INTRODUÇÃO: A determinação do genótipo RHD fetal no plasma materno é um teste de diagnóstico pré-natal não invasivo oferecido a gestantes RhD negativo que apresentam potencial de sensibilização e/ou Doença Hemolítica Perinatal. Atualmente, este exame é realizado de rotina em diversos países, mas não no Brasil. A Clínica Obstétrica do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (HCFMUSP) oferece atendimento terciário a gestantes RhD negativo, com monitorização dos títulos de anticorpos irregulares, administração da imunoglobulina anti-D e/ou terapêutica fetal, quando necessários. OBJETIVO: Avaliar a acurácia do teste semiautomatizado para determinação do genótipo RHD fetal no plasma materno. METODOLOGIA: Foram coletadas prospectivamente amostras de sangue de 220 gestantes RhD negativo, com idade gestacional entre 8-28 semanas. O plasma foi obtido em no máximo 2 horas após a coleta, e uma alíquota de 1 mL foi submetida à extração de ácidos nucléicos no equipamento automatizado MagNA Pure Compact (Roche), empregando o kit Large Volume. O DNA extraído foi submetido a PCR em tempo real (Step One Plus - Applied Biosystems), usando o protocolo do grupo SAFE, que tem como alvos os éxons 5 e 7 do gene RHD. RESULTADOS: Ocorreu exclusão de 35 amostras devido a problemas pré-analíticos, aborto ou desconhecimento do fenótipo do recém-nascido. Entre as 185 amostras analisadas, 130 (70,2%) foram genotipadas como RHD+ e 55 (29,8%) RHD-. Os resultados obtidos foram comparados com a fenotipagem do cordão umbilical, e houve concordância completa (100%). Sete amostras exibiram amplificação exclusiva para o éxon 7. Essas amostras foram submetidas aos protocolos em PCR convencional, e PCR em tempo real específico para o pseudogene RHD. Ambos os ensaios apresentaram os mesmos resultados: cinco positivos e dois negativos. Nesses mesmos 7 casos, após extração da camada de leucócitos materna, os protocolos foram repetidos, e o resultado confirmou que cinco mães eram RHD. As duas amostras com resultado negativo foram submetidas ao protocolo Multiplex, envolvendo os éxons 3-9 do gene RHD, com resultados negativos, confirmando que as mães são verdadeiramente RHD- portanto o sinal do éxon 7 é provindo dos fetos que são D variantes. CONCLUSÃO: O método para a determinação do RHD fetal no plasma materno descrito demonstrou ser rápido, de fácil execução, alta precisão e reprodutível, além de indicar possíveis variantes RHD em nossa população / BACKGROUND: Fetal RHD genotype determination in maternal plasma is a noninvasive prenatal diagnostic test performed in RhD negative pregnant women at risk of alloimmunization and/or Hemolytic Disease of Fetus and Newborn. Currently, this test is routinely performed in many countries but not in Brazil. The Department of Obstetrics at Hospital das Clínicas, São Paulo University Medical School provides tertiary antenatal care for RhD negative pregnant women including anti-D immunoglobulin administration, antibody levels monitoring and intrauterine treatment if necessary. AIMS: To validate the accuracy of a semi-automated test for fetal RHD genotype determination in maternal plasma. METHODS: Two-hundred and twenty blood samples were prospectively collected between 8 and 28 weeks of gestational age. Plasma processing was performed within 2 hours after blood collection, and nucleic acids were extracted from 1mL aliquots with an automated extraction platform (MagNA Pure Compact Roche) and the Large Volume kit. RHD gene exons 5 and 7 were amplified with real-time PCR (Step One Plus - Applied Biosystems) using the SAFE group protocol. RESULTS: Thirty-five samples were excluded due to pre-analytical problems, miscarriage and missing follow-up. In the remaining 185 samples, 130 (70.2%) were genotyped as RhD+ and 55 (29.8%) RhD-. Comparison with umbilical cord blood group phenotype showed 100% concordance. Seven samples showed amplification for exon 7 only. These were further investigated with conventional and real-time PCR with an specific protocol for RHD? pseudogene: 5 were positive and 2, negative. In these 7 cases, maternal buffy-coat DNA analysis also confirmed that 5 women were RHD?. In the remaining 2 cases, a multiplex protocol directed at RHD gene exons 3-9 confirmed that both mothers were truly RhD negative so exon 7 signal comes from the fetuses, further found to harbor D variants. CONCLUSION: The present study demonstrates that fetal RHD determination in maternal plasma is a fast, easy-to-perform and reproducible technique with high accuracy in our population. Moreover, it helps in the identification of possible RHD variants in our population

