are discussed. The 2 volumes contain articles that cover a wide spectrum of subject matter such as different aspects of the development of scaffolds and coatings with enhanced performance and bioactivity, including investigations of material surface-cell interactions

    Structure-function relationships in transmembrane transporters

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    Οι μεταφορείς είναι διαμεμβρανικές πρωτεΐνες που πραγματοποιούν την επιλεκτική μεταφορά ουσιών μέσω των μεμβρανών. Τα μέλη της οικογένειας μεταφορέων NAT (Nucleobase Ascorbate Transporter family) είναι συμμεταφορείς H+ ή Na+ ειδικοί για την πρόσληψη είτε πουρινών και πυριμιδινών είτε L-ασκορβικού οξέος [1]. Παρά το γεγονός ότι αρκετά μέλη έχουν μελετηθεί εκτενώς σε γενετικό, βιοχημικό και κυτταρικό επίπεδο και οι δομές μελών από το βακτήριο Escherichia coli and τον μύκητα Aspergillus nidulans έχουν δημοσιευθεί παρέχοντας μια πληθώρα δεδομένων για τον μηχανισμό λειτουργίας, τα υπάρχοντα δεδομένα δεν είναι ικανά να εξηγήσουν πλήρως το πως καθορίζεται η εκλεκτικότητα των υποστρωμάτων [2,3]. Καλά χαρακτηρισμένα μέλη από τα βακτήρια, τους μύκητες και τα φυτά μεταφέρουν ειδικά πουρίνες η/και πυριμιδίνες ενώ τα θηλαστικά και άλλα σπονδυλωτά διαθέτουν μέλη που είναι ειδικά για L-ασκορβικό οξύ (SVCT1/2) αλλά και μέλη ειδικά για νουκλεοτιδικές βάσεις (π.χ. rSNBT1) [4,5]. Τα σπονδυλωτά διαθέτουν ένα επιπλέον παράλογο άγνωστης λειτουργιας (SVCT3) [5]. Οι δομές από δυο μέλη της NAT οικογένειας είναι γνωστές [2,3,6]. Αυτές είναι η δομή του μεταφορέα ουρακίλης της E.coli UraA και του μεταφορέα ουρικού οξέος-ξανθίνης του A. nidulans UapA. Και οι δυο πρωτεΐνες αποτελούνται από 14 διαμεμβρανικά τμήματα που χαρακτηρίζονται από δύο ανεστραμμένες επαναλήψεις (7+7) που αντιστοιχούν σε δύο επικράτειες, την επικράτεια πυρήνα (core domain) και την επικράτεια διμερισμού (dimerization domain). Και οι δύο πρωτεΐνες σχηματίζουν διμερή, ο σχηματισμός των οποίων είναι απαραίτητος για την λειτουργία των μεταφορέων. Ο UapA είναι ένας συμμεταφορέας ουρικού οξέος-ξανθίνης/H+ του μύκητα A. nidulans και θεωρείται το πρότυπο, ευκαρυωτικό μέλος αυτής της οικογένειας επειδή είναι ένας από τους πιο εκτενώς χαρακτηρισμένους ευκαρυωτικούς μεταφορείς σε ότι αφορά τις σχέσεις-δομής λειτουργίας, την εκλεκτικότητα υποστρώματος, την ρύθμιση της έκφρασης και την υποκυτταρική διακίνηση [7]. Όλοι οι ΝΑΤ μεταφορείς περιέχουν ένα συντηρημένο μοτίβο στο 10ο διαμεμβρανικό τμήμα που ονομάστηκε ιστορικά σαν αλληλουχία-αναγνώρισης ΝΑΤ (NAT signature motif) το οποίο περιλαμβάνει κατάλοιπα που είναι απαραίτητα για την δέσμευση υποστρώματος και την εκλεκτικότητα ή για την κατάλυση της μεταφοράς [1,8]. Προηγούμενες μελέτες στον UapA έδειξαν ότι οι περισσότερες μεταλλαγές που επηρεάζουν την εκλεκτικότητα του, που έχουν προκύψει από τυχαίες μεταλλαξιγενέσεις, βρίσκονται εκτός της θέσης δέσμευσης υποστρώματος και της αλληλουχίας-μοτίβου ΝΑΤ [9–11]. Από αυτές οι πιο γνωστές μεταλλαγές αφορούν τα κατάλοιπα Arg481, Thr526 και Phe528 που εντοπίζονται κατά μήκος της πορείας κύλισης της επικράτειας πυρήνα πάνω στο διμερές. Επιπλέον, πρόσφατες μελέτες έδειξαν ότι συγκεκριμένες αληλλεπιδράσεις του UapA με μεμβρανικά λιπίδια στην επιφάνεια διμερισμού είναι απαραίτητες για τον σχηματισμό ή/και την σταθερότητα των λειτουργικών διμερών [12]. Πιο συγκεκριμένα, κατά την διαδικασία απομόνωσης του UapA συγκατακρημνίζονται και λιπίδια και η απομάκρυνση αυτών των λιπιδίων οδηγεί σε διάλυση του διμερούς συμπλόκου σε μονομερή. Προσθήκη φωσφο-ινοσιτιδίων (PIs) ή φωσφατιδυλοαιθανολαμίνης (PEs) οδήγησε στον επανασχηματισμό του διμερούς. Προσομοιώσεις μοριακής δυναμικής (MDs) προέβλεψαν την ύπαρξη μιας ειδικής θέσης δέσμευσης λιπιδίων στην επιφάνεια διμερισμού που αποτελείται από τρία κατάλοιπα αργινίνης Arg287, Arg478 και Arg479. Η αντικατάσταση αυτών των καταλοίπων οδήγησε σε πλήρη απώλεια λειτουργίας η οποία οφείλεται στην απώλεια σχηματισμού του λειτουργικού διμερούς σε μεγάλο ποσοστό της πρωτεΐνης όπως αποδείχτηκε από φασματομετρία μάζας (native MS) και δοκιμασίες εντοπισμού πρωτεϊνικών αλληλεπιδράσεων με το σύστημα BiFC. H παρούσα διατριβή είναι χωρισμένη σε τρία κεφάλαια. Στο πρώτο ερευνήθηκε η μοριακή βάση της εξειδίκευσης υποστρώματος στην ΝΑΤ οικογένεια και μελετήθηκε η εξέλιξη των μεταφορέων ασκορβικού πραγματοποιώντας αρχικά μια εκτενή φυλογενετική ανάλυση και στη συνέχεια μεταλλαγές στο μοτίβο ΝΑΤ του UapA. Την παραπάνω συστηματική μεταλλαξιγένεση ακολούθησε ορθολογικά σχεδιασμένος συνδυασμός υποκαταστάσεων ενώ απομονώθηκαν νέες επιπλέον υποκαταστάσεις μέσω τυχαίων μεταλλαξιγενέσεων. Τα αποτελέσματα συνολικά υποστηρίζουν ότι ο ρόλος κάποιων μερικώς συντηρημένων καταλοίπων του μοτίβου NAT στην εξειδίκευση του μεταφορέα UapA εξαρτάται από την ύπαρξη συγκεκριμένων αμινοξέων σε άλλες θέσεις. Επιπλέον παρουσιάζονται νέα δεδομένα για το πώς το κατάλοιπο Phe528, που βρίσκεται εκτός της θέσης πρόσδεσης υποστρώματος, μπορεί να επηρεάζει την εκλεκτικότητα του UapA. Tο δεύτερο μέρος αυτής της διατριβής αφορά τον ρόλο των αλληλεπιδράσεων του UapA με λιπίδια στη λειτουργία, τη σταθερότητα και τη μεταφορά του στη μεμβράνη. Πιο συγκεκριμένα, εξετάστηκε περαιτέρω ο ρόλος των αλληλεπιδράσεων στην επιφάνεια διμερισμού και διερευνήθηκε ο πιθανός ρόλος άλλων αλληλεπιδράσεων, που έχουν προβλεφθεί από MDs, στην