Effectively treating infectious diseases often requires a multi-step approach to target different components involved in disease pathogenesis. Similarly, the COVID-19 pandemic has become a global health crisis that requires a comprehensive understanding of Severe Acute Respiratory Syndrome Corona Virus 2 (SARS-CoV-2) infection to develop effective therapeutics. One potential strategy to instill greater immune protection against COVID-19 is boosting the innate immune system. This boosting, termed trained immunity, employs immune system modulators to train innate immune cells to produce an enhanced, non-specific immune response upon reactivation following exposure to pathogens, a process that has been studied in the context of and clinical studies prior to the COVID-19 pandemic. Evaluation of the underlying pathways that are essential to inducing protective trained immunity will provide insight into identifying potential therapeutic targets that may alleviate the COVID-19 crisis. Here we review multiple immune training agents, including Bacillus Calmette-Guérin (BCG), β-glucan, and lipopolysaccharide (LPS), and the two most popular cell types involved in trained immunity, monocytes and natural killer (NK) cells, and compare the signaling pathways involved in innate immunity. Additionally, we discuss COVID-19 trained immunity clinical trials, emphasizing the potential of trained immunity to fight SARS-CoV-2 infection. Understanding the mechanisms by which training agents activate innate immune cells to reprogram immune responses may prove beneficial in developing preventive and therapeutic targets against COVID-19.

Recent advances in the field of host immunity against parasitic nematodes have revealed the importance of macrophages in trapping tissue migratory larvae. Protective immune mechanisms against the rodent hookworm Nippostrongylus brasiliensis (Nb) are mediated, at least in part, by IL-4-activated macrophages that bind and trap larvae in the lung. However, it is still not clear how host macrophages recognize the parasite. An in vitro co-culture system of bone marrow-derived macrophages and Nb infective larvae was utilized to screen for the possible ligand-receptor pair involved in macrophage attack of larvae. Competitive binding assays revealed an important role for β-glucan recognition in the process. We further identified a role for CD11b and the non-classical pattern recognition receptor ephrin-A2 (EphA2), but not the highly expressed β-glucan dectin-1 receptor, in this process of recognition. This work raises the possibility that parasitic nematodes synthesize β-glucans and it identifies CD11b and ephrin-A2 as important pattern recognition receptors involved in the host recognition of these evolutionary old pathogens. To our knowledge, this is the first time that EphA2 has been implicated in immune responses to a helminth.

No description available.

Während der Entwicklung von Früchten der Tomate (Sorte Micro-Tom) wurde der Kohlenhydrat-Stoffwechsel untersucht. Es wurde ein Unterschied zwischen dem Metabolismus im Perikarp und dem des Plazenta-Gewebes gefunden. Stärke wurde in der Plazenta langsamer abgebaut als im Perikarp, während lösliche Zucker im Perikarp stärker akkumulierten. Die Aktivitäten der glykolytischen Enzyme tendierten zu einem Maximum 40 Tage nach der Blüte. Weiterhin wurde die Expression einiger plastidärer Transporter untersucht. Sowohl der Triosephosphat-Tranporter (TPT) als auch der Glucose-6-phosphat-Transporter wurden am stärksten in grünen Früchten exprimiert, während der Reife nahm die Expression ab. Der ATP/ADP-Transporter wurde während der Fruchtentwicklung nur schwach exprimiert.Es besteht die Hypothese, daß die Rolle der drei Enzyme plastidäre Fructose-1,6-Bisphosphatase (cp-FBPase), ADP-Glucose Pyrophosphorylase (AGPase) und Glucan Wasser Dikinase (GWD) darin besteht, die Stärke-Akkumulation in der frühen Entwicklung der Tomaten-Frucht zu beeinflussen. Diese Hypothese wurde unter Verwendung der Antisense-Technik für die plastidären FBPase (unter der Kontrolle des B33 Promoters), sowie für die AGPase und die GWD (beide unter der Kontrolle des CaMV 35S-Promoters) in der Tomaten-Kultivar Moneymaker untersucht. Die Repression von plastidärer FBPase oder AGPase in der Frucht der Tomate scheint die Metaboliten-Konzentrationen nicht so stark wie in den Blättern zu beeinflussen. Der Grund hierfür ist wahrscheinlich, daß jede Veränderung durch die Fähigkeit der Frucht, Zucker zu importieren, abgepuffert wird. Auf der anderen Seite hatte die Repression des GWD Proteins in der Frucht der Tomate starke Effekte auf die Metaboliten-Konzentrationen. / Carbohydrate metabolism was studied during the development of fruits of the tomato cultivar Micro-Tom. The metabolism of the pericarp and placental tissues was found to be different. Starch being degraded more slowly in the placenta than in the pericarp, while soluble sugars accumulated to a greater extent in the pericarp. The activities of glycolytic enzymes tended to peak at 40 days after flowering. The expression of some plastidial transporters was also studied. Both the triose phosphate transporter (TPT) and Glucose-6-Phosphate (Glc-6-P) transporter were expressed greatest in green fruits, before declining. The expression of the triose phosphate transporter (TPT) was greater than that of Glc-6-P transporter. The ATP/ADP transporter was expressed to a low level throughout fruit development. The role of three enzymes Chloroplastic Fructose-1,6-bisphosphatase (cp-FBPase), ADP-glucose Pyrophosphorylase (AGPase) and Glucan Water Dikinase (GWD) protein are thought to influence the accumulation of starch in early development in tomato fruit were studied in normal sized tomatoes of the cultivar Moneymaker using antisense technique under the control of the patatin B33 promoter in the case of cp-FBPase, and the CaMV 35S promoter in the case of AGPase and GWD protein. It appears that repression of cp-FBPase and AGPase in tomato fruits does not influence metabolite levels as greatly as it does in leaves, possibly because any alterations are buffered by the ability of the fruit to import sugars. On the other hand, the repression of GWD protein in tomato fruits has a strong effect on metabolite levels.