Les travaux présentés dans cette thèse font suite à celle de Melle LE GOFF. Ils se concentrent sur la technologie HIFI brevetée et développée pendant ses travaux. Une première partie du travail présenté dans ce manuscrit concerne le test HIFI Blood 96™ et plus particulièrement les améliorations et les évolutions apportées au test afin d'en faire un véritable outil de génotypage, multiparamétrique et haut-débit pouvant être installé dans les banques de sang dans le but de constituer des inventaires de sang génotypé de façon étendue, participant ainsi à améliorer la sécurité transfusionnelle. Il permet de caractériser 96 échantillons sur 15 polymorphismes (divisés en deux panels) associés aux groupes sanguins en approximativement 4h30. Cette plateforme a fait l'objet d'une étude de validation à moyenne échelle sur 583 donneurs pour le panel 1 et 190 donneurs pour le panel 2. La deuxième partie des travaux décrit l'adaptation de la technologie HIFI appliquée au diagnostic des pathologies respiratoires, avec le développement d'une autre plateforme, ReSynPlex, en partenariat avec 3 équipes de recherche de Grenoble / The work reported in this thesis follows the one undertaken by Ms LE GOFF. It is focused on HIFI technology, which is patented and developed during her thesis. The first part of this work concerns the HIFI Blood 96™ test, and particularly the improvements and developments adduced to the test to make it a real diagnostic tool, multiparametric and high-throughput which can be implemented in blood banks in order to constitute negative antigen inventories, thus contributing to improve blood safety. It allows to characterize 96 samples on 15 polymorphisms (divided in two panels) associated to blood group systems in approximately 4.5 hours. A mesoscale validation study has been conducted on 583 samples for panel 1 and 190 samples for panel 2. The second part of this work describes the adaptation of HIFI technology applied to diagnosis of respiratory tract infections, with the development of another platform, ReSynPlex, in partnership with 3 research teams in Grenoble

The evolutionary history of H. neanderthalensis, interwoven with that of H. sapiens, has always fascinated the scientific world. Recent adavncess in paleogenetics shedds new light on the phylogenetic relationship between Neandertals and modern humans. The studies developed in this thesis intend principally to control the contaminants through the development of an anti-contamination protocol for decreasing the human contamination in pre-laboratory phases. We designed a PCR-based method specific for reducing human contamination during the laboratory analysis, and we analyzed the fragmentation pattern of the ancient sequences by massively parallel sequencing technologies. Furthermore, we studied two nuclear genes, TAS2R38 -associated to bitter taste perception- and ABO blood group system –involved in natural immunity- that provide specific information on aspects of the Neanderthal phenotype and adaptation. / La historia evolutiva d’H. neanderthalensis, imbricada amb la d’H. sapiens, ha fascinat sempre el món científic. Avenços recents en paleogenètica aporten una nova llum sobre la rel•lació filogenètica entre els neandertals i els humans moderns. Els treballs d’aquesta tesi intenten principalment controlar els contaminants mitjançant el desenvolupament d’un protocol d’anti-contaminació que disminueixi la contaminació humana de les mostres en la fase de pre-laboratori. Hem desenvolupat un mètode basat en la PCR específic per a reduïr els contaminants humans durant l’anàlisi en el laboratori, i hem analitzat el patró de fragmentació de les seqüències antigues amb tècniques de seqüenciació massiva en paral•lel. A més a més, hem estudiat dos gens nuclears, el TAS2R38 –associat a la percepció del gust amarg- i el grup sanguini ABO –implicat en la immunitat natural- que proporcionen informació específca sobre aspectes del fenotip i de les adaptacions dels neandertals.