περιφέρεια της επικράτειας πυρήνα του UapA. Βρέθηκε πως διακριτές αλληλεπιδράσεις του UapA με μεμβρανικά λιπίδια είναι απαραίτητες για τον εξαρχής σχηματισμό διμερών στο ενδοπλασματικό δίκτυο, ή την έξοδο από αυτό και την περαιτέρω στόχευση του στη μεμβράνη. Επιπλέον, μέσω τυχαίων μεταλλαξιγενέσεων απομονώθηκαν μεταλλαγές που επαναφέρουν τον σχηματισμό διμερών ή/και τη στόχευση στη μεμβράνη. Τέλος, στο τρίτο μέρος, χρησιμοποιώντας αποτελέσματα της παρούσας διατριβής έγινε για πρώτη φορά λειτουργική ετερόλογη έκφραση μιας NAT ομόλογης πρωτεΐνης από τα θηλαστικά στον A. nidulans.Transporters are transmembrane proteins that mediate the selective translocation of solutes across biological membranes. Members of the ubiquitous Nucleobase Ascorbate Transporter (NAT) family are H+ or Na+ symporters specific for the cellular uptake of either purines and pyrimidines or L-ascorbic acid [1]. Despite the fact that several members have been extensively characterized at a genetic, biochemical or cellular level, and crystal structures of NAT members from Escherichia coli and Aspergillus nidulans have been determined pointing to a mechanism of transport, the current knowledge cannot explain how substrate selectivity is determined [2,3]. Functionally characterized NATs from bacteria, fungi and plants are specific for nucleobases, but rather surprisingly, mammals and other vertebrates possess NAT homologues that are specific for L-ascorbate transport (SVCT1/2) in addition to nucleobase specific members (i.e. rSNBT1) [4,5]. Vertebrates also include a third distinct paralogue of unknown function called SVCT3 [5]. High-resolution crystal structures from two NAT members have been obtained [2,3,6]. These are the UraA uracil transporter of E. coli and the UapA uric acid-xanthine transporter of A. nidulans. Both proteins are composed of 14 transmembrane segments characterized by a 7 helix inverted repeat (7+7) forming a core and a dimerization domain. Additionally, these proteins exist as dimers, the formation of which is essential for transport activity. UapA is considered the prototypic eukaryotic member of this family as it is one of the most extensively studied eukaryotic transporters in respect to structure-function relationships, substrate specificity, regulation of expression and subcellular trafficking. All NATs include a highly conserved motif in TMS10 historically referred as the NAT signature motif which includes residues critical for substrate binding and specificity or transport catalysis [1,8]. Previous studies on UapA reported that most specificity substitutions in UapA, selected by direct genetic screens, map outside the major substrate binding site and the NAT signature motif [9,10,13]. The most prominent specificity substitutions concerned residues Arg481, Thr526 or Phe528, which are located along the proposed sliding trajectory of the core domain in the UapA dimer. Moreover, it has been shown recently that specific interactions with plasma membrane phospholipids at the dimer interface of UapA are essential for the formation and/or stability of functional dimers [12]. More specifically, UapA co-purifies with lipid and delipidation results in dissociation into monomers. Addition of PIs or PEs resulted in the re-formation of the UapA dimer. MDs predicted a specific lipid binding site at the dimer interface that is formed by three arginine residues Arg287, Arg478 and Arg479. Replacement of these arginines by alanine residues led to total loss of UapA function and both native MS and bifluorescence complementation (BiFC) assays indicated that a major fraction of UapA cannot dimerize. This study is divided in three distinct chapters. In the first one the molecular aspects of NAT substrate specificity and the evolution of ascorbate transporters were investigated by making at first an extensive phylogenetic analysis and then a mutational analysis on the NAT signature motif of UapA. This mutational analysis was coupled with a rational combination of substitutions while new substitutions were also isolated by novel genetic screens. Overall, the results revealed cryptic context-dependent roles of partially conserved residues in the NAT signature motif in determining the specificity of the UapA transporter. Additionally we provided novel findings concerning how Phe528, a residue outside the substrate binding site, might function as a key amino acid in determining UapA specificity. The second part of the study was focused on the role of lipid interactions in UapA function, stability and trafficking. In particular, the role of UapA-lipid interactions at the dimer interface was further examined and the possible role of other predicted, by MDs, interactions at the membrane facing regions of the core domain of the UapA dimer was identified. We found that distinct interactions of UapA with membrane lipids are essential for ab initio formation of functional dimers in the ER, or ER exit and further subcellular trafficking. Additionally, through genetic screens, we identified substitutions that restore defects in dimer formation and/or trafficking. Finally, in the third part of the present study, using knowledge acquired from this work we achieved for the first time the functional expression of a mammalian NAT homologue in A. nidulans

