Nanomechanics of cell-derived matrices as a functional read-out in collagen VI-related congenital muscular dystrophies

Changes in the mechanical properties of the extracellular matrix (ECM) are a hallmark of disease. Due to its relevance, several in vitro models have been developed for the ECM, including cell-derived matrices (CDMs). CDMs are decellularized natural ECMs assembled by cells that closely mimic the in vivo stromal fibre organization and molecular content. Here, we applied atomic force microscopy-force spectroscopy (AFM-FS) to evaluate the nanomechanical properties of CDMs obtained from patients diagnosed with collagen VI-related congenital muscular dystrophies (COL6-RDs). COL6-RDs are a set of neuromuscular conditions caused by pathogenic variants in any of the three major COL6 genes, which result in deficiency or dysfunction of the COL6 incorporated into the ECM of connective tissues. Current diagnosis includes the genetic confirmation of the disease and categorization of the phenotype based on maximum motor ability, as no direct correlation exists between genotype and phenotype of COL6-RDs. We describe differences in the elastic modulus (E) among CDMs from patients with different clinical phenotypes, as well as the restoration of E in CDMs obtained from genetically edited cells. Results anticipate the potential of the nanomechanical analysis of CDMs as a complementary clinical tool, providing phenotypic information about COL6-RDs and their response to gene therapies

Resilience through a multisystemic perspective: analyzing individual, family, and community systems

In this article, we approach resilience from a multisystemic point of view in which individual, family, and community characteristics play a part. The main goal of the study is to analyze how the different systems of resilience (individual, family, and community) work in times of uncertainty and great societal challenges, such as the COVID-19 pandemic. Our study uses a mixed methods approach with a sample of 1,436 Spaniards during the COVID-19 crisis. After grouping the descriptions of their living experiences into positive and negative, the study builds and operationalizes the different systems of resilience. Furthermore, the study uses structural equation modeling to analyze the role of each system on how individuals face the future in a situation of uncertainty. The study indicates that family resilience, followed by individual and community resilience, helps individuals to face the future with high optimism. In contrast, individual resilience helps to face the future with less pessimism. In addition, all systems of resilience—individual, family, and community—significantly impact an optimistic view of the future through the mediation of positivity. Our article contributes to the ongoing debates regarding the role of resilience in building a stronger society that is capable of facing challenges and recovering from adversity

Extent and dynamics of the remunicipalisation of public services

Remunicipalisation of local public services has attracted the interest of scholars from different fields of the social sciences in recent years. Whereas earlier studies into this phenomenon were dominated by case-based analysis there has been a noteworthy increase in quantitative studies in recent years. There is now, abundant evidence on the drivers of remunicipalisation especially the influence of pragmatic and political motivations. Other topics, such as the effects of remunicipalisation, however, remain underdeveloped. We provide an overview of the scope and dynamics of remunicipalisation, recent advances in its research, and discuss where future research should be directed

Bilingual (Greek-Latin) Metrical Inscriptions from the Roman Western world. Corpus and Analysis