Les norovirus sont l'une des causes principales de gastroentérite. Depuis 2002, des variants de norovirus GII.4 successifs ont circulé dans la population par cycle de 2-3 ans, ce qui suscite des interrogations quant au rôle de leurs ligands, les antigènes tissulaires de groupes sanguins (HBGA), dans leur évolution. Nous avons analysé l'interaction entre des variants de GII.4 représentatifs et des HBGA, et déterminé le rôle d’acides aminés (aa) clés. Par mutagénèse dirigée, nous avons montré qu’une configuration stricte des aa directement impliqués dans l’accroche est indispensable. La suppression de la thréonine 395, caractéristique des variants après 2002, confère la capacité de se lier à Lex et Si-Lex, démontrant que les aa en dehors du site de liaison peuvent modifier les propriétés d’attachement. L'analyse de l'accroche de VLP de 6 variants isolés de 1987 à 2007 à des échantillons de salive phénotypés et des HBGA synthétiques montre que tous les variants sont capables de s’attacher à la salive des sécréteurs indépendamment du phénotype ABO et aux oligosaccharides propres au phénotype sécréteur. Deux variants récents ont pu également s’accrocher aux sucres présents dans la salive des nonsécréteurs Le(+). Nos données suggèrent que la capacité de se lier à Lex et Si-Lex serait une conséquence de la variation génétique des aa situés à proximité du site de liaison. L'analyse des propriétés d’attachement par résonance plasmonique de surface a montré que seuls les variants après 2002 présentent une affinité forte pour les antigènes A et B, suggérant que l’accélération évolutive des GII.4 pourrait être liée à une affinité accrue des variants pour les HBGA après 2002. / Noroviruses are one of the leading causes of gastroenteritis worldwide. Since 2002 successive GII.4 variants have circulated in the population before being replaced every 2-3 years, which raises questions about the role of their histo-blood group antigen (HBGAs) receptors in their evolution. We analyzed the interaction between representative GII.4 variants and HBGAs and determined the role of selected amino acids (aa) in the binding profiles. By mutagenesis, we showed that there was a strict structural requirement for the aa directly implicated in HBGA bindings. The ablation of the threonine 395 residue, an epidemiological feature of the post 2002 variants, allowed to gain the capacity to bind to the Lewis x and sialyl-Lewis x antigens, demonstrating that aa residues outside the HBGA binding site can modify the binding properties. The analysis of the attachment of VLPs from 6 variants isolated from 1987 to 2007 to phenotyped saliva samples and synthetic HBGAs shows that all variants could attach to saliva of secretors irrespective of the ABO phenotype and to oligosaccharides characteristic of the secretor phenotype. Interestingly, two recent variants additionally bound to carbohydrates present in the saliva of Lewis-positive non-secretors. Our data suggest that GII.4 binding to Lex and Si-Lex antigens might be a by-product of the genetic variation of the aa located in the vicinity of the binding site. Analysis of the binding properties by surface plasmon resonance showed that only post 2002 variants presented a strong affinity for A and B antigens, suggesting that the GII.4 evolution could be related to an increased affinity for HBGAs for the post 2002 variants.

No description available.