    LOW-ENERGY ELECTRON DAMAGE IN DNA

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    In this thesis, Photo-Electron Spectroscopy (PES) and Photo-Electron Imaging (PEI) have been exploited to study low-energy electron and photon-driven damage in DNA derivatives. After an introduction on photoelectron spectroscopy and DNA, an instrumental overview, together with a brief explanation of the theoretical methods used, is given. The results section is divided according to the different chemical systems that have been considered. First, the viability of a dipole-bound state, which are electronic non-valence states that play an important role in electron transfer in DNA, has been studied in a model molecule: despite the presence of an alkyl chain directly poking into it, the dipole-bound state is retained in all cases. Secondly, the possibility of achieving intra-molecular charge transfer as probe for low-energy electron damage has been explored in a carboxylated adenosine analogue. Although no conclusive evidence of charge-transfer from the carboxylic acid to the nucleobase has been observed, this approach has then been applied to different DNA derivatives. The object of the third section of the results chapter is, in fact, the photophysics of the doubly-deprotonated dianion of adenosine-5’-triphosphate, which exhibits electron tunneling through the Repulsive Coulomb Barrier (RCB) upon irradiation at 266 nm; excited states calculation and RCB simulations have been performed to support these findings. Lastly, the photophysics of other doubly-deprotonated di- and tri-phosphorylated purine dianions have been explored in the last section: only one of them, adenosine diphosphate ([ADP–H2]2–), shows evidence of intra-molecular charge transfer, however further research is needed to corroborate this hypothesis

    Cellular and Organismal Ramifications of de novo Purine Synthesis Dysregulation

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    Purines are a class of nitrogenous bases and are essential small molecules to life. Purines are used within the cell as genetic information carriers, energy currency, signaling molecules, and cofactors for multiple processes. They are formed through de novo and salvage pathways found in cells across the phylogenetic tree. The substrates of enzymes within de novo purine synthesis are known to influence other processes within the cell, such as energy homeostasis. In humans, de novo purine synthesis disorders are rare, with around 100 people identified. These patients exhibit a range of phenotypes, with varying degrees of mental retardation, seizure activity, facial and body dysmorphic features, autistic features, respiratory failure, and congenital blindness. To date, the explanation of phenotypes associated with these disorders remains elusive and as such, no effective therapeutic has been identified. Rare disorders are often caused by a single genetic mutation and studying rare disorders can providing key insight into processes regulated by that specific enzyme. In this body of work, I use transcriptomic profiling techniques to provide cellular and organismal process characterization of a novel cellular model of de novo purine deficiency in three CRISPR generated HeLa cell lines. I examine the de novopurine synthesis enzymes ADSL, GART, and ATIC. Processes identified influenced by de novopurine dysregulation identified are focused around neural, embryonic, organ, and placental development, epithelial to mesenchymal transition, fatty acid and inflammatory response, muscle function, tumorigenesis, oxidative stress responses, as well as TGFβ/SMAD signaling among others. Metabolomic profiling was employed to bolster transcriptomic findings, with aberrations of metabolic pathways involved in energy production, vitamin B6 and B5 metabolism, oxidative stress responses, lipids, amino acids, among others. My findings highlight areas in which de novo purine synthesis enzymes influence cellular processes responsible for cellular and organismal function and represent novel avenues of continued research

    Identification of copper metabolism as a KRAS-specific vulnerability in colorectal cancer