[eng] This doctoral dissertation consists of a corpus of inscriptions discovered in the Western Mediterranean which contain a metrical and bilingual text in Greek and Latin. Specifically, the corpus includes inscriptions that are either bilingual or exhibit bilingual phenomena, where at least one of the texts is metrical or contains metrical reminiscences. The main corpus comprises eighty-two inscriptions categorised into three sections: inscriptions with metrical parts in both languages (“Additional Poetical Bilingualism”), inscriptions with a prose text in one language and a carmen in the other (“Functional Bilingualism in the Western Half of the Roman empire”), and inscriptions where one part is written in one language but using sometimes the alphabet of another (“Transcribed Text”). Regarding the inscriptions that are not included in the main corpus, twenty-two epigraphs have been classified as dubia for various reasons: their fragmentary state, metrical irregularities or commatica, metrical parts due to stereotyped formulae, questionable bilingual-metrical intention, and inscriptions from potentially different ages. Furthermore, fifteen epigraphs have been relegated to the appendices for several reasons, including defixiones with metrical reminiscences, having a literary intention but isolated rhythmic sequences, tabulae opisthographicae, being inscriptions of different centuries, being published together despite being different inscriptions found at the same location, displaying bilingual phenomena resulting from a stonecutter’s mistake, and being bilingual because the author of the text (not the commissioner) signed the inscription in Greek and Latin. The corpus analyses epigraphs dating from the first century BCE to the sixth century CE. The limes referenced are those established during the division of the Roman Empire into its Western and Eastern halves. Therefore, this (main) corpus includes bilingual metrical inscriptions from Rome, Regio I, III, VIII, and XI, Sicilia, Sardinia, Germania Superior, Dalmatia, Pannonia, Gallia Narbonensis, Gallia Lugdunensis, Africa proconsularis, Mauretania Caesariensis, Hispania citerior, and Lusitania. Rome stands out with forty-nine examples, underscoring its central role as the epicentre of Roman culture and administration. Regio I, a region adjacent to Rome with a Greek background, provides nine examples. Single examples from Regio III, Regio VIII, and Regio XI illustrate the small presence of such inscriptions in other regiones of Italy. Similarly, the islands of Sicilia and Sardinia each provide one example. Germania Superior, on the northern frontier, also offers one example, while in Dalmatia there have been preserved two bilingual carmina epigraphica. Pannonia, bordering province, provides three examples of this sort of inscriptions. The provinces of Gallia Narbonensis and Gallia Lugdunensis offer five and one examples respectively. Africa proconsularis provides three examples: an epitaph, an honorific inscription, and a mosaic with texts. In Mauretania Caesariensis a sole bilingual metrical epitaph has been detected. Finally, the Iberian Peninsula is represented by two examples from Hispania Citerior and one inscription from Lusitania. In terms of epigraph typology, funerary inscriptions remain the most prevalent category of texts. The second most numerous group consists of votive inscriptions, while honorific and musive inscriptions represent the categories with the fewest examples. Each scheda within the study meticulously examines the onomastics of individuals named in the inscription. Besides, attention has been given to the reason for the language choice in the piece commissioned. Additionally, linguistic features, stylistic nuances, thematic elements, and metrics have been thoroughly analysed and discussed.[cat] Aquesta tesi doctoral consisteix en un corpus d’inscripcions descobertes al Mediterrani Occidental que contenen textos mètrics i bilingües en grec i en llatí. El corpus inclou cent divuit inscripcions bilingües o que mostren fenòmens bilingües, en les quals almenys un dels textos és mètric o conté reminiscències mètriques. El corpus principal comprèn vuitanta-dos inscripcions categoritzades en tres seccions: inscripcions amb parts mètriques en ambdós idiomes (“Additional Poetical Bilingualism”), inscripcions amb un text en prosa en un idioma i un carmen en l’altre (“Functional Bilingualism in the Western Half of the Roman empire”), i inscripcions on una part està escrita en un idioma però utilitzant a vegades l’alfabet de l’altre (“Transcribed text”). Pel que fa a les inscripcions excloses al corpus principal, vint-i-dos s’han classificat com a dubia per diversos motius: estat fragmentari, irregularitats mètriques, parts mètriques a causa de fórmules estereotipades, intenció bilingüe-mètrica dubtosa i inscripcions probablement de diferents èpoques. A més, quinze epígrafs s’han col·locat als appendices per diversos motius: defixiones amb reminiscències mètriques, intenció literària però amb seqüències rítmiques aïllades, tabulae opisthographicae, inscripcions de diferents segles, diferents peces d’un jaciment publicades juntes, bilingüisme a causa d’un error, i ser bilingües perquè l’autor del text signa la inscripció en grec i llatí. El corpus analitza epígrafs que daten del segle I aC al segle VI dC. Els limes referits són els establerts durant la divisió de l’Imperi Romà en les seves meitats occidental i oriental. Per tant, aquest corpus principal inclou inscripcions bilingües mètriques de Roma, Regio I, III, VIII i XI, Sicilia, Sardinia, Germania Superior, Dalmatia, Pannonia, Gallia Narbonensis, Gallia Lugdunensis, Africa proconsularis, Mauretania Caesariensis, Hispania Citerior i Lusitania. Roma destaca amb quaranta-nou exemples, seguida de la Regio I, amb nou inscripcions. Quant a la tipologia d’inscripcions, els epígrafs funeraris continuen sent el grup més nombrós. El segon grup més abundant consisteix en inscripcions votives, mentre que les inscripcions honorífiques i musives representen les categories amb menys exemples. Cada scheda examina minuciosament l’onomàstica de les persones esmentades a la inscripció. A més, s’ha prestat atenció a la raó de la elecció de l’idioma en la peça encarregada. Així mateix, s’han analitzat i discutit detalladament els aspectes lingüístics, estilístics, temàtics i la mètrica

Improvements in functioning and workplace productivity with esketamine nasal spray versus quetiapine extended release in patients with treatment resistant depression: Findings from a 32-week randomised, open-label, rater-blinded phase IIIb study