Made available in DSpace on 2014-06-11T19:32:55Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-06Bitstream added on 2014-06-13T21:05:14Z : No. of bitstreams: 1 vasconcelos_afd_dr_rcla.pdf: 873436 bytes, checksum: e2a838b6410dbd9cdc2bc380f1261e4e (MD5) / Exopolissacarídeos do tipo β-glucanas são polímeros produzidos por uma grande variedade de microrganismos e podem possuir diferentes propriedades físicas, químicas e aspectos estruturais. Esses biopolímeros apresentam atividades biológicas interessantes (antitumor, antiviral, anticoagulante) e aplicações comerciais como produtos em alimentos, cosméticos e farmacêuticos. Entretanto, para a aplicação dessas moléculas, é necessário primeiramente o conhecimento de suas estruturas químicas. Assim, o objetivo deste trabalho foi a produção, caracterização química de quatro exopolissacarideos (EPSGRAVIOLA, EPSMANGA, EPSPINHA e EPSLARANJA) de isolados de Botryosphaeria obtidos de frutas tropicais em decomposição e crescidos em sacarose como única fonte de carbono, determinando o melhor EPS para realizar testes de atividade anticoagulante. A homogeneidade de cada EPS foi determinada por cromatografia de filtração em gel, os quais eluíram como um único pico. Hidrólise ácida total e análise por HPAEC/PAD mostrou glucose como constituinte básico. Dados de metilação e RMN de 13C indicaram que os EPSMANGA, EPSPINHA e EPSLARANJA são glucanas lineares unidas por ligações do tipo β(1®6) e o EPSGRAVIOLA é uma glucana com ligações β(1®3) e com ramificações em C-6 de resíduos glucopiranosídicos. O espectro de FT-IR mostrou uma banda em 891 cm-1, e a espectroscopia de 13C NMR mostrou que todas as ligações eram do tipo β. Estudos realizados com o corante Vermelho Congo indicaram que os EPS possuem conformação em tripla hélice. O EPSLARANJA, uma b- D-(1®6)-glucana, foi submetido a sulfatação visando induzir a atividade anticoagulante e melhorar a solubilidade da molécula em solução, importante para a atividade biológica. Espectros de FT-IR mostraram bandas em 808 and 1252 cm-1, indicando a entrada dos grupos sulfato e as análises de RMN de 13C mostraram... / Exopolysaccharides (EPS) as β-glucans are polymers produced by a great variety of microorganisms and can possess different physical and chemical properties, and structural features. These biopolymers having interesting biological activities (antitumor, anti-viral, anticoagulant), and commercial applications in foods, cosmetics and pharmaceutical products. However, for the applications of these macromolecules, it is first necessary to understand their chemical structures. Therefore the goal of that study was the production, chemical characterization and biological activity of four exopolysaccharides (EPSGRAVIOLA, EPSMANGO, EPSPINHA and EPSORANGE) obtained from Botryosphaeria strains isolated from rotting tropical fruit grown on sucrose as carbon and the best EPS was used for the anticoagulant activity The homogeneity of each EPS was determined by gel filtration chromatography, which was eluted as a single peak. Total acid hydrolysis and HPAEC/PAD analysis of each EPS yielded only glucose. Data from methylation analysis and 13C NMR spectroscopy indicated that the EPSMANGO, EPSPINHA and EPSORANGE consisted of a linear chain of (1-6)- linked glucopyranosyl residues and EPSGRAVIOLA consisted of a main chain of glucopyranosyl (1-3) linkages substituted at O-6. FTIR spectra showed one band at 891 cm-1, and 13C NMR spectroscopy showed that all glucosidic linkages were of the β-configuration. Dye-inclusion studies with Congo Red indicated that each EPS existed in a triple-helix conformational state. The EPSORANGE, a β-(1®6)- D-glucan was submitted to a sulfation to induce anticoagulant activity and also to make this EPS more soluble, which is in favor to its biological action. The FT-IR spectrum showed bands at 808 and 1252 cm-1 indicating insertion of sulfonyl groups and the 13C NMR analysis showed that the sulfonyl groups were inserted mainly in C-4 of the b(1®6)-D-glucan... (Complete abstract click electronic access below)

Le genre Pneumocystis désigne un groupe de champignons opportunistes présentant une étroite spécificité d'hôte. Il détermine lors d'immunodépression sévère une infection pulmonaire grave, la pneumonie à Pneumocystis (PPC). La transmission de Pneumocystis par voie aérienne d'un hôte développant une PPC à un hôte susceptible a été démontrée à l'aide des modèles murins. Les travaux menés chez la souris ont montré également que des sujets immunocompétents colonisés par Pneumocystis murina peuvent transmettre le champignon à des souris immunodéprimées qui développeront une PPC ultérieurement. Les individus colonisés par Pneumocystis sp., ainsi que ceux développant une PPC, participeraient au réservoir du champignon. La survenue de cas groupés