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    KRAS est parmi les gènes les plus fréquemment mutés dans les cancers humains, tel que ~ 45% des cancers colorectaux (CCR). Malgré les efforts déployés pour réduire son potentiel oncogénique, KRAS muté est fréquemment associé à la résistance aux médicaments et est extrêmement difficile à cibler sur le plan thérapeutique. Les protéines à la surface cellulaire sont souvent dérégulées dans les cancers et sont des cibles thérapeutiques attrayantes en raison de leur accessibilité aux anticorps. Nous avons séquençé les ARNm de cellules épithéliales intestinales exprimant KRAS muté et observé que ces dernières présentaient des changements importants dans les gènes codant pour des protéines de surface cellulaire. Par conséquent, notre objectif était d'identifier de nouvelles cibles thérapeutiques exprimées à la surface de cellules transformées par l’oncogène KRAS. En utilisant une approche de pointe en protéomique de surface cellulaire, nous avons identifié plusieurs protéines différentiellement exprimées dans les cellules avec KRAS muté par rapport à leurs homologues de type sauvage. Nous avons ensuite effectué un crible CRISPR/Cas9 basé sur les protéines de surface cellulaire, qui a révélé que la perte de la protéine Atp7a affectait de manière différentielle les cellules épithéliales intestinales, en fonction de leur statut KRAS. De façon intéressante, nous avons constaté que ATP7A était régulé à la hausse dans les cellules avec KRAS muté par rapport à leurs homologues de type sauvage. ATP7A a un double rôle dans les cellules; alors qu'il est essentiel pour la maturation des enzymes dépendantes du cuivre (Cu), ATP7A protège les cellules d'une toxicité excessive induite par le Cu (cuproptose). Chez l'homme, les mutations dans ATP7A entraînent des troubles caractérisés par des déficiences systémiques dans le transport et les niveaux de Cu. Chez les animaux et dans les modèles de culture cellulaire, tel que les cellules épithéliales intestinales, les niveaux intracellulaires de Cu sont directement corrélés avec l'abondance post-transcriptionnelle d'ATP7A. Dans le même ordre d'idées, nous avons observé que les cellules de CCR avec KRAS muté avaient relativement plus de Cu intracellulaire, et la surexpression d'ATP7A protégeait les cellules KRAS muté de la cuproptose, par rapport à leurs homologues de type sauvage. Nous avons également observé que la croissance in vivo des xénogreffes KRAS mutées était réduite lorsque les souris étaient nourries avec un régime pauvre en Cu. Le Cu est utilisé par plusieurs enzymes qui régulent des fonctions cellulaires critiques, notamment la respiration mitochondriale, la motilité cellulaire et la prolifération. Nous montrons que les cellules mutantes KRAS étaient plus sensibles au chélateur de Cu, ammonium tetrathiomolybdate (TTM), par rapport aux cellules de type sauvage. De plus, les cellules avec KRAS muté traitées avec le TTM ont présenté des activités réduites de MEK1/2 dépendant du Cu et de l'enzyme de la chaîne de transport d'électrons mitochondriale, cytochrome c oxidase (CCO). Nous avons été surpris de constater que le transporteur de Cu de haute affinité, CTR1, est régulé à la baisse dans les cellules avec KRAS muté, et avons donc émis l'hypothèse que les cellules KRAS mutées doivent absorber le Cu par d'autres moyens. Ainsi, nous avons constaté que la macropinocytose agit comme une voie non canonique d'approvisionnement en Cu dans les cellules avec KRAS muté. Le traitement de cellules in vivo avec l'inhibiteur de la macropinocytose, EIPA, a inhibé l'expression d'ATP7A et diminué le Cu biodisponible dans les xénogreffes KRAS mutées. En conclusion, nos résultats montrent que les cellules avec KRAS muté augmentent les niveaux de Cu et d'ATP7A pour soutenir la tumorigenèse en augmentant l'activité cuproenzymatique et diminuant la cuproptose. Cette étude est pertinente pour le cancer, car les tissus tumoraux contiennent fréquemment des niveaux de Cu plus élevés que les tissus normaux. Des études récentes ont mis en évidence un potentiel de repositionnement du chélateur de Cu TTM, qui est disponible en clinique et utilisé pour traiter les troubles du Cu. Nos résultats démontrent que la biodisponibilité du Cu pourrait être exploitée pour traiter le CCR avec KRAS muté avec de tels inhibiteurs. Les travaux futurs comprennent l'identification de stratégies combinatoires qui peuvent être améliorer les effets anti-cancéreux de la chélation du Cu.KRAS is amongst the most frequently mutated genes driving human cancers, including ~ 45% of colorectal cancers (CRC). Despite intense efforts to curb its oncogenic potential, mutant KRAS is frequently associated with drug resistance and is extremely challenging to target therapeutically. Cell-surface proteins are often spatially dysregulated in cancers and are attractive therapeutic targets due to their easy accessibility. We performed RNA sequencing of mutant KRAS-expressing intestinal epithelial cells and observed that cells undergoing transformation exhibited dramatic changes in cell surface-coding genes. Therefore, our goal was to identify novel druggable targets expressed at the cell surface of mutant KRAS-transformed cells. Using a cutting-edge cell surface proteomics approach, we identified several differentially expressed proteins at the surface of KRAS-mutant cells compared to wild-type counterparts. We then performed a cell surface based CRISPR/Cas9 screen, which revealed that loss of the copper exporter Atp7a differentially affected the fitness of intestinal epithelial cells, depending on their KRAS status. Interestingly, we found that ATP7A was upregulated in KRAS-mutant cells compared to wild-type counterparts. ATP7A has a dual role in cells; while it is essential for maturation of copper (Cu)-dependent enzymes, ATP7A protects cells from excess Cu-induced toxicity (cuproptosis). In humans, ATP7A mutations result in disorders characterized by systemic deficiencies in Cu transport and levels. In animals and in tissue culture models, including intestinal epithelial cells, intracellular Cu levels are directly correlated with the post-transcriptional abundance of ATP7A. In line with this, we observed that KRAS-mutant CRC cells and tissues had relatively more intracellular Cu, and ATP7A-overexpression protected KRAS-mutant cells from cuproptosis, compared to wild-type counterparts. We also observed that in vivo growth of KRAS-mutant xenografts was reduced when mice were fed a Cu-deficient diet. Cu is utilized by several enzymes that regulate critical cellular functions including mitochondrial respiration, cell motility and proliferation. We show that KRAS-mutant cells were more sensitive to the Cu chelating drug ammonium tetrathiomolybdate (TTM), compared to wild-type cells. Moreover, TTM-treated KRAS-mutant cells displayed reduced activities of Cu-dependent MEK1/2 and mitochondrial electron transport chain enzyme, cytochrome c oxidase (CCO). We were surprised to find that the high-affinity CTR1 importer is downregulated in KRAS-mutant cells, and so we hypothesized that KRAS cells must uptake Cu through alternate means. In accordance with this, we found that macropinocytosis acts as a non-canonical Cu-supply route in KRAS-mutant cells. In vivo, treating cells with the macropinocytosis inhibitor EIPA, inhibited the expression of ATP7A and decreased bioavailable Cu in KRAS xenografts. In conclusion, our results show that KRAS-mutant cells increase Cu and ATP7A levels, likely to support tumorigenesis by elevating cuproenzymatic activity and parallelly dealing with cuproptosis. This study is relevant to cancer as tumor tissues and patients contain higher Cu levels than normal controls. Recent studies have highlighted a potential for repurposing the clinically available copper chelator TTM, which is used to treat Cu disorders. Our results demonstrate that copper bioavailability could be exploited to treat KRAS-mutated CRC with such inhibitors. Future work includes identification of combinatorial strategies that may be synthetic lethal to copper chelation