Patients with treatment resistant depression (TRD) experience a greater negative impact on their functioning and productivity at home and in the workplace versus treatment-responsive patients. Here, we report the effects of esketamine nasal spray (NS) versus quetiapine extended release (XR) on functioning, work productivity and activity impairment. ESCAPE‑TRD (NCT04338321) was a 32-week randomised, open‑label, rater‑blinded, active‑controlled phase IIIb study comparing the efficacy and safety of esketamine NS versus quetiapine XR, both alongside an ongoing selective serotonin reuptake inhibitor or serotonin norepinephrine reuptake inhibitor (SSRI/SNRI), in patients with TRD. Patient functioning was assessed via the Sheehan Disability Scale (SDS; functional remission ≤6). Absenteeism, presenteeism, work productivity loss and activity impairment over time were assessed using the Work Productivity and Activity Impairment: Depression (WPAI:D) questionnaire. Results were cumulated over the entire study duration. Esketamine NS-treated patients (N=336) experienced 43.2% more weeks with functional remission versus quetiapine XRtreated patients (N=340) over the 32-week study period (difference: 2.0 weeks [95% CI: 0.7, 3.3]; p=0.0023 [ANCOVA models]). Up to Week 32, esketamine NS-treated patients experienced an 11.9% reduction in productivity loss due to absenteeism (difference: −1.1 weeks [95% CI: −2.9, 0.7]; p=0.2285) and a 14.2% reduction inoverall work productivity loss (difference: –2.3 weeks, 95% CI: [–3.9, –0.7] p=0.0045) versus quetiapine XR-treated patients, based on mixed models for repeated measures.Patients receiving esketamine NS experienced greater improvements in functioning and productivity over 32 weeks versus quetiapine XR. These improvements demonstrate the clinical and functional benefit of treatment with esketamine NS for patients with TRD

Javier Cercas, anatomía íntima de un intelectual

[spa] La tesis que presento es un ensayo de interpretación de la faceta intelectual del escritor Javier Cercas a partir esencialmente de su articulismo hasta el término del año 2024. El trabajo reposa sobre tres grandes pilares: periodismo, literatura y política. En el primero, tras esbozar el perfil de autor transfronterizo que es hoy, exploro los mimbres que fundamentan su vertiente de escritor de periódicos desde que diera los primeros pasos en el oficio de columnista reivindicando un espacio en el diario en el que expresar una verdad que fuera más allá de lo meramente factual, hasta trazar un boceto tanto de la moral que alienta su prosa como del mecanismo de funcionamiento de la imprescindible ironía que marca el compás del conjunto de su poética. En el segundo ahondo en su dimensión de crítico cultural, tanto de la obra ajena —sea o no sea literaria, con especial énfasis en la influencia del cine— como de la propia, que el autor aborda con una hiperconciencia de novelista a la que no le es posible renunciar, y que ofrece como resultado un trabajo muy original en cuanto a formas y contenidos que plasma sin esconder su experiencia lectora o vital, y que por eso mismo en conjunto puede leerse a modo de solvente autobiografía. Acabo con la semblanza de un ciudadano que se fue insertando en el debate público sobre todo a partir de la crisis de 2008; de un socialdemócrata pragmático y escéptico que nunca ha dejado de reclamar un gran pacto de Estado con el que blindar los cimientos de la democracia española; de un federalista cuyas críticas apuntan sobre todo a la propia izquierda liberal, a la que sin embargo no ha dejado de apoyar; y, en fin, de un europeísta que concibe la creciente ola nacionalpopulista como la amenaza más peligrosa a la que todo demócrata, como hijo de la razón, ha de enfrentarse en nuestros días.[cat] La tesi que presento és un assaig d’interpretació de la faceta intel·lectual de l'escriptor Javier Cercas a partir essencialment del seu articulisme fins al terme de l'any 2024. El treball reposa sobre tres grans pilars: periodisme, literatura i política. En el primer, després d'esbossar el perfil de l’autor transfronterer que és avui, exploro els vímets que fonamenten la seva vessant d'escriptor de periòdics des que fes els primers passos en l'ofici de columnista reivindicant un espai en el diari en el qual expressar una veritat que fos més enllà d’allò merament factual, fins a traçar un esbós tant de la moral que encoratja la seva prosa com del mecanisme de funcionament de la imprescindible ironia que marca el compàs del conjunt de la seva poètica. En el segon aprofundeixo en la seva dimensió de crític cultural, tant de l'obra aliena —sigui o no sigui literària, amb especial èmfasi en la influència del cinema— com de la pròpia, que l'autor aborda amb una hiperconsciència de novel·lista a la qual no li és possible renunciar, i que ofereix com a resultat un treball molt original pel que fa a formes i continguts que plasma sense amagar la seva experiència lectora o vital, i que per això mateix en conjunt pot llegir-se a manera de solvent autobiografia. Acabo amb la semblança d'un ciutadà que es va anar inserint en el debat públic sobretot a partir de la crisi de 2008; d'un socialdemòcrata pragmàtic i escèptic que mai ha deixat de reclamar un gran pacte d'Estat amb el qual blindar els fonaments de la democràcia espanyola; d'un federalista les crítiques del qual apunten sobretot a la mateixa esquerra liberal, a la qual no obstant això no ha deixat de secundar; i, en fi, d'un europeista que concep la creixent ona nacionalpopulista com l'amenaça més perillosa a la qual tot demòcrata, com a fill de la raó, ha d'enfrontar-se en els nostres dies.[eng] The thesis I present here is an interpreting essay on the intellectual side of the writer Javier Cercas, essentially based on his articles up to the end of the year 2024. The work rests on three main pillars: journalism, literature and politics. In the first, after sketching the profile of the cross-border author that he is today, I explore the elements that underpin his work as a newspaper writer since he took his first steps as a columnist, claiming a space in the newspaper in which to express a truth that goes beyond the merely factual, to outline both the moral that drives his prose and the functioning mechanism of the essential irony that marks the compass of his poetics as a whole. In the second, I delve into his dimension as a cultural critic, both of the work of others —whether literary or not, with special emphasis on the influence of cinema— and of his own, which the author approaches with a novelist’s hyper- awareness that he cannot renounce, and which offers as a result a highly original work in terms of form and content that he captures without hiding his reading or life experience, and which can therefore be read as a whole as a solvent autobiography. I end with a portrait of a citizen who has been inserting himself into the public debate especially since the 2008 crisis; of a pragmatic and skeptical social democrat who has never ceased to call for a great pact of State with which to shield the foundations of Spanish democracy; of a federalist whose criticisms are aimed above all at the liberal left itself, which he has nevertheless never ceased to support; and, finally, of a Europeanist who sees the rising tide of national populism as the most dangerous threat that any democrat, as a child of reason, has to face today