de PPC en milieu hospitalier est en faveur de la transmission interindividuelle de Pneumocystis jirovecii (P.jirovecii) chez l’homme. La détection de l'ADN de P.jirovecii dans l'air exhalé par les patients développant une PPC suggère que cette transmission se fait par voie aérienne. La caractérisation des populations infectées par P.jirovecii et la caractérisation génotypique du champignon au sein de son réservoir humain constituent la base de ce travail de recherche. Nous avons montré que la prévalence de la colonisation par P.jirovecii est faible chez les patients atteints de mucoviscidose et suivis dans notre CHU. La participation de ces patients au réservoir de P.jirovecii à Brest serait donc marginale. Cette faible prévalence pourrait être le reflet d'une faible circulation du champignon dans les communautés humaines dans notre région. Nous avons évalué le dosage du ß-1,3-D glucane sérique pour dépister les populations infectées. Ce dosage couplé à la détection de P.jirovecii dans les prélèvements respiratoires par la microscopie et la PCR, permet de différencier les patients développant une PPC et les patients présentant une colonisation pulmonaire par P.jirovecii. De plus, les premières données sur le ß-1,3-D glucane au cours de la primo-infection chez le nourrisson ont été obtenues.En termes de caractérisation de P.jirovecii dans notre région, l'analyse du locus dihydropteroate synthase (DHPS) a montré que: i) le lieu habituel de résidence plutôt que le lieu de diagnostic de l’infection à P.jirovecii serait un facteur prédictif d’infection par un mutant, ii) P.jirovecii pourrait circuler en France d’une région à une autre via des voyageurs infectés, iii) la prévalence de mutants potentiellement résistants chez les patients vivant effectivement à Brest était de 0%. L'analyse des séquences des "internal transcribed spacers" (ITS) 1 et 2 de P. jirovecii conforte l'hypothèse que les patients développant une PPC et les patients colonisés sont infectés par des populations fongiques présentant des caractéristiques identiques. Tous les patients, quelle que soit la présentation clinique de leur infection, constitueraient un réservoir unique et commun de P.jirovecii. Les travaux de génotypage ont constitué l'étape préalable nécessaire à l'analyse de cas groupés d'infections à P.jirovecii survenus chez des patients transplantés rénaux au CHU de Brest. Nous avons apporté des données originales sur le rôle des patients colonisés en tant que source potentielle de P. jirovecii dans un contexte d'acquisition et de transmission nosocomiales du champignon. Par ailleurs, la concordance partielle ou complète des génotypes ITS et DHPS dans les couples "prélèvements d'air–LBA" réalisés chez des patients développant une PPC est compatible avec l’exhalation du champignon et sa diffusion aérienne dans l’environnement hospitalier. Ces données apportent des arguments pour l'application de mesures de prévention des infections nosocomiales à P. jirovecii. Les précautions "gouttelettes" recommandées par la Société Française d'Hygiène Hospitalière devraient être appliquées a minima aux patients développant une PPC. Nous proposons leur extension aux patients colonisés par le champignon. / The genus Pneumocystis represents a group of opportunistic fungi that show strong host specificity. It is the cause of severe pneumonia (Pneumocystis Pneumonia [PCP]) in immunocompromised subjects. Pneumocystis transmission from a host with PCP to another susceptible host via the airborne route has been demonstrated in rodent models. Moreover, it has been established that Pneumocystis murina can be transmitted from immunocompetent mice, transiently colonized by the fungus, to immunocompromised susceptible mice that subsequently develop PCP. Colonized subjects and those developing PCP may be part of the fungus reservoir. Reports of PCP case cluster in hospital strongly suggest that Pneumocystis jirovecii (P.jirovecii) transmission in humans may also occur. P.jirovecii DNA detection in the air surrounding PCP patients is consistent with the transmission of P.jirovecii via the airborne route.Our goals were to characterize human populations infected with P.jirovecii and to characterize P.jirovecii within its human reservoir. We showed that P.jirovecii was rarely involved in pulmonary colonization in patients with cystic fibrosis monitored in the Brest Hospital. Thus this patient population was not part of the human reservoir of the fungus in our region (Brittany, Western France). This low prevalence of colonization may reflect a low level of P.jirovecii circulation within human communities in Brittany. In order to improve the identification of patients infected with P.jirovecii, we evaluated ß-1,3-D glucan detection in serum samples. We showed that serum ß-1,3-D glucan levels