    Low-Energy Electron Interactions with Radiosensitisers and Hydrated Biomolecular Clusters

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    Interactions of free electrons with kinetic energies between 0 eV and 100 eV with isolated biomolecules, (hydrated) radiosensitisers and doped neon clusters were studied, with focus on associative and dissociative electron attachment (AA and DEA, respectively) processes and electron ionisation. Radiosensitisers are applied in radiotherapy to enhance the ratio of damage to malignant compared to healthy cells. The principle of action on a physico-chemical stage is yet unknown and was studied within this thesis. Clusters are an intermediate state of matter between gaseous and solid state. In this thesis, cluster size dependencies and effects in doped clusters were studied. Two different mass spectrometry setups were used. Data from electron interactions with both gas-phase biomolecules and doped neon clusters was taken at the experimental apparatus in Innsbruck. A hemispherical electron monochromator enables high energy resolution measurements and is combined with a quadrupole mass filter. For the acquisition of hydrated radiosensitiser data, the setup in Prague was used. There, an electron gun is combined with a time-of-flight mass analyser. The clusters were in both cases produced via supersonic expansion. The radiosensitisers studied include 5-selenocyanato-2’-deoxyuridine (SeCNdU), nimorazole and misonidazole. All of them exhibit efficient electron capturing characteristics. SeCNdU is a potential radiosensitiser for highly proliferating cells and exhibits (SeU-yl) and CN as strongest fragment anions upon DEA, formed already at virtually 0 eV kinetic energy of the incoming electron. The formation of highly reactive species reinforce SeCNdU as a promising candidate. Nimorazole and misonidazole both exhibit an intense ion signal for the parent anion upon electron attachment. The absolute cross section at 0 eV electron energy was determined for nimorazole and is in the order of 3 1018 m2. For misonidazole, the absolute cross section is estimated to be of the same order of magnitude. The fragmentation channels upon DEA are at least one order of magnitude weaker, with NO 2 being the most intense among them. They are further quenched upon hydration of the agent. It is suggested that the radiosensitising action is caused by the associative attachment channel and the DEA products only play a minor role, opposing previous assumptions. In case of the doped neon clusters, indications for the formation of a conduction band were found in the form of an energy barrier for incoming electrons: Comparing previous results of electron attachment to pure carbon dioxide clusters with neon clusters doped with CO2 results in a blue-shift of the resonance positions by up to 0.8 eV. The effect depends on the size of the neon cluster. For molecular oxygen as the dopant, evidence was found that an incoming electron can first react with the neon cluster via an excitation event and subsequently attach to the dopant cluster. Both the study of radiosensitisers and of neon clusters should be continued. Further radiosensitisers should be studied in order to implement a model in which the effects on a physico-chemical stage are investigated and compared to radiosensitisers already in use. From such a study, potential new radiosensitisers can be derived. In the study of doped neon clustes, the solvation of different complexes with neon was investigated. Particularly, the formation of a conduction band, neon cluster - dopant interactions, and quenching of molecular processes in dopants by neon as a collision partner are analysed. The results require further investigations of additional dopants to unambiguously explain those effects