Exposure and Health Effects of Microplastics in Humans

[eng] Microplastic (MP) refers to plastic particles having diameter of 5 mm along their longest dimension. In the last decade, extensive research efforts have taken to identify and quantify MP in different environmental compartments and recently also in human samples as blood, urine, stool, lung, and placentae tissue proving human intake of MP. Because of our exposure to MP via inhalation and ingestion, this Thesis focuses in applying different analytical methods, both spectroscopic and thermal, to identify and quantify MP in air as well as bottled water to carry out a human exposure assessment through these pathways of exposure. Moreover, a migration study was done to assess the potential release of MP from face masks into water after potential incorrect waste disposal, finding that PP, PA, PES, and cellulose fibres shred from the masks. Air samples were collected both indoors from private and public settings, and outdoors using active sampling for 3 hours until 1.8 m3 of air were filtered and the particles were analysed with micro-Fourier Transform Infrared (μ-FTIR) spectroscopy to identify the presence of plastic polymers and to assess any potential differences in the concentration of MP between the areas considered and their potential sources. The areas considered were a urban area (Barcelona, Spain) and a rural area (Sassari, Italy). Indoor air samples (n=10) were collected from private houses, whilst outdoor air samples (n=11) were collected during rush hours in the main roads. Results reveal that indoor levels were higher in Sassari, while outdoor concentrations were greater in Barcelona. In Barcelona, MP comprised 2-15% indoors and 0-20% outdoors of the total particulate count (TPC), while Sassari showed 0-10% indoors and 8-18% outdoors. The predominant indoor polymers were polyacrylonitrile and nylon 6,6, used as fiber in clothing, home furnishing, while outdoor samples included PAN, nylon 6,6, and various polyethylene copolymers all used in clothing as well as automobile industry. The same technique was applied to assess the potential release of MP from face masks (n=4) into water during a migration study to mimic their behaviour when their disposal is mismanaged, or they are directly discarded in the environment. Results showed that MP can release polypropylene, polyamide, and polyester into the aquatic environment. Despite the advantages in using μ-FTIR, the major drawback is that it is size dependent and can be used to analyse particles having diameter >20 μm. Therefore, a pyrolysis-GC/MS method was developed to overcome the size-limitation and analyse MP in air and water samples with high degree of accuracy and precision in a rapid manner. This method enabled the target analysis of the several plastic polymers including polyethylene, polypropylene, polystyrene, polycarbonate, polyvinyl chloride, nylon 6,6, polyethylene terephthalate, polymethylmethacrylate, nitrile rubber, acrylonitrile butadiene styrene. Air samples collected from public libraries (n=14) in the urban area of Barcelona showed that high-density polyethylene was the only plastic polymer present, probably released from textiles, containers, and appliances. Bottled water samples (n=40) from 5 different European countries (Spain, Italy, France, Portugal, and Greece) were also studied and it was shown that high-density polyethylene was present in 33 out of 40 samples. The presence of polyethylene can be reconducted to the mechanical abrasion of the bottle cup. An in vitro toxicological test on human hepatocellular carcinoma (HepG2) cells was performed to assess the potential hepatotoxicity of leachates from high density polyethylene using both medium and organic solvent to extract the chemicals. HepG2 cells were exposed to both extractions at concentration of 1 μg μl-1 and no toxicity was observed when using cell viability and reactive oxygen species (ROS) generation as toxicological endpoints. Moreover, literature review concerning MP exposure through air (indoors and outdoors), bottled water, soft drinks, alcoholic beverages, and food was done to estimate the total daily intake of MP for adults, children and toddler. Additionally, bibliographic research about human biomonitoring and presence of MP in different samples including blood, urine, stool, placentae and lung tissues among others is presented to highlight that the widespread of MP has led to human intake. To conclude, the Thesis served (i) to verify that face masks could represent a potential source of MP in water bodies, (ii) to develop analytical methods to identify and quantify different MP polymers in air and water samples, (iii) to apply the results to assess human exposure through inhalation and ingestion and (iv) to investigate in vitro hepatotoxicity of HDPE leachates.[cat] El terme "microplàstic" (MP) es va encunyar l'any 2004 per referir-se a partícules de plàstic amb una mida igual o inferior a 5 mm en la seva dimensió més llarga. Des d'aleshores, s'han realitzat nombrosos estudis per analitzar la seva presència en diferents medis ambientals i avaluar els seus possibles efectes tant en els ecosistemes com en la salut humana. El present treball s'ha centrat en avaluar l'exposició humana als MP a través de la inhalació i la ingestió. Per fer-ho, s'han utilitzat tècniques analítiques avançades, tant espectroscòpiques com tèrmiques, que permeten identificar i quantificar amb precisió aquests contaminants en mostres d'aire i aigua embotellada. A més, s'ha dut a terme un estudi específic per analitzar l'alliberament de MP a partir de màscares facials quan aquestes es gestionen incorrectament com a residus, amb resultats que demostren com aquests productes poden alliberar fibres de polipropilè, poliamida, polièster i cel·lulosa al medi ambient. Pel que fa a l'anàlisi de l'aire, es van recollir mostres tant en entorns interiors com exteriors, comparant una zona urbana (Barcelona) amb una àrea rural (Sassari). Els resultats van revelar diferències en la concentració de MP entre ambdós entorns. A l'interior, les concentracions eren més elevades a Sassari, amb valors que oscil·laven entre el 0% i el 10% del total de partícules, mentre que a Barcelona aquestes xifres es situaven entre el 2% i el 15%. En canvi, a l'exterior, Barcelona presentava nivells més alts, amb un màxim del 20% de MP respecte al total de partícules, enfront del 8% al 18% observat a Sassari. Els polímers més freqüents a l'interior van ser el poliacrilonitril i el niló 6,6, associats a tèxtils i mobiliari, mentre que a l'exterior, a més d'aquests, també es van detectar diversos copolímers de polietilè, relacionats tant amb la indústria tèxtil com amb la automobilística. Una limitació important de la tècnica μ-FTIR utilitzada és que només permet detectar partícules superiors a 20 μm. Per superar aquest obstacle, es va desenvolupar un mètode alternatiu basat en piròlisi acoblada a espectrometria de masses (Pyr-GC/MS), que no només permet analitzar partícules més petites sinó que també ofereix una major precisió i rapidesa en la identificació dels polímers. Aplicant aquesta tècnica, es van analitzar mostres d'aire recollides en biblioteques de Barcelona, on es va detectar predominantment polietilè d'alta densitat, probablement procedent de tèxtils i envasos. Paral·lelament, en l'estudi d'aigua embotellada procedent de cinc països europeus, es va trobar que el polietilè d'alta densitat estava present en 33 de les 40 mostres analitzades, un fet que es pot atribuir a l'abrasió mecànica de les pròpies ampolles durant el seu ús i transport. Per avaluar els possibles efectes sobre la salut humana, es van realitzar assajos in vitro amb cèl·lules HepG2 exposades a lixiviats de polietilè d'alta densitat. Els resultats no van mostrar efectes tòxics significatius en termes de viabilitat cel·lular o generació d'espècies reactives d'oxigen en les condicions d'estudi, tot i que aquests resultats no descarten la necessitat d'investigar més profundament els possibles efectes a llarg termini o en altres models cel·lulars. Com a complement a aquests estudis experimentals, es va dur a terme una revisió de la literatura científica sobre l'exposició humana als MP, analitzant tant les principals vies d'exposició com la presència d'aquests contaminants en mostres biològiques humanes. Aquesta revisió no només confirma la ingesta i absorció de MP per part de la població, sinó que també posa en relleu com aquest fenomen s'ha generalitzat a escala global. En conclusió, aquesta tesi ha permès avançar en la comprensió de la problemàtica dels microplàstics des de múltiples perspectives. D'una banda, ha confirmat el paper de les màscares facials com a font potencial de contaminació quan no es gestionen adequadament. D'altra banda, ha contribuït al desenvolupament de mètodes analítics més precisos per a la detecció i quantificació d'aquests contaminants. Finalment, els resultats obtinguts han permès avaluar amb major precisió l'exposició humana als MPs i els seus possibles efectes