combined with P.jirovecii detection in pulmonary samples using microscopic examination and a PCR assay make it possible to distinguish between PCP and pulmonary colonization. Moreover the first data on ß-1,3-D glucan levels during primary infection were obtained.In order to characterize P.jirovecii in our region, we performed the typing of P.jirovecii isolates from infected patients monitored at Brest hospital, using the dihydropteroate synthase (DHPS) and the internal transcribed spacer (ITS) 1 and 2 locus analysis. DHPS typing showed that i) the usual city of patient residence rather that the city in which the diagnosis of P.jirovecii infection has been made is a predictor of mutants, ii) mutants can be imported from one region to another through infected visitors, iii) the prevalence of mutants potentially resistant to sulfonamides was 0% in patients who effectively lived in the Brest geographic area. Results of ITS analysis in PCP patients and colonized patients are consistent with the hypothesis that these 2 patient groups are infected with similar P.jirovecii populations. All infected patients, whatever their clinical presentation, may be part of a common and unique reservoir of the fungus. We investigated an outbreak of P.jirovecii infections in 18 renal transplant recipients using the same typing method combined with patient encounter analysis. The results provided evidence of the role of colonized patients as potential sources of P.jirovecii. The same typing method was applied to pairs of pulmonary samples and room air samples of PCP patients. Full or partial matches of P.jirovecii types in pulmonary and air sample pairs were observed. These results are consistent with P.jirovecii exhalation by PCP patients in their close environment. These data support arguments for applying droplet precautions, at least to PCP patients, to prevent P.jirovecii transmission, as recommended by the "Société française d'hygiène hospitalière". We suggest extending droplet precautions to colonized patients to achieve the prevention of P.jirovecii nosocomial infections.

Activation of the immune system during photodynamic therapy (PDT) and improvement of the effector functions of mAbs - these are the ways to use and enhance the potential of the immune system to fight cancer. Tumor cells lack β-glucan as a surface compo¬nent and can‘t trigger complement receptor 3-dependent cellular cytoto¬xicity and initiate tumor-killing activity during PDT. So, it gave rise to the hypothesis that β-glucan in combination with PDT will produce more effective killing of by iC3b fragment opsonized tumor cells. The human Fc portion is essential for the recruiting of human effector immune cells to produce antitumor effect. Therefore, connection of Fv portion of murine anti-CD7 antibody with Fc portion of human IgG1 can be helpful for such protein to obtain ideal feathers. However, each modification of the monoclonal antibody can cause the lost or decrease in the rate of protein expression and antigen-binding properties. Monoclonal antibody products are unique in their molecules. Because of post-translational modifications that often occur during the fermentation process, the final product is heterogeneous. Therefore, careful characterization of monoclonal antibodies is required in order to assess their identity, purity, potency and safety. Response of Lewis lung carcinoma tumor to PDT modulated by β-glucan was assessed in mice and functional characteristics of novel purified chimeric anti-CD7 antibody was tested in this study. / Imuninės sistemos aktyvinimas taikant fotosensibilizacinę terapiją (FNT) ir monokloninių antikūnų efektorinių funkcijų gerinimas yra vienas iš būdų, kaip naudoti ir sustiprinti organizmo imuninės sistemos veiksnių potencialą kovai su naviku. Navikinės ląstelės membranos paviršiuje nėra β-gliukano, todėl FNT metu negali būti aktyvinamas nuo komplemento receptoriaus 3 priklausomas ląstelės citotoksiškumo mechanizmas bei sukeliama navikinės ląstelės žūtis. Todėl galima tikėtis, kad β-gliukanas ir FNT, taikomi kartu, gali aktyvinti iC3b frag¬mento opsonizuotų navikinių ląstelių žūtį. Norint aktyvinti žmogaus imunines ląsteles priešnavikiniam poveikiui sukelti, pelės monokloniniam antikūnui būtina žmogaus imunoglobulino Fc dalis. Todėl pelės antikūno prieš CD7 vienos grandinės Fv fragmento sujungimas su žmogaus imunoglobulino G1 Fc dalimi baltymui gali suteikti idealias savybes. Tačiau, bet kokia monokloninio antikūno modifikacija gali keisti jo savybes ir, išskiriant baltymą, dėl daugiapakopio proceso ji gali keistis. Todėl, norint įvertinti chimerinio CD7-Fc antikūno identiškumą, grynumą, veiksmingumą bei saugumą, būtina atlikti tyrimus, kuriais būtų galima nustatyti, ar antikūnas išlaiko specifiškumą antigenui, kaip aktyviai jungiasi prie jo, ar aktyvina imuninius mechanizmus. Šio tyrimo metu buvo vertintas pelėms įskiepytos Luiso plaučių karcinomos atsakas į FNT moduliaciją β-gliukanu ir išgrynintas chimerinis antikūnas prieš CD7 bei ištirtos jo funkcinės savybės in vitro.