    Singlet Oxygen Oxidation of Guanine, 9-Methylguanine and Guanine-Cytosine Base Pair: Dynamics and Kinetics Revealed by Parallel Gas- and Solution-Phase Experiments and Computations

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    Singlet oxygen (1O2) oxidatively generated damage of DNA gives rise to mutagenesis, carcinogenesis, and cellular death. Guanine is the most susceptible DNA target of 1O2. The related process has been studied over three decades but the mechanism has remained elusive. My thesis research has focused on reaction mechanism, dynamics and kinetics of 1O2 oxidation of guanine, 9-methylguanine and guanine-cytosine base pair, from the gas-phase bare ions, through hydrated clusters, to aqueous solution. Various techniques have been adapted in the work, including 1O2 generation and detection, guided-ion beam tandem mass spectrometry, gas-phase ion-molecule scattering, and on-line spectroscopy and mass spectrometry measurement of solution kinetics. Experimental measurements, corroborated by electronic structure calculations, Rice-Ramsperger-Kassel-Marcus (RRKM) theory and direct dynamics trajectory simulations, have provided insights into the 1O2 oxidation chemistry of guanine. Four projects have been completed, and each of which is described below. In the first project, ion-molecule scattering mass spectrometry was utilized to capture unstable endoperoxides in the collisions of hydrated guanine ions (protonated or deprotonated) with 1O2 at ambient temperature. Theoretical calculations have strongly supported an intermediate structure of 5,8-endoperoxide rather than 4,8-endoperoxide was proposed in literature. Protonation and deprotonation of reactants in the gas phase, vis-à-vis acidic and basic media in solution reactions, lead to different oxidation chemistries starting from initial stage. This project has pieced together reaction mechanisms and dynamics data concerning the early stage of 1O2 induced guanine oxidation, which is missing from conventional condensed-phase studies. In the second experiment of this thesis, gas-phase dry and monohydrated 9-methylguanine (9MG) was utilized as a model compound to examine the early stage oxidation mechanism and dynamics of the guanine nucleoside. Different levels of theory, including Multi-referential CASSCF and CASMP2, were applied for a reliable description of the early-stage reaction potential surface (PES). The oxidation of protonated 9MG is initiated by the formation of a 5,8-endoperoxide via a concerted cycloaddition as protonated guanine. In contrast, the initial stage of deprotonated 9MG oxidation switches to an addition of O2 to the C8 position only. The comparison between the 1O2 oxidation of ionized guanine and 9-methylguanine indicates that the N9-substitution not only affects the reaction mechanism but inhibits the reactivity of guanine toward 1O2. In the third project, a solution-phase kinetic and mechanistic study of 1O2 oxidation of guanine and 9MG was examined at pH 3.0, 7.0 and 10.0, respectively. Oxidation products and the branching ratio were determined, with each structure inferred from collision-induced dissociation (CID) mass spectra. In basic and neutral solutions, the oxidation products of guanine and 9MG are dominated by spiroiminodihydantoin (Sp), whereas in acidic solution guanidinohydantoin (Gh) is the favored product, showing strong pH dependence of oxidation. gem-diol intermediate, which serves as the precursor for the formation of Gh, was detected. On the basis of solution compositions at each pH, first-order rate constants for individual oxidizable species were extracted. That is 3.2 - 3.6 ´ 106 M-1∙s-1 for deprotonated guanine, 1.1 ´ 106 and 4.6 - 4.9 ´ 107 M-1∙s-1 for neutral and deprotonated 9MG, respectively. Guided by density functional theory-calculated reaction potential energy surfaces, transition state theory (TST) was applied to evaluate the kinetics of the 1O2 addition to guanine and 9MG. The comparison of TST predictions with experiment assures that initial 1O2 addition is the rate-limiting for oxidation, and all of the end products evolve from ensuring endoperoxides and/or peroxides which form at an efficiency of £ 2.5% based on previous measurements of the same systems in the gas phase. In the last project, an experimental and trajectory study was reported, focusing on the 1O2 oxidation of gas-phase deprotonated guanine-cytosine base pair [G·C – H]– that is composed of 9HG·[C – H]– and 7HG·[C – H]– (pairing 9H- or 7H-guanine with N1-deprotonated cytosine), and 9HG·[C – H]–_PT and 7HG·[C – H]–_PT (formed by intra-base-pair proton transfer from guanine N1 to the N3 of [C – H]–). Guided-ion-beam mass spectrometry was used to measure the conformer-averaged product and cross section for [G·C – H]– + 1O2. 1O2 collision dynamics with each of the four conformers was simulated at B3LYP/6-31G(d), to explicate conformation-specific reactivities and changes upon and after oxidation. Trajectories showed that 9HG-containing base pairs favor stepwise formation of 4,8-endoperoxide of guanine, whereas 7HG-containing base pairs prefer concerted formation of guanine 5,8-endoperoxide. Oxidation entangles with intra-base-pair proton transfer, and prefers to occur during the time when the base pair adopts a proton-transferred structure. Guided by trajectories, reaction PESs were established using spin-projected density functional theory. PESs indicate that proton-transferred base-pair conformers have lower barriers for oxidation than non-proton-transferred counterparts

    Molecular basis of the primary and secondary radiation damage to DNA/RNA nucleobases