Enhanced mineralization of pharmaceutical residues at circumneutral pH by heterogeneous electro-Fenton-like process with Cu/C catalyst

Conventional electro-Fenton (EF) process at acidic pH ∼3 is recognized as a highly effective strategy to degrade organic pollutants; however, homogeneous metal catalysts cannot be employed in more alkaline media. To overcome this limitation, pyrolytic derivatives from metal-organic frameworks (MOFs) have emerged as promising heterogeneous catalysts. Cu-based MOFs were prepared using trimesic acid as the organic ligand and different pyrolysis conditions, yielding a set of nano-Cu/C catalysts that were analyzed by conventional methods. Among them, XPS revealed the surface of the Cu/C-A2-Ar/H2 catalyst was slightly oxidized to Cu(I) and, combined with XRD and HRTEM data, it can be concluded that the catalyst presents a core-shell structure where metallic copper is embedded in a carbon layer. The antihistamine diphenhydramine (DPH), spiked into either synthetic Na2SO4 solutions or actual urban wastewater, was treated in an undivided electrolytic cell equipped with a DSA-Cl2 anode and a commercial air-diffusion cathode able to electrogenerate H2O2. Using Cu/C as suspended catalyst, DPH was completely degraded in both media at pH 6–8, outperforming the EF process with Fe2+ catalyst at pH 3 in terms of degradation rate and mineralization degree thanks to the absence of refractory Fe(III)-carboxylate complexes that typically decelerate the TOC abatement. From the by-products detected by GC/MS, a reaction sequence for DPH mineralization is proposed

Unraveling injury and repair mechanisms in alcohol-associated liver disease: From neutrophils to organoid-based modeling