Les flavonoïdes glycosylés sont des métabolites secondaires d’origine végétale, qui présentent de nombreuses propriétés physico-chimiques et biologiques intéressantes pour des applications industrielles. La glycosylation accroît généralement la solubilité de ces flavonoïdes mais leurs faibles niveaux de production dans les plantes limitent leur disponibilité. Ces travaux de thèse portent donc sur le développement de nouvelles voies de gluco-diversification des flavonoïdes naturels, en mettant à profit l’ingénierie des protéines. Deux transglucosylases recombinantes, structurellement et biochimiquement caractérisées, l'amylosaccharase de Neisseria polysaccharea et la glucane-saccharase de branchement α-(1→2), forme tronquée de la dextran-saccharase de L. Mesenteroides NRRL B-1299, ont été sélectionnées pour la biosynthèse de nouveaux flavonoïdes, possédant des motifs originaux d’α-glycosylation, et potentiellement une solubilité accrue dans l'eau. Dans un premier temps, une librairie de petite taille de mutants de l’amylosaccharase, ciblée sur le site de liaison à l’accepteur, à été criblée en présence de saccharose (donneur d’unité glycosyl) et de lutéoline comme accepteur. Une méthode de screening a donc été développée, et a permis d’isoler des mutants améliorés pour la synthèse de nouveaux glucosides de lutéoline, jusqu’à 17000 fois plus soluble dans l’eau que la lutéoline aglycon. Afin de glucosyler d’autres flavonoïdes, la glucane-saccharase de branchement α-(1→2), a été préférentiellement sélectionnée. Des plans expérimentaux alliés à une méthodologie en surface de réponse ont été réalisés pour optimiser la production de l’enzyme sous forme soluble et éviter la formation de corps d’inclusion. Cinq paramètres ont été ainsi analysés : le temps de culture, la température, et les concentrations en glycérol, lactose (inducteur) et glucose (répresseur). En appliquant les conditions optimales prédites, 5740 U.L-1 de culture d’enzyme soluble ont été produites en microplaques, alors qu’aucune activité n’était retrouvée dans la fraction soluble, lors de l’utilisation de la méthode de production précédemment utilisée. Finalement, Une approche de modélisation moléculaire, structurellement guidés par l’arrimage de flavonoïdes monoglucosylés dans le site actif de l’enzyme, a permis d’identifier des cibles de mutagenèse et de générer des libraries de quelques milliers de variants. Une méthode rapide de criblage sur milieu solide, basée sur la visualisation colorimétrique d’un changement de pH, a été mise au point. Les mutants encore actifs sur saccharose ont été sélectionnés puis analysés sur leur capacités à glucosyler la quercétine et la diosmétine. Une petite série de 23 mutants a ainsi été retenue comme plate-forme d’enzymes améliorées dédiées à la glucosylation de flavonoïdes et a été évalués pour la glycosylation de six flavonoïdes distincts. La promiscuité, remarquablement générée dans cette plateforme, à permis d’isoler quelques mutants beaucoup plus efficaces que l’enzyme sauvage, produisant des motifs de glucosylation différents et fournissant des informations intéressante pour le design et l’amélioration des outils enzymatiques de glucosylation des flavonoïdes. / Flavonoid glycosides are natural plant secondary metabolites exhibiting many physicochemical and biological properties. Glycosylation usually improves flavonoid solubility but access to flavonoid glycosides is limited by their low production levels in plants. In this thesis work, the focus was placed on the development of new glucodiversification routes of natural flavonoids by taking advantage of protein engineering. Two biochemically and structurally characterized recombinant transglucosylases, the amylosucrase from Neisseria polysaccharea and the α-(1→2) branching sucrase, a truncated form of the dextransucrase from L. Mesenteroides NRRL B-1299, were selected to attempt glucosylation of different flavonoids, synthesize new α-glucoside derivatives with original patterns of glucosylation and hopefully improved their water-solubility. First, a small-size library of amylosucrase variants showing mutations in their acceptor binding site was screened in the presence of sucrose (glucosyl donor) and luteolin acceptor. A screening procedure was developed. It allowed isolating several mutants improved for luteolin glucosylation and synthesizing of novel luteolin glucosides, which exhibited up to a 17,000-fold increase of solubility in water. To attempt glucosylation of other types of flavonoids, the α-(1→2) branching sucrase, naturally designed for