Tesis por compendio / [ES] Los oligosacáridos de la leche humana (OLHs) y la parte glicana de los glicoconjugados son hidrolizados por las glicosil hidrolasas (GHs), las cuales son expresadas por la microbiota intestinal de niños lactantes promoviendo el establecimiento de una microbiota intestinal con beneficios para su salud. El objetivo de esta Tesis Doctoral consistió en la caracterización funcional de GHs de la microbiota intestinal de niños lactantes capaces de metabolizar OLHs y glicoconjugados, y el estudio de su relevancia biológica y su potencial biotecnológico. Se aislaron cepas bacterianas a partir de heces de niños lactantes.Solo las cepas del género Bifidobacterium metabolizaron alguno de los OLHs testados. Bifidobacterium infantis Y538 consumió eficientemente todos los OLHs testados, mientras que las dos cepas de Bifidobacterium dentium Y510 y Y521 solo metabolizaron lacto-N-tetraosa (LNT) y lacto-N-neotetraosa (LNnT). Se caracterizaron dos ß-galactosidasas de B. dentium Y510; Bdg42A mostró la mayor actividad en LNT, hidrolizándolo en galactosa y lacto-N-triosa (LNTII), mientras que Bdg2A mostró actividad contra lactosa, 6'-galactopiranósil-N-acetilglucosamina, N-acetillactosamina y LNnT. También se aislaron cepas bacterianas con actividad glicosidasa extracelular de las heces de lactantes. B. infantis E17 y E18, y Enterococcus faecalis E8 y E41 exhibieron actividad de endo-ß-N-acetilglucosaminidasa, liberando N-glicanos de glicoproteínas. La endo-ß-N-acetilglucosaminidasa EndoE de E. faecalis E8 deglicosiló eficientemente la proteína S1 del coronavirus 2 del síndrome respiratorio agudo severo (SARS-CoV-2). Tanto la EndoE silvestre como un mutante catalíticamente inactive mostraron actividad lectina frente a la proteína S1 y actividad neutralizante frente a la infección de un virus pseudotipado que presenta la proteína S de SARS-CoV-2 expresada. También se identificaron GHs putativas a través del análisis metagenómico de las heces de niños lactantes, pertenecientes a los géneros Bifidobacterium, Bacteroides, Ruminococcus, Actinomyces, Klebsiella, Phocaeicola y Streptococcus. Se seleccionaron diez ¿-L-fucosidasas GH29 (Fuc18, Fuc19A, Fuc30, Fuc35A, Fuc35B, Fuc39, Fuc193, Fuc1584, Fuc2358 y Fuc5372). Las ¿-L-fucosidasas Fuc18, Fuc19A, Fuc35B, Fuc39 y Fuc1584 mostraron actividad hidrolítica frente a enlaces de fucosa ¿-1,3/4 y Fuc35A, Fuc193 y Fuc2358 mostraron actividad enlaces de fucosa ¿-1,2/3/4/6. Fuc30 mostró actividad sobre la enlaces de fucosa ¿-1,6 mientras que Fuc5372 mostró preferencia por los enlaces ¿-1,2. Fuc2358 mostró actividad frente a glicoconjugados con lacto-N-fucopentaosa II, lacto-N-fucopentaosa III y contra la mucina. Fuc18, Fuc19A y Fuc39 eliminaron fucosa de neoglicoproteínas y de la glicoproteína ¿-1 ácida. Las ¿-L-fucosidasas aisladas fueron evaluadas por su capacidad para sintetizar fucosil-oligosacáridos (FUS) a través de reacciones de transfucosilación. Fuc2358 produjo rendimientos del 35% de 2'-fucosillactosa (2'FL) y también 3'-fucosillactosa (3'FL) y 1-fucosillactosa (1FL). Fuc5372 sintetizó 2'FL, 3'FL y 1FL, con una proporción más alta de 3'FL. Se llevó a cabo mutagénesis dirigida para aumentar los rendimientos de transfucosilación. Los mutantes Fuc2358-H132F, Fuc2358-F184H, Fuc2358-R301Q, Fuc2358-K286R y Fuc5372-R230K mostraron una mayor relación entre la 2'FL producida y el pNP-Fuc hidrolizado que sus respectivas enzimas silvestres. Además, se observó que los residuos F184 de Fuc2358 y W151 de Fuc5378 afectan a la regioselectividad de la transfucosilación, la fenilalanina aumentando la selectividad por los enlaces ¿-1,2 y el triptófano aumentando la selectividad por los enlaces ¿-1,3. Los resultados presentados muestran la diversidad de GHs presentes en la microbiota intestinal de niños lactantes y expanden el conocimiento sobre su especificidad, contribuyendo al conocimiento del posible papel de las GHs en la colonización bacteriana del tracto gastrointestinal y, además, muestra su potencial biotecnológico / [CA] Els oligosacàrids de la llet humana (OLHs) i la part glicana de glicoconjugats són hidrolitzats per les glicosil hidrolases (GHs), les quals són expressades per la microbiota intestinal de xiquets lactants, promovent l'establiment d'una microbiota intestinal amb beneficis per a la seua salut. L'objectiu d'aquesta Tesi Doctoral va ser la caracterització funcional de GHs de la microbiota intestinal de xiquets lactants capaços de metabolitzar OLHs i glicoconjugats i l'estudi de la seua rellevància biològica i el seu potencial biotecnològic. Es van aïllar