[eng] Alcohol-associated liver disease (ALD) is one of the leading causes of disability and premature mortality worldwide, with few therapeutic treatments. Therefore, it is important to understand the underlying mechanisms of the disease in order to develop new therapies. Neutrophil infiltration into the liver is a hallmark of chronic liver disease, particularly alcohol-associated hepatitis (HA), the most severe form of ALD. In addition, neutrophils are known to orchestrate tissue repair, especially after acute injury. However, the characteristics and role of neutrophils in chronic liver diseases, as well as their contribution to the regression of chronic damage, remain unknown. In this thesis we hypothesize that neutrophils adapt their recruitment dynamics, phenotype and function in response to signals from the liver microenvironment, playing different roles in the progression and resolution of the disease. Therefore, we wanted to characterize neutrophils and investigate their contribution to chronic liver damage and tissue repair by using human samples and murine models. On the other hand, the study of ALD is limited by the lack of optimal preclinical models. Although organoids show great potential, liver organoids derived from patient samples are limited to the dissociation of tissues obtained in advanced stages of the disease and to a cholangiocyte phenotype, which limits their usefulness. Therefore, the second hypothesis we formulated is that liver organoids can be generated from needle biopsies taken at different stages of ALD, which would allow modeling the disease and testing drugs in a personalized way. Our objective was to generate and characterize these organoids in order to discover potential therapeutic targets and test drugs. With respect to the first objective, histological analyses revealed a population of ductular reaction-associated neutrophils (DRANs) that interact closely with biliary epithelial cells and increase as the disease progresses. We demonstrated by intravital microscopy and neutrophil labeling assays in CDD-treated mice that DRANs remain immobilized around the biliary epithelium for prolonged periods. In addition, hepatic neutrophils showed a unique phenotypic and transcriptomic profile. Neutrophil depletion using an anti-Ly6G antibody as well as inhibition of neutrophil recruitment using a CXCR1/2 inhibitor reduced DRANs, ductular reaction, liver fibrosis, and angiogenesis. Finally, neutrophils deficient in PAD4 and ELANE were not able to promote cell proliferation biliary, highlighting the role of extracellular traps and elastase in disease progression. During the resolution of chronic liver damage, we observed a reduction and delocalization of DRANs, accompanied by hepatocyte proliferation and a regression of fibrosis and expansion of the biliary tree. Neutrophil depletion during a resolution period of 5 days affected liver regeneration, showing a lower proliferation of hepatocytes, regression of fibrosis and decreased ductular reaction. The hepatic neutrophils obtained during the resolution phase showed a distinctive transcriptomic profile, enriched in pro-resolutive mediators and immature neutrophil characteristics. In addition, we were able to observe how inhibition of mature neutrophil recruitment during the resolving phase still allowed the regression of liver damage, suggesting that immature neutrophils participate in liver regeneration. Regarding the second objective, we were able to successfully generate liver organoids from intact biopsies - called b-Orgs - from patients at different stages of ALD. The phenotypic and functional analysis of the b-Orgs showed typical characteristics of hepatocytes. In addition, single-cell transcriptomic sequencing revealed a heterogeneous cellular composition that mirrors the epithelial composition of the liver of patients with ALD. In addition, the b-Orgs preserved the characteristics of the disease, identifying the association of ELF3 with loss of hepatocyte identity and disease progression. Finally, when stimulated with AHD inducers, b-Orgs exhibited pathophysiological characteristics typical of HA, which were mitigated by prednisolone, thus demonstrating the potential of b-Orgs for personalized medicine and the identification of molecular factors in ALD. In summary, this thesis provides new experimental models for the study of ALD, and relevant knowledge about the mechanisms underlying the disease, which could have a possible application in other chronic liver diseases, as well as contribute to the development of new therapeutic strategies to slow down the progression of AHD and promote tissue regeneration.