acceptor reaction, was preferred. Experimental design and Response Surface Methodology were first used to optimize the production of soluble enzyme and avoid inclusion body formation. Five parameters were included in the design: culture duration, temperature and concentrations of glycerol, lactose inducer and glucose repressor. Using the predicted optimal conditions, 5740 U. L-1of culture of soluble enzyme were obtained in microtiter plates, while no activity was obtained in the soluble fraction when using the previously reported method of production. A structurally-guided approach, based on flavonoids monoglucosides docking in the enzyme active site, was then applied to identify mutagenesis targets and generate libraries of several thousand variants. They were screened using a rapid pH-based screening assay, implemented for this purpose. This allowed sorting out mutants still active on sucrose that were subsequently assayed for both quercetin and diosmetin glucosylation. A small set of 23 variants, constituting a platform of enzymes improved for the glucosylation of these two flavonoids was retained and evaluated for the glucosylation of a six distinct flavonoids. Remarkably, the promiscuity generated in this platform allowed isolating several variants much more efficient than the wild-type enzyme. They produced different glucosylation patterns, and provided valuable information to further design and improve flavonoid glucosylation enzymatic tools.

Tesis por compendio / [ES] El objetivo de esta tesis doctoral ha sido proporcionar conocimientos fundamentales y prácticos relacionados con la valorización de la biomasa de setas, para comprender la relación estructura-funcionalidad de extractos acuosos ricos en ß-glucanos, en términos de capacidad inmunorreguladora, antioxidante y antiviral, así como las propiedades tecnológicas (gelificantes y emulsionantes). El objetivo de esta tesis doctoral también ha sido explorar la viabilidad de dicha biomasa en el desarrollo de materiales compostables para el envasado de alimentos. Inicialmente, se aplicó un proceso de extracción secuencial, que implicaba varios tratamientos consecutivos tanto acuosos con y sin temperatura como alcalinos, aplicados a setas de gran consumo (P. ostreatus, L. edodes y G. frondosa), con la finalidad de comprender cómo las diferencias iniciales en la composición y la arquitectura de la pared celular afectaban a la extracción de ß-glucano. A partir de los resultados obtenidos, se analizó en profundidad la aplicación potencial de extractos acuosos, ricos en ß-glucanos, del género Pleurotus, explorando cómo la composición de los extractos y la complejidad estructural de ß-glucanos afectaban a su capacidad inmunorreguladora. Los resultados evidenciaron que tanto Pleurotus ostreatus como sus estipes mostraron los mejores resultados, con una actividad inmunoestimulante mucho mayor que las otras especies de Pleurotus exploradas. En la segunda parte de esta tesis, se evaluaron las propiedades funcionales (antivirales y antioxidantes) y tecnológicas (gelificantes y emulsionantes) de los extractos acuosos de ß-glucano, purificados y sin purificar, de Pleurotus ostreatus y sus estipes. El proceso de purificación, como era de esperar, incrementó el porcentaje en carbohidratos (con un mayor aumento en los estipes), lo que se tradujo en una mayor viscosidad y capacidad gelificante. Además, el extracto obtenido de los estipes mostró una fuerte actividad antiviral frente a norovirus murino, probablemente atribuida a la mayor complejidad estructural de sus ß-glucanos. Aunque la presencia de proteínas en los extractos acuosos de ß-glucanos potenció sus propiedades emulsionantes, esta propiedad fue dependiente de la accesibilidad de la proteína para adsorberse en la interfase O/W, lo que también afectó a la viscosidad de las emulsiones resultantes. La quitina y los ß-glucanos son dos de los principales carbohidratos de las setas, y tienen un gran potencial en la formación de materiales de envasado. En la última parte de esta tesis, se investigó la viabilidad de la biomasa de residuos de setas para desarrollar materiales de envasado de alimentos biodegradables y compostables. Los resultados mostraron que la composición de la biomasa de champiñón y las temperaturas de procesado tuvieron un impacto en las propiedades fisicoquímicas de los films desarrollados y, todas fueron biodesintegrables en condiciones de compostaje según la norma ISO 20200. Por lo tanto, esta tesis doctoral representa un importante avance en la valorización de la biomasa de setas (seta entera y estipes) y pone de relieve su idoneidad para desarrollar nuevos ingredientes funcionales y materiales de envasado para aplicaciones alimentarias y de envasado de alimentos. / [CA] L'objectiu d'aquesta tesi ha sigut proporcionar els