soques bacterianes a partir de les femtes de xiquets lactants. Només els soques del gènere Bifidobacterium van metabolitzar algun dels OLHs testats. Bifidobacterium infantis Y538 va consumir eficientment tots els OLHs testats. Les dos soques de Bifidobacterium dentium Y510 i Y521 sol van metabolitzar lacto-N-tetraosa (LNT) i lacto-N-neotetraosa (LNnT).Es van caracteritzar dos ß-galactosidasas de B. dentium Y510; Bdg42A va exhibir la major activitat enfront de LNT, hidrolitzant-la en galactosa i lacto-N-triosa (LNTII), mentre que Bdg2A va mostrar activitat enfront de lactosa, 6'-galactopiranósil-N-acetilglucosamina, N-acetillactosamina i LNnT. També es van aïllar soques bacterianes amb activitat glicosidasa extracelul·lar. B. infantis E17 i E18, i Enterococcus faecalis E8 i E41 van exhibir activitat endo-ß-N-acetilglucosaminidasa, alliberant N-glicans de glicoproteïnes. La endo-ß-N-acetilglucosaminidasa EndoE de E. faecalis E8 va deglicosilar eficientment la proteïna S1 del coronavirus 2 del síndrome respiratori agut greu (SARS-CoV-2).Tant la EndoE salvatge com un mutant catalíticament inactiu van mostrar activitat lectina enfront de la proteïna S1 i activitat neutralitzador enfront de la infecció d'un virus pseudotipat que presenta la proteïna S de SARS-CoV-2 expressada. També es van identificar GHs putatives a través de l'anàlisi metagenómic de la femta de xiquets lactants, pertanyents als gèneres Bifidobacterium, Bacteroides, Ruminococcus, Actinomyces, Klebsiella, Phocaeicola i Streptococcus. Es van seleccionar deu ¿-L-fucosidasas GH29 (Fuc18, Fuc19A, Fuc30, Fuc35A, Fuc35B, Fuc39, Fuc193, Fuc1584, Fuc2358 i Fuc5372). Les ¿-L-fucosidasas Fuc18, Fuc19A, Fuc35B, Fuc39 i Fuc1584 van mostrar activitat hidrolítica enfront d'enllaços de fucosa ¿-1,3/4 i Fuc35A, Fuc193 i Fuc2358 van mostrar activitat enllaços de fucosa ¿-1,2/3/4/6. Fuc30 va mostrar activitat enfront d'enllaços de fucosa ¿-1,6 mentre que Fuc5372 va mostrar preferència pels enllaços ¿-1,2. Fuc2358 va mostrar activitat enfront de glicoconjugats amb lacto-N-fucopentaosa II, lacto-N-fucopentaosa III i contra la glicoproteïna de la mucina. Fuc18, Fuc19A i Fuc39 van eliminar fucosa de neoglicoproteïnes i de la glicoproteïna ¿-1 àcida. Les ¿-L-fucosidasas aïllades van ser avaluades per la seua capacitat per a sintetitzar fucosil-oligosacàrids (FUS) mediant reaccions de transfucosilació. Fuc2358 va produir rendiments del 35% de 2'-fucosillactosa (2'FL) i també 3'-fucosillactosa (3'FL) i 1-fucosillactosa (1FL). Fuc5372 va sintetitzar 2'FL, 3'FL i 1FL, amb una proporció més alta de 3'FL. Es va dur a terme mutagénesis dirigida per a augmentar els rendiments de transfucosilación. Els mutants Fuc2358-H132F, Fuc2358-F184H, Fuc2358-R301Q, Fuc2358-K286R i Fuc5372-R230K van mostrar una major relació entre la 2'FL produïda i el pNP-Fuc hidrolitzat que els seus respectius enzims salvatges.A més, els residus F184 de Fuc2358 i W151 de Fuc5378 afecten la regioselectivitat de la transfucosilación; la fenilalanina augmenta la selectivitat pels enllaços ¿-1,2 i el triptòfan augmenta la selectivitat pels enllaços ¿-1,3. Els resultats presentats mostren la diversitat de GHs presents en la microbiota intestinal de xiquets lactants i expandixen el coneixement sobre la seua especificitat, contribuint al coneixement del possible paper de les GHs en la colonització bacteriana del tracte gastrointestinal i, a més, mostra el seu potencial biotecnològic. / [EN] Human milk oligosaccharides (HMOs) and the glycan portion of glycoconjugates are hydrolyzed by glycoside hydrolases (GHs) that are expressed by the neonatal intestinal microbiota, promoting the establishment of an intestinal microbiota with health benefits for infants. The objective of this Doctoral Thesis consisted of the functional characterization of GHs from the intestinal microbiota of breastfed infants capable of metabolizing HMOs and glycoconjugates and the study of their biological relevance and their biotechnological potential. Bacterial