[spa] La enfermedad hepática asociada al alcohol (EHA) es una de las principales causas de discapacidad y mortalidad prematura a nivel mundial, con escasos tratamientos terapéuticos. Por lo tanto, es importante comprender los mecanismos subyacentes de la enfermedad para desarrollar nuevas terapias. La infiltración de neutrófilos en el hígado es una característica distintiva de la enfermedad hepática crónica, particularmente de la hepatitis asociada a alcohol (HA), la forma más severa de la EHA. Además, se sabe que los neutrófilos orquestan la reparación tisular, especialmente después de una lesión aguda. Sin embargo, las características y el papel de los neutrófilos en las enfermedades hepáticas crónicas, así como su contribución a la regresión del daño crónico siguen siendo desconocidos. En esta tesis hipotetizamos que los neutrófilos adaptan su dinámica de reclutamiento, fenotipo y función en respuesta a las señales del microambiente hepático, desempeñando roles distintos en la progresión y resolución de la enfermedad. Por ello, quisimos caracterizar a los neutrófilos e investigar su contribución al daño hepático crónico y la reparación tisular mediante el uso de muestras humanas y modelos murinos. Por otro lado, el estudio de la EHA está limitado por la falta de modelos preclínicos óptimos. Aunque los organoides muestran un gran potencial, los organoides hepáticos derivados de muestras de paciente están limitados a la disociación de tejidos obtenidos en etapas avanzadas de la enfermedad y a un fenotipo de colangiocito, lo que limita su utilidad. Por ello, la segunda hipótesis que formulamos es que los organoides hepáticos pueden generarse a partir de biopsias de aguja tomadas en diferentes estadios de la EHA, lo que permitiría modelar la enfermedad y testar fármacos de forma personalizada. Nuestro objetivo fue generar y caracterizar estos organoides con el fin de descubrir potenciales dianas terapéuticas y testar fármacos. Con respecto al primer objetivo, los análisis histológicos revelaron una población de neutrófilos asociados a la reacción ductular (DRANs) que interactúan estrechamente con las células epiteliales biliares y aumentan a medida que avanza la enfermedad. Demostramos mediante microscopía intravital y ensayos de marcaje de neutrófilos en ratones tratados con DDC que los DRANs permanecen inmovilizados alrededor del epitelio biliar durante períodos prolongados. Además, los neutrófilos hepáticos mostraron un perfil fenotípico y transcriptómico único. La depleción de neutrófilos utilizando un anticuerpo anti-Ly6G así como la inhibición de su reclutamiento mediante un inhibidor de CXCR1/2 redujo los DRANs, la reacción ductular, la fibrosis hepática y la angiogénesis. Por último, los neutrófilos deficientes en PAD4 y ELANE no fueron capaces de promover la proliferación de células biliares, lo que destaca el papel de las trampas extracelulares y la elastasa en la progresión de la enfermedad. Durante la resolución del daño hepático crónico, observamos una reducción y deslocalización de los DRANs, acompañada por la proliferación de hepatocitos y una regresión de la fibrosis y expansión del arbol biliar. La depleción de neutrófilos durante un período de resolución de 5 días afectó a la regeneración hepática, mostrándose una menor proliferación de hepatocitos, regresión de la fibrosis y disminución de la reacción ductular. Los neutrófilos hepáticos obtenidos durante la fase de resolución mostraron un perfil transcriptómico distintivo, enriquecido en mediadores pro-resolutivos y características de neutrófilo inmaduro. Además, pudimos observar cómo la inhibición del reclutamiento de neutrófilos maduros durante la fase resolutiva permitió aun así la regresión del daño hepático, lo que sugiere que los neutrófilos inmaduros participan en la regeneración hepática. Respecto al segundo objetivo, pudimos generar con éxito organoides hepáticos a partir de biopsias intactas - denominados como b-Orgs - de pacientes en diferentes estadios de la EHA. El análisis fenotípico y funcional de los b-Orgs mostró características típicas de los hepatocitos. Además, la secuenciación transcriptómica de célula única reveló una composición celular heterogénea que refleja la composición epitelial del hígado de los pacientes con EHA. Además, los b-Orgs preservaron las características de la enfermedad, identificando la asociación de ELF3 con la pérdida de identidad de los hepatocitos y la progresión de la enfermedad. Finalmente, cuando se estimularon con inductores de la EHA, los b-Orgs exhibieron características patofisiológicas típicas de la HA, las cuales fueron mitigadas por la prednisolona, demostrándose así el potencial de los b-Orgs para la medicina personalizada y la identificación de factores moleculares en la EHA. En resumen, esta tesis proporciona nuevos modelos experimentales para el estudio de la EHA, y conocimientos relevantes sobre los mecanismos subyacentes a la enfermedad, los cuales podrían tener una posible aplicación en otras enfermedades hepáticas crónicas, así como contribuir al desarrollo de nuevas estrategias terapéuticas para frenar la progresión de la EHA y promover la regeneración del tejido