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    La interacción radiación-materia ha sido una de las grandes preocupaciones científicas de los últimos tiempos, y es que es clave para entender la vida en la Tierra tal y como la conocemos. En este contexto, son de especial relevancia los procesos inducidos en sistemas celulares por radicación externa, en particular, aquellos localizados en los ácidos nucleicos, ya que éstos son los portadores de la información genética de todos los seres vivos. Sólo la comprensión de la competitividad entre procesos inocuos y lesivos inducidos por radiación llevará al entendimiento del sutil equilibrio entre integridad y cambio, salud y enfermedad, y en términos más globales, inmovilidad y evolución. La Tesis Doctoral que nos ocupa se ha centrado en estudiar los mecanismos de daño a las bases nitrogenadas del ADN/ARN inducidos por radiación UV y/o ionizante a partir de sus principios fundamentales, mediante el uso de modernas metodologías químico-cuánticas. Los avances en el entendimiento del daño primario causado por radiación UV se han centrado, en primer lugar, en el estudio del decaimiento del estado excitado de tipo pi,pi* de la timina mediante los métodos multiconfiguracionales CASSCF y CASPT2, y en segundo lugar, en el estudio de la competición entre los procesos de formación de excímeros entre moléculas de citosina y la transferencia de hidrógeno entre guanina y citosina, usando como modelo el trímero guanina-citosina/citosina. Por otro lado, los procesos de daño secundario abordados son de dos tipos: oxidativos (causados por radiación UV e ionizante) y reductivos (causados por radiación ionizante). Los procesos oxidantes están mediados en su mayoría por el radical OH, que es capaz de adicionarse a los dobles enlaces de las nucleobases. Estas adiciones térmicas se han estudiado haciendo uso de metodologías basadas en el funcional de la densidad, en la teoría de los clústeres acoplados y de tipo multiconfiguracional, mientras que la espectroscopia y la fotoquímica de los correspondientes aductos se ha caracterizado con precisión mediante el protocolo CASPT2//CASSCF. Por otro lado, los procesos reductivos por adición de átomos de hidrógeno al enlace C5=C6 de las pirimidinas se han abordado mediante la fotogeneración del correspondiente aducto en posición C6 y posterior caracterización experimental y teórica, haciendo también uso del protocolo CASPT2//CASSCF, de sus propiedades ópticas en el rango UV-Vis. Por último, los procesos reductivos mediados por adición de electrones de baja energía, que dan lugar a fragmentaciones de las bases de ADN/ARN, se han estudiado en base a la determinación teórica de las afinidades electrónicas, los umbrales de energía de las reacciones de deshidrogenación, los valores mínimos de energía del estado electrónico pi2- y del mapeo de las superficies de energía potencial de los estados electrónicos relevantes a lo largo de las coordenadas de reacción N-H

    Structural and functional studies of AT-Rich DNA ligands and their effect on trypanosoma brucei