strains were isolated from breastfed infant faeces, showing that only Bifidobacterium genus strains metabolized any of the HMOs tested. Bifidobacterium infantis Y538 efficiently consumed all tested HMOs, while the two strains isolated from Bifidobacterium dentium Y510 and Y521 only metabolized lacto-N-tetraose (LNT) and lacto-N-neotetraose (LNnT). Two ß-galactosidases from B. dentium Y510 were characterized; Bdg42A exhibited the highest activity on LNT, hydrolyzing it into galactose and lacto-N-triose (LNTII), while Bdg2A displayed activity against lactose, 6'-galactopyranosyl-N-acetylglucosamine, N-acetyllactosamine and LNnT. Bacterial strains with extracellular glycosidase activity were also isolated from breastfed infant faeces. B. infantis E17 and E18, and Enterococcus faecalis E8 and E41 exhibited endo-ß-N-acetylglucosaminidase activity, releasing N-glycans from glycoproteins. The endo-ß-N-acetylglucosaminidase EndoE from E. faecalis E8 efficiently deglycosylated the spike S1 protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Both the EndoE wild-type and a catalytically inactive mutant exhibited lectin activity towards the S1 protein and neutralizing activity against SARS-CoV-2 S pseudotyped virus infection. Putative GHs were also identified through metagenomic analysis of breastfed infant faeces, belonging to Bifidobacterium, Bacteroides, Ruminococcus, Actinomyces, Klebsiella, Phocaeicola, and Streptococcus genera. Ten ¿-L-fucosidases GH29 (Fuc18, Fuc19A, Fuc30, Fuc35A, Fuc35B, Fuc39, Fuc193, Fuc1584, Fuc2358, and Fuc5372) were selected. The ¿-L-fucosidases Fuc18, Fuc19A, Fuc35B, Fuc39, and Fuc1584 showed hydrolytic activity on ¿-1,3/4-linked fucose and Fuc35A, Fuc193 and Fuc2358 showed activity on ¿-1,2/3/4/6-linked fucose. Fuc30 displayed activity only on ¿-1,6-linked fucose, and Fuc5372 showed a preference for ¿-1,2-linked fucose. Fuc2358 displayed activity against glycoconjugates carrying lacto-N-fucopentaose II, lacto-N-fucopentaose III and against the mucin glycoprotein. Fuc18, Fuc19A, and Fuc39 removed fucose from neoglycoproteins and human ¿-1 acid glycoprotein. The isolated ¿-L-fucosidases were evaluated for their capacity to synthesize fucosyl-oligosaccharides (FUS) through transfucosylation reactions. Fuc2358 produced 35 % yields of 2'-fucosyllactose (2'FL) and also 3'-fucosyllactose (3'FL) and 1-fucosyllactose (1FL). Fuc5372 synthesized 2'FL, 3'FL and 1FL, with a higher proportion of 3'FL. Site-directed mutagenesis was conducted to increase the transglycosylation yields. Mutants Fuc2358-H132F, Fuc2358-F184H, Fuc2358-R301Q, Fuc2358-K286R and Fuc5372-R230K showed a higher ratio between 2'FL yields and hydrolyzed pNP-Fuc than their respective wild-type enzymes. The transfucosylation activity results also showed that the residues F184 of Fuc2358 and W151 of Fuc5378 affect transfucosylation regioselectivity, with phenylalanine increasing the selectivity for ¿-1,2 linkages and tryptophan for ¿-1,3 linkages. The results presented in this doctoral thesis illustrate the diversity of GHs in the intestinal microbiota of breastfed infants and have expanded our knowledge of their specificities, which could contribute to a better understanding of the possible role of GHs in the bacterial colonization of the infant gastrointestinal tract and presents significant biotechnological potential. / This work is part of the Grant PID2020-115403RB (C21 and C22) funded by the Spanish Ministry of Science and Innovation (MICIN)/Spanish State Research Agency (AEI)/10.13039/501100011033. The study was also supported by Valencian Government grant AICO/2021/033. EMM-G was supported by the Grant PRE2018-085768 funded by MICIN/AEI/10.13039/501100011033 and by “ESF Investing in your future”. / Moya Gonzálvez, EM. (2024). Isolation, Functional Characterization and Biotechnological Applications of Glycoside Hydrolases from the Intestinal Microbiota of Breastfed Infants [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/203171 / Compendio