coneixements fonamentals i pràctics relacionats amb la valorització de la biomassa de bolets, per a comprendre la relació estructura-funcionalitat d'extractes aquosos rics en ß-glucans, en termes de capacitat immunoreguladora, antioxidant i antiviral, així com de les propietats tecnològiques (gelificants i emulsionants). L'objectiu d'aquesta tesi ha estat també l'exploració de la viabilitat d'aquesta biomassa pel desenvolupament de materials compostables per a l'envasat d'aliments. Inicialment, es va aplicar un procés d'extracció seqüencial, que implicava diversos tractaments consecutius, tant aquosos amb temperatura i sense, com alcalins, aplicats a bolets de gran consum (P. ostreatus, L. edodes i G. frondosa), per tal de comprendre com les diferències inicials en la composició i l'arquitectura de la paret cel·lular afectaven l'extracció de ß-glucà. A partir dels resultats obtinguts, es va analitzar en profunditat la potencial aplicació d'extractes aquosos, rics en ß-glucans, del gènere Pleurotus, explorant així com la composició dels extractes i la complexitat structural dels ß-glucans afectaven a la seua capacitat immunoreguladora. Els resultats van evidenciar que tant Pleurotus ostreatus com les seues estipes mostraren els millors resultats, amb una activitat immunoestimulant molt més gran que la obtinguda per a les altres espècies de Pleurotus explorades. A la segona part d'aquesta tesi, es van avaluar les propietats funcionals (antivirals i antioxidants) i tecnològiques (gelificants i emulsionants) dels extractes aquosos de ß-glucà, purificats i sense purificar, de Pleurotus ostreatus i les seues estipes. El procés de purificació, com calia esperar, va incrementar el percentatge en carbohidrats (amb un major augment en els estipes), cosa que es va traduir en una major viscositat i capacitat gelificant. A més, l'extracte obtingut dels estipes va mostrar una forta activitat antiviral contra norovirus murí, probablement atribuïda a la complexitat estructural més gran dels seus ß-glucans. Tot i que la presència de proteïnes en els extractes aquosos de ß-glucans va potenciar les seues propietats emulsionants, aquesta propietat va ser dependent de l'accessibilitat de la proteïna per adsorbir-se a la interfase O/W, cosa que també va afectar la viscositat de les emulsions resultants. La quitina i els ß-glucans són dos dels principals carbohidrats dels bolets, i tenen un gran potencial en la formació de materials d'envasament. Així, a la darrera part d'aquesta tesi, es va investigar la viabilitat de la biomassa de residus de bolets per desenvolupar materials biodegradables i compostables d'envasat d'aliments. Els resultats van mostrar que la composició de la biomassa de bolets i les temperatures de processament van tenir un impacte en les propietats fisicoquímiques de les pel·lícules desenvolupades i, totes elles van ser biodesintegrables en condicions de compostatge segons la norma ISO 20200. Per tant, aquesta tesi doctoral representa un important pas endavant en la valorització de la biomassa de bolets (sencers i estipes) i posa en relleu la seua idoneïtat per desenvolupar nous ingredients funcionals i materials d'envasament per a aplicacions alimentàries i d'envasat d'aliments. / [EN] This doctoral thesis was aimed at providing fundamental and practical knowledge related to the valorisation of underexploited mushroom biomass, in order to understand the structure-function relationship of ß-glucan aqueous extracts in terms of immunoregulatory, antioxidant and antiviral capacity as well as technological (gelling and emulsifying) properties. It was also the aim of this PhD thesis to explore the feasibility of mushroom biomass to develop compostable food packaging materials. Initially, a sequential fractionation process, involving several consecutive cold or hot aqueous and alkaline treatments, was applied to widely consumed mushrooms (P. ostreatus, L. edodes and G. frondosa), in order to understand how the initial differences in composition and cell wall architecture affected ß-glucan extraction. Based on the results, the potential application of ¿-glucan aqueous extracts from Pleurotus genus was deeply analysed, thus exploring how the composition of the extracts and structural complexity of ¿-glucans affected their immunoregulatory capacity. The results evidenced that both Pleurotus ostreatus and its stipes showed the best results, with a much higher immunostimulant activity than the other explored Pleurotus species. In the second part of the thesis, functional (antiviral and antioxidant) and technological (gelling and emulsifying) properties of purified and unpurified¿¿-glucan aqueous extracts from Pleurotus ostreatus and its stipes were evaluated. The purification process, as expected, increased the carbohydrates content (greater in