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    AT-rich sequences confer unique properties to DNA, such as high polymorphism and flexibility. The abundance of AT-rich DNA in several pathogens' genomes and the ability of specific molecules to selectively target AT base pairs have prompted studies on ligands that interact with the minor groove of high AT content DNA. Of special interest are kinetoplastid parasites, such as Trypanosoma brucei, the causative agent of sleeping sickness, which are distinguished by the presence of a very AT-rich mitochondrial DNA structure called kinetoplast. Minor groove binding ligands have offered critical information on DNA molecular recognition, providing clinically useful strategies against diseases. Thus, the binding affinity and structural characteristics of AT-rich oligonucleotides in complex with different ligands, specifically with HMG proteins and bisimidazolinium compounds, has been chosen as the object of study. The `High Mobility Group¿ (HMG) is a family of architectural proteins that bind to DNA and influence a myriad of essential cellular processes. This research work has focused in two HMG subfamilies: HMGA and HMGB. They bind to the minor groove of the DNA by means of AT-hook (HMGA) or HMG-box (HMGB) domains. HMGA1a(50-91), HMGB1 box B and HMGB1 box AB have been expressed and purified. High similar binding affinity to an AT-rich DNA sequence containing [AATAAT_ATTATT] has been found by SPR¿biosensor experiments for both proteins. A d[CCAATAATCGCGATTATTGG]2-HMGB1 box B complex was crystallized. The diffraction patterns of the crystal at 2.68 Å resolution presented well-defined spots revealing two diffraction orientations. A series of derivatives of FR60 [4-((4,5-dihydro-1H-imidazol-2-yl)amino)-N-(4-((4,5-dihydro-1Himidazol-2-yl)amino)phenyl)benzamide] have been proved to be high affinity DNA binders with a preference for AT over GC-rich DNA, showing slight selectivity towards sequences containing [AATT] versus [(AT)4] or [AATAAT_ATTATT]. Furthermore, competition assays have demonstrated that JNI18 competes with HMGA1a and HMGB1 for binding to DNA and it is able to displace the proteins from their DNA binding sites. This last interaction is of prime importance, as related proteins have been found to be essential in kinetoplastid parasites. The structure of the bis(2-aminoimidazoline) compound CDIV32 with the oligonucleotide d[AAATTT]2 partially solved at 3.10 Å resolution, displays DNA columns of stacked oligonucleotides forming apseudo-continuous helix packed in a crossed column configuration of DNA helices that are at ~90° to each other. The presence of the drug CDIV32 modulates the organization of duplexes. The crystal structure of the complex of the oligonucleotide d[AAATTT]2 with the lead compound FR60, solved at atomic resolution of 1.25 Å (PDB-ID: 5LIT) by X-ray crystallography, is constituted of stacked oligonucleotides organized as infinite continuous parallel columns, packed in a pseudo-tetragonal configuration. The structure revealed that the drug interacts with the central [AATT] region, covers the minor groove of DNA, displaces bound water and interacts with neighboring DNA molecules as a crosslinking agent. Finally, a functional analysis has been performed on the effect of different bis(2-aminoimidazolines) on T.brucei (>70% AT kDNA) to assess whether parasite DNA was a target for these compounds. By a combination of flow cytometry and imaging techniques such as fluorescence microscopy and TEM, it was demonstrated that these compounds have a clear effect on the S-phase of T. brucei cell cycle by inflicting specific damage on the kinetoplast. It can be concluded that the studied DNA binding compounds FR60 and JNI18 are powerful trypanocides that act directly on the kinetoplast DNA. As the compounds show 100% curative activity in a mouse model of T. b. rhodesiense infection, they are potentially an effective chemotherapeutic agent for the treatment of sleeping sickness.Las secuencias ricas en AT le confieren al ADN propiedades únicas como un alto polimorfismo y flexibilidad. Su abundancia en el genoma de varios patógenos y la selectividad de unión a secuencias AT que presentan ciertas moléculas, han llevado al estudio de ligandos que interactúan con el surco estrecho de DNA con alto contenido en AT. De especial interés son los parásitos kinetoplástidos, como el Trypanosoma brucei, agente causante de la enfermedad del sueño, los cuales se distinguen por la presencia de una estructura de ADN mitocondrial muy rica en AT llamada kinetoplasto. Los ligandos de unión al surco estrecho han ofrecido información primordial sobre el reconocimiento molecular del ADN, proporcionando estrategias terapéuticas útiles. Por ello, se ha elegido como objeto de estudio complejos de ADN ricos en AT con diferentes ligandos, específicamente con proteínas HMG y compuestos bisimidazolinio. Las HMG son una familia de proteínas arquitectónicas que se unen al ADN e influyen en numerosos procesos celulares esenciales. En este trabajo se han estudiado dos subfamilias de las HMG: HMGA y HMGB. Ambas se unen al surco estrecho del ADN mediante diferentes motivos de unión: AT-hook (HMGA) y HMG-box (HMGB). Se han expresado y purificado las formas HMGA1a(50-91), HMGB1 box B y HMGB1 box AB. Mediante SPR, ambas proteínas presentaron una afinidad de unión alta y similar hacia un ADN conteniendo la secuencia [AATAAT_ATTATT]. Se cristalizó el complejo d[CCAATAATCGCGATTATTGG]2- HMGB1 box B. La difracción a una resolución de 2.68 Å presentó reflexiones bien definidas que indicaban dos orientaciones preferenciales. Una serie de derivados del compuesto FR60 [4-((4,5-dihidro-1H-imidazol-2-il)amino)-N-(4-((4,5-dihidro-1H-imidazol-2-il)amino)fenil)benzamida] han demostrado ser ligandos de alta afinidad por secuencias AT con respecto a GC, mostrando cierta preferencia hacia secuencias con [AATT] comparado con [(AT)4] o [AATAAT_ATTATT]. Además, se ha demostrado que el JNI18 compite con la HMGA1a y la HMGB1 en su unión al ADN y es capaz de desplazar a dichas proteínas de sus sitios de unión al ADN. Este hecho es de especial relevancia, ya que se han encontrado proteínas relacionadas que son esenciales en parásitos kinetoplástidos. La estructura del compuesto de bis(2-aminoimidazolinio) CDIV32 con el oligonucleótido d[AAATTT]2 ha sido parcialmente resuelta a una resolución de 3.10 Å. Se encontraron columnas de oligonucleótidos apilados formando una hélice pseudo-continua, empaquetada en una configuración de columnas cruzadas perpendicularmente. La presencia del fármaco CDIV32 modula la organización de las hélices de ADN. Se ha resuelto la estructura cristalográfica del complejo d[AAATTT]2-FR60 a resolución atómica de 1.25 Å (PDB-ID: 5LIT). Se encontraron los oligonucleótidos apilados organizados en columnas infinitas y paralelas en una configuración pseudo-tetragonal. El fármaco interacciona con la región central [AATT], ocupa el surco estrecho del ADN, desplaza las moléculas de agua presentes e interactúa con moléculas de ADN vecinas como un agente entrecruzador. Finalmente, se ha realizado un análisis funcional del efecto de diferentes compuestos bis(2-aminoimidazolinio) en T. brucei (con >70% de AT en su kDNA) para evaluar si el ADN del parásito es una diana para estos compuestos. Se ha estudiado su efecto in vitro mediante una combinación decitometría de flujo y técnicas como microscopía de fluorescencia y TEM. Los resultados permitieron demostrar que estos compuestos tienen un efecto claro sobre la fase S del ciclo celular de T. brucei al dañar específicamente el kinetoplasto. Se ha podido concluir que los compuestos FR60 y JNI18 son potentes tripanocidas que actúan directamente sobre el ADN del kinetoplasto. Ya que los compuestos muestran una actividad curativa del 100% en un modelo de ratón infectado por T. b. rhodesiense, representan un agente quimioterapéutico potencialmente eficaz para el tratamiento de la enfermedad del sueño.Postprint (published version
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