Efecte dels polifenols sobre assaigs espectrofotomètrics per a la determinació de sucres totals

Treballs Finals de Grau de Química, Facultat de Química, Universitat de Barcelona, Any: 2024, Tutores: Sònia Sentellas Minguillón, Mercè Granados JuanPolyphenols, naturally present in plants and some foods, exhibit beneficial effects on human health due to their antioxidant capacity, acting as reducing species. Within the context of circular economy, polyphenols are being recovered from agri-food by-products. In these recovery processes, it is necessary to track the target compounds, but also possible residues, being sugars the principal concern. This project is focused on the impact of polyphenols on spectrophotometric methods to quantify sugars. Most of these methods are based on redox reactions, with sugars acting as reducing agents. This work assesses the effect of the presence of polyphenols in sugar assays in this context. DNS and BCA methods were tested showing sugar and polyphenol responses. BCA showed low LOD and LOQ compared to DNS but presented a short linearity range. Also, it has been observed that polyphenols are contributing to sugar response on both colorimetric assays. The interference of polyphenols is important when polyphenol concentration is higher or even similar to sugars concentration so, the obtained results reveal none of the methods are suitable to quantify sugars when extract purification is performed. When analysing extracts from plant samples, the comparison of results from spectrophotometric methods with those obtained by HPLC confirms that the presence of polyphenols can lead to an overestimation of total sugar