those obtained from the stipes), which resulted in a greater viscosity and gelling capacity. Furthermore, the extract obtained from the stipes showed a strong antiviral activity against murine norovirus, probably ascribed to the higher structural complexity of ß-glucans. Although the presence of proteins in the ¿-glucan aqueous extracts enhanced their emulsifying properties, it depended on the accessibility of the protein to adsorb at the O/W interphase, which also affected the viscosity of the resulting emulsions. Chitin and ß-glucans are two of the major carbohydrates of mushrooms, having a great potential in the formation of packaging materials. Thus, in the last part of this thesis, the feasibility of mushroom waste biomass to develop biodegradable and compostable food packaging materials was investigated. The results showed that the composition of mushroom biomass and the processing temperatures had an impact on the physicochemical properties of the developed films and, all of them were biodisintegrated under composting conditions according to ISO 20200. Therefore, this PhD thesis represents a significant step forward in the understanding of the discarded mushroom biomass (whole biomass and stipes) for valorisation purposes and highlights its suitability to develop new cost-efficient functional ingredients and packaging materials for food and food packaging applications. / This work was performed with the financial support of the CIEN project BIOPRO from “Centro para el Desarrollo Tecnológico Industrial” (CDTI), Ministry of Science and Innovation, Government of Spain. The Accreditation as Center of Excellence Severo Ochoa CEX2021-001189-S funded by MCIN/AEI/10.13039/501100011033 is also fully acknowledged. his study forms part of the AGROALNEXT programme and was supported by MCIN with funding from European Union NextGenerationEU (PRTR-C17. I1) and by Generalitat Valenciana. Synchrotron experiments were performed at NCD beamline at ALBA Synchrotron with the collaboration of ALBA staff (proposal 2,022,025,569). IF was supported by a postdoctoral contract grant for the requalification of the Spanish university system from the Ministry of Universities of the Government of Spain, financed by the European Union (NextGeneration EU) (MS21-006). / Pérez Bassart, Z. (2024). Fungal Biomass Valorization for Obtaining Functional Food-Related Materials [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203841 / Compendio

Regular starches contain two principal types of glucan polymers: amylopectin and amylose. The structure of amylopectin is characterized according to the unit chain length profile and the nature of the branching pattern, which determine the alignment of glucan chains during biosynthesis. The organization of glucan chains in amylopectin and their impact on the structure of starch are still open to debate. The location of amylose and its exact contribution to the assembly of crystalline lamellae in regular and high-amylose starch granules also remain unknown. The primary focus of this thesis is the organization and flexibility of glucan chains in crystalline lamellae. The organization and flexibility of glucan chains in native, annealed (ANN), and heat-moisture treated (HMT) normal, waxy, hylon V, hylon VII, and hylon VIII corn starches were examined. This study has shown for the first time that increased amounts of apparent amylose in B-type starches hinder the polymorphic transition (from B to A+B) during HMT. The research has also demonstrated that an iodine-glucan complex transformed the B-type polymorphic pattern of hylon starches into a V-type pattern. The differential scanning calorimetry (DSC) results showed that ANN- and HMT-induced changes were most pronounced in hylon starches. These findings suggest that the glucan tie chains influences the assembly of crystalline lamellae in high-amylose starches. The relationship between the internal unit chain composition of amylopectin, and the thermal properties and annealing of starches from four different structural types of amylopectin was investigated by DSC. The onset gelatinization temperature (To) correlated negatively with the number of building blocks in clusters (NBbl) and positively with the inter-block chain length (IB-CL). The enthalpy of gelatinization (∆H) correlated positively with the external chain length of amylopectin. Annealing results showed that starches with a short IB-CL were most susceptible to ANN, as evidenced by a greater increase in the To and Tm. The increase in enthalpy was greater in starches with long external chains and IB-CLs. These data suggest that the internal organization of glucan chains in amylopectin determines the alignment of chains within the crystalline lamellae and thereby the thermal properties